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PROJECT SYNOPOSE S
EUR 20495
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EUROPEAN COMMISSION
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European Commission
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ANTIMICROBIAL
RESISTANCE
RESEARCH
in the Quality Of Life Programme
Editor:
Dr. Anna Lönnroth
European Commission
Life sciences, genomics and biotechnology for health
EUR 20495
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Luxembourg: Office for Official Publications of the European Communities, 2003
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Contents
Dynamics of the evolution of antimicrobial drug resistance
10
European Resistance intervention study - reducing resistance in respiratory tract pathogens in children
12
Development and evaluation of nucleic acid amplification methods for the detection of respiratory pathogens
in community acquired pneumonia
14
Pneumococcal disease in Europe
16
Severe Streptococcus pyogenes disease in Europe
18
Microbiological and structural strategies for the diagnostics and epidemiology of Pseudomonas aeruginosa infections
20
Structural and functional genomics of Mycobacterium tuberculosis
22
New strategies for treatment and prevention of Mycobacterial diseases
24
Inhibitors of the non-mevalonate pathway of isoprenoid biosynthesis as drugs against tuberculosis
26
Novel drug targets specific to persistent (latent) tuberculosis infection: crystallisation,
structure determination and functional studies
28
Development of integratable sensors for screening of antibiotic resistance in Mycobacterium
30
Characterisation of Mycobacterium tuberculosis populations during infection:
a longitudinal study on drug resistance development
32
New generation genetic markers and techniques for the emidemiology and control of tuberculosis
34
Surveillance of tuberculosis in Europe
36
Improved diagnosis, drug resistance detection and control of tuberculosis in Latin America
38
Development of antimicrobial peptides as novel anti-infective drugs
40
Bacterial two-component systems as targets for the development of novel antibacterials and anti-infectives
42
Replication initiation protein as new targets for bacterial growth inhibition
44
Molecular mechanism of resistance: application in drug development
46
Type IV secretion systems as targets for anti-infectious therapies
48
New antimicrobial targeting translation in bacteria and fungi
50
Towards new antibiotics
52
Novel non-antibiotic treatment of staphyloccal diseases
54
Development of novel anti-bacterials and anti-infectives that target programmed bacterial cell death
56
Comparative analysis of proteome modulation in human pathogenic bacteria for the identification of new vaccines,
diagnostics and antibacterial drug targets
58
Glycosylation engineering for novel antibiotics
60
Metabolic engineering of glycopeptide antibiotics : technology, optimisation and production
62
Novel sources of Actinomycete Diversity for Detection of Antimicrobial Agents with Pharmaceutical Applications
64
Combinatorial biosynthesis: generation of novel therapeutic substances
by combining genes from actinomycetes and cyanobacteria
66
Designing and improving health and food related production processes using filamentous fungal cell factories
68
Discovery of a new class of bioactive compounds: Bacterial conjugation inhibitors
70
Novel inhibitors of adhesin/receptor interactions involved in microbial infection at mucosal surfaces
72
Treatment and prevention of bacterial infections by anti-adhesion compounds
74
Control of lung infection, allergy and inflammation:
Assessment of the therapeutic potential of recombinant forms of the lung surfactant proteins SP-A and SP-D
76
Development of a photodynamic treatment to eradicate and control the current spread
of infectious antibiotic resistant microorganisms in man
78
Antimicrobial endotoxin neutralising peptides to combat infectious diseases
80
Screening assays for new bacterial inhibitors based on targets active in septation
82
Rapid antibiotoc susceptibility testing using dielectrophoresis
84
Differential diagnosis of infectious lung diseases
86
Development of strategies for control and prevention of antibiotic resistance in European hospitals
88
Scientific evaluation on the use of antimicrobial agents in human therapy
90
Towards controlling antimicrobial use and resistance in low-income countries - an intervention study in Latin America
92
Antibiotic Resistance surveillance & control in the Mediterranean region
94
TREAT-a system for balancing antibiotic treatment against development of drug resistance
96
Self-medication with antibiotics and resistance levels in Europe
98
TV-film: “The battle against antibiotic resistant bacteria”
100
Network for automated bacterial strain fingerprinting in Europe
102
Impact of meningococcal epidemiology and population biology on public health in Europe
104
Strengthening international Salmonella surveillance through strain typing and differentiation
106
Human enteric pathogen surveillance network
108
European antimicrobial resistance surveillance system
110
European network of nosocomial infections (implementation phase II)
112
Novel approaches for the control of fungal disease
114
Novel approaches to combat multidrug resistance (MDR) in pathogenic yeast
116
Exploiting yeast cell wall for high throughput screening of antimicrobial agents
118
New tools to investigate and suppress HIV drug resistance
120
HIV reverse transcriptase inhibitor and its consequences for viral virulence
122
Rational approaches towards understanding and overcoming HIV protease inhibitor resistance
124
Strategy to control spread of HIV drug resistance
126
The EuroSIDA network 2000-2004. Clinical and virological outcome of European patients infected with HIV
128
Emerging variants of Hepatitis B virus: New tools for epidemiological survey, diagnosis of infection,
and monitoring of drug resistance
130
Analysis of the molecular mechanisms of Hepatitis C Virus (HCV) resistance to antiviral therapy
132
Adhesive interactions in malaria: new targets for intervention
134
Malarial chemotherapy targeting Plasmodial phospholipid biosynthesis:
implementation of a prodrug strategy for orally active compounds
136
Genetic analysis of the chloroquine drug resistance and the accelerated-resistance-to-multiple-drugs
phenotypes in the human malarial parasite Plasmodium falciparum
138
Development of a malaria resistance DNA chip as a public health tool for the management
of Plasmodium falciparum malaria drug resistance
140
Miltefosine for Leishmaniasis: Molecular basis of mechanisms of action, resistance and combination therapy
142
Molecular tools for monitoring emergence and spreading of drug resistance among natural populations of Leishmania
144
Praziquantel resistance in African Schistosomiasis
146
Praziquantel: its central role in the chemotherapy of schistosome infection
148
Antimicrobial resistance transfer from and between gram-positive bacteria of the digestive tract
and consequences for virulence
150
Antibiotic resistance in bacteria of animal origin - II
152
Pathology and ecology of the genus Clostridium in humans, animals and foodstuffs:
Identification, epidemiology and prophylaxis
154
Rapid antibiotic detection for illegal and unlicenced substances in animal feedingstuffs
156
Defining and validating gut health criteria in young pigs, based on digestive physiology,
microbiology and mucosal immunology investigations for testing alternative strategies to in-feed antibiotics
158
Spore probiotics: an alternative to antibiotics in animal husbandry
160
Development of a competitive exclusion product for poultry meeting the regulatory requirements in the European Union
162
Multi-residue determination of (flouro) quinolone antibiotics in foodstuffs from animal origin by LC-MS-MS
164
Cartridges with molecularly imprinted recognition elements for antibiotic residues monitoring in milk
166
Biosafety evaluation of probiotoc lactic acid bacteria used for human consumption
168
Antimicrobials Online
170
Safety evaluation of horizontal gene transfer from genetically modified organisms to the microflora
of the food chain and human gut
172
An ecologically safe selection system for transgenic crops based on modified plant-tubulin genes
174
Sea lice resistance to chemotherapeutants: diagnosis, mechanisms, dynamics and control
176
Improved procedures for flatfish larval rearig through the use of probiotic bacteria
178
Hazard analysis of antimicrobial resistance associated with Asian aquacultural environments
180
Resist on resistance: Mobilising the research efforts for combating multi-resistance against antibiotics
182
5
Acknowledgements
The preparation of this catalogue would not have been possible without the contribution of all project coordinators
responsible for the projects presented. Considerable support in compiling the material has been provided by
Mrs. Tuija Jansson, Ms. Christine Dell’Osso, Mr. Thomas Jussen. Dr. Alain Vanvossel, head of unit for Major Diseases of
DG RTD/F-2 at the European Commission provided supervision and guideance. The help and support of numerous
other colleagues at DG RTD, DG SANCO, DG INFSO and the Joint Research Centre are also highly acknowledged.
6
Foreword
The growing threat of antimicrobial resistance is an issue of great
concern to public health. The EU Council of health ministers has recognised
that preservation of the effectiveness of drugs for the treatment of infections can not be achieved by national initiatives alone. It rather requires a
common approach and coordinated action at EU and international level, as
concluded in the Council Resolution on antimicrobial resistance of June
1999 "A strategy against the microbial threat". The EU firstly took action to
reduce and phase out the use of antibiotics as growth promoters in farm animals. In November 2001, the Council went on to focus its attention to improved medical use of antimicrobials
through its “Recommendation on the prudent use of antimicrobial agents in human medicine”. Research
constitutes an essential component in all aspects of the EU’s strategy to address these issues.
Antimicrobial resistance is a multi-faceted problem that requires a coordinated and multi-disciplinary response. The genomic revolution has generated a wealth of microbial and human genome information
with novel approaches to an improved understanding of basic molecular mechanisms of resistance, monitoring of resistance and development of drugs and diagnostic tests. The time is now ripe to start mobilising this
new knowledge towards more focused strategies and targeted clinical applications. Europe offers unique
expertise and resources to address these issues in an effective way. What is first needed, however, is coordination of already existing activities and resources. This is why the fight against antimicrobial resistance has
obtained a prominent position in the first priority “Genomics and biotechnology for health” in the Sixth
Framework Programme for Research and Technological Development (2002-2006), launched in December
2002. The Sixth Framework Programme enables the mobilisation of a critical mass of resources and knowledge in networks of excellence and integrated projects that hold promise to de-fragment the field and to
bring Europe to the front line of world class research.
In October 2001, as a step in the preparation of research priorities for the Sixth Framework
Programme, DG Research published an inventory of ongoing research projects addressing various aspects of
antimicrobial resistance, funded by the Quality of Life programme of the Fifth Framework Programme. This
catalogue described 38 projects with a total EU contribution of € 50 million. It helped to raise awareness of
who is doing what in research on antimicrobial resistance in Europe, to stimulate the debate on future orientations in the field, and to serve as a tool to identify potential partners for new collaborative initiatives. The
feed-back from the scientific community and other stakeholders was so positive that I thought it useful to
publish an updated edition that presents a more comprehensive set of projects in this field, launched throughout the whole Fifth Framework Programme up to its closure at the end of 2002.
This revised inventory encompasses research projects relevant to antimicrobial resistance, most of
which are funded by the Quality of Life programme (key actions 1, 2, 3, 5 and generic activities), but some also
by the International co-operation programme and Information Society Technologies programme. Furthermore,
the catalogue describes an important activity in this area carried out at the Commission’s Joint Research
Centre in Geel, Belgium. The present publication comprises over 80 research projects with a total Commission
contribution of more than € 100 million.
In addition to the described research projects, the Commission’s service for Health and Consumer
Protection (DG SANCO) is funding projects on surveillance and public perception issues related to antimicrobial resistance. This revised edition includes information about seven DG SANCO funded projects of high relevance to the fight against antimicrobial resistance. The Sixth Framework Programme has also put in place specific measures to provide scientific support for monitoring antimicrobial resistance in the context of the
Community network on the epidemiological surveillance of communicable diseases.
It is our common responsibility in Europe to secure the availability of effective antimicrobials for
future generations. It is therefore essential to ensure that our efforts to tackle antimicrobial resistance will be
growing faster than the challenges posed by these escalating problems. I hope that this revised inventory of
EU funded research projects on antimicrobial resistance will be a valuable tool to both scientists and policy
makers in order to accelerate European action to combat resistance. Effective collaboration in the field will
bring us yet another step closer to realising a true European Research Area, where the research component
is becoming increasingly integrated with other EU policies.
Philippe Busquin
7
Introduction
Drug resistance in microbes is a natural phenomenon that arises as an evolutionary response to the
exposure of anti-infective drugs. Careless use of these precious drugs over a long time has now resulted in a
situation where many of them are rapidly losing their efficacy. Not only are antibiotics often used against
minor bacterial infections, but also against viral infections against which they are totally inefficient. In developing countries, the problem is rather under-use due to lack of access to good quality drugs or because
patients can not afford a full antibiotic regimen, all of which are factors that contribute to the emergence of
drug resistance. In addition to human medical and veterinary use, anti-microbials have also been extensively
consumed for the sole purpose of accelerating livestock fattening and antibiotic resistance genes have been
used as selection markers in certain transgenic crops. In recent years, however, there has been a raising awareness of the link between the use of anti-infectives and the emergence of resistance.
Although these lessons have been learned almost exclusively in relation to the use of anti-bacterials, the correlation of use and resistance applies also to the other types of anti-infective drugs, i.e. the antiviral, anti-fungal and anti-parasitic drugs.
This publication gives an overview of the vast scope of EU supported research projects that have
been launched in this area during the years 1999-2002, corresponding to the duration of the Fifth Framework
programme for Research and Technological Development. The projects presented here cover a broad range of
objectives and approaches, but their common denominator is that they all, directly or indirectly, aim to contribute to the fight against antimicrobial resistance.
This catalogue is an updated and extended edition of a first version that was published in October
2001. All the previously presented 38 projects re-appear again here, but another 42 research projects have
been added. Some of these newly added projects were launched after the publication of the previous edition
of the catalogue. Others have been added because this new version has an extended scope across a broader
range of programmes. For instance, several projects targeting specific conditions in other regions of the world
have been included from the International co-operation programme. Also included is one project funded by
DG INFSO on the development of an informatics tool for prescribers and a core activity of the European
Commission’s Joint Research Centre on the measurement of antibiotic residues in food. The total EU contribution of the presented projects amounts to over € 100 million in research support.
Research can not act in isolation. Therefore, the currently ongoing public health activities funded by
DG Health and Consumer Protection in the area of antimicrobial resistance have also been added for a more
complete overview. These projects address the link between consumption of antimicrobials and resistance,
surveillance of specific groups of pathogens, as well as public perception issues and they represent important supplements to the research actions funded by DG Research.
EU funded projects in this area cover a large number of different bacterial, fungal, protozoan and
viral target pathogens known to generate drug resistance problems of public health importance. These pathogens include among others Streptococcus pneumoniae, Staphylcoccus aureus, Mycobacterium tuberculosis,
Candida sp., Plasmodium falciparum and HIV.
Many projects focus on the development of new classes of antimicrobials and alternative strategies,
and on the development of new diagnostic and susceptibility tests. Other projects investigate basic mechanisms of resistance, modes of transmission or novel intervention strategies in the hospital or the community.
The previous edition identified a total lack of projects on resistance in farm animals and aquaculture. These areas were better covered towards the end of the programme.
However, in order not to distort the overview, the Commission’s substantial efforts to support vaccine research, although recognised as a powerful tool to indirectly reduce use of antimicrobials through infection control, have not been included in this review.
Fierce action is needed at many fronts, but most significantly, the field must be advanced through
addressing the excessive fragmentation and heterogeneity that currently characterises it. The challenge taken
on board by the future European research strategy on antimicrobial resistance is to channel the wide scope
of novel opportunities offered in human and microbial genomics towards the development of new antimicrobial drugs, diagnostic tests and new tools for surveillance, that build on new sustainable structures for future coordinated research actions. The Sixth Framework Programme will aim to further these efforts towards clinical applications.
Anna Lönnroth
Brussels, October 2003
DYNAMICS OF THE EVOLUTION OF
ANTIMICROBIAL DRUG RESISTANCE
SUMMARY
This project aims to generate knowledge concerning the rates and trajectories of the evolution, spread and
stability of antibiotic resistance. The project targets resistance in bacteria constituting significant health risks
as well as economic costs such as Streptococcus pneumoniae, Helicobacter pylori, Salmonella typhimurium and
Escherichia coli, but offers deliverables that should have a general applicability to any bacteria and resistance.
PROBLEM
The evolution and spread of antibiotic resistance has partly been caused by an extensive use and overuse of antibiotics and suboptimal infection control practices within
EU as well as worldwide. Due to the rapid spread of resistance, accelerated by population migration and travelling, infections that were previously treatable might no
longer be so, or might require treatment with more expensive or less efficient drugs.
Thus, antibiotic resistance represents a major public health concern and economic
problem. Whether we can slow down, reverse or prevent this resistance development
is unclear. A major obstacle is that current knowledge is insufficient to describe, predict or prevent the development of antimicrobial drug resistance in a rational manner.
This is due to a poor understanding of the different microbial, host and treatment
parameters that determine the rates and trajectories of the evolution, spread and stability of resistance.
AIM
1. Examine the importance of mutator genes and the host on the rate of resistance development
2. Examine the effect of different dosage regimes on resistance development.
3. Determine fitness costs of resistance and how these costs can be genetically compensated
EXPECTED RESULTS
1. To provide experimental measurements of some of the parameters that set the rates of antibiotic resistance development in bacteria.
2. To develop and examine specific strategies to prevent and reverse antibiotic resistance, by in
particular, alternative treatment regimes.
3. To provide the knowledge base required to develop novel diagnostic test systems for resistant bacterial clones with a high risk of resistance development; e.g. high mutation rates, low
costs, efficient compensation.
4. To provide part of the knowledge required to predict resistance development and the value
of intervention strategies.
10
Acronym : DEAR
Project number : QLK2-2001-00873
EC contribution : 1.300.000 €
Duration : 36 months
Starting date : December 1st 2001
Contract Type : Shared cost
Keywords : antibiotic resistance, infectious disease, pathogenic bacteria, biological cost, genetic compensation, mutation rate, antibiotic
treatment,
POTENTIAL APPLICATIONS
The results obtained from this network of researchers will (1) provide the basis for mathematical modeling and risk assessment for the development, and spread of resistance to any given
antibiotic. The achievements made here will also form (2) the basis for assessing the success
potential of any intervention strategy against resistance development made in the society.
Likewise, this project contributes to the (3) base knowledge required to formulate and to interpret intervention strategies attempting to achieve reversibility of an already existing resistance
problem in the society. Only by understanding the correlation between mode of treatment
regime and resistance development will it be possible to provide solid information to those regulatory bodies that give out treatment recommendations to acting physicians. The approaches
suggested in this project will also be useful for pharmaceutical companies and drug-licencing
agencies when they are to assess the potential risk for resistance development towards both
new and established antibiotics. Finally, the methodology and approaches will make it possible to (4) identify particular attributes in resistant bacteria (e.g. clones that have no fitness cost
of being resistant) allowing for their rapid dissimination in the community, and hence provide
the basis for early warning diagnostic systems forecasting rapidly spreading resistant bacterial clones. Resistant bacteria that through compensation of fitness cost have developed an
altered virulence in humans have to be identified, and constantly monitored in the society to
prevent the spread of infectious disease in Europe with novel and potentially devastating
effects. By identifying attributes in sensitive bacteria predisposing for later resistance development (e.g. presence of mutators) this project promises to provide the knowledge base on
which to develop diagnostic systems forecasting resistance development within sensitive bacterial communities.
PROJECT CO-ORDINATOR:
Dan I Andersson
Swedish Institute for Infectious Disease
Control
Nobels väg 18 - SE-171 82 Solna - Sweden
T: +46 8 4572432; F: +46 8 301797
Email: [email protected],
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PARTNERS:
Fernando Baquero
Department of Microbiology at the Ramón y
Cajal Hospital
Carretera de Colmenar Km. 9.100
ES-28034 Madrid - Spain
Otto Cars M.D
Department of Infectious Diseases and
Clinical Microbiology
Head of Antibiotic Research Unit
Uppsala University Hospital
Uppsala Sweden
Lars G Engstrand
Control
Jurgen Reden
European Federation for the Pharmaceutical
Industry Association (EFPIA)
Scientific and Regulatory Affairs
Avenue Louise 250 - BE-1050 Bruxelles - Belgium
Regine Hakenbeck
University of Kaiserslautern
Department of Microbiology
Paul Ehrlich Strasse - DE-67663 Kaiserslautern
Germany
Birgitta Henriques
Microbiology and Tumorbiology Center (MTC)
and Swedish Institute for Infectious Disease
Control
Diarmaid Hughes
Department of Cell and Molecular Biology
Biomedical Center
Box 596 - Uppsala University - SE-751 24 Uppsala
Sweden
Staffan Normark
Microbiology and Tumorbiology Center (MTC)
Karolinska Institutet
SE-171 77 Stockholm
Sweden
Niels Frimodt-Møller
Dept. of Clinical Microbiology
Statens Serum Institut
5 Artillerivej - DK-2300 Copenhagen S - Denmark
11
EUROPEAN RESISTANCE INTERVENTION
STUDY REDUCING RESISTANCE
IN RESPIRATORY TRACT PATHOGENS
IN CHILDREN
SUMMARY
The aim of EURIS is to introduce and test the efficacy of intervention strategies by which the prevalence of
resistant strains of Streptococcus pneumoniae colonizing children attending day-care centers (DCCs) could be
reduced. The interventions and strain collections will be carried out by centers located in four countries. Three
additional collaborating units will assist as reference centers for harmonization of methods in clinical
microbiology; molecular epidemiology of drug resistance genes and clones; data management and mathematical
modelling (including risk assessment) of epidemiological aspects of EURIS. The final deliverable will include a
bioinformatic tool to disseminate the conclusions and predictive models identified.
PROBLEM
Antibiotic-resistant S. pneumoniae is one of the major community-acquired pathogens with
global annual mortality rates of over one million. The ecology and diseases caused by S. pneumoniae are strongly associated with children. The nasopharynx of children also appears to be
the major global reservoir of this species namely of antibiotic resistant strains, associated
almost exclusively with capsular serotypes unique to the paediatric flora. In most developed
countries an increasing proportion of children of pre-school age are recruited into DCCs producing social/epidemiological structures in which the children are together in close physical
proximity greatly increasing the chances of transfer of drug-resistant pneumococci.
AIM
Four European centers will introduce and test several intervention strategies to lower carriage
of drug-resistant pneumococci in the nasopharynx of children attending DCCs. Isolates will be
collected and characterized– before, during and after intervention to evaluate the impact of the
following:
1. reduced antibiotic use.
2. optimization of antibiotic dosing during therapy.
3. improvement of hygienic conditions in DCCs.
4. infection control measures involving mandatory notification of drug-resistant strains and isolation of carriers.
All demographic, microbiological and epidemiological data will be deposited in a common data
base for statistical analysis and identification of risk factors. This wealth of information will be
analyzed by an internet interfaced Knowledge Engine which will incorporate artificial intelligence based modeling tools - to evaluate alternative management scenarios for the control of
the epidemiology of antibiotic resistance carriage and disease.
Clone identification by pulsed field gel electrophoresis and cluster analysis
Disk diffusion test
12
EXPECTED RESULTS
1. Establishment of completely uniform microbiological and molecular typing techniques and
data management tools.
2. Use of harmonized methods to obtain a comprehensive view of the frequency and clonal
types of drug resistant pneumococci colonizing children in four European countries.
3. Evaluation and comparison of the impacts of the different interventions on carriage rates.
4. All data will be fed into a Knowledge Engine and analysed by artificial intelligence based
tools to construct models for the epidemiology of drug-resistance carriage and disease. The
resulting web-based tool is being designed to enable later development of global epidemiological monitoring system based on the submission of typing profiles.
Acronym : EURIS
Starting date : September 1st 2000
Keywords : Pneumococcalcarriage, drug-resistance,
day-care centers, intervention to reduce resistance, molecular
fingerprinting of drug-resistant pneumococcal clones, Knowledge Engines.
We anticipate that the results of the project will give an important contribution to the control
and containment of resistance and on the relative power of several different intervention
methods in reducing the carriage of resistant pneumococci. Such information is invaluable
for decision-makers in public health in designing policies to reduce the rate of antimicrobial
resistance in Europe (and elsewhere). The results to be obtained may be adapted and extended to other countries in the future and the best strategies might lead to recommendations to
be adopted by the European Union. The development of global epidemiological information
systems are the necessary response to a problem that is global in nature. The clinical studies
and the bioinformatic tools developed by EURIS have that long term goal in mind.
Herminia de Lencastre
Molecular Genetics Laboratory (ITQB)
Universidade Nova de Lisboa (UNL) –
ITQB/UNL
Rua da Quinta Grande 6 - Apartado 127
PT-2780 Oeiras Cedex - Portugal
T: +351 21 44 69 870; F: +351 1 442 8766
[email protected]
Web-site: www.itqb.unl.pt/
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PARTNERS:
Thorolfur Gudnason (PC), Vilhjalmur A.
Arason
Department of Pediatrics
National University Hospital
P.O. Box 1465 - 121 Reykjavik
Iceland
T: +354 560 1000; F: +354 560 1055
Email: [email protected]
Jonas Almeida
Biomathematics Group
Instituto de Biologia Experimental e
Tecnolológica (IBET)
Apartado 12 - PT-2781-901 Oeiras
Portugal
T: +351 21 446 9852; F: +351 21 442 8766
Email: [email protected];
Web-site: www.itqb.unl.pt/
Karl G. Kristinsson
Department of Microbiology National
University Hospital
P.O. Box 1465 - 121 Reykjavik
Iceland
T: +354 560 1952; F: +354 560 1957
Regine Hakenbeck
University of Kaiserslautern
Paul Ehrlich Strasse, Geb. 23
DE-67663 Kaiserslautern
Germany
T: +49 631 205 2353; F: +49 631 205 3799;
Web-site: www.uni-kl.de/FB-Biologie/AGHakenbeck/
Claude Carbon (PC), Didier Guillemot
C.H.U. Bichat - Claude-Bernard
EMI9933 (INSERM)
46, Rue Henri Huchard
FR-75877 Paris Cedex 18 - France
T: +33 1 4025 7001; F: +33 1 4025 8845
Karl Ekdahl (PC), Birgitta Henriques
Swedish Institute for Infectious Diseases
Control
SE-171 82 SOLNA
Sweden
T: +46 8 457 2379; F: +46 8 300 626;
Alexander Tomasz
The Rockefeller University
1230 York Avenue
New York
NY 10021 - U.S.A;
T: +1 212 327 8277; F: +1 212 327 8688
Email:[email protected]
Web-site:www.rockefeller.edu
13
DEVELOPMENT AND EVALUATION
OF NUCLEIC ACID AMPLIFICATION METHODS FOR
THE DETECTION OF RESPIRATORY PATHOGENS
IN COMMUNITY ACQUIRED PNEUMONIA
SUMMARY
Appropriate DNA (PCR) and RNA (both NASBA and PCR) based amplification techniques will be developed for the
detection of bacteria and viruses responsible for atypical pneumonia i. e. Mycoplasma pneumoniae, Chlamydia
pneumoniae, Legionella pneumophila, and Bordetella pertussis, RSV, rhino-, entero-, influenza-, parainfluenza-,
and coronaviruses. Primers will be selected using sequence information available and if necessary through
additional sequencing of relevant DNA.
The analytical specificities and sensitivities of the reactions will be determined on sets of reference strains and
dilutions thereof. Clinical samples will be collected for the evaluation of the clinical sensitivity and specificity of
the tests. Multiplex formats combining the simultaneous detection of several etiologies will be developed. The
performance of PCR and NASBA will be compared by interlaboratory exchange of both sets of reference strains
and clinical specimens.
Quality assurance reagents will be prepared. The multiplex reactions will be applied on samples
collected prospectively from patients with documented community acquired pneumonia.
The clinical and laboratory data will be analyzed to define diagnostic algorithms for the syndrome.
PROBLEM
The development and transnational spread of antibiotic resistance is an increasing source of
concern in modern medicine but can be reduced if proper diagnostic tests can be made available. The development of more rapid diagnostic methods for bacterial infections, in this case
atypic pneumonia, might allow for better targeting of antimicrobial treatments with minimisation of the unnecessary use of these drugs.
AIM
• To develop mono and multiplex nucleic acid amplification assays (NASBA and
PCR) for the detection of the agents responsible for atypical community
acquired pneumonia.
• To apply and validate the newly developed tests on clinical Community acquired
pneumonia (CAP) specimens.
• To prepare reagents and proficiency panels for quality control.
EXPECTED RESULTS
• Development of multiplex real time amplification tests
• Comparison of multiplex NASBA and multiplex PCR applied to respiratory specimens from patients with community acquired pneumonia.
• Preparation of suitable quality control reagents that are intended to be used on a broader and
even on an international scale.
• Optimised strategy for the etiologic diagnosis of CAP
• Algorithms in the detection of pathogens causing CAP
14
Acronym : NAACAP
EC contribution : 1,191,106 €
Starting date : October 1st 2000
Keywords : atypical pneumonia, laboratory diagnosis, molecular
techniques, real time PCR, real time NASBA, multiplex reactions,
quality assurance, algorithms.
The end results of the project are ready-to-use molecular amplification tests including: procedures and standardised methodologies, a profiency panel, guidelines and algorhitms.
Through the expertise generated during the project, the collaborating laboratories will
become reference centres for the diagnosis of CAP. On this basis they will provide education
and training for diagnostic laboratories in the EU, improving their performance.
The development of performant real time multiplex systems may lead to a competitive advantage for future applications of this technology and given the high incidence of CAP there is a
considerable market for these diagnostic tests. Basic reagents that have been used to set up
the assays, will be made commercially available by the industrial partner in a socalled open
tests format. Information on the most optimal primers and probes for the pathogens will be
made available in the public domain as described above.
The end user groups of these results are clinical laboratories. The distribution of proficiency
panels will allow laboratories involved in the etiologic diagnosis of CAP, to test their procedures for sensitivity and specificty. The associated reporting and information exchange
process will disseminate data which will allow comparison of different test protocols at
national and trans-national levels.
Prof. Dr. M. Ieven
Universitaire Instelling
Antwerpen/Universitair
Ziekenhuis Antwerpen
Department of Medical Microbiology
Wilrijkstraat 10 - B-2650 Edegem - Belgium
T:+3238213644 ; F: 3238254281
• •
•
PARTNERS:
Dr. J. D. Fox
University of Wales College of Medicine
Heath Park - CF144XN Cardiff - United Kingdom
T: 44(0)2920743583 ; F: 44(0)2920742161
Dr. C. Van Eekelen
Organon Teknika BV.
Business area Nucleic Acid
Diagnostics/NucliSens Group
Boseind 15 PO Box: 84 - 5280 AB Boxtel
The Netherlands
T: 31411654524 ; F: 31411654311
Prof. Dr. Willy Spaan
Universiteit Leiden/Leiden University
Medical Center
Department of Virology
Albinusdreef 2 PO Box 9600
2300 RC Leiden - The Netherlands
T: 31(71)5261652 ; F: 31(71)5266761
15
PNEUMOCOCCAL DISEASE
IN EUROPE
PROBLEM
Streptococcus pneumoniae (Pnc) causes a substantial burden of disease in infants and adults in Europe.
With the imminent licensing of new Pnc conjugate vaccines, it is imperative to establish the pre-vaccination
epidemiology of Pnc infection to enable policy makers to implement the most appropriate vaccination strategy.
AIM
We collect data with standardised methodology to determine the actual incidence of Pnc carriage and Pnc invasive disease in a range of European countries and the risk factors for Pnc
invasive disease. We also develop new laboratory methodology to measure serological correlates of natural and vaccine induced immunity.
EXPECTED RESULTS
Utilising this data in tailored mathematical models, we will predict the impact of a variety of
vaccination strategies on Pnc infection and disease and undertake an economic evaluation of
these alternative interventions to enable the design of an optimal vaccination strategy.
16
Acronym : PNC-EURO
Project number : QLG4-2000-00640
Keywords : Streptococcus pneumoniae, conjugate vaccines,
vaccination policy
We foresee as an extended outcome, the development of an optimal Pnc vaccination strategy
to be used by public health authorities in revisiting their vaccination policies.
Dr Terhi Kilpi
Department of Vaccinology
National Public Health Institute
Mannerheimintie 166, FIN-00300 Helsinki, Finland
T: (+358) 9 4744 8678; F: (+358) 9 4744 8675
•
•
•
•
•
PARTNERS:
Prof. Reinhard Kurth
Robert Koch-Institut
PF 650280 Nordufer 20, D-13353 Berlin,
Germany
T: (+49) 1887 542000; (+49) 1888 7542 61/0
Dr Vittorio Demigheli
Servizio Sovrazonale di Epidemiologica
Regione Piemonte Azienda Sanitaria Locale
Via S. Caterina 30, 1-15100 Alessandria, Italy
T: (+39) 013 1307821; F: (+39013 1307847
Mrs Andrea Tinson
City University
Northampton Square, EC1V 0HB London, UK
T: (+44) 207 4778069; F: (+44) 207 4778975
Prof. Giuseppe Penagiano
Laboratory of Epidemiology and Biostatistics
Istituto Superiore di Sanità
Viale Regina Elena 299, I-00161 Roma,
Italy
T: (+39) 064 9902693; F: (+39) 064 9387118
Dr Barbara Byth
Institute of Child Health and Great Ormond
Street Hospital NHS Trust
University College London
30 Guilford Street, WC1N 1EH London, UK
T: (+44) 207 2428789 / 8290; F: (+44) 207 8310488
Prof. Georg Sandberger
Institut für Medizinische Biometrie
Eberhard-Karls-Universität Tübingen
55 Westbahnofstrasse, D-72070 Tübingen,
Germany
T: (+49) 707 1297795/0; F: (+49) 707 1295784
Prof. Paul Boseley
Communicable Disease Surveillance Centre
Public Health Laboratory Service
61 Colindale Avenue, NW9 5EQ London, UK
T: (+44) 208 20012953200
Dr Nils Strandberg Pedersen
Artillerivej 5, DK-2300 Copenhagen,
Denmark
T: (+45) 32683212; F: (+45) 32683795
Mr Detlef Klimpe
Institute for Medical Microbiology
Aachen University of Technology
Pauwelsstr. 30, D-52057 Aachen, Germany
T: (+49) 241 8088045; F: (+49) 241 8888506
17
SEVERE STREPTOCOCCUS PYOGENES
DISEASE IN EUROPE
SUMMARY
The Strep-EURO is a research project and surveillance programme for severe group A streptococcal disease (GAS).
The aim of the project is to enhance understanding of the epidemiology of GAS invasive disease in Europe.
Public health institutes from Sweden, Germany, the United Kingdom, the Czech Republic, Greece, Italy, Finland,
Denmark, Romania, and Cyprus will participate. We apply modern state of the art molecular methods for typing
and clonal identification of clinical isolates. Strains are analysed for antibiotic susceptibility, and resistance
determinants are traced. Furthermore, pathogenic aspects of severe streptococcal disease, such as proteolytic
activation of the contact system, and selected virulence factors, such as surface proteins and superantigens,
are investigated.
PROBLEM
Severe invasive group A streptococcal (Streptococcus pyogenes) disease includes life-threatening sepsis and/or necrotising fasciitis often afflicting previously healthy subjects. Severe
streptococcal disease is not notifiable in most European countries. Estimates based on clinically and bacteriologically documented cases, however, suggest that during the last decade the
minimum incidence in Europe ranged between 1.0 - 6.0 cases per 100,000 inhabitants per year.
Despite surgical, antibiotic and intensive care treatment, mortality rates have ranged between
30-60%.
AIM
• To improve the understanding of the epidemiology of severe group A streptococcal disease in Europe.
• To develop a pan-European epidemiological perspective on severe GAS disease
in Europe
• To analyse and compare invasive GAS isolates from seven EU and three associated countries
• To develop and apply molecular methods for typing and clonal identification of
clinical isolates
• To determine antibiotic susceptibility and detect genetic resistance markers
• To detect bacterial superantigens and their role in the invasive process
• To study pathogenic aspects specific to invasive GAS disease, such as proteolytic mechanisms
18
Acronym : Strep-EURO
EC contribution : 1 200.000 €
Keywords : Streptococcus pyogenes, invasive infections, molecular
typing, epidemiology
EXPECTED RESULTS
• Enhanced surveillance of severe GAS disease in Europe
• Characterisation of prevalent strains of GAS causing severe invasive disease by M and emmtype, clonal relatedness and virulence gene distribution
• Evaluation of molecular methods for clonal identification and comparison of isolates
• Establishment of a network of specialist reference and research laboratory centres for streptococci within EU and some associated countries
• Elucidation of pathogenic mechanisms in severe GAS disease
Results of the project should have an impact on the design of future immunoprophylactic
measures against group A streptococcal disease.
Claes Schalen
Dep. of Medical Microbiology and Infection,
University of Lund
Soelvegatan 23 22362 Lund, Sweden
T: (+46) 46173284; F: (+46) 46135936
PARTNERS:
Rudolph Luetticken
Institute of Medical Microbiology
University Hospital RWTH
Pauwelsstrasse 30, D-52074 Aachen, Germany
T: (+49) 2418089510; (+49) 2418082483
Androulla Efstratiou
PHLS,CPHL
61 Colindale Avenue, London NW9 5HT, UK
T: (+44) 2082001295; F: (+44) 2082056528
•
•
•
•
••
•
Paula Kriz
National Institute of Public Health
Srobarova 48, 10042 Prague, Czech Republic
T: (+420) 267082259; F: (+420) 267311454
•
•
Jaana Voupio-Varkila
Mannerheimintiae 166, 00300 Helsinki, Finland
T: (+358) 947448240; F: (+358) 947448238
Helle B Konradsen
Artillerivej 5, DK-2300 Copenhagen, Denmark
T: (+45) 32683158; F: (+45) 32683862
Monica Straut
Cantacuzino Institute
Splaiul Independentei 103, R-70100 Bucharest,
Romania
T: (+40) 1 4113800/206; F: (+40) 1 4115672
Nicolas J Legakis
National and Kapodistrian University
Mikras Asias 75, 11527 Athens, Greece
T: (+30) 2107462011; F: (+30) 2107462124
Birgitta Henriques Normark
Swedish Institute for Infectious
Disease Control
S-17182 Solna, Sweden
T: (+46) 845722413; F: (+46) 8302566
Graziella Orefici
Istituto Superiore di Sanita
Viale Regina Elena 299, 00161 Roma, Italy
T: (+39)0649902333; F: (+39) 0649902036
Maria Koliou
Archbishop Makarios Hospital
Nicosia, Cyprus
T: (+357) 22315520; F: (+357) 22499747
•
19
MICROBIOLOGICAL AND STRUCTURAL
STRATEGIES FOR THE DIAGNOSTICS AND
EPIDEMIOLOGY OF PSEUDOMONAS
AERUGINOSA INFECTIONS
SUMMARY
Pseudomonas aeruginosa is a leading cause of nosocomial (hospital-acquired) infections associated with high
morbidity and mortality. Our project is a global effort from clinicians, microbiologists, pharmacists and structural
biologists to develop new strategies in order to reduce the incidence and improve the treatment of infections due
to P. aeruginosa. The experience collected would not be limited to P. aeruginosa but could be largely applied to a
great majority of human pathogens.
PROBLEM
P. aeruginosa is an important cause for nosocomial infections. It is particularly a problem in
intensive care units, burn units, neonatal units, and wards housing leukaemia and other cancer
patients. Nosocomial pneumonia is the second most common hospital-acquired infection and
P. aeruginosa is the most prevalent etiological agent for both poly- and mono-microbial pneumonia. On clinical grounds, it is extremely difficult to distinguish whether P. aeruginosa is still
only an innocent coloniser or has become an invasive pathogen and no diagnostic tool is available to discern the state of activation of the bacteria. Empirical treatment for high clinical suspicion is common, leading in a significant amount of patients to unnecessary treatment associated with serious drug toxicity and extra costs and risk of selection of resistant strains. Hence
any measures of prevention and surveillance of (nosocomial) infections can substantially
reduce morbidity and costs.
AIM
Pseudomonas aeruginosa
6 264 403 bp
The central focus of this scientific project is the identification of cellular functions essential for
virulence, with the objectives:
• Develop diagnostic tests based on DNA chip technology that allow surveillance and infection
control;
• Identify the genomic and expression signatures of particularly virulent strains for future
pathotyping of P. aeruginosa;
• Produce crystal structures of active efflux systems of P. aeruginosa involved in mechanism
such as multidrug resistance
• Find potential inhibitors of signal peptide proteases.
• Identify new virulence factors using the new host system C. elegans
EXPECTED RESULTS
The expected results will be:
1 New diagnostic test systems and new risk assessment methods based on:
SNP genotyping and pathotyping
Standardised and validated criteria for resistance monitoring
2 Crystal structures of efflux pumps of P. aeruginosa with methodological developments that
could be applied to other membrane proteins.
3 Inhibitors of proteases responsible for the proper assembly of efflux pumps and other cell
surface components.
4 Evaluate the impact of efflux pumps deficient P. aeruginosa strains on virulence and identify
new virulence factors using the new host system C. elegans.
20
Acronym : Pseudomonas virulence
Keywords : Diagnostic tests, antibiotic resistance, epidemiology,
efflux pumps, antimicrobial agents, structural biology, virulence factors,
Pseudomonas aeruginosa
The combination of bacterial efflux pump inhibitors with existing antibiotics could increase
the susceptibility of P. aeruginosa to such antibiotics thereby lowering required therapeutic
doses.
The experience collected would not be limited to P. aeruginosa but could be largely applied to
a great majority of human pathogens. Thereby both the clinical and financial impact of a commercial implementation of this new technology is extremely high.
Prof. Arnaud Ducruix
Laboratoire de Cristallographie et RMN
Biologiques - UMR 8015 CNRS
Faculté de Pharmacie
4, Avenue de l'Observatoire
FR-75270 Paris cedex 06 - France
T: +33 1 53 73 98 36; F: +33 1 53 73 99 25
PARTNERS:
Alain Brisson
Institut Européen de Chimie et Biologie
CNRS FRE 2247 - Université Bordeaux
16, Avenue Pey Berland
FR-33607 Pessac Cedex - France
T: +33 5 57 96 34 58
F: +33 5 57 96 22 26
Christian Van Delden
University of Geneva
Faculty of Medecine
Dept of Genetics and Microbiology, CMU
9 Av. de Champel - CH-1211 Geneva
Switzerland
T: +41 22 702 5111; F: +41 22 7025702
•
• •
•
Fernando Rojo
Centro Nacional de Biotecnologia,
CSIC - Campus de la Universidad Autonoma
Cantoblanco - ES-28049-Madrid, - Spain
T: +34 91 585 45 39; F: +34 91 585 45 06
Burkhard Tümmler
Klinische Forschergruppe
PO Box 30623
Carl Neunberg Strasse 1
D-30625 Hanovre - Germany
T: +49 511 532 2920; F: +49 511 532 6723
Alain Filloux
Laboratoire d'Ingenierie des Systemes
Macromoleculaires
IBSM - CNRS
31, Chemin Joseph Aiguier
FR-13402 Marseille Cedex 20 - France
T: +33 4 91 16 41 27; F: +33 4 91 71 21 24
Bernard Roques
Pharmaleads
16 avenue de Bouvines
FR-75011 Paris - France
T: +33 1 53 73 96 88; F: +33 1 43 26 69 18
Guillaume L'Hermite
SARL BioXtal
La Pépinière d’Entreprises
BP 76` - FR-91193 Gif sur Yvette - France
T: +33 1 64 86 58 21; F: +33 1 64 86 58 23
21
STRUCTURAL AND FUNCTIONAL
GENOMICS OF MYCOBACTERIUM
TUBERCULOSIS
SUMMARY
The general objective of the X-TB project is to use an integrated, multidisciplinary approach combining
proteomics with structural and functional genomics to define the proteome of the tubercle bacillus with the
explicit aims of identifying novel drug targets and developing new chemotherapeutic compounds to treat
tuberculosis. Attempts will be made to identify lead compounds for new drugs that might help to reduce the
duration of therapy through their greater potency and show activity against the current multidrug resistant strains
of Mycobacterium tuberculosis.
PROBLEM
Real time NASBA
sense RNA
oli go P 1
RNase H
oli go P 1
Reverse
Transcriptase
RNase H
pri mer P2
Reverse
Transcri ptase
Reverse Transcriptase
primer P2
Reverse Transcriptase
anti-sense
RNA
T7 RNA polymerase
T7 RNA
polymerase
Given the current global socio-political climate, the importance of curing
and preventing tuberculosis cannot be overstated. While the development
of a new vaccine to replace BCG is a laudable if long-term objective, the
design of new drugs is a more tangible goal. Although directly observed
short course chemotherapy (DOTS) exists to treat the disease, this treatment has not been improved for over 30 years. DOTS is singularly inefficient
by the standards of today’s pharmaceutical industry in terms of drug activity and toxicity, and its efficacy is threatened by increasingly widespread
drug resistance. Here, we wish to employ state-of-the-art post-genomics
technology to initiate the development of enhanced tuberculosis
chemotherapy for the new millenium. In particular, we would like to harness the powerful new tools of structural and functional genomics for drug
and drug target discovery.
AIM
The principal objective is to further the development of new drugs to combat the growing menace of tuberculosis by using a post-genomics approach. The proteome of Mycobacterium tuberculosis will be intensively studied to expand our knowledge base and identify proteins or
enzymes that could serve as targets for new antibiotics. Essentiality will be determined by creating appropriate mutants and specificity appraised by bioinformatics. Mycobacterial proteins
that could serve as novel targets for chemotherapy will be identified, and inhibitors will either
be isolated from libraries using novel screens of natural or (semi)synthetic products or, by using
three dimensional structures as templates for drug design.
EXPECTED RESULTS
1. Biochemically and structurally characterised potential drug targets of known function
2. High-throughput production of unknown proteins for library screening and structural studies
3. Function predictions and refined protein families from advanced bioinformatics
4. Definition of the proteome under different physiological conditions
5. Linkage maps for intraproteome and host-pathogen protein-protein interactions
6. Databases for proteomics and structural genomics
7. Confirmation of essentiality by gene replacement
8. Protein structures determined by NMR and X-ray crystallography
9. Novel automated screens using fluorescent two-hybrid systems
10. Screens of chemical and natural product libraries to identify potential lead compounds
11. Leads for drugs from rational design and molecular modelling
22
Acronym : X-TB
Keywords : Protein structure, proteomics and linkage maps,
chemical library screenings, rational design of inhibitors,
bioinformatics, drug targets, comparative genomics, protein
and nucleotide kinases, cytochromes P450, mycolic acid pathway.
This project will lead to better definition of the proteins present in M. tuberculosis together
with detailed understanding of their activities, regulation, structures and interactions. It is
probable that many of these proteins play essential roles and therefore represent attractive
drug targets. Screening of chemical and natural product libraries will identify potential ligands
and inhibitors that could correspond to lead compounds for the development of new drugs to
treat tuberculosis, in all its forms.
http://www.pasteur.fr/X-TB/
Prof. Stewart T. Cole
Unité de Génétique Moléculaire Bactérienne
Institut Pasteur
25-28, rue du Docteur Roux
T: +33 1 45 68 84 46 ; F: +33 1 40 61 35 83
Web-site:
http://www.pasteur.fr/recherche/unites/Lgmb/
Web-site: http://www.pasteur.fr/units/Lgmb
• •
•
• •
•
PARTNERS:
Prof. Stefan H. E. Kaufmann
Max-Planck-Institut for Infection Biology
Monbijoustrasse 2 DE-10117 Berlin- Germany
T: +49 30 28 460 502 ; F: +49 30 28 460 501
Dr. Ida Rosenkrands
Statens Seruminstitut
Bacterial Vaccine Department
5 Artillerivej DK-2300 Copenhagen S
Denmark
Phone: +45 32 68 37 21 ; Fax: +45 32 68 30 35
Dr. Ute Möllmann
Hans-Knöll-Institut für Naturstoff-Forschung
Beutenbergstr. 11 - DE-07745 Jena - Germany
T: +49 3641 65 6656 ; F: +49 3641 65 6652
Dr. Andrew Munro
University of Leicester,
University Road- UK- Leicester LE1 7RH - United
Kingdom
T: +44 116 252 3464 ; F: +44 116252 3369
Dr. Michael Arand
Institute of Toxicology - University of Mainz
Obere Zahlbacher Str. 67- DE-55131 MainzGermany
T: +49 6131 3934376 ; F: 49 6131 230506
Prof. Kai Johnsson
Institute of Organic Chemistry - University of
Lausanne
CH-1015 Lausanne - Switzerland
T: +41 21 692 3956; F: +41 21 692 3965
Dr. Nicholas Keep
Department of Crystallography,
Birkbeck College, University of London
Malet Street - London WC1E 7HX - United Kingdom
T: +44 20 7631 6852; F: +44 20 7631 6803
Dr. Keith S Wilson
Structural Biology Laboratory
Department of Chemistry - University of York
Heslington, York YO1 5DD
United Kingdom
T: +44 1904 432519; F: +44 1904 410519
Dr. Herman Van Tilbeurgh
AFMB, CNRS UPR9039
31, Chemin Joseph Aiguier
FR-13402 Marseille Cedex 20 - France
T: +33 4 91 82 86 24; F: +33 4 91 82 86 21
Dr. Alwyn Jones
Department of Cell and Molecular Biology Biomedical Center
Uppsala University
Box 596
SE-751 Uppsala - Sweden
T: +46 18 4714982; F: +46 18 536971
23
NEW STRATEGIES FOR
TREATMENT AND PREVENTION
OF MYCOBACTERIAL DISEASES
SUMMARY
The overall objective of the Cluster project is to establish a European framework to develop new
strategies for treatment of mycobacterial diseases. 1. Study of the mycobacterial cell wall structure to
overcome the permeability barrier mediating innate drug resistance, 2. characterization of mycobacterial targets
for future drug design. Integration of these activities, with the participation of leading research groups in Europe,
is essential for completion of the goal of developing novel approaches for control of infections with
Mycobacterium tuberculosis.
PROBLEM
Tuberculosis, the white plague of former times, represents a major health problem worldwide.
It still is the single largest cause of death amongst all bacterial infections: 3 million people die
each year of tuberculosis, and one third of the world’s population is being infected with
Mycobacterium tuberculosis, the causative bacterial agent. A particularly threatening development has been the emergence of strains of Mycobacterium tuberculosis which are resistant to
all of the front-line antituberculous drugs. There is thus an urgent need to tackle the problem
of treatment with infections with M. tuberculosis, both with an understanding of drug resistance mechanisms and towards development of new drugs with antimycobacterial activity.
One of the major problems in developing antibiotics with antimycobacterial activity is the
unusual mycobacterial cell wall with intrinsically low permeability, making these microorganisms resistant to most commonly used antibacterial agents. This permeability barrier serves as
a rate-limiting step in drug uptake and is controlled by properties of the cell envelope.
Many of the existing antituberculous drugs target the synthesis of the mycobacterial cell wall,
and the unusual structure of the cell wall makes this a particularly fertile area for developing
new antituberculosis agents. More recently, the availability of the M. tuberculosis genome
sequence has provided unprecedented insights into physiological processes which may point
toward completely novel drug targets.
AIM
Structure of the Cluster
Project 1
Cell Wall Structure and
Permeability
Project 2
Novel Drug Targets
Core activities
Porins and Antibacterial
Peptides
Essential Cofactors
Crystallography
Targeted Gene
Inactivation
Cell Wall structure
Dormancy
Animal
Experiments
Drug Transport
Rapid progress in the fields of biochemistry and mycobacterial genetics provides exciting new opportunities for developing new strategies for treatment of
mycobacterial diseases. The TB Prevention cluster provides a multidisciplinary
European approach including the areas of biochemistry, genetics, crystallography, lipid chemistry and microbiology, and is based on an integrated goal-oriented strategy. The Cluster combines two areas of fundamental importance to
tackle the problem of tuberculosis: (1) cell wall structure, permeability and
innate drug resistance, (2) novel drug targets.
Ribosome
The general aims of the areas of work are:
1. to understand the architecture of the M. tuberculosis cell wall to overcome the inefficient
transport of many antibiotics across the mycobacterial cell wall and to investigate the
involvement of cellular transport and permeability in innate and acquired drug resistance.
2. to identify, produce and characterize novel drug targets, to carry out structural analysis as a
requisite for developing new inhibitors and to prioritize these novel drug targets to be taken
forward for collaborations within a commercial environment who have expertise in synthetic
and combinatorial chemistry and structure-activity based drug design.
24
Acronym : TB prevention cluster
Contract Type : Shared cost (cluster)
Keywords : Tuberculosis, Drug Development, Treatment, Prevention,
Drug Resistance, Molecular Genetics, Crystallography, Biochemistry
EXPECTED RESULTS
- Identification of novel potential and promising candidate enzymes involved in the synthesis
of the mycobacterial cell wall as drug target
- Characterization of mycobacterial structures for future drug design
- Role of membrane components in drug resistance
- Strategies to overcome innate mycobacterial drug resistance
Strategies fueling into optimal use of existing antimycobacterial agents, means to overcome
innate mycobacterial drug resistance and the identification of novel targets for anti-infective
drugs will have a major impact on tuberculosis control.
Prof. Erik C. Böttger
Institut für Medizinische Mikrobiologie
Universität Zürich
Gloriastrasse 30/32 - CH-8028 Zürich - CH
T: +41 1 634 26 60; F: +41 1 634 49 06
PARTNERS:
Dr. Mats Andersson
Dept. Medical Biochemistry and Biophysics Karolinska Institutet
SE-17177 Stockholm - Sweden
T: +46 8 728 76 99; F: +46 8 337 46 2
Dr. Alexander Apt
Laboratory for Immunogenetics
Central Institute for Tuberculosis
Yauza Alley 2 - 107564 Moscow- Russia
T: +7 095 268 78 10; F: +7 095 144 56 18
•
•
•
• •
•
•
Dr. M. Jo Colston
National Institute for Medical Research
Division of Mycobacterial Research
The Ridgeway Mill Hill - UK-NW7 1AA London
United Kingdom
T: +44 208 959 36 66 ext. 23 54;
F: +44 208 913 85 28
Dr. Bruno Curti
Dipartimento di Fisiologia e Biochimica
Generali - Università degli Studi di Milano
Milano - Italy
T: +39 02 7064 4504 ; F: +39 02 2362 451
Dr. Mamadou Daffe
Institut de Pharmacologie et de Biologie
Structurale - CNRS
205, Route de Narbonne FR-31077 Toulouse
France
T: +33 5 61 17 55 69; F: +33 5 61 17 59 93
•
Dr. Brigitte Gicquel
Unité de Génétique Mycobactérienne
Institut Pasteur
25, Rue du Dr. Roux - FR-75724 Paris Cedex 15 /
France
T: +33 1 45 68 88 28; F: +33 1 45 68 88 43
Dr. Giulio Magni
Instituto de Biochimica
Facultà di Medicina e Chirurgia
Università di Ancona
Via Ranieri - IT-60100 Ancona - Italy
T: +39 071 22 04 678 ; F: +39 071 28 02 117
Dr. Carlos Martin
Facultad de Medicina
Dpto Microbiologia y Salud Publica
Universidad de Zaragoza
C/Domingo Miral s/n - ES-50009 Zaragoza - Spain
T: +34 9 76 76 17 59; F: +34 9 76 76 16 64
Dr. John Joe McFadden
Molecular Microbiology Group
School of Biological Sciences
University of Surrey
Guildford - UK-Surrey GU2 5XH - United Kingdom
T: +44 483 300 800 / 2671; F: +44 483 300 374
Dr. Michael Niederweis
Lehrstuhl für Mikrobiologie
Universität Erlangen
Staudtstr. 5 - DE-91058 Erlangen - Germany
T: +49 9131 852 88 02; F: +49 9131 852 80 82
Prof. Giovanna Riccardi
Dipartimento di Genetica e Microbiologia
Università di Pavia
Via Ferrata, 1 -27100 Pavia Italy
T: +39 0382 505574; F: +39 0382 528496
Dr. Menico Rizzi
Università di Piemonte Orientale "A.
Avogadro"
Viale Ferrucci 33 - IT-28100 Novara - Italy
T: +39 0321 657 632; F: +39 0321 657 641
Dr. Edda de Rossi
Dipartimento di Genetica e Microbiologia
Università degli Studi di Pavia
Via Abbiategrasso 207 - IT-27100 Pavia - Italy
T: +39 0382 505 575 ; F: +39 0382 528 496
Dr. Douglas Young
Wright Fleming Institute
Dept. of Medical Microbiology
St. Mary’s Hospital Medical School
Norfolk Place 1PG - UK-London W2 - UK
T: +44 171 594 39 62; F: +44 171 262 62 99
25
INHIBITORS OF THE NON-MEVALONATE
PATHWAY OF ISOPRENOID BIOSYNTHESIS
AS DRUGS AGAINST TUBERCULOSIS
SUMMARY
A new drug against tuberculosis (TB) will be developed based on the inhibition of the 1-deoxy-D-xylulose
5-phosphate (DOXP) pathway. The DOXP pathway supplies Mycobacterium tuberculosis with essential isoprenoids. In mammals, the DOXP pathway is absent and isoprenoids are synthesised by the mevalonate pathway.
Therefore, inhibitors of the DOXP pathway are non-toxic for the human host. Inhibitors of DOXP reductoisomerase
(DXR), a key enzyme of the DOXP pathway, are to be developed as potential anti-TB drugs. In addition, the crystal
structure of the mycobacterial GcpE enzyme will be solved to provide a basis for further drug development.
PROBLEM
Tuberculosis is a highly contagious infectious disease of humans caused by the bacterium
Mycobacterium tuberculosis, and easily spread by airborne transmission. To date, about 2 to 3
million people world-wide succumb to the disease each year, and there are some 8 million new
TB cases per year, over a quarter of a million of which occur in Eastern Europe. There is an
urgent need for new safe and efficient anti-TB drugs since with multidrug-resistant pathogens,
the course of treatment increases from normally 6 to 18-24 months, and cure rates drop from
nearly 100 % to less than 60 %.
AIM
The project aims at developing a new drug against tuberculosis based on a novel mode of
action. No cross-resistance against such a new drug with existing antimycobacterial agents is
expected. Since the molecular target is not present in humans the new drug will be well tolerated and applicable to long-term therapy.
EXPECTED RESULTS
It is expected that a lead compound active against M. tuberculosis in the mouse model will
emerge during the funding period.
26
Acronym : New Antimycobacterials
Starting date : : September 1st 2002
Keywords : crystal structure, 1-deoxy-D-xylulose 5-phosphate, DOXP,
drug design, isoprenoid, MEP, 2-C-methyl-D-erythritol-4-phosphate,
Mycobacterium tuberculosis, reductoisomerase
The identified lead compound will be further developed into an anti-TB drug, which also may
be useful for the treatment of leprosy and infections with atypical mycobacteria.
Dr. Hassan Jomaa
Jomaa pharmaka GmbH
Frankfurter Strasse 50
D-35392 Giessen, Germany
T: +49 641 7970 717; F: +49 641 7970 710
PARTNERS:
Dr. Tanya Parish
St. Bartholomew's and the Royal London School of
Medicine and Dentistry
Queen Mary and Westfield College, University of
London
Turner Street
London E1 2 AD, United Kingdom
T: +44 20 7377 0444; F: +44 20 7247 3428
Dr. Gerhard Klebe
Institut für Pharmazeutische Chemie
Philipps-University Marburg
Marbacher Weg 6
D-35032 Marburg, Germany
T: +49 6421 28 21313; F +49 6421 28 28994
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Dr. Martin Schlitzer
Department for Pharmacy
Center for Pharmacological Research
Ludwig-Maximilians University Munich
Butenandtstr. 5-13
D-81377 Munich, Germany
T: +49 89 2180 7804
Dr. Mamadou Daffé
Institut de Pharmacologie et de Biologie
Structurale
UMR CNRS-Université Paul Sabatier 5089
205, Route de Narbonne
F-31077
Toulouse Cedex 04, France
T: +33 5 6117 5569; F: +33 35 6117 5994
[email protected]
Dr. Jan Langemans
Department of Parasitology
Biomedical Primate Research Centre
Lange Kleweg 151
NL-2288 GJ Rijswijk, The Netherlands
T: +31 15 284 2861; F: +31 15 284 3986
[email protected]
Dr. Francois Jehl
Faculté de Medecine
Institut de Bactériologie
University of Strasbourg
3, Rue Koeberlé
FR-67000 Strasbourg, France
T: +33 3 9024 3781; F: +33 3 8825 1113
[email protected]
Dr. Serge von Calenbergh
Laboratorium voor Medicinale Chemie (FFW)
Ghent University
Harelbekestraat 72
B-9000 Ghent, Belgium
T: +32 9 264 8124; F: +32 9 264 8192
27
NOVEL DRUG TARGETS SPECIFIC TO PERSISTENT
(LATENT) TUBERCULOSIS INFECTION:
CRYSTALLISATION, STRUCTURE DETERMINATION
AND FUNCTIONAL STUDIES
SUMMARY
The aim of our project is to solve the 3-D structure of several sterilising drug targets, some of which have been
identified by us recently. These specific drug targets are associated with persistence of Mycobacterium
tuberculosis. Our approach would target proteins that are active in dormant cells. The structural elucidation of
such drug targets would lead to the development of totally novel antibiotics, with completely new modes of
action, which are active against stationary phase persistent Mycobacterium tuberculosis. These antibiotics will
shorten the duration of chemotherapy and/or synergise with existing antibiotics in humans by reducing bacterial
sub-populations, which are tolerant to conventional antimicrobials and thus control reactivation of TB.
PROBLEM
M. tuberculosis remains the leading cause of death worldwide from an infectious agent and is
responsible for nearly three million deaths every year. About one-third of the world population
is infected with M. tuberculosis, 10 % of which will develop the disease at some point in their
lives. Currently available antibiotics are completely ineffective against persistent bacteria.
AIM
The approach is to focus on persister-associated proteins and to determine the structure of
these proteins. The main thrust of this project is in drug design, based on the molecular 3dimensional structure of drug targets for latent TB.
EXPECTED RESULTS
The know-how on drug targets for latent tuberculosis generated in our project is expected to
result in the development of specific inhibitors/antibiotics for tuberculosis. These antibiotics
will shorten the duration of chemotherapy and/or synergise with existing antibiotics in humans
by reducing bacterial sub-populations, which are tolerant to conventional antimicrobials.
28
Acronym : Persistent TB
EC contribution : 800.000 €
Keywords : Latent-tuberculosis; Drug-targets; 3 D-Structure;
Inhibitors; Drug design
It is anticipated that the information generated will be used for drug design and development
in collaboration with the pharmaceutical industry.
Prof. Mahavir Singh
Lionex GmbH
Mascheroder Weg 1b, 38124 Braunschweig,
Germany
T: (+49) 531 260 12 66; F: (+49) 531 260 11 59
Web-site: www.lionex.de
•
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PARTNERS:
Prof. Anthony Coates
Medical Microbiology
St George’s Hospital Medical School
Cranmer Terrace, SW17 0RE, London
T: (+44) 208 725 5725 ; F: (+44) 208 672 0234
Prof. Gunter Schneider
Department of Medical Biochemistry and
Biophysics (MBB)
Karolinska Institutet,
Scheeles väg 2, 171 77 Stockholm, Sweden
T: (+46) 8 08 728 76 75 ; F: (+46) 8 08-32 76 26
Dr Paul C. Driscoll
Department of Biochemistry and Molecular
Biology - University College London
Gower Street, WC1E 6BT, London, U.K.
T : (+44) 207 679 7035 ; F : (+44) 207 679 7193
Dr. Han-Jürgen Hecht
Struckturforschung
GBF-German Research Center for Biotechnology
Mascheroder Weg 1, 38124 Braunschweig, Germany
T: (+49) 531 6181 369 ; F: (+49) 531 6181 355
29
DEVELOPMENT OF INTEGRATABLE SENSORS FOR
SCREENING OF ANTIBIOTIC RESISTANCE IN
MYCOBACTERIUM
SUMMARY
The overall aim of this project is the development of multi-sequence DNA PCR-ELISA and sensor arrays for the
identification of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis.
The methodologies developed will allow assessment from clinical samples within hours, in comparison to the
laborious and time-consuming methods presently in use. The use of a 3 x 3 final configuration sensor array
allows for the development of a disposable, rapid, simple and one-step measurement system.
PROBLEM
In the last decade, TB has re-emerged as one of the leading causes of death
(nearly 3 million deaths annually). The estimated 8.8 million new cases
every year correspond to 52,000 deaths per week or more than 7,000 each
day, which translates into more than 1,000 new cases every hour, every day.
These death rates, however, only partially depict the global TB threat; more
than 80% of TB patients are in the economically productive age of 15 to 49
years. Especially in resource poor countries, there is an increasing incidence
of tubercle bacteria resistant to the most effective drug. A major public
health problem worldwide, TB is now a global emergency. In 1993 the WHO
initiated a Global Programme on Drug Resistance in 1994 and in 1998
announced improved treatment regimens.
It seems, therefore, reasonable, that a detection device for mutations
responsible for MDRTB is chosen in this project as a demonstration of a
molecular diagnostics tool that can be applied in all types of DNA diagnostics, using the same generic technology. Thus, with a relatively simple, disposable array the concept will be demonstrated and a useful diagnostic tool
will be developed.
Rifampicin (RIF), first introduced in 1972 as an anti-tubercular drug, is extremely effective
against M. tuberculosis. Resistance to rifampicin is increasing because of widespread application and results in selection of mutants resistant to other components of short-course
chemotherapy. In this project the aim is to tackle some of the problems of detecting antibiotic
resistance of M. tuberculosis, using rifampicin as a surrogate marker for MDRTB, allowing more
effective treatment of the disease.
AIM
This project focuses on the resistance of M. tuberculosis to rifampicin.
Resistance to this front line anti-mycobacterial agent is conferred by mutations
in the rpoB gene, encoding the b subunit of RNA polymerase. The work will centre on a subset of the mutations, which confer rifampicin resistance to M. tuberculosis and will establish a set of protocols for release, amplification and detection of PCR product.
Platform for clinical diagnostics
30
Acronym : DISSARM
Keywords : Tuberculosis, multi-drug resistance, rifampicin,
sensor array, molecular biology methods.
EXPECTED RESULTS
1 Rapid DNA release, <20 minutes, from clinical samples with no use of enzymes, organic solvents of purification columns
2 Sensitivity of suitable level to be able to detect M. tuberculosis from smear positive clinical
samples and culture
3 PCR amplification specific for M. tuberculosis.
4 Hybridisation detection of wild type sequence and the 4 most prevalent mutations causing
rifampicin resistance.
5 Simultaneous detection of wild type and all mutations in a multi-sequence sensor array with
hand-held instrument for signal readout. PCR-ELISA as gold standard of mutation detection
and for validation of sensor arrays.
The system under development is based on a generic technology with application in a number of different diagnostic fields. We have chosen as a model system determination of multidrug resistant strains of M. tuberculosis. Methodologies and findings will be published in
peer-reviewed journals. Technologies will be tested under laboratory conditions with a view
to establishing sensitivity, specificity, usability and rapidity. A marketing plan will assess further commercial routes. The results will be used by a number of different groups, including
molecular biology, and medical microbiology diagnostic and research laboratories and by laboratories specialising in diagnosis of infectious diseases.
http://www.nmrc.ie/projects/dissarm/
Bill Lane (Helen Berney)
National Microelectronics Research Centre
Prospect Row - Cork - Ireland
T: +353 21 4904010; F: +353 21 4270271
Web-site:http://www.nmrc.ie
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PARTNERS:
Ioannis Katakis
Universitat Rovira i Virgili
Carretera de Salou S/N - ES-43006 Tarragona - Spain.
T: +34 977 559655; F: +34 977 559667
Web: http://www.etse.urv.es/BBG
Rosario Cospedal
Pharmagen S. A. Alcarria7
Poligono Industrial De Coslada
ES-28820 Coslada Madrid - Spain
T: +34 91 6748990; F: +34 91 6748991
Web-site: http://www.pharmagen.es/
Ken Forbes
Polwarth Building
Foresterhill - UK-Aberdeen AB25 2ZD - UK
T: +44 1224 663123 Extension 54953;
F: +44 1224 685604
Web-site: http://www.abdn.ac.uk/
Wolfgang Künnecke
TRACE Biotech AG
Mascheroder Weg, 1B
DE-38124 Braunschweig - Germany
T: +49 531 261 33 30; F: +49 531 261 33 38
Web-site: http://www.trace-ag.de
Hedda Steingrimsdottir
Microzone Ltd
112 Malling Street - UK-Lewes BN7 2RJ - UK
T: +44 1273 483349; F: +44 1273 483391
Web-site: http://www.microzone.co.uk/
Brian Watt
Lothian University Hospitals NHS Trust
Scottish Mycobacteria Reference Laboratory
Greenbank Drive - UK-Edinburgh EH10 5SB -UK
T: +44 131 5366357; F: +44 131 5366152
Web-site: http://www.phls.co.uk/
•
31
CHARACTERISATION OF MYCOBACTERIUM
TUBERCULOSIS POPULATIONS DURING
INFECTION: A LONGITUDINAL STUDY ON
DRUG RESISTANCE DEVELOPMENT
SUMMARY
Drug resistance arises in Mycobacterium tuberculosis (MTB) generally by mutation of chromosomal genes, but
despite combined drug therapy the spread of multi-drug-resistant (MDR) strains is alarmingly increasing. The
LONG-DRUG project is a longitudinal study, carried out in an area with high MDR-TB prevalence, which aims to
document the generation of multi drug resistant MTB strains through the use of novel molecular techniques. The
combined use of a variety of techniques on longitudinally obtained samples and primary cultures will enable to
generate clinically significant knowledge on the evolution of multi drug resistance in Mycobacterium.
MTB positive clinical samples will be collected in the central TB Hospital of Abkhazia (former Soviet Union), an
area of increased MDR-TB prevalence, by Médecins Sans Frontières (MSF). An already ongoing medical aid
programme of MSF guarantees for longitudinal sample collection. After sample shipment and routine micro
biological examination the minimal inhibitory concentration to primary and secondary anti-tuberculosis drugs will
be determined. The determination of the resistance phenotype will target the qualitative molecular analysis to
resistance related genes. Real time PCR and RFLP-PCR will be used to screen for heterogeneous MTB populations.
Quantitative real time PCR using molecular beacons on sequenced single nucleotide polymorphisms will finally
elucidate prevalence of single resistant clones in bacterial populations. The genetic relationship of all significant
strains will be analysed by IS-RFLP and spoligotyping. Centralized data management and correlation of clinical,
bacteriological and molecular data should enable to elucidate the contribution of resistant subpopulations to the
development of MDR-TB and thus the clinical significance of drug-resistant TB clones in mixed populations.
PROBLEM
High prevalence of drug resistance in TB in various geographical settings suggests that MDRTB may become a more significant problem in the future even for industrialised countries. The
fact that no significant novel first line anti-TB drugs have been developed over the last 40 years
does contribute to the significance of the problem.
AIM
The first aim of the LONG-DRUG consortium is the generation of a collection of longitudinally
collected samples from patients with MDR-TB. Diverse molecular tools will be generated and
validated for an in-depth characterisation of these samples. Primary objectives are (i) the
molecular characterisation of multiple drug resistance development in MTB populations over
time, (ii) the clarification of the epidemiological relationship of the strains and sub-clones identified in the study population, and (iii) the elucidation if the generation of resistant sub-populations of MTB are of clinical relevance.
Detection of single nucleotide
polymorphisms (SNPs) in mixed bacterial
populations using FRET probes in real time
PCR. 10-fold reciprocal dilutions of two target
DNA, which differed for one SNP, was
assayed. The melting temperature peak of
67.5 °C corresponds to the DNA homologous
to the probe sequence while the Tm peak
of 59 °C identifies those sequences
which have a SNP.
32
Acronym : LONG-DRUG
Keywords : tuberculosis, resistance, multi drug resistance,
molecular typing, MDR-TB, SNP, single nucleotide polymorphisms
EXPECTED RESULTS
Generate knowledge on (i) the resistance development in a bacterial population infecting over
time the human host, (ii) the fait of the different resistant subpopulations generated during
this process (iii) and the clinical relevance of these microbial populations.
ACHIEVED RESULTS
Over 300 samples were already collected to which the minimal inhibitory concentration MIC
for the classical first line anti TB drugs is presently being determined. For about 200 of these,
resistant to at least one drug, the determination of the MIC for second line anti TB drugs is in
progress. The collection of samples contains already an extensive follow-up from over 15
MDR-TB patients.
With this information insight will be gained into the M. tuberculosis strains and genotype families involved in transmission of tuberculosis and outbreaks of this disease.
Dr Marco R. Oggioni
LAMMB, Dept. Biol Mol.
Università di Siena
LAMMB, Policlinico Le Scotte 1S
Viale Bracci 53100 Siena Italia
T: (+39) 0577 233101; F: (+39) 0577 233334
Web-site: http://www.unisi.it/
PARTNERS:
Dr Francesco Checchi
EPICENTRE
Rue Saint Sabin 8, 75011 Paris, France
T: (+33) 1 4021 2904; F: (+33) 1 40212803
Dr Graziella Orefici
Laboratory of Bacteriology and Medical
Mycology
Viale Regina Elena 299, 00161 Roma, Italia
T: (+39) 06 49902333; F: (+39) 06 4938 7112
Web-site: http://www.iss.it/english/home.htm
Prof. Peter Andrew
Department of Microbiology and Immunology
University of Leicester
PO Box 138, University Rd LE1 9HN, Leicester, UK
T: (+44) 116252 2941; F: (+44) 116 252 5030
Web-site:http://www-micro.msb.le.ac.uk/
•
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Prof. Heinz Rinder
Department of Infectious Diseases and
Tropical Medicine
Universität München
Leopoldstr. 5, 80802 München, Deutschland
T: (+49) 89 21803618; F: (+49) 89 336112
Web-site: http://www.tropinst.med.uni-muenchen.de/tropi_e/
Dr. Sabine Rüsch-Gerdes
National Reference Centre for Mycobacteria
Research Centre Borstel
Parkallee 18, 23845 Borstel, Deutschland
T: (+49) 4537 188213; F: (+49) 4537 188311
Web-site: http://www.fzborstel.de/Referenzzentrum/RefZentr.html#Referen
zzentrum
Dr. Germano Orrù
Dip. Scienze Odontostomatologiche
Università di Cagliari
Via Binaghi 4, 9121 Cagliari, Italia
T: (+39) 070 537420
Web-site: http://www.unica.it/
Dr. Thierry Jarosz
3Es (Essai clinique Evaluation Epidemiologie
Statistiques)
49 rue A Lancon, 75013 Paris, France
T: (+33) 1 5380 3495
Dr. Francis Varaine
Médecins Sans Frontières
Rue Saint Sabin 8, 75011 Paris, France
T: (+33) 1 4021 2935
Web-site: http://www.paris.msf.org/
33
NEW GENERATION GENETIC MARKERS AND
TECHNIQUES FOR THE EPIDEMIOLOGY AND
CONTROL OF TUBERCULOSIS
SUMMARY
DNA fingerprint techniques have been developed to type Mycobacterium tuberculosis isolates for studying the
molecular epidemiology of tuberculosis. The previous concerted action projects have promoted optimal
communication between EU partners, thus enabling standardization of these methods and meaningful
interpretation and comparison of results from epidemiological studies in different areas. In this project we plan to
maintain the established network in Europe and to use the DNA fingerprint database to trace transmission of
(resistant) strains internationally and to study the natural history and control of tuberculosis. Furthermore, we
aim to test the potential of novel genetic markers, derived from the recently established genome sequence of
M. tuberculosis, and to explore the DNA chip- and microarray technology to allow the use of multiple genetic
markers with different molecular clocks.
PROBLEM
The treatment of a multidrug resistance tuberculosis case is not only very costly, but also an
enormous burden for the concerned patient because of the long term (often years of ) treatment, the required compliance, the severe side effects caused by the use of multiple drugs, and
the containment required at the infectious stage of the disease. Furthermore, treatment is not
always successful. Prevention is therefore of vital importance, and also surveillance of transmission of (resistant) strains is crucial.
AIM
One of the most important aims within this EU project is to establish an international database
of DNA fingerprints of multidrug resistant M. tuberculosis isolates. This database will be linked
to the EURO-TB surveillance register to enable a meaningful interpretation of the typing results.
Dendrogram of IS6110 restriction fragment
length polymorphism patterns (left) and
spoligopatterns (right) of 24 M. tuberculosis
strains of the Beijing genotype, with a
picture of Ziehl Neelsen-stained
M. tuberculosis bacteria as background.
The Beijing genotype strains constitute a
genetically highly conserved group of
M. tuberculosis complex bacteria, and have
been found significantly associated with
drug resistance in several areas.
34
Acronym : Mol. Epidemology TB
Contract Type : Concerted Action
Keywords : Tuberculosis, DNA fingerprinting, molecular epidemiology,
multidrug resistance, database.
EXPECTED RESULTS
With this information insight will be gained into the M. tuberculosis strains and genotype families involved in transmission of tuberculosis and outbreaks of this disease.
Dr. Dick van Soolingen
National Institute of Public health and the
Environment (RIVM)
P.O. BOX 1 - NL-3720 BA Bilthoven
The Netherlands
T: + 31 30 2742363 ; F: +31 30 2744418
Web-site: www.rivm.nl
PARTNERS:
Argentina: Instituto de Biotechnologia
Austria: University of Innsbruck
Belgium: Institut Pasteur de Bruxelles, Institute of
Tropical Medicine
Czech Republic: National Institute of Public
Health
The Netherlands: National Institute of Public
Health and the Environment, Royal Netherlands
Tuberculosis Association
UK: London School of Hygiene and Tropical
Medicine, University of Surrey, University College
USA: Stanford University, Centers for Disease
Control and Prevention
Denmark: Statens Serum Institute
France: Institute Pasteur, Hospital Saint Louis,
Institut de Biologie de Lille, Institute de Veille
Sanitaire
Germany: University of Heidelberg,
Forschungszentrum Borstel
Guadeloupe: Institute Pasteur
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Israel: Institute Pasteur
Italy: University of Milan
Norway: National Institute of Public Health
Portugal: University of Lisbon, Instituto Nacional
de Saude Ricardo Gorge
South Africa: University of Stellenbosch
Spain: University of Zaragoza, Instituto de Salud
de Carlos III, Servicio Canario de Salud, Institute
Municipal de Salut Publica
•
Sweden: Swedish Institute for Infectious Disease
Control
35
SURVEILLANCE OF TUBERCULOSIS IN EUROPE
SUMMARY
The aim of the EuroTB project is to improve the contribution to epidemiological surveillance to TB control in
Europe. As part of the project, consensus recommendations on surveillance of anti-TB drug resistance (DRS) have
been issued. Based on those recommendations, information is collected yearly from the national correspondents
of the 51 countries of the WHO European Region: laboratory practices, characteristics of national DRS and results
of drug susceptibility testing (DST) at the start of treatment for isoniazid, rifampicin, ethambutol and pyrazinamide for culture positive human TB cases notified or included in specific studies. DST results can be provided as
part of the individual anonymous data file on TB cases or as aggregate tables by previous anti-TB treatment status
and geographic origin. After validation, data are published in the annual project report in scientific journals,
available on the project web-site and provided to WHO for inclusion in global anti-TB drug resistance reports.
PROBLEM
Anti-TB drug resistance is an increasingly recognised world-wide man made public health problem which decreases the effectiveness of standard anti-TB treatments, the mainstay of TB control. The level of drug resistance in a TB case population constitutes an indicator of the overall
adequacy and quality of anti-TB treatment.
AIM
EuroTB intends to provide an overall, comparable picture of the level and distribution of antiTB drug resistance in Europe and to monitor time trends in resistance.
All the 51 countries of the WHO European Region participate in the project through an official
correspondent, usually the person responsible for tuberculosis surveillance at national level.
EuroTB methods are based on consensus recommendations issued by working groups including experts from European countries, WHO and the International Union against Tuberculosis
and Lung Disease (IUATLD) and approved by country correspondents.
The core activity of the project is the yearly collection, validation, analysis and publication of
standardised data on tuberculosis notifications and, since 1998, on anti-tuberculosis drug
resistance. Each country provides individual or, if not possible, aggregate data by age, sex, geographic origin, previous history of tuberculosis, site of disease, bacteriological results and drug
susceptibility testing results at start of treatment. Information is also collected on the organisation of surveillance at national level. Since the year 2000, the collection and validation of
aggregate data are organised jointly with WHO.
ACHIEVED RESULTS
Already achieved results include the set up of a standardised surveillance system at the
European level. In year 2000, data were provided from 41 countries, of which more than 20 representing exhaustive data on all culture confirmed TB cases. Coordination of international surveillance with WHO was also achieved.
EuroTB publishes a free annual surveillance report, maintains a website and disseminates data
through participation in congresses and publications in scientific journals.
EXPECTED RESULTS
Description of time trends in anti-TB drug resistance; impact on national surveillance practices
(methods, definitions).
36
Data are increasingly used by the participating countries and the EC to prioritise and harmonise
actions to prevent and control drug resistance in Europe. In an era of increased availability of
international funding for TB control, comparable international data constitute a useful baseline
information for deciding priorities of action and allocation of funds.
Keywords : human tuberculosis, drug resistance, multidrug resistance,
surveillance, Europe, M. tuberculosis, isoniazid, rifampicin, ethambutol,
pyrazinamide
Acronym : EuroTB (Funded by DG SANCO)
http://www.eurotb.org
Dr Andrea Infuso
12, rue du Val d'Osne
F-94415 Saint-Maurice Cedex
France
Tel: +33 1 41 79 68 05
Fax: +33 1 41 79 68 02
Institut de Veille Sanitaire (InVS)
PARTNERS:
Albania : Dr Hafizi Hazan, Sanatorium
[email protected]
Andorra : Dr Coll Margarida Armangue
Ministry of Health and Welfare
[email protected]
Bosnia and Herzegovina :
Prof Dizdarevic Zehra
Clinic of Pulmonary Diseases and TB
"Podhrastovi"
[email protected]
Dr Stefanovic Biljana
Public Health Institute
[email protected]
Bulgaria : Prof Alexandrov Stoyan
Ministry of Health
[email protected]
Croatia : Dr Gjenero Margan Ira
Croatian National Institute of Public Health
[email protected]
Armenia : Prof Safarian Marina
[email protected]
Czech Republic : Dr Mazankova Vlasta
Institute of Health Information and
Statistics
[email protected]
Austria : Dr Klein Jean-Paul
Bundesministerium für soziale Sicherheit
und Generationen
[email protected]
Denmark : Dr Andersen Peter
Statens Serum Institute
[email protected] http://www.ssi.dk
Azerbaïjan : Eioub Aliev
Ministry of Health
[email protected]
Estonia : Dr Vahur Hollo
Estonian National TB Register
[email protected]
Belarus : Prof Borstchevsky Valentin
Institute of Pulmonology and Phthisiatry
[email protected]
Finland : Dr Ruutu Petri
[email protected]
Belgium : Dr An Aerts
Vlaamse Vereniging Respir.
Gezondheidszorgs TB
[email protected]
Dr Wanlin Maryse
Oeuvre Nationale Belge
de Défense Contre la TB
[email protected]
France : Dr Decludt Bénédicte
Institut de Veille Sanitaire (InVS)
[email protected]
http://www.invs.sante.fr
Georgia : Prof Khechinashvili George
Institute of Phthisiology and Pulmonology
[email protected]
Germany : Dr Haas Walter
Robert Koch-Institut
[email protected]
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Greece : Dr Spala Georgia
National Centre for
Surveillance and Intervention
(NCSI)
[email protected]
http://www.ncsi.gr
Italy : Dr Caraffa de Stefano Dina
Ministero della Sanità
[email protected]
Kazakhstan : Dr Rakishev G.A
Kazak Tuberculosis Research Institute
[email protected]; [email protected]
Kyrgyzstan : Dr Alisherov Avtandil
Shermamatovitch 0
National Tuberculosis &
Lung Diseases Inst.
[email protected]
Latvia : Dr Leimans Janis
States Centre of TB &
Lung Diseases of Latvia
[email protected]
Lithuania : Prof Davidaciene Edita
Lithuanian Center Pulmonology &
Tuberculosis
[email protected]
Luxembourg : Dr Huberty-Krau Pierrette
[email protected]
Direction de la Santé
Macedonia : Dr Simonovska Biljana
Institute for Lung Diseases and
Tuberculosis
[email protected]
Malta : Dr Micallef Malcolm P
Department of Public Health
[email protected]
Moldova : Dr Viktor Burinski
Phtisiopulmonology Institute
[email protected]
Monaco : Dr Nègre Anne
Ministère d'Etat, Département de
l'Intérieur
Netherlands : Dr Paul Van Gerven
Royal Netherlands Tuberculosis
Association
[email protected]
http://www.artsen.net/kncv
Phthisiopulmonology
[email protected] [email protected]
San Marino : Dr Sorcinelli Antonella
Ospedale di Stato di San Marino
[email protected]
Slovakia : Dr Rajecova Eva
National Institute of TB and Respiratory
Diseases
[email protected]
Slovenia : Dr Sorli Jurij
University Clinic of Respiratory and Allergic
Diseases
[email protected]
Spain : Dr Diez Mercedes
[email protected]
Dr Tello Anchuela Odorina
[email protected]
Instituto de Salud Carlos III
http://www.iscii.es/unidad/sge/uit/cuit.ht
ml
Sweden : Dr Romanus Victoria
Control
[email protected]
http://www.smittskyddsintitutet.se
Switzerland : Dr Helbling Peter
Swiss Federal Office of Public Health
[email protected]
Tajikistan : Prof Sirodjiddinova Umriniso I.
Tajikistan Medical University
[email protected]
Turkey : Dr Kibaroglu Emel
Ministry of Health
[email protected]
Turkmenistan : Prof Akhundov N.A.
Central Tuberculosis Control Hospital
Ukraine : Dr Melnik Vasil Mihailovich
[email protected]
Institute of Tuberculosis & Pulmonology
Portugal : Dr Fonseca Antunes Antonio
Directorate - General of Health
[email protected]
http://www.dgsaude.pt/
United Kingdom : Jim McMenamin
Scottish Centre for Infection
and Environnmental Health
[email protected]
Dr Smyth Brian
NI
[email protected]
Dr Watson John
[email protected]
PHLS
Ireland : Dr O'Flanagan Darina
Sir Patrick Dun's Hospital
[email protected]
Romania : Prof Corlan Emil
Institute of Pneumophtisiology
"Marius Nasta"
[email protected]
Uzbekistan : Dr Uzakova Gulnoz T.
Scientific Research Institute of Phtisiology
& Pulmonology
[email protected]
Israel : Dr Chemtob Daniel
Ministry of Health
[email protected].
gov.il
Russian Federation Prof Perelman
Mikhail l.
Prof Shilova Margarita Victorovna
Russian Research Institute of
Yugoslavia : Prof Popovac Dusan
Municipal Institute Lung Disease
& Protection against TB
[email protected]
Hungary : Dr Imre Vadasz
Dr Kozma Deszo
"Koranyi" National Institute TB
& Pulmonology
[email protected]
Iceland : Dr Blöndal Thorsteinn
Reykjavik Health Care Centre
[email protected]
Norway : Dr Heldal Einar
Norwegian Institute of Public Health
[email protected]
Poland : Dr Korzeniewska-Kosela Maria
[email protected]
37
IMPROVED DIAGNOSIS, DRUG RESISTANCE
DETECTION, AND CONTROL OF TUBERCULOSIS
IN LATIN AMERICA
SUMMARY
This concerted action will develop, adapt and evaluate some newer, more appropriate tools for diagnosing tuberculosis (TB) and detecting drug resistance. It will build upon collaborations developed between a network of
European and Latin American TB researchers. This project will test the new diagnostic tools in multi-center pilot
studies, modify them when necessary and run prospective clinical trials in high endemic TB areas for accuracy,
cost, ease of implementation and impact on TB control.
PROBLEM
A strategic obstacle for the improvement of TB control is the lack of inexpensive and facile techniques that can replace the slow and laborious diagnostic methods currently available.
AIM
The project plans to:
• Develop, standardize, and evaluate new methods for TB diagnosis, including improvements
in microscopy, rapid culture techniques, serological markers, and rapid speciation.
• Develop, standardize, and evaluate new methods for the detection of rifampicin resistance as
surrogate marker for multi-drug resistant (MDR) TB.
• Develop simple, rapid, and inexpensive methods for drug susceptibility testing of M. tuberculosis.
• Extend initial molecular and traditional epidemiological studies on Latin American MDR-TB
strains in order to delineate transmission patterns.
The project comprises two main areas of activities: diagnosis and drug resistance detection.
The following activities will be undertaken:
• Development and evaluation of a simple and inexpensive method of cultivation employing a
thin layer of agar.
• Application of a simple and direct method of cultivation appropriate for field conditions.
• Development of a rapid molecular method for species identification.
• It will also investigate additional diagnostic strategies such as an improved microscopical
detection and various M. tuberculosis antigens for their diagnostic value in a serological test.
• Development and evaluation of methods for the rapid detection of drug resistant TB, such as
rifoligotyping for direct use with clinical samples; RIF-thin layer agar and luciferase phage
test as a rapid direct test for RIF resistance. It will also evaluate faster, easier and accurate
methods for drug susceptibility testing, including second-line and newer anti-tuberculosis
drugs: rapid colorimetric methods, and conventional culture techniques adapted to a multiwell plate.
• A database of MDR genotypes for Latin America will be created and will include traditional
epidemiological information, so that transmission patterns could be delineated.
EXPECTED RESULTS
• Development of newer techniques into inexpensive, facile, accurate and reproducible formats.
• Performing multi-center pilot studies for standardization as necessary.
• Running of prospective clinical trials in TB endemic areas.
• Recommendation of methods for improved TB diagnosis, drug resistance detection, and drug
susceptibility testing
• Delineation of transmission patterns of MDR-TB in Latin America.
The project aims at applications of new inexpensive, easy-to-use, accurate and reproducible
diagnosis and resistance detection in high MDR-TB endemic areas of Latin America.
38
Acronym : Upgrade Diagno MDR-TB
Project number : ICA4-2001-10087
Starting date : January 1st 2002
Françoise Portael
Microbiology Institute of Tropical Medicine
Nationalestraat 155, 2000 Antwerp, Belgium
T: (+32) 3 2476317; F: (+32) 3 2476333
Email: [email protected], [email protected]
Clara Ines Leon
Laboratorio de Micobacterias
Instituto Nacional de Salud AA 80080, Av. El
Dorado con carrera 50, Santa Fe de Bogota,
Colombia T: (+57) 1 222 0577 Ext. 435;
F: (+57) 1 222 0194 Email: [email protected]
PARTNERS:
Dick van Soolingen LIS: Mycobacteria
National Inst. of Public Health and the Environment
P.O. BOX 1, antonie van Leeuwenhoeklaan 9, 3720
BA Bilthoven, The Netherlands
T: (+31) 30 2742363, F: (+31) 30 2744418
Patricia Del Portillo
Mycobacteriology Corpogen
Carrera 5 No. 66-68, Santa Fe de Bogota, Colombia
T: (+57) 1 348 4606; F: (+57) 1 348 4607
Carlos Martin
Microbiologia, Medicina Preventiva y Salud
Publica Universidad de Zaragoza
c/Domingo Miral, 50009 Zragoza, Spain
T: (+34) 976 761759; F: (+34) 976 761664
Peter Hermans Laboratory of Pediatrics
Erasmus University Rotterdam
P.O. Box 1738 Dr.Molewaterplein 50, 3000 DR
Rotterdam, The Netherlands
T: (+31) 10 408 8224, F: (+31) 10 408 9486
Brigitte Gicquel Génétique Mycobactérienne
Institut Pasteur
25, rue du Dr. Roux, 75724 Cedex 15, Paris, France
T: (+33) 1 45688828; F: (+33) 1 45688843
Maria Jesus Garcia Medicina Preventiva
Universidad Autonoma de Madrid
Arzobispo Morcillo s/n, 28029 Madrid, Spain
T: (+34) 91 3975440; F: (+34) 91 3975353
Jaime Robledo Bacteriology, Mycobacteriology
Corporacion para Investigaciones Biologicas
Carrera 72A No. 78B-141, Medellin, Colombia
T: (+57) 4 4410855; F: (+57) 4 4415514
Howard Takiff
Microbiologia y Biologia Celular
IVIC - Apto. 21827, 1020A Caracas, Venezuela
T: (+58) 212 504 1439; F: (+58) 212 504 1382
Jacobus de Waard Laboratory of Tuberculosis
Instituto de Biomedicina 4043, Carmelitas, Caracas
Al lado del Hospital Vargas
San Jose, 1010A Caracas, Venezuela
T: (+58) 212 8607095 Ext. 4307;
F: (+58) 212 8611258
Sylvia Leao
Microbiologia, Inmunologia y Parasitologia
Universidade Federal de Sao Paulo
Rua Botucatu, 862 3o, Andar 04023-062, Sao
Paulo, Brazil T: (+55) 11 5576 4537;
Viviana Ritacco Bacteriology – Mycobacteria
INEI-ANLIS Av. Velez Sarsfield 563, 1281 Buenos
Aires, Argentina T: (+54) 11 4602 7635,
Nora Morcillo
Mycobacteria Reference Laboratory
Hospital Cetrangolo - Italia 1750, 1602 Buenos
Aires, Argentina T: (+54) 114 756-4164;
F (+54) 11 4 762-0622
Angel Cataldi Laboratorio de Biotecnologia
INTA Los Reseros y las Cabañas, 1712, Buenos
Aires, Argentina T: (+54) 11 4621 1447 Ext. 148;
F: (+54) 11 4481 2975
Amelia Bernardelli Micobacterias DILACOT
Av. Alexander Fleming 1653,
1640, Martinez-Bs.As., Argentina
T: (+54) 11 4836 1992; F: (+54) 11 4836 1992
•
•
••
Philip Suffys Lab. Molecular Bioogy and
Infectious Diseases Oswaldo Cruz Institute
P.O. Box 926, Av. Brasil 4365,
21045-900 Rio de Janeiro, Brazil
T: (+55) 21 2598 4289; F: (+55) 21 2270 9997
Maritza Velasco Health-Section Mycobacteria
Instituto de Salud Publica
P.O. BOX 48, Av. Marathon 1000, Santiago de Chile,
Chile T: (+56) 2 3507 409; F: (+56) 2 239 7400
Eliana Roxo Laboratorio de tuberculose
Instituto Biologico P.O. Box 12898, Av. Conselheiro
Rodrigues Alves, 1252, 04014-002 Sao Paulo, Brazil
T: (+55) 11 5087 1774; F: (+55) 11 5087 1791
Maria Alice Telles Setor de Micobacterias
Instituto Adolfo Lutz Av. Dr. Arnaldo 351, 01246-902
Sao Paulo, Brazil T: (+55) 11 3068 2895;
Clara Espitia Ins. Investigaciones Biomedicas
Universidad Autonoma de Mexico
P.O. Box 70228, Circuito Universitaroi, 04510
Mexico D.F, Mexico
T: (+52) 55 5622 3884; F: (+52) 55 5622 3369
Julieta Luna Inmunologia
Inst. Politecnico Nacional - Prol. Carpio y Plan de
Ayala, 11340 Mexico D.F, Mexico
T: (+52) 55 729 6000 EXT. 62371;
F: (+52) 55 5396 3503
Ernesto Montoro
Mycobacteriology ReferenceLab.
Insittuto Pedro Kouri
601, Marianao 13, Autopista Novia del Mediodia
Km. 6 1/2, La Lisa 11300, La Habana Cuba:
T: (+53) 7 2020 448; F: (+53) 7 2046051
[email protected]
Luis Chacon
Mycobacteria Reference Laboratory
Centro Nacional de Diagnostico y Referencia
P.O. Box 2900 Complejo Nacional de Salud,
Managua, Nicaragua
T: (+505) 289 7723; F: (+505) 289 7723
Pedro Almeida da Silva Patologia
Universidade do Rio Grande
Rua. Engenheiro Alfredo Huch 475,
96201-900 Rio Grande, Brazil
T: (+55) 53 2311222 Ext. 189;
F: (+55) 53 232 8941
Afranio Kritski
Tuberculosis Research Unit
Universidade Federal de Rio de Janeiro
Av. Brigadiero Trompowsky s/n,
21941590 Rio de Janeiro , Brazil
T: (+55) 21 562 24 26; F: (+55) 21 550 69 03
Jeannete Zurita
Lab. Microbiologyospital Vozandes
P.O. BOX 17-17-691 Villalengua
Oe 2-37, Quito, Ecuador
T: (+593) 02 262142; F: (+593) 02 269234
Mirtha Camacho
Lab. Tuberculosis
INLASA
M10019 Mayor Zubieta Final, La Paz Bolivia
T: (+591) 2 2226048; F: (+591) 2 2228254
39
DEVELOPMENT OF ANTIMICROBIAL PEPTIDES
AS NOVEL ANTI-INFECTIVE DRUGS
SUMMARY
The rapid spread of antibiotic resistance and the development of new resistance mechanisms are growing health
threats. The search for alternative anti-infective drugs and new targets for antibiotic therapy is currently of high
priority. Promising, but so far unexploited candidates are antimicrobial peptides (AMPs) which are abundant in
nature and which have been conserved throughout evolution from bacteria to mammals as effective defence tools.
They interfere with vital functions of microbial membranes, a mechanism of action that is not exerted by currently
used antibiotics and thus circumvents the problem of pre-existing resistance mechanisms. The objective of this
project is to evaluate AMPs for therapeutic use against infections by bacteria and fungi and to bring selected
AMPs to the level of preclinical development.
PROBLEM
The alarming increase of antibiotic resistance is a problem of worldwide dimension. This problem is aggravated by a relative stagnation in the production of novel antibiotics by pharmaceutical companies. The health risks associated with the rapid spread of antimicrobial resistance
and the development of new resistance mechanisms in an increasing number of pathogens are
all too obvious. To name but a few problematic areas which urgently need improvements: (1)
methicillin-resistant S. aureus (MRSA) which can only be treated with glycopeptides and these
last-resort antibiotics are clearly loosing power (VISA strains); (2) there is an overwhelming frequency of penicillin-resistant pneumococci in some countries, and there is the danger of
importing resistant mycobacteria; (3) there are some very common infectious diseases such as
infections with Helicobacter pylori or Chlamydia for which better drugs need to be designed.
Thus, the development of new classes of antibiotics with different mechanisms of action is in
high demand.
AIM
The aim of the project is to systematically develop potent
AMPs by modification of natural peptides, knowledge-based
individuation of novel peptides, de novo peptide design, random combinatorial generation and high throughput screening
for therapeutic use against infections caused by bacteria and
fungi. The project concept is focussed on the exploitation of a
new microbial target structure. It will provide AMP-based antiinfective drugs on the level of preclinical development.
EXPECTED RESULTS
We expect to identify at least one potent AMP or AMP/antibiotic combination per group of pathogens which are most
prominent as to the development of antibiotic resistance
(MRSA, VRE, pneumococci, mycobacteria, multi-resistant
gram-negative rods such as enterobacteria and pseudomonads, yeast and fungi) and to provide material meeting the criteria regarding potency, toxicity, availability and purity for preclinical development. Moreover, the project will provide new
biotechnology (cost-effective and large-scale production systems for AMPs, High-Throughput technologies) and bioinformatic tools.
40
Acronym : PANAD
Keywords : Antimicrobial peptides, AMPs, antibiotic combinations,
multiresistant bacteria, rational design of AMPs, directed evolution of
AMPs, High-throughput screening of AMPs, bioproduction technologies
for AMPs
The research will generate exploitable results in two areas:
1 Technology and tools: This will include expression systems for AMPs, including hosts and
cloning vectors, novel screening tools, tools for computational analysis of AMPs etc.
2 Novel antibiotics
It is a common practice for pharmaceutical industries to buy know-how at the aimed level of
preclinical development for further in-house development.
http://www.bbcm.univ.trieste.it/~PANAD/links.html
Prof. Dr. Hans-Georg Sahl
University of Bonn
Institute for Medical Microbiology and
Immunology
Sigmund-Freud-Strasse 25 - DE-53127 Bonn
Germany
T: + 49 228 287 5704; F: + 49 228 287 4808
PARTNERS:
Prof. Dr. Åsa Ljungh
University of Lund
Department of Medical Microbiology,
Dermatology and Infection
Sölvegatan 23 - SE-223 62 Lund - Sweden
T: + 46 46 173283; F: + 46 46 152564
••
•
•
Dr. Alessandro Tossi
University of Trieste
Department of Biochemistry, Biophysics and
Macromolecular Chemistry
Via Giorgieri 1 - IT-34127 Trieste - Italy
T: + 39 040 6763673; F: + 39 040 6763691
Prof. Yechiel Shai, PhD
Weizmann Institute of Science
Department of Biological Chemistry
76100 Rehovot - Israel
T: + 972 89342711; F: + 972 89344112
Hans-Henrik Kristensen, PhD
Novozymes A/S
Krogshoejvej 36 - DK-2880 Bagsvaerd - Denmark
T: + 45 4442 1823; F: + 45 4498 0246
http://www.novozymes.com
Prof. Dr. Ingolf F. Nes
Agricultural University of Norway
Department of Chemistry and Biotechnology
Laboratory of Gene Technology
P.O. Box 5051 - NO-1432 Ås
Norway
T: + 47 64949471; F: + 47 64941465
•
•
41
BACTERIAL TWO-COMPONENT SYSTEMS AS
TARGETS FOR THE DEVELOPMENT OF NOVEL
ANTIBACTERIALS AND ANTI-INFECTIVES
SUMMARY
Microbial drug resistance imposes an increasing threat to human and animal health and the development of new
drugs to control bacterial infections is therefore urgently needed. The overall objective of this project is to exploit
a new class of bacterial targets called two-component systems (TCS) for the development of novel antibacterials
and drugs that specifically target the mechanisms by which bacteria establish infection in the host. This objective
will be achieved by focusing critical mass, expertise and support on our understanding of the structure, function
and biological diversity of TCS and the cellular processes that they control in the model organism S. pneumoniae.
By the end of the project we will expect to have selected appropriate targets and have developed selective assays
for inhibitors of those targets that can be used for high throughput screening in industry.
PROBLEM
The frequency of resistance to antibiotics among community acquired pathogens and the number of drugs to which they are resistant are increasing throughout Europe and the rest of the
world. Microbial drug resistance is an ever increasing threat to human and animal health and
imposes a heavy cost to the European economy. Resistance to antibiotics is linked to the widespread use of antibiotics in human healthcare and in veterinary medicine and is spreading
because of population migration and extensive travelling by citizens of the European
Community. The severity of the situation is exemplified by the increasing resistance of S. pneumoniae to common antibiotics which is currently undermining our ability to treat communityacquired pneumococcal disease. It is likely that the situation will get worse because of demographic trends in the number of elderly and immunocompromised European citizens at risk and
because of an increase in the use of in-dwelling devices in clinical medicine; use of these
devices increases the risk of infection. The development of new drugs to control bacterial infections is therefore urgently needed.
AIM
The overall aim of this project is to exploit bacterial two-component systems (TCS) as a new
class of targets for the development of novel pharmacological interventions to control infectious disease and combat antimicrobial drug resistance.
EXPECTED RESULTS
We expect to be able to identify the genes (and pathways) regulated by TCS that are essential
for growth and survival of bacteria in vitro and in vivo as these represent potential targets for
novel antimicrobials. We fully expect to find TCS that are important for survival or pathogenesis inside the host that could be explored as targets for anti-infective or adjunct therapies.
These might also be conserved among bacteria that cause similar disease syndromes (i.e.
meningitis or pneumonia). We anticipate that the knowledge we will gain about the function
and structure of these target proteins will enable us to develop selective assays for HTS of compounds that can be further developed to control bacterial infections and prevent bacterial disease in man and animals.
42
Acronym : TCS-Targets
Starting date : November 1st 2000
Keywords : Two-component systems, antimicrobials,
anti-infectives, Streptococcus pneumoniae, signal transduction,
Worldwide revenues from the sale of anti-infectives (which includes antimicrobials) is in
excess of £14 billion per annum representing the 5th largest category of worldwide pharmaceutical sales. This project is expected to generate validated drug targets and selective assays
for inhibitors that are amenable to high throughput screening. Moreover the detailed knowledge of the structure and function of the selected targets generated in the project will facilitate the iterative process of lead drug improvement through rational drug design and combinatorial chemistry. The projects focus on a novel and very promising class of targets is expected to create a competitive advantage for further development and exploitation. The industry
‘push’ behind this project is European and European industry will be at the forefront of this
exploitation. The exploitation and development process may still be lengthy taking up to five
years to register a new product. This will involve the screening of thousands of compounds
that have been designed on the lead inhibitors structure, biological studies, toxicology, manufacture and registration and clincal trials. This will involve links between industry and create
numerous employment opportunities in the European biotechnology and pharmaceutical
industry. It is anticipated that successful new drugs will have an enormous market and provide considerable opportunities for further wealth creation and employment.
Dr Jerry WELLS
Head of Bacterial Infection & Immunity
Institute of Food Research,
Norwich Research Park - Norwich, NR4 7UA
United Kingdom
T +44 1603 255250; F: +44 1603 507723
Web-site: http://www.ifr.bbsrc.ac.uk
PARTNERS
Prof Gianni Pozzi
Instituto di Microbiologica,
Universita di Siena
Via Laterina, 8 - IT-53100 Siena - Italy
T: +39 0577 233910; F: +39 0577 233870
Dr Paloma Lopez
Centro de Investigaciones Biológicas
Vélazquez 144 - ES-28006 Madrid
Spain
T: +34 91 5611800 Ext:4203;
F: +34 91 5627518
Dr Jean-Pierre Claverys
Laboratoire de Microbiologie et Génétique
Moléculaire
CNRS-UPR 9007, Université Paul Sabatier
118 route de Narbonne
FR-31062 Toulouse Cedex - France
T: +33 5 61 33 59 11; F: +33 5 61 33 58 86
•
•
•
Prof Tim Mitchell
Division of Infection and Immunity
Institute of Biomedical and Life Sciences,
Joseph Black Building
University of Glasgow
UK-Glasgow G12-8QQ - United Kingdom
T: +44 141 330 3749; F: +44 141 330 3727
•
43
REPLICATION INITIATION PROTEINS
AS NEW TARGETS FOR BACTERIAL
GROWTH INHIBITION
SUMMARY
The development of the new classes of antibacterial drugs to combat drug resistant strains of pathogens is
urgently needed. This project aims at exploring the possibilities offered by targeting the proteins of initiation of
DNA replication (IDR). Novel assays will be developed to highlight inhibition of IDR proteins.
PROBLEM
There is an urgent need for the discovery of novel antibacterial compounds to combat deadly
new multi-drug resistant strains.
AIM
Structure of the RepA homohexameric DNA
helicase. The complex of the
pot-shaped RepA hexamer with six modeled
ATP (blue stick models) located in clefts between the monomers is seen from the ``top''.
The N terminus of each monomer embraces
the adjacent
monomer. The central hole has a van der
Waals diameter of ~17 Å.
The DnaB-DnaC complex at 26 Å resolution.
Superimposed surface renderings of the
DnaB-DnaC reconstruction obtained from
cryo-electron microscopy images. The blue
one accounts for the 100% of the expected
mass, whereas the yellow one, which highligths the regions of higher density, accounts
only for the 40%. The hexameric helicase is
in the bottom part of this view, forming a
ring-shaped structure. On top of this ring,
three dimers of DnaC interlock with the DnaB
monomers.
44
Our targets are the proteins of initiation of DNA replication (IDR). Since they differ substantially in bacteria and eukaryotes the bacterial IDR proteins constitute an attractive and unexplored
family of targets for development of new antibiotics which is the essence of our project. The
novelty of this approach lies in the development of assays to highlight the inhibition of IDR proteins and in the identification and/or design of compounds, which target IDR proteins. Our
objective is to engineer bacterial strains for in vivo screening and in vitro biochemical detection
methods for the identification of novel inhibitors of DNA replication initiation. High-throughput
screening (HTS) and detailed structure-activity knowledge of the IDR protein inhibitors will be
used for synthetic modifications to enhance the specificity of targeting
EXPECTED RESULTS
Initiation of DNA replication (IDR) is a poorly exploited target, hence we aimed to develop cellular assays to screen for inhibitors of the IDR machinery that are robust, easy to run, cheap,
and address finding inhibitors that cross the cell membrane(s). We will validate the highlighted
positive hits in vitro. We will engineer bacterial strains for HTS to enable searches for possible
antimicrobials that inhibit the formation of macromolecular complexes of the IDR-proteins and
their DNA targets (primary screening). Biochemical assays will be established to ascertain the
mode of action of any identified inhibitor and confirm their function as inhibitors of the IDR
machinery. Evaluation and validation of these newly developed assays will allow the industrial
partner to search for new inhibitors based on work of this consortium. A natural compound,
that selectively inhibits replication fork DNA helicases, will be characterised and it will form the
basis for the lead structure used in the development of more potent and selective agents.
Biochemical and biophysical methodologies will be use to yield data on the details of the interactions of the system components for possible use in rational selection of compounds and tailored drug design. In addition, structure determination at medium and high resolution of individual IDR proteins will be carried out to provide further information for the industrial partner
to exploit a rational approach aimed to finding and improving the desired antimicrobials.
Acronym : DNA REPLICATION INHIBITORS
Keywords : Antimicrobials, High-throughput screening,
replication initiation proteins, replication origin, replisome organizer,
replicative helicase, helicase loader, DNA primase, DNA polymerase
Development of a new class of antibacterial compounds.
http://biocomp.cnb.uam.es/replication
Juan C. Alonso
Departamento de Biotecnología Microbiana
Centro Nacional de Biotecnología, CSIC
Campus Universidad Autónoma de Madrid
Cantoblanco, 28049 MADRID, SPAIN
T: (34) 91585 4546
F: (34) 91585 4506.
PARTNERS
Dr. R. Díaz-Orejas
Centro Investigaciones Biologicas, CSIC,
Madrid, ES
T: (+34) 91 5644562 – 4351, F: (+34) 91562 7518, email: [email protected]
Prof. W. Saenger,
Institut fuer Chemie/Kristallographie Freie
Universitaet,
Berlin, DE
T: (+49) 30838 3412, F: (+49) 30838 6702,
e-mail: [email protected]
Dr. J. M. Carazo
Centro Nacional de Biotecnología, CSIC,
Madrid, ES
T: (+34) 91 5854543, F: (+34) 91 5854506,
Prof. D. Rice,
The Krebs Institute, The University of
Sheffield,
Sheffield, UK
T: (+44) 114222 4242, F: (+44) 114272 8697,
Prof. Kirsten Skarstad
Inst. Cancer Research, The Norwegian
Radium Hospital, Oslo, NO
T: (+47) 2293 4255, F: (+47) 2293 4580
•
• •
•
Dr. E. Lanka,
Max-Planck-Institut Mol. Genetik,
Berlin, DE
T: (+49) 308413 1696, F: (+49) 308413 1130,
Prof. R. Boelens
Bijvoet Center for Biomolecular Research,
Utrecht University, Utrecht, NL
T: (+31) 30 2534035, F: (+31) 30 2537623,
Dr. J. F. Garcia Bustos
GlaxoSmithKline Research Centre,
Tres Cantos, ES
T. (+34) 91807 0606, F: (+34) 91807 0550,
•
45
MOLECULAR MECHANISM OF MACROLIDE
ANTIBIOTIC ACTION AND RESISTANCE:
APPLICATION IN DRUG DEVELOPMENT
SUMMARY
Bacterial pathogens with multiple antibiotic resistance have developed to previously unseen levels, and are
presently one of the greatest threats to human health. To tackle this problem, it is of crucial importance to
develop new classes of antibiotics. The most effective way to develop new antibiotics is to design them by
rational approaches, but in order to do this the molecular mechanisms of antibiotic action and resistance must
first be understood. We aim to elucidate the mechanisms of the action and resistance for the macrolide class of
antibiotics, as well as for the latest generation of macrolide derivatives, the ketolides, which exhibit activity
against macrolides-resistant bacterial strains. Both macrolides and ketolides interact with the RNA component of
ribosomes and thereby inhibit protein synthesis. We propose to employ a combination of genetic, biochemical
and structural tools, that are operational in our different research groups, and elucidate the molecular details of
the interaction and resistance to these drugs. The structural data will form the basis for the rational development
of new therapeutic agents..
PROBLEM
Resistance enzymes that modify the antibiotic or the biological targets have compromised the
clinical utility of many antibiotics. The molecular mechanisms of macrolide antibiotic interaction with its targets are still largely unknown. The elucidation of these mechanisms, and the
identification of new targets on the ribosome for novel therapeutic agents is a priority.
AIM
The development of antibiotics will be facilitated by an understanding of their molecular mechanism of action and associated resistance. Our specific aim is to understand, at the molecular level, the drug-rRNA interactions that are responsible for
macrolide and ketolide activity, and the structural basis for resistance to these drugs.
These antibiotics are similar to many other antibiotics in current clinical use, in that
they bind to ribosomal RNA (rRNA) and inhibit protein synthesis. Resistance is conferred to these drugs by modification of specific rRNA nucleotides within the drugbinding site. Nucleotide modification is achieved by methylation or by a substitution
mutation. The ketolides show improved interaction with the rRNA, which is reflected
in their ability to bind and inhibit the ribosomes of some macrolide-resistant bacterial pathogens. We believe that the ketolides are capable of further improvement, once
the details of their ribosome interactions are known in detail. Another approach that
we are following is to attack the source of the resistance phenomenon. This can be
achieved by blocking the pathogen’s ability to methylate its rRNA, which would reinstate the clinical utility of a range of macrolides drugs.
EXPECTED RESULTS
The results will describe the molecular basis of the recognition of macrolide and ketolide antibiotics on their rRNA target. The structure of a minimal RNA substrate in complex with a methyltransferase enzyme will give a precise view of the molecular mechanisms involved by which the
rRNA target is recognised and modified. Definition of the precise configuration of nucleotides
in the RNA motif will facilitate the rational design of drugs that can block the RNA binding site
within the methyltransferases. Such drugs would eliminate the ability of the methyltransferases to modify the rRNA target, and thereby reduce the capacity of bacteria to become resistant
to antibiotics.
46
Acronym : MOL-MECH-MAC
EC contribution : 661827 €
Keywords : antibiotic resistance, bacterial pathogens, ribosome,
macrolide, ketolide, rRNA
The results are predicted to enable further refinement of existing macrolide and ketolide
drugs. Secondly, the identification of new potential rRNA targets will facilitate the design of
new antibiotics. Furthermore, the potential to block an important bacterial drug resistance
mechanism will make it possible to reinstate several classes of drugs that are presently clinically compromised. An industrial collaborator (Aventis, France) will have the benefit of access
to the material and information gained from the project. The medical community will greatly
benefit from an increase in the arsenal of drugs with which bacterial infections can be combated.
Dr Dominique Fourmy
Laboratoire de RMN
ICSN-CNRS
Bât 27, 1 Ave de la Terrasse
FR-91198 Gif-sur-Yvette
France
T: +33 1 69 82 37 76; F: +33 1 69 82 37 84
•
•
Prof. Remme Jaanus
University of Tartu
Institute of Molecular and Cell Biology
Riia 23
51010 Tartu
Estonia
T: +372 7 375031; F: +372 7 420286
PARTNERS:
Dr Stephen Douthwaite
Department of Biochemistry and Molecular
Biology
University of Southern Denmark
University of Odense
Campusvej 55
DK-5230 Odense M
Denmark
T: +45 65 50 23 95; F: +45 65 93 27 81
Web-site:
http://www.sdu.dk/nat/bmb/faculty/srd.html
•
47
TYPE IV SECRETION SYSTEMS AS TARGETS
FOR ANTI-INFECTIOUS THERAPIES
SUMMARY
New targets for anti-infective intervention: We believe that the eukaryotic and prokaryotic molecules identified
as components or effec-tors in bacterial pathogenicity are targets for new drugs.
As proof of principle, we will identify molecules inhibiting Type IV secretion systems, a new target
for antimicrobials.
PROBLEM
Bacterial resistance to antibiotics is a major factor in the currently recognised resur-gence of
infectious diseases. It is a worldwide threat, which requires not only the imple-mentation of
prevention strategies but also the discovery of new drugs. The spectacular progress of the last
few years in dissecting the molecular basis of bacterial virulence has not yet been efficiently
directed to the area of drug discovery.
AIM
Subversion of eukaryotic hosts by bacterial pathogens requires specialised secretion systems
delivering virulence factors either into the environment or directly into host cells. Four major
macromolecule secretion pathways have been identified and designated as types I-IV. Export
pathways comprising conjugative DNA transfer systems and pathogenicity-related secretion
systems that are evolutionary and functionally related have been classified as type IV secretion
systems (TFSS). Transport of macromolecules across bacterial and eukaryotic membrane barriers is a complex process requiring multi-component machineries spanning the bacterial cell
wall. Examples of such systems are the Ptl, (Pertussis toxin export) system of Bordetella pertussis, the cag system of Helicobacter pylori, the VirB system in Brucella and the Dot/Icm system of Legionella pneumophila. Genome sequencing projects have also identified Type IV
secretion systems in pathogens including Rickettsia prowazekii, Actinobacillus actinomycetemcomitans and Bartonella henselae. Type IV secretion systems and the effector molecules that they transport are potentially ideal targets for new classes of anti-infective molecules.
We will design high-throughput screening methods for the identification of inhibitors of type IV
secretion from recombinatorial libraries. Fundamental research will be carried out in parallel to
increase our understanding of the structure and function of Type IV secretion systems in human
pathogens. This data will be essential for the optimisation of screening methods, the comprehension of the mechanisms of action of the inhibitors identified, and the modification and
improvement of first generation inhibitors. We will use the expertise in the consortium with
Type IV secretion systems in human pathogens, but will also use our considerable experience
with model systems such as conjugal plasmid transfer in E. coli or T-DNA transfer in
Agrobacterium to advance our com-prehension of bacterial virulence.
EXPECTED RESULTS
The molecules identified will allow the development of strategies for the optimisation of the
use of anti-infective agents, since the aim of these inhibitors is to disarm the pathogen by
blocking the export/translocation of essential virulence factors, and thus make it sensitive to
the host defences. In this way, this strategy circumvents the selective pressure imposed by the
bactericidal or bacteriostatic antimicrobial agents presently in use and may therefore prevent
the rapid development of resistant strains.
48
Acronym : TFSS
Keywords : Discovery of new drugs. Identification of new classes
of antimicrobials. Development of new antimicrobial approaches.
Bacterial virulence factors.
Success in this project will give birth to a new con-cept for development of antimicrobials, and
will stimulate the research for a whole new generation of molecules based on the inhibition
of bacterial virulence factors.
Dr. Werner Pansegrau,
Immunobiological Research Institute of
Siena (IRIS)
Chiron Vaccines S.p.A
Via Fiorentina, 1 - IT-53100 Siena
Italy
T: +39 0577 243383; F: +39 0577 243564
PARTNERS:
Prof. Antonello Covacci
Immunobiological Research Institute of
Siena (IRIS)
Chiron Vaccines S.p.A.
Via Fiorentina 1 - IT-53100 Siena
Italy
T: +39 0577 243235; F +39 0577 243564
Dr. David O'Callaghan
INSERM U431, Montpellier/Nîmes, France.
Université de Montpellier II
Faculté de Médecine
Place E. Battaillon - Avenue Kennedy
FR-34905 Montpellier – FR-30900 Nîmes
France
T: +33 4 66 23 48 99; F: +33 4 66 23 49 28
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Dr. Juan M, Garcia-Lobo
Depto. de Biologia Molecular
Facultad de Medicina
Universidad de Cantabria,
ES-39011 Santander
Spain
T: + 34 942 201 948; F + 34 942 201 945
Prof. Günther Koraimann
Institut für Mikrobiologie
Universitätsplatz 2
AT-8010 Graz
Austria
T:+43 316 380 5626; F:+43 316 380 98986
Dr. Christian Baron
Lehrstuhl für Mikrobiologie der Universität
München
Maria-Ward-Str. 1a
DE-80638 München
Germany
T: +49 89 2180 6138; F: +49 89 2180 6122
Prof. Paul Hookyaas
Leiden University
Institute of Molecular Plant Sciences
Clusius Lab
Wassenaarseweg 64
NL-2333 AL Leiden
The Netherlands
T: +31 71 5274933; F: +31 71 5274999
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49
NEW ANTIMICROBIALS TARGETING
TRANSLATION IN BACTERIA AND FUNGI
SUMMARY
The identification of new targets for antimicrobial and antifungal therapy and the development of inhibitors for
these targets are of eminent therapeutic importance. The project aims at pursuing these goals by addressing partial reactions of protein synthesis on the ribosome. Advanced high-throughput screening assays will be combined
with crystal structure determination of inhibitor complexes with potential targets (ribosomes, parts of ribosomes,
translation factors) to identify new lead compounds. Inhibitors will be selected from different compound libraries
obtained by chemical modification of known antibiotics and newly developed lead compounds, combinatorial
chemistry, or isolation from microbial extracts. Inhibition potential and mechanisms will be studied in established
bacterial and fungal model systems and in new assay systems derived from pathogenic microorganisms.
Emphasis will be on multiresistant organisms causing infections that present particular public health problems.
PROBLEM
The increasing frequency of nosocomial infections due to multi-resistant pathogens associated
with a decline in the immune defences due to age, invasive surgical procedures or other ailments, calls for novel and better antibiotics, effective especially for life-threatening infections.
The social changes brought about by an ageing population, new migratory fluxes and improved
surgical procedures often favour the establishment of hard-to-eradicate bacterial infections.
Examples of such bacterial infections include hospital-acquired infections, such as those
caused by Staphylococci, enterobacteria and others. In many of these cases, existing antibiotics are not effective anymore. Similarly, immunosuppression during treatment of AIDS
patients or accompanying organ transplantation has increased the number of opportunistic
and nosocomial fungal infections. Mycoses in these patients are often severe, rapidly progressing and difficult to treat. Different strains of Aspergillus, in particular A. fumigatus, are
found among the most infectious organisms. The spectrum of available antifungal drugs is
small, and resistances reduce their effectivity. The discovery of new and more effective antibiotics is thus a major challenge.
AIM
Direct location of antibiotic binding sites
on the 30S subunit of the bacterial ribosome
by x-ray crystallography.
(a) Ribbon diagram of the 30S subunit structure,
outlining the central region in which the
antibiotics bind,
(b) the central region expanded to show the
locations of streptomycin (blue), spectinomycin
(green), paromomycin (red), tetracycline
(orange), pactamycin (cyan), and hygromycin
B (magenta).
The general strategy of the project is to use the translation apparatus as target for the development of new, effective antimicrobials that are free of cross-resistances to existing antibiotics.
Major objectives are the following: Inhibitory substances will be selected from chemical
libraries, from collections of substances obtained by modification of known antibiotics or other
lead substances, and from microbial extracts. New inhibitory compounds will be isolated by
screening libraries of various kinds. By characterizing the inhibition mechanism of selected
compounds, new targets among the many components of the translation apparatus will be
identified. Whenever feasible, functional screening of compound libraries will be complemented with structure-based rational design based on lead compounds of known structure, target
and inhibition mechanism.
EXPECTED RESULTS
Expected achievements are the identification of inhibitors of bacterial and fungal translation
that may be developed for antimicrobial therapy. It seems realistic to expect several promising
compounds to emerge from the three screening approaches taken in parallel, i.e. screening of
microbial extracts, screening of combinatorial libraries, and rational design based on structures
of lead compounds of which several are already known. New and versatile assays of translation
adapted to high-throughput screening will be implemented for selection. Inhibition mechanisms will be clarified by structural and functional studies, and selectivity against the targeted
translation apparatus will be determined. Structures of inhibitor-target complexes will be determined and will form the basis for designing new inhibitors circumventing the problem of resistance. Results from the project are also expected to be of major scientific interest.
50
Acronym : Ribosome inhibitors
Keywords : Antibiotics, Translation inhibitors, Ribosomes, Protein
synthesis, Multiresistant micro-organisms, Combinatorial libraries,
Microbial extracts, High-throughput screening, Rational inhibitor
design, Crystal structure
The expected results will form the basis for the development of efficient methods for screening compound libraries which will lead to the identification of new potential drugs which further on may enter testing in animal models and clinical trials. The successful completion of
the project is expected to lead to the development of new products and increase market
shares. Given the large market for antibiotics against infectious diseases of both humans and
animals, the results are expected to significantly improve the competitiveness of the
European pharmaceutical industry. The impacts of the long-term aspect of this project, in the
design and selection of new antibiotic molecules, are potentially strong in the social and economic sphere.
www.uni-wh.de
Prof. Dr. Wolfgang Wintermeyer
Institute of Molecular Biology,
University of Witten/Herdecke
D-58448 Witten, Germany
Phone (+49 2302) 669140/1; Fax (+49 2302) 669117
e-mail: winterme@uni-w
website: www.uni-wh.de
PARTNERS:
Prof. Dr. Diarmaid Hughes
Dept. of Cell and Molecular Biology,
Uppsala University
PO Box 596, S-75124 Uppsala, Sweden
Phone (+46 18) 4714354; Fax (+46 18) 530396
website: www.uu.se
Prof. Dr. Claudio O. Gualerzi
Dip. di Biologia MCA, University of Camerino
via Camerini 2, I-62032 Camerino, Italy
Phone (+39 0737) 403240; Fax (+39 0737) 403243
website: www.unicam.it
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•
Prof. Dr. Roger A. Garrett
Institute of Molecular Biology,
University of Copenhagen
Soelvgade 83H, DK-1307K Copenhagen, Denmark
Phone (+45 35) 22010; Fax (+45 35) 322040
website: www.ku.dk
Prof. Dr. Jens Nyborg
Dept. of Molecular and Structural Biology,
Aarhus University
Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark
Phone (+45) 89425257; Fax (+45) 86123178
website: www.au.dk
Dr. Venkitaraman Ramakrishnan
Laboratory of Molecular Biology, MRC
Hills Road, Cambridge CB2 2QH, United Kingdom
Phone (+44 1223) 402213; Fax (+44 1223) 213556
website: www.mrc-lmb.cam.ac.uk
Dr. Fredrik Björkling
Dept. of Medicinal Chemistry,
Leo Pharmaceutical Products Ltd
Industriparken 55, DK-2750 Ballerup, Denmark
Phone (+45) 44923800; Fax (+45) 44945510
website: www.leo-pharma.com
Prof. Dr. Juan P. Ballesta
Molecular Biology Center, CSIC
Canto Blanco, E-28049 Madrid, Spain
Phone (+3491) 3975076; Fax (+3491) 3974799
website: www.csic.es
Dr. Roscoe Klinck
Ribotargets Ltd.
Granta Park, Abington, Cambridge CB1 6GB, United
Kingdom
Phone (+44 1223) 895555; Fax (+44 1223) 895556
website: www.ribotargets.com
Prof. Dr. Anders Liljas
Dept. of Molecular Biophysics, Lund University
PO Box 124, S-22100 Lund, Sweden
Phone (+46 46) 2224681; Fax (+46 46) 2224692
website: www.lu.se
Dr. Stefano Donadio
Biosearch Italia SpA.
via R. Lepetit 34, I-21040 Gerenzano, Italy
Phone (+39 0296) 474243; Fax (+39 0296) 47423
website: www.biosearch.it
51
TOWARDS NEW ANTIBIOTICS
SUMMARY
This project will result in the production of cell factories that will mediate improved routes for the production of
antibiotics and beta-lactamase inhibitors. The Cell Factories will utilise modified versions of naturally occurring
enzymes involved in beta-lactam biosynthesis and modification.
PROBLEM
The major mode of bacterial resistance to beta-lactam antibiotics including the penicillins and
cephalosporins is mediated via beta-lactamase enzymes that destroy the beta-lactam ring. The
most successful strategy to overcoming this threat has been the use of the antibiotic in combination with a ‘guardian angel’ compound that neutralises the beta-lactamase by inhibition.
Clavulanic acid is one such compound and in combination with amoxycillin (as Augmentin) it
has been a highly successful pharmaceutical. Interestingly, relatively little resistance has been
observed in the combination therapy. However, a drawback to the use of clavulanic acid is that
it is only effective against one sub-family of the beta-lactamases, the Class A enzymes, which
are selective for penicillins. It is ineffective, for example, in protecting the cephalosporins that
are primarily susceptible to the Class C enzymes. This is important since cephalosporins, along
with other beta-lactam families including carbapenems, are important antibiotics used in hospitals. Although beta-lactamase inhibitors that are active against both Class A and C inhibitors
have been developed, none have progressed to clinical use, in part because of cost of production issues associated with a combination therapy.
AIM
The overall aim of this project is to use engineered micro-organisms to provide efficient routes
for (i) production at templates for modification into antibiotics and b-lactamase inhibitors, (ii)
clean synthesis routes to existing antibiotics which would otherwise have to be produced via
costly and environmentally unfriendly procedures, (iii) making possible the commercial production of broad spectrum b-lactamase inhibitors which are otherwise too costly to produce.
EXPECTED RESULTS
The work will involve an integrated program involving detailed structural, mechanistic, analytical and engineering studies on the relevant enzymes. The results will be combined to design
and generate the engineered micro-organisms or Cell Factories which will utilise modified version of naturally occurring enzymes involved in biosynthesis.
A view derived from the crystal structure clavaminic acid synthase
complexed to iron (in pink). It shows part of
the active site of the
enzyme substrate (NAA/SAKA2) complex
before (in green) and after the
addition of NO (in yellow) which acts as an
analogue for the natural
co-substrate dioxygen.
52
Acronym : TNA
Project number : QLK3- 2000-00513
Starting date : January 1st, 2001
Keywords : antibiotic, antimicrobial drug resistance, beta-lactamase,
cephalosporin, clavulanic acid, fermentation, enzyme inhibitors,
oxygenase, penicillin.
The scientific results will be implemented by the construction of engineered micro-organisms.
These will be used as starting points for industry to develop new production routes to the
desired target compounds.
Prof. Christopher J. Schofield,
The Oxford Centre for Molecular Sciences
and The Dyson Perrins Laboratory
Oxford University , UK-Oxford OX1 3QY, UK
T: (+44) 1865 275625; F: (+44) 1865 275625
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•
PARTNERS:
Prof. Janos Hajdu
Laboratory of Cellular and Molecular
Biophysics
Department of Biochemistry
Uppsala University
Box 576, SE-751 23 Uppsala, Sweden
T: (+46) 18 4714449; F: (+46) 18 511755
Prof. Inger Andersson,
Department of Molecular Biology
Swedish University of Agricultural Sciences
Uppsala Biomedical Centre
Box 590, SE-751 24 Uppsala, Sweden
T: (+46) 18 471 4288; F: (+46) 18 536971
Prof. Jean-Marie Frère
Centre for Protein Engineering (CIP)
Faculté des Sciences, Institut de Chimie B6
Université de Liege au Sart Tilman
BE-4000 Liege 1, Belgium.
T: (+32) 4 366 33 98; F: (+32) 4 366 33 64
Dr. Clive Halliday
AMURA Limited
Compass House
Vision Park
Chivers Way, Histon, Cambridge, CB4 9ZR UK
T: (+44 ) 0 1223 266782; F: (+44) 0 1223 266781
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53
NOVEL NON-ANTIBIOTIC TREATMENT
OF STAPHYLOCOCCAL DISEASES
SUMMARY
The work of this project aims to explore the proteolytic system of Staphylococcus aureus and to generate
compounds that have suitable pharmacological properties for treatment of staphylococcal diseases through
selective interference with staphylococcal proteases affecting the bacterium viability or attenuating its virulence.
Furthermore, included in the project is the evaluation of targets and substances in animal models as proof of
concept.
PROBLEM
Currently, S. aureus is a leading etiologic agent of nosocomial infections world-wide, causing
an astonishing variety of diseases ranging from superficial abscesses, osteomyelitis, endocarditis, toxic shock syndrome and, of course, life threatening bacteremia. Taking into account
the rapid spread of antibiotic resistance in hospitals worldwide, it is apparent that S. aureus
constitutes a serious danger. In this critical situation a comprehensive understanding of pathogenicity pathways, together with a complete description of virulence factors and how they are
regulated, is urgently needed to design new therapeutic and preventive treatments.
AIM
The overall objective is to characterise the complement of important proteases of S. aureus and
to develop them as novel drug targets to improve our fight against disease. Our specific objectives are to:
• Establish an integrated programme of basic and applied research, using state of the art
approaches, to determine the role of the proteases of S. aureus in disease.
• Address specific work packages to define protease targets, characterise the target enzymes
at the molecular level and design and produce inhibitors for anti-staphylococcal and pharmacological evaluation.
• Create a fully coordinated, international, synergistic, research consortium with a common
goal to improve human health via a clearly defined research programme.
• Provide an ideal academia/industry interface to allow rapid application of discoveries into
novel therapeutic approaches based on the proteases as targets.
• Maintain and enhance European leadership and excellence in basic and applied research
into microbial pathogenicity.
EXPECTED RESULTS
In order to achieve these aims, this consortium incorporates a strong basic research program
aimed at the elucidation of the mechanisms regulating the expression and processing of virulence factors at the molecular level. Information gained will be fed back to our rational drug
design program reinforced by crystallographic studies of the selected targets. In addition to a
significant broadening of our knowledge of S. aureus physiology and the molecular basis of
pathogenicity, a successful outcome to this project will provide compounds with potency for
treatment of staphylococcal infections using a novel therapy, which may replace classical
antibiotics. Moreover, this approach may set a new paradigm to treat infectious diseases that
are resistant to classical antibiotics by targeting pathogen-encoded proteases. Although this
project with its three-line structure represents the early research phase in drug development
we expect to deliver at least two drug candidates effective in treatment of experimental antibiotic resistant staphylococcal infections in several animal models. Such successful outcome will
be intensively exploited by the SME in the consortium to bring a new protease inhibitor-based
antistaphylococcal therapy into clinical trials.
54
Acronym : ANTISTAPH
Project number : QLKT-2002-01250
st
Starting date : September 1 , 2002
Keywords :Staphylococcus, protease, inhibitor
Publication of results obtained in the early work packages of the project and their dissemination has the highest priority. The up-coming drug candidates will represent late stages of
basic research and will be shuttled directly into pre-clinical and subsequently clinical testing.
The participating SME, 4SC, intends to exploit the results by patenting the discovered compounds or used synthesis methods/technologies, by continuing research on these compounds in pre-clinical and clinical studies and by subsequent licensing of these inventions to
industrial partners for marketing and further application in treatment of staphylococcal diseases.
Dr Magnus Abrahamson
Lund University
Dept Clinical Chemistry
University Hospital, 22185 Lund, Sweden
T: (+46) 46 173445; F: (+46) 46 130064
Web-site: www.klinkem.lu.se
PARTNERS:
Dr Matthias Bochtler
International Institute of Molecular
and Cell Biology
Dept of Structural Biology
ul. Ks. J. Trojdena 4, 02-109 Warsaw, Poland
T: (+48) 22 6685193; F: (+48) 22 6685288
Web-site: www.iimcb.gov.pl
Prof. Simon Foster
The University of Sheffield
Dept Molecular Biology and Biotechnology
Firth Court, Western Bank, Sheffield, S10 2TN, UK
T: (+44) 114 2224411; F: (+44) 114 2728697
Web-site: www.sheffield.ac.uk
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Prof. Timothy Foster
Trinity College Dublin
Dept Microbiology
Moyne Institute of Preventive Medicine
Dublin 2, Ireland
T: (+353) 1 6082014; F: (+353) 1 6799294
Web-site: www.tcd.ie
Prof. Jan Potempa
Jagiellonian University Krakow
Dept Microbiology
Inst. Mol. Biol. & Biotechnol.
ul. Gronostajowa 7,30-387 Krakow, Poland
T: (+48) 12 2526343; F: (+48) 12 2526902
Web-site: www.uj.edu.pl
Prof. Andrej Tarkowski
University of Gothenburg
Dept. Rheumatology
Guldhedsgatan 10, 41346 Gothenburg, Sweden
T: (+46) 31 3424056; F: (+46) 31 823925
Web-site: www.microbio.gu.se
Dr Stefan Strobl
4SC AG
Dept. Biology
Am Klopferspitz 19, 82152 Planegg-Martinsried,
Germany
T: (+49) 89 7007630; F: (+49) 89 70076329
Web-site: www.4sc.com
55
DEVELOPMENT OF NOVEL ANTI-BACTERIALS
AND ANTI-INFECTIVES THAT TARGET
PROGRAMMED BACTERIAL CELL DEATH
SUMMARY
As a novel strategy to combat bacterial multidrug resistance we have selected as targets naturally occurring
bacterial apoptotic systems (BASs). As their key elements, they have a stable toxin (T) and an unstable anti-toxin
(A) that may be chromosomally encoded or encoded by multidrug-resistance plasmids. Typically, the TA pair form
a complex that prevents the activity of the toxin. Increased proteolysis of the chromosomally-encoded anti-toxin
can lead to an apparent loss of cell viability and thus these systems have been referred to as bacterial apotosis
systems (BAS) leading to programmed cell death. Apoptotic systems differ in bacteria and eukaryotes, thus TA
interactions and their checkpoint elements are an attractive and a so-far unexplored family of targets for the
development of antibiotics. Our novel approach lies in the development of assays to highlight the deregulation of
the TA systems with a subsequent triggering of cell death. High-throughput screening (HTS) and detailed
structure-function knowledge of the TA systems will be used for drug design and will enable the design of synthetic modifications necessary to enhance the specificity of targeting.
PROBLEM
The need to discover novel antibacterial compounds to combat new multi-drug resistant strains
is urgent, because of the rapid rise in the number of pathogenic bacteria that have acquired
resistance to all commonly used drugs. This problem requires immediate action along new
paths. We therefore decided to exploit TA systems as a new and unexplored class of targets for
the development of novel anti-microbial compounds and anti-infective interventions.
AIM
A model of E. coli RelBE-mediated cell death.
The antitoxin (RelB) neutralises the toxin
(RelE) by forming a TA complex.
The TA complex binds to the operator in the
promoter region and represses transcription.
A cellular protease degrades the antitoxin
thereby leaving an activated toxin.
The question mark indicates that it is not yet
known if a free toxin or a TA with a different
stoichiometry binds to the target.
To engineer bacterial strains for in vivo screening and to develop in vitro methods to identify
novel compounds that may deregulate the TA interactions. To develop cellular assays to monitor the activity of the BASs and to render them suitable for HTS of chemical libraries and natural compounds collections. We will also identify the regulatory circuits and targets of toxins for
several different TA systems using DNA microarrays and proteomics. Biochemical and biophysical approaches will be used to characterize the details of toxin-antitoxin protein complexes.
Additionally we aim to solve the structure of selected TA proteins to assist with the rational
design of inhibitors.
EXPECTED RESULTS
The work will involve an integrated programme as follows: i) the production of novel, robust,
easy to run, and cheap in vivo assays to screen for the deregulation of the TA interactions; ii)
the identification of bacterial growth inhibitors that cross the cell membrane(s); iii) medium to
high resolution structural information on individual toxin-antitoxin proteins, and mechanistic,
analytical, and engineering studies on the relevant TA systems; iv) the identification of selected cellular targets of selected TA toxins; v) the development of biochemical and biophysical
methodologies will provide data on the interactions of the system components, which will be
used in the rational selection of compounds and tailored drug design, and vi) the validation of
the highlighted positive hits in vitro. The results will be combined to design engineered bacteria which will be used to identify lead compounds. Employment of biochemical and biophysical
methodologies yielding data on the details of the interactions of the system components for
possible use in rational selection of compounds and tailored drug search will be pursued.
56
Acronym : BAS anti-microbials
Starting date :December 1st 2001
Keywords : toxin-antitoxin interactions, bacterial apoptotic systems,
checkpoint elements, de-regulation of toxin-antitoxin systems,
bacterial cell death
The identification of the TA’s checkpoint elements and the biochemical and biophysical analysis of the toxin / target interactions will lead to an understanding of their functional mechanisms. Based upon this knowledge, the means to activate the toxins leading to bacterial cell
death will be developed. The scientific results will be implemented for the construction of
engineered bacterial strains that will be used as starting points for industry to identify new
chemical leads compounds that blocks cell proliferation. The validated compounds would be
used to design new active substances that target BASs, so that they may constitute the next
generation of antimicrobials. The industrial participant has the expertise and commitment to
develop this kind of molecules.
http://www.biocomp.cnb.uam.es/BAS
Prof. Manuel Espinosa
Protein Structure & Function
Consejo Superior de Investigaciones Científicas
Velázquez, 144, E-28006 Madrid, Spain
T: (+34) 915611800; F: (+34) 915627518
Web-site: http://www.cib.csic.es
PARTNERS:
Prof. Juan Carlos Alonso
Microbial Biotechnology
Consejo Superior de Investigaciones Científicas
Centro Nacional Biotecnología
Universidad Autónoma Cantoblanco
E-28049 Madrid, Spain
T: (+34) 91585 4546; F: (+34) 91585 4506
Web-site: http://www.cnb.uam.es
Prof. Kenn Gerdes
Biochemistry & Molecular Biology
South Denmark University
South Denmark University
Campusvej 55, DK-5230 Odense, Denmark
T: (+45) 65502413; F: (+45) 65932781
Web-site: http://www.sdu.dk/Nat/bmb/
•
Dr. Dolors Balsa
Biochemistry
Laboratorios Salvat
Gall,, 30 – 36, E-08950,
Esplugues de Llobregat, Spain
T: (+34) 933718600; F: (+34) 933732292
Email: bioquí[email protected]
Web-site: http://www.salvat-lab.es
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Dr. Ehud Gazit
Molecular Microbiology & Biotechnology
Tel-Aviv University
69978 Tel-Aviv, Israel
T: (+972) 3640750; F: (+972) 36409407
Dr. Jeremy Wells
Division of Food Safety Science
Institute of Food Research
Norwich Research Park,
Colney NR4 7UA, Norwich, UK
T: (+44)- 603255250; F: (+44) 1603255037
Web-site: http://www.ifr.bbsrc.ac.uk
Prof. Wolfram Saenger
Institut für Kristallographie
Freie Universität Berlin
Institut für Kristallographie
Takustrasse, 6, D-14195 Berlin, Germany
T: (+49) 3083853412; F: (+49) 3083856702
Web-site: http://userpage.chemie.fu-berlin.de
Prof. Heinz Welfle
MAX-DELBRUECK-CENTER FOR MOLECULAR
MEDICINE
Robert-Roessle, 10, D-13092 Berlin-Buch, Germany
T: (+49) 3094062840, F: (+49) 3094062840
•
57
COMPARATIVE ANALYSIS OF PROTEOME
MODULATION IN HUMAN PATHOGENIC
BACTERIA FOR THE IDENTIFICATION
OF NEW VACCINES, DIAGNOSTICS AND
ANTIBACTERIAL DRUG TARGETS
SUMMARY
The EBP network intends to discuss common experimental approaches and criteria for building a prototype
database of bacterial proteomes, and proteome modulation in human bacterial pathogens, while performing at
the same time pilot studies on a few selected bacterial species. The ultimate scope is that of exploring how the
implementation of state-of-the-art proteomic techniques, and the availability of databases of protein expression
profiles in bacterial pathogens, can speed up the identification of new protein candidates for future health-care
product development.
PROBLEM
Several bacterial infections are still representing a clinical problem, because there are no effective vaccines or only partially effective vaccines are available, or because of an observed
increasing frequency of antibiotic resistant strains, or because of inadequate diagnostic procedures. Considering the current availability of a constantly increasing number of fully sequenced
genomes, and the powerful technology today available for the analysis of complex protein samples it seems logic to assume that new vaccines and druggable targets for antibacterial therapy should be ultimately found by an adequate screening of all the proteins potentially encoded by a pathogenic bacterial species.
AIM
To perform – as a preliminary demonstration exercise – pilot experiments of proteome analyses
on a panel of 6 selected pathogens, using different technical approaches according to the problems posed by the biology of the individual bacteria. Accordingly, different partners perform
proteome analyses with diverse technical approaches: annotation of 2DE protein maps by
mass spectrometry techniques, 2D immunoblotting, transcript profiling analyses using genomic DNA microarrays, and generation of a collection of proteome specific antibodies by highthroughput cloning and expression. In particular the network encourages comparative studies
of protein expression profiles displayed in diverse physiologically relevant conditions, proteome changes in mutants with different pathogenicity phenotype, and the identification of
(outer)membrane proteins, and/or immunogenic proteins.
To discuss common standards and collect data into a database with a user-friendly interface for
data mining.
EXPECTED RESULTS
An experimental assessment of the current efficacy of proteomic approaches for the identification of new health care products in the field of human bacterial infections.
Establishment of a prototype public Database of Protein Expression Profiles in Human Bacterial
Pathogens. Such a database is meant to provide a basic tool for future searches of useful
molecular targets through data mining.
58
Acronym : EBP
Starting date : Dec 1st 2000
Contract Type : Thematic network
Keywords : Bacteria, proteomics, proteome expression profiles,
genomic transcript profiles, vaccines, druggable targets, bacterial
pathogenicity, human infectious disease, Databases, Data Mining
Besides the prospected public use of the EBP database outlined above, the studies promoted and facilitated by EBP may in fact already identify candidate protein molecules to be
selected for better focussed studies and future development of new health care products.
http://www.mpiib-berlin.mpg.de/2D-PAGE/ (EBP Database Project)
http://www.ebpnet.it (only for registered users)
Dr Giulio Ratti
IRIS Research Centre, Chiron Vaccines
Via Fiorentina, 1 – Siena 53100 –Italy
T: (+39) 0577 243 239; F (+39) 0577 243564
Philip Marsh
CAMR, Centre for Applied Microbiology
& Research
Porton Down, Salisbury, Wiltshire, SP4 0JG, UK
T: (+44) 1980 612100; F; (+44) 1980 612731
PARTNERS:
Guido Grandi
IRIS Research Centre
Chiron Vaccines
Via Fiorentina 1 - 53100 Siena, Italy
T: (+39) 0577 243506
Siv Andersson
The Linnaeus Centre for Bioinformatics
Department of Molecular Evolution
Uppsala University
"Norbyvägen 18 D, S-752 36 UPPSALA", Sweden
T: (+46) 18 4714379; F: (+46) 18 4716404
Web-site:
http://artedi.ebc.uu.se/molev/staff/staf/people/si
v.html
Rino Rappuoli
IRIS Research Centre, Chiron Vaccines
Via Fiorentina 1 - 53100 Siena, Italy
T: (+39) 0577 243414
Christoph Dehio
Biozentrum of the University of Basel
Klingelbergstrasse 70 CH-4056 Basel, Switzerland
T: (+41) 612672140; F: (+41) 612672118
Web-site:
http://www.biozentrum.unibas.ch/Research/Micro
biology/microbiology.html
•
• •
• •
•
•
•
Joel Vandekerckhove
Department of Medical Protein Research
University Gent
"A. Baertsoenkaai 3, B-9000 GENT", Belgium
T: (+32) 9 3313317F: (+32) 9- 313597
Gunna Christiansen
Svend Birkelund
Department of Medical Microbiology and
Immunology
The Bartholin Building - University of Aarhus,
8000 Aarhus C, Denmark
T: (+45) 8942 1749; F: (+45) 86196128
Web-site: http://www.gram.au.dk (temporarily
behind a firewall)
Xavier Nassif
Université René Descartes
Faculté de Medicine NECKER
Laboratoire de Microbiologie INSERM U 411
156, rue de Vaugirad, 75730 Paris Cedex 15, France
T: (+33) 1 40615375; F: (+33) 1 40 615592
Email: http://www.necker.fr/u570/
Stefan H.E. Kaufmann
Max Planck Institute for Infection Biology
Schumannstrabe 21/22 - 10117 Berlin, Germany
T: (+49) 30 28460 502; F: (+49) 30 28460501
Web-site: http://www.mpiib-berlin.mpg.de/institut/immunology.htm
Peter Jungblut
Schumannstrabe 21/22, 10117 Berlin, Germany
T: (+49) 30 28460 133
Web-site: http://www.mpiib-berlin.mpg.de/2DPAGE/
Thomas F. Meyer
T: (+49) 30 28460 400; F: (+49) 30 28460401
Web-site: http://www.mpiib-berlin.mpg.de/institut/molecular.htm
Mark Achtman
Max Planck Institut fur Infektionsbiologie
T: (+49) 30 28460751; F: (+49) 30 28460750
related link - Infection, Genetics and Evolution:
Web-site: http://www.elsevier.nl/locate/meegid
Luca Bini, Marco Oggioni
Universita’ degli Studi di Siena
Via Fiorentina 1, 53100 Siena, Italy
T: (+39) 0577 234937/8; F: (+39) 0577 234903
Web-site: http://www.bio-mol.unisi.it/2d/2d.html
Web-site: http://www.unisi.it/ricerca/prog/pepsacmimic/
Pierre-Alain Bintz Denis Hochstrasser
Hopital Cantonal Universitaire Genevois
Departement de Biochimie medicale
24, rue Micheli-du-Crest, CH-1211 Geneve 14,
Switzerland
T: (+41) 22 3727357
Web-site: http://www.expasy.ch/PierreAlain.Binz.html
Jeremy Wells,
Institute Food Research
Norwich Research Park
Colney, Norwich NRR4 77UA, U.K.
T: (+44) 1603 255250; F: (+44) 1603 507723
Web-site: http://www.micron.ac.uk/People/jerrywells/wells.html
59
GLYCOSYLATION ENGINEERING
FOR NOVEL ANTIBIOTICS
SUMMARY
Medically important polyketide antibiotics produced by streptomycetes often contain deoxysugar residues
essential for their function. Altering these sugars would be a powerful route to new antibiotics. Increasing
microbial resistance makes this an urgent need. This project aims to study production of novel glycosylated
polyketides, using knowledge of the biosynthetic pathways to deoxyhexoses (6-DOH).
PROBLEM
Antimicrobial resistance is known to be sensitive to the pattern and type of sugar substitution
on macrolide antibiotics. Current methodologies using chemical modification are too slow to
make changes to this glycosylation, and too inefficient and non-specific.
AIM
Most of the antibiotics in clinical use are produced by actinomycete bacteria. The polyketides
in particular are a structurally diverse group of antibiotics, which include compounds with antibacterial, anti-fungal and other bioactivities. Their diversity is further increased by the attachment of sugar residues, which have a decisive influence on bioactivity. The continuing problem
of microbial resistance to existing antibiotics means that there is an urgent need to use information about these biosynthetic pathways in actinomycetes to accelerate the production of
new antibiotic compounds. Fortunately, even small structural changes may provide significant
therapeutic gains. The objectives of this project are to use a range of both in vivo and in vitro
approaches to produce polyketide antibiotics bearing altered sugar residues.
We aim to develop generic technologies based on genetic manipulation of the antibiotic-producing genes, that make a real difference to the speed with which sugar modifications on
antibiotic polyketide aglycones can be accomplished. Our objectives include the development
of gene cassettes for production of activated sugars in heterologous hosts, and also in vitro.
EXPECTED RESULTS
At least 10 new hybrid polyketide macrolides and the development of the first
in vitro systems for alternative combinatorial glycosylation of macrolide templates.
A heterologous glycosyltransferase
gene and a plasmid encoding an entire
activate sugar biosynthetic pathway are
introduced into a suitable bacterial cell,
and the recombinant cell is used as a
cell factory for bioconversion of added
aglycones to new compounds.
60
Acronym : GENOVA
Starting date : February 1st 2000
Contract Type : Shared-Cost R D
Keywords : Streptomyces; macrolide; antimicrobial; resistance;
glycosylation; polyketide; actinomycete; antifungal;
glycosyltransferase; deoxyhexose
The results will be implemented in several ways: (i) in in-house developments in
GlaxoSmithKline’s research on antimicrobials; (ii) for further R & D in partners’ laboratories
towards hybrid antibiotics; (iii) through the start-up company Biotica Technology (Cambridge)
which is associated to GENOVA to build a platform technology for glycosylation engineering;
and though the start-up company Combinature AG which is not formally associated but which
has as co-founder Professor A Bechthold who is a GENOVA partner.
Prof. Peter F Leadlay FRS
University Professor of Molecular
Enzymology and BBSRC Research Professor
Department of Biochemistry,
University of Cambridge
80 Tennis Court Road, UK-Cambridge CB2 1GA, UK
T: (+44) 1223 333656; F: (+44) 1223 766091
Web-site:
http://www.bioc.cam.ac.uk/UTOs/Leadlay.html
••
PARTNERS:
Prof. Andreas Bechthold
Albert-Ludwigs-Universitaet Freiburg
Pharmazeutische Biologie
Stefan-Meier-Strasse 19, DE-79104 Freiburg,
Germany
T: (+49) 761 2032802; F: (+49) 761 2032803
Professor Wolfgang Piepersberg
Bergische Universität GH
FB 9, Chemische Mikrobiologie
DE-42097 Wuppertal, Germany.
T: (+49) 202 439 2521; F: (+49) 202 439 2698
Dr Jesús Cortés
GlaxoSmithKline Research and
Development Ltd,
Medicines Research Centre
Gunnels Wood Road
Stevenage, UK-Hertfordshire, SG1 2NY, UK
T: (+44) 1 438 763781; F: (+44) 1438 764473
Dr Lothar Elling
Institut für Enzymtechnologie der HeinrichHeine-Universität
Düsseldorf im Forschungszentrum Jülich
DE-52426 Jülich, Germany
T: (+49) 2461 613348; F: (+49) 2461 612490
Dr Patrick Caffrey
University College Dublin
Department of Industrial Microbiology
Belfield, Dublin 4, Ireland
T: (+44) 3 531 706 1396; F: (+44) 3 531 706 1183
Prof. José Salas
Departamento de Biologia Funcional
Universidad de Oviedo
ES-33006 Oviedo, Spain
T: (+34) 985 103652; F: (+34) 985 103148
•
•
61
METABOLIC ENGINEERING OF GLYCOPEPTIDE
ANTIBIOTICS: TECHNOLOGY, OPTIMISATION
AND PRODUCTION
SUMMARY
The project is directed at generating novel glycopeptide antibiotics and effective tools for their development.
PROBLEM
The increasing frequency of nosocomial infections due to multi-resistant pathogens exerts a
significant toll in the industrialised world and calls for novel and better antibiotics, which need
to be developed in a fast and effective manner.
AIM
The aim of this project is to integrate industrial and academic laboratories in a combined effort
of research and development directed at generating new glycopeptide antibiotics and effective
tools for their faster development. Technological tools for the manipulation of the glycopeptide
pathway and of the producing strains will be developed. Glycopeptide enzymes will be overproduced and used in biotransformation of pathway intermediates obtained by gene disruption
and of precursors obtained by chemical synthesis. These novel glycopeptides will be evaluated
against significant pathogens, including vancomycin-resistant strains. Once interesting structures are identified, suitable producer strains will be engineered. The project focuses on a semisynthetic derivative of the natural glycopeptide A40926, produced by a Nonomuria sp., currently under clinical development. The objectives of here are: 1) to establish tools for efficient
manipulation of glycopeptide structures and producer strains; 2) to produce selected glycopeptides with improved properties in comparison with existing molecules; 3) to develop an
efficient process for the production of the glycopeptide A40926 by the original Nonomuria producer and by an engineered Streptomyces lividans host.
EXPECTED RESULTS
The expected results of this work are the establishment of tools for efficient manipulation of
glycopeptide structures and producer strains; the production of selected glycopeptides with
improved properties in comparison with existing molecules; the development of an efficient
process for glycopeptide production by the native and engineered producer
62
Acronym : Mega-Top
Keywords : actinomycetes, artificial chromosomes, combinatorial
biosynthesis, fermentation
New antibiotics for human health
Stefano Donadio
Biosearch Italia
Via R. Lepetit 34, IT-21040 Gerenzano (VA), Italy
T: (+39) 02 96474 243; F: (+39) 02 96474 238
Web-site: www.biosearch.it
PARTNERS:
Prof. Lubbert Dijkhuizen
University of Groningen
Microbial Physiology
P.O. Box 14, Kerklaan 30, NL-9751 NN Haren
The Netherlands
T: (+31) 50 3632153; F: (+31) 50 3632154
Prof. Jens Nielsen
Technical University of Denmark
Center for Process Biotechnology
Department of Biotechnology
Building 223, DK-2800 Lyngby , Denmark
T: (+45) 45 252696; F: (+45) 45 884148
•
•
•
•
•
Prof. John Robinson
University of Zuerich
Institute of Organic Chemistry
Winterthurerstrasse 190; CH-8057 Zuerich ,
Switzerland
T: (+41) 1 6354242; F: (+41) 1 6356812
Prof. Wolfgang Wohlleben
Eberhard-Karls Universitaet Tuebingen
Biologisches Institut
Auf def Morgenstelle 28, DE-72076 Tuebingen ,
Germany
T: (+49) 07071 2976944; F: (+49) 07071 295979
Prof. Anna Maria Puglia
Università di Palermo
Dip. Biologia Cellulare e Sviluppo
Parco d'Orleans II, IT-90128 Palermo , Italy
T: (+39) 091 6573712; F: (+39) 091 420361
63
NOVEL SOURCES OF ACTINOMYCETE DIVERSITY
FOR DETECTION OF ANTIMICROBIAL AGENTS
WITH PHARMACEUTICAL APPLICATIONS
SUMMARY
We aim to reverse the trend of declining discovery rate of antimicrobial compounds by exploiting biodiversity
within the uncultured microbial populations in the environment and clone habitat meta-genomes using bacterial
artificial chromosomes. To evaluate the cloning procedure, habitat diversity is analysed at the functional and taxonomic level. Targeted assays are used to identify antimicrobial activities of clones for use in control of infections,
as transdermal applications and as preservatives for pharmaceuticals. We aim to combine chemical novelty with
novelty in application.
PROBLEM
Novel anti-microbial agents are needed to overcome problems of resistance and provide protection against infection. The control of microbial growth is also an essential part of product
preservation in the cosmeceutical industry. Despite high throughput screening the discovery
rate of novel antimicrobial compounds has declined.
AIM
The problems of selective culture will be solved whilst simultaneously critically evaluating alternative approaches to exploiting bacterial diversity in natural environments, thus obtaining
novel antimicrobial agents. This will be achieved by taking a systematic approach to recovering
genetic diversity from soil both by cultivation and cultivation independent means. The availability of streptomycete artificial chromosome now provides the opportunity to clone high
molecular weight DNA from total community extracts. The biosynthetic potential of uncultured
actinomycete groups will be evaluated. Cloned pathways provide considerable advantages due
to ease of detection and the use of hosts such as S.lividans, which will be selected, for ease of
handling. Novel molecules will be characterised and tested for diverse uses in systemic, dermal
and transdermal applications and as biocides for preservation of pharmaceutical products.
EXPECTED RESULTS
Development of a molecular toolkit for evaluating diversity within actinomycete populations. A
strategy for cloning high molecular weight DNA from soil and stable integration into the chromosome of S.lividans host. Rapid methods for detection of altered fermentation profiles of
clone libraries using MALD-MS. Provision of molecular structure and activity profiles.
64
Acronym : ACTAPHARM
Starting date : August 1st 2001
Keywords : Actinomycetes, antibiotics, anti-infectives
Development of novel applications for use of antimicrobial agents as anti-infectives, dermal
patch treatments and biocidal agents.
Prof. E M H Wellington
Department of Biological Sciences
University of Warwick
CV4 7AL Coventry, UK
T: (+44) 024 7652 3184; F: (+44) 024 7652 3701
PARTNERS:
Flavia Marinelli
Microbial Fermentation
Biosearch Italia SpA
Via R. Lepetit, 34
21040 Gerenzano (Varese), Italy
T: (+39) 2 9647 4239; F: (+39) 2 9647 4400
Prof. Dr Hans-Peter Fiedler
Mikrobiologisches Institut
Universität Tübingen
Auf der Morgenstelle 28
72076 Tübingen, Germany
T: (+49) 7071 2972 079; F: (+49) 7071 295 999
Prof. Dr L. Dijkhuizen
Microbiology
PO Box 14
9750 AA Aaren, The Netherlands
T: (+31) 50 3632 150; F: (+31) 50 3632 154
Joachim Vater
Max-Volmer-Institut für Biophysikalische
Chemie und Biochemie
Technische Universität Berlin
Franklinstr. 29, 10587 Berlin, Germany
T: (+49) 30 3142 5609; F: (+49) 30 3142 4783
• •
•
•
65
COMBINATORIAL BIOSYNTHESIS:
GENERATION OF NOVEL THERAPEUTIC
SUBSTANCES BY COMBINING GENES FROM
ACTINOMYCETES AND CYANOBACTERIA
SUMMARY
The research aims to find novel therapeutic substances. This will be achieved by 1) screening of bacteria,
actinomycetes and cyanobacteria with modern fingerprinting techniques and by 2) combinatorial biosynthesis.
The latter approach includes the creation of expression systems for genes derived from cyanobacteria. The
compounds obtained from wild type bacteria and those derived from genetically engineered strains are screened
through antimicrobial, antitumour and receptor based systems. Biopharmaceutical evaluation of the hits that will
be found in the project leads to optimisation of the promising target molecule in the early phase of drug development process. Seven European groups, four academic and three SMEs form the multidisciplinary project team.
PROBLEM
Antimicrobial resistance is increasing fare more rapidly than current drug development rates
can meet through the production of new antibiotics.
AIM
The major aim of the project is to find a novel lead molecule for drug development. To achieve
the goal, two strategies are used:
• screening of 600 bacteria by fingerprinting to obtain novel natural products and
• combinatorial biosynthesis
(combination of biosynthetic routes of metabolites by gene technology).
Therefore, the other objectives are:
1. a screening model for natural products
2. cloning of eight different biosynthetic gene clusters representing three different metabolite
classes
3. combining genes in cassettes already available to join the chemical patterns of three distinct
metabolites
4. expressing the biosynthetic genes derived from cyanobacteria in streptomycetes
5. determining the chemical structures and bioactivities of compounds and
6. biopharmaceutical evaluation of promising molecules.
The final aim of the project is a novel lead molecule for drug development. The project focuses
on products of two bacteria, actinomycetes and cyanobacteria, which are known producers of
secondary metabolites with important bioactivities. Six hundred bacterial strains will be analyzed, using genetic and chemical fingerprinting techniques.
Based on the bioinformatics obtained, the bacterial strains producing distinct metabolite patterns will be selected for further studies:
1. the extracts of these strains for bioactivity screens
(antimicrobial, antitumor and receptor based assays) and
2. the genetic material for combinatorial biosynthesis.
Based on the bioactivity data, the metabolites of selected strains will be isolated. Chemical
nature of novel molecules is determined by spectroscopy. Molecular biology groups use gene
cassettes already available in their laboratories and they start combinatorial biosynthesis using
genes already cloned. Random and desired gene combinations generate bacterial strains producing extraordinary molecular structures. Combinatorial biosynthesis in the project is focused
on combining building blocks derived from different compound classes thus creating a model
to produce compound libraries of high diversity. Chemical and bioactivity nature of each compound generated by combinatorial biosynthesis will be analyzed. As soon as the first promising compounds are available, biopharmaceutical evaluation will take place to direct the further
modification of the target molecule. Modification will be achieved by gene technology.
66
Acronym : Cyanomyces
Keywords : Actinomycetes, Cyanobacteria, antimicrobials
EXPECTED RESULTS
Screening strategy obtained by DNA and chemical fingerprinting of secondary metabolites
will be published. As 600 bacteria will be characterized for screening purposes, the well-characterized bacterial strains are considered as deliverables in the consortium. During the project several cloned and identified genes for bioactive metabolites will be delivered in the consortium together with expression cassettes for generation and modification of lead compounds. Most important deliverables are bioactive molecules either natural or semi-natural
origin for further development to drug candidates.
Development of new therapeutic substances
Kristiina Ylihonko
Galilaeus Oy
Kairiskulmantie 10, 20781 Kaarina, Finland
T: (+358) 2 274 1450; F: (+358) 2 273 1460
PARTNERS:
Thomas Börner
Department of Genetics
University of Humboldt
Institute of Biology
10115 Berlin, Germany
T: (+49) 30 2093 8142; F: (+49) 30 2093 8141
Email: thomas=boerner@
Web-site: biologie.hu-berlin.de
Pekka Mäntsälä
University of Turku, 20014, Turku, Finland
T: (+358) 2 333 6850; F: (+358) 2 333 6860
•
•
Hans von Döhren
Max-Volmer Institute
Technical University of Berlin
T: (+49) 30 3142 2697; F: (+49) 30 3142 4783
Francisco Malpartida
Centro Nacional de Biotechnologia
Campus Universidad Autonoma de Madrid
28049 Madrid, Spain
T: (+34) 91 585 4548; F: (+34) 91 585 4506
Juha-Matti Savola
Juvantia Pharma Ltd
20520 Turku, Finland
T: (+358) 2 333 7684; F: (+358) 2 333 7680
Sveinbjorn Gizurarson
Lyfjathroun Hf
101 Reykjavik, Iceland
T: (+354) 511 2020; F: (+354) 511 2021
•
•
67
DESIGNING AND IMPROVING HEALTH AND FOOD
RELATED PRODUCTION PROCESSES USING
FILAMENTOUS FUNGAL CELL FACTORIES
SUMMARY
Filamentous fungi are known for their ability to produce metabolites important for human health (antibiotics) and
enzymes used in the food and feed industry. They are promising hosts for the production of pharmaceutical
proteins, antibodies and vaccines and of novel non-ribosomal peptide antibiotics. The project designs versatile
filamentous fungal cell factories for different products by pinpointing and eliminating the major bottlenecks,
which limit their efficiency. A first objective is to improve the ability of fungi to make foreign proteins by better
understanding the secretion process. The second objective is to increase fungal production rates and improve
substrate utilisation. Thirdly, new fungal antibiotics are developed to replace existing antibiotics for which
bacterial resistance appears.
PROBLEM
Despite the potential of filamentous fungi to secrete proteins in large quantities, heterologous
proteins are often made in low yield. A major bottleneck is the inefficient folding of foreign proteins in the endoplasmatic reticulum leading to unfolded protein response and stress.
AIM
In order to address these problems, a comprehensive understanding of the cellular reactions
involved is necessary to identify essential factors. These can then be engineered and tested in
production systems. Three proteins of medical interest have been selected as target. Additional
improvement is sought by intelligent screening after mutagenesis and identification of the
genes involved. To produce industrial enzymes at low costs, focus is on upgrading waste products e.g. hemicellulose from the agro-industry. The complex structure of the central metabolism
requires research on regulation of gene expression, sugar metabolism and quantification of the
relative fluxes through various pathways. Pathway modelling and flux analysis is used to
improve strains by genetic engineering.
The project aims at designing and improving fungal production processes for different product
categories with medically important heterologous proteins, enzymes useful in food- and feed
applications and novel non-ribosomal peptide antibiotics. Novel non-ribosomal peptides will
be identified by novel screening methods or made by combinatorial approaches and screened
for their bioactivity. Novel peptide biosynthesis will be integrated in a fungal cell factory optimised for a high flux through secondary metabolism.
EXPECTED RESULTS
Demonstrating how to obtain heterologous proteins at a gram/litre scale would be a major
achievement providing the European biotechnology industry new options. Obtaining new
antibiotics by combinatorial biology and/or by discovery based on genetic diversity is highly
innovative and necessary to fight increased bacterial resistance.
A Fungal Industrial Platform of 17 members is associated with the programme to exploit the
results and to stimulate commercial activities as a consequence. The benefits for the society
are economic but also directly relate to health and nutrition. Furthermore, fungal gene technology provides alternatives for transgenic animal technology.
68
Acronym : Eurofund
Dr Jaap Visser Wageningen University
Section Molecular Genetics of Industrial
Mircoorganisms Dreijenlaan 2, 6703 HA
Wageningen, The Netherlands
T: (+31) 317 482 865; F: (+31) 317 484 011
PARTNERS:
Dr Cornelis Van den Hondel
Leiden University Institute of
Molecular Plant Sciences
Clusius Laboratory Wassenaarseweg 64, 2300 RA
Leiden, The Netherlands
T: (+31) 30 694 4461; F: (+31) 30 694 4466
Dr Miguel A. Peñalva
Departemento de Microbiología Molecular
Velázquez 14, 28005 Madrid, Spain
T: (+34) 91 564 4562 ext. 4358;
F: (+34) 91 562 7518
Dr Jens Nielsen
Technical University of Denmark
Center for Process Biotechnology
Building 223, DTU, 2800 Lyngby, Denmark
T: (+45) 45 2525 2696; F: (+45) 45 884 148
Dr Merja.E. Penttilä
VTT Technical Research Center for Finland
Biotechnology and Food Research
P.O. Box 1500, 02044 VTT Espoo, Finland
T: (+358) 9 456 4504; F: (+358) 9 455 2103
Dr Hans von Döhren
Technical University Berlin
Institut für Biochemie und Molekulare Biologie
Franklinstrasse 29; 10587 Berlin, Germany
T: (+49) 30 3142 2697; F: (+46) 30 3142 7695
•
• ••
••
•
•
•
Dr David Archer
Institute of Food Research
Genetics and Microbiology Deptartment
Norwich Research Park, Colney, Norwich NR4 7UA,
UK T: (+44) 1603 255 256; F: (+44) 1603 507 723
Dr Roland Contreras
University of Gent
Laboratory of Molecular Biology
K.L. Ledenganckstraat 35, 9000 Gent, Belgium
T: (+32) 9 264 5136; F: (+32) 9 264 5348
Dr Anthony P.J.Trinci
The Victoria University of Manchester
The School of Biological Sciences
1800 Stopford Building Oxford Road
Manchester M13 9PL United Kingdom
Tel: +44-161-275 3893
Fax: +44-161-275 5656
Dr Christian P. Kubicek
Technische Universität Wien
Institut für Biochemische Technologie und
Mikrobiologie Abteilung für Mikrobielle Biochemie
P.O. Box 172-5 1060 Wien, Austria
Tel: +43-1-588 014 707 Fax: +43-1-581 6266
Dr Joop C. Van der Laan
Genencor International BV P.O. Box 218
2300 AE Leiden The Netherlands
Tel: +31-71-5686 126/133 Fax: +31-71-5686 130
Dr Susan Madrid
Danisco A/S Danisco Biotechnology
P.O. Box 17 1001 Copenhagen K, Denmark
Tel: +45-32-662 242 Fax: +45-32-662 167
Dr Cor Dijkema
Wageningen Agricultural University
Department of Molecular Physics
P.O. Box 8128 6700 ET Wageningen
The Netherlands
Tel: +31-317-484 314 Fax: +31-317-482 725
Dr Kim Hansen
Novo Nordisk A/S Dept. of Microbial Physiology
Molecular Biotechnology, Enzyme Research
Novo Alle, 1L 2880 Bagsvaerd, Denmark
Tel: +45-444-22 460 Fax: +45-444-26 645
Dr Axel Brakhage
Darmstadt University of Technology
Institute of Microbiology and Genetics
Schnittspahnstrasse 10 64287 Darmstadt, Germany
Tel: +49-6151-165 566 Fax: +49-6151-162 956
Dr Juan F. Martin
University of Léon Department of Ecology,
Genetics and Microbiology Faculty of Biology
Campus de Vegazana, S/N 24071 Leon, Spain
Tel: +349-87-291 505 Fax: +349-87-291 506
Dr Arnold J.M. Driessen
Kerklaan 30 9751 NN Haren The Netherlands
Tel: +31-50-363 2164 Fax: +31-50-363 2154
Dr Udo Gräfe Hans-Knöll-Institute
of Natural Products Research
Beutenbergstrasse 11 07745 Jena, Germany
Tel: +49-3641-656 701 Fax: +49-3641-656 705
Dr Geoffrey Turner The University of Sheffield
Dr Albert de Graaf Research Center Jülich
Institut für Biotechnologie 52425 Juelich, Germany
Tel: +49-2461-613 969 Fax: +49-2461-612 710
Department of Molecular Biology and Biotechnology
Dr Herbert Arst
The Imperial School of Medicine
Dpt. of Infectious Diseases and Bacteriol.
Ducane Road 150 London W12 0NN
United Kingdom Tel: +44-181-383 3436
Fax: +44-181-383 3394 E-mail: [email protected]
Dr Marcel Erhard BrainTec GmbH
Im Biotechnologiepark,
14943 Luckenwalde, Germany
Tel: +49-3371-681 126 Fax: +49-3371-681 127
Dr Beatrice Felenbok
Dr Claudio Scazzocchio
Université Paris-Sud XI
Institut de Génétique et Microbiologie
Batiment 409, Centre d’Orsay
15, rue Georges Clemenceau
91405 Orsay Cédex, France
Tel: +33-1-6915 6356-28 Fax: +33-1-6915 7808
Dr Daniel Ramon Vidal
Consejo Superior de Investigaciones Cientificas
Instituto de Agroquímica y
Technología de Alimentos
P.O. Box 73, Burjassot 46100 Valencia, Spain
Tel: +34-96-390 0022 Fax: +34-96-363 6301
Western Bank Sheffield S10 2UH United Kingdom
Tel: +44-114-222 6211 Fax: +44-114-272 8697
Dr Hans van den Brink
Chr. Hansen A/S 10-12 Boege Allee
2970 Horsholm, Denmark
Tel: +45-45-748 455 Fax: +45-45-748 994
Dr John Chapman
Unilever Research Laboratorium
URL Vlaardingen P.O. Box 114
3130 AC Vlaardingen The Netherlands
Tel: +31-10-460 5247 Fax: +31-10-460 5383
Dr Thierry Dauvrin
Frimond S.A. Rue Bourrie 12
5300 Andenne, Belgium
Tel: +32-85-823 250 Fax: +32-85-823 260
69
DISCOVERY OF A NEW CLASS
OF BIOACTIVE COMPOUNDS:
BACTERIAL CONJUGATION INHIBITORS
SUMMARY
Acquisition of antibiotic resistance (AbR) genes occurs primarily by the mechanism of bacterial conjugation.
Preventing conjugation should markedly limit the dissemination of AbR. The ultimate aim of this project is to
devise the most effective strategies to inhibit conjugation in or around the bacterial targets where resistance is
undesirable. For this purpose, we need to isolate and characterize conjugation inhibitors (COINS). No specific
COIN has been previously described.
PROBLEM
Bacteria usually respond to antimicrobial agents not by mutation, but by spreading genes that
carry determinants of resistance. Thus, killing sensitive bacteria not only fails to destroy the
determinants of resistance, but rather acts to select and propagate them among bacterial populations. Fighting conjugative systems of plasmids and transposons DIRECTLY is the only effective way to avoid the spread, and to some extent the evolution, of antibiotic resistance genes.
AIM
We search for COINS by screening an existing large library of over 50,000 microbial extracts
proven to be a rich source of potentially active and diverse natural products. Experiments are
carried out under laboratory conditions using a novel automated assay for conjugation in general followed by system-specific tests using standard procedures.
EXPECTED RESULTS
1. Develop an automated assay for bacterial conjugation
2. Screen for COINS with an existing large library of natural microbial metabolites (primary
screen)
3. Characterize the chemical structures of COINS
4. Define the range of biological activity of COINS (secondary screens)
ACHIEVED RESULTS
The relevant plasmids and bacterial strains have been constructed. The conjugation assay was
developed and has been validated for high throughput screening. A first run using 2,400
extracts has resulted in 22 dereplicated hits, which contain potential COINS. They are presently being subjected to chemical fingerprinting and fractionation, to obtain pure compounds for
further analysis.
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Acronym : COINS
Starting date : April 1st 2001
Keywords : Bacterial conjugation, conjugation inhibitor, conjugation
assay, high throughput screening, antimicrobial drug resistance,
antibiotic resistance transmission
The industrial partner, Cubist Pharmaceuticals (UK) Limited, will use its extensive expertise in
the field of commercial exploitation and protection of intellectual property to capture value
from the project outputs.
The objects of exploitation may include, but not be limited to:
• The use of high throughput screening methods to monitor bacterial conjugation or related
processes,
• The know-how derived from the physico-chemical, biological or other analysis of the small
compounds inhibiting their activities, and
• The in vivo therapeutic or other activities of the inhibitor molecules.
http://grupos.unican.es/genetica/COINS
Prof. Fernando De La Cruz
Dept. de Biología Molecular
Universidad de Cantabria
C. Herrera Oria s/n
ES-39011 Santander, Spain
T: (+34) 942 201942; F: +(34) 942 201945.
Web-site: http://www.unican.es/
PARTNERS:
Dr. Miquel Coll
Instituto de Biología Molecular, CSIC
Jordi Girona 18, ES-08034 Barcelona, Spain
T: (+34) 93 400 6149; F: (+34) 93 204 5904
Dr. Steven M. Martin
Cubist Pharmaceuticals (UK) Ltd
545, Ipswich Road
UK-Slough SL1 4EQ, UK
T: (+44) 1753 706878; F: (+44) 1753 706808
Dr. Manuel Espinosa
Departamento de Estructura y Función de
Proteínas
Centro de Investigaciones Biológicas, CSIC
Velázquez, 144, ES-28006 Madrid, Spain
T: (+34) 91 561 1800 ext. 4209; F: (+34) 91 562 7518
Dr. Ellen L. Zechner
Institute of Molecular Biologie
Biochemistry and Microbiology
University of Graz, Universitaetsplatz 2
AT-8010 Graz, Austria
T: (+43) 316 380 5624; F: (+43) 316 380 9898
Dr. Erich Lanka
Max-Planck-Institut fuer Molekulare Genetik
Ihnestrasse 73, Dahlem, DE-14195 Berlin, Germany
T: (+49) 30 8413 1696; F: (+49) 30 8413 1130
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Dr. Walter Keller
Institut für Chemie
Karl-Franzens-Universität Graz, Heinrichstrasse 28
AT-8010 Graz, Austria
T: (+43) 316 380 5423; F: (+43) 316 380 9850
Prof. Soeren Molin
Molecular Microbial Ecology Group
Technical University of Denmark, DK-2800 Lyngby
Denmark
T: (+45) 4525 2513; F: (+45) 4588 7328
71
NOVEL INHIBITORS OF ADHESIN/RECEPTOR
INTERACTIONS INVOLVED IN MICROBIAL
INFECTION AT MUCOSAL SURFACES
SUMMARY
The aim of this project is to develop novel antimicrobial therapies that combat infection of mucosal surfaces by
blocking the initial attachment of pathogenic microorganisms to host tissue and stimulate clearance by
mechanisms of innate immunity. Attachment or adhesion to the host mucosa is an essential early event in
microbial infection and involves specific interaction between microbial cell surface molecules (adhesins) and
surface molecules of the host (receptors). We are targeting common infections of the gastric (Helicobacter pylori),
intestinal (rotavirus) or vaginal (Candida albicans) mucosa and expect to produce three types of adhesionblocking molecule:
1 adhesin analogues: synthetic peptides or recombinant proteins that competitively inhibit adhesion by binding
to host receptors.
2 monoclonal antibodies or antibody fragments that are directed towards adhesins and prevent adhesion and in
the former case can enhance phagocytosis.
3 receptor analogues: complex carbohydrates that competitively inhibit adhesion by binding to the microbial
adhesin.
Delivery of adhesion inhibitors to mucosae by the use of genetically engineered strains of commensal bacteria
will be investigated and their efficacy will be determined using in vivo models of infection.
PROBLEM
The spread of antibiotic resistance has stimulated investigation of new approaches to antimicrobial therapy. Blocking microbial adhesion which is an early step in infection is an attractive
approach which may be less likely to induce resistant microorganisms since, in contrast to
antibiotic treatment, it does not impose sustained selective pressure. Adhesion-blocking molecules have been used in a limited number of clinical trials with some success; however, if this
form of treatment is to be applied to a variety of infections, techniques are required for identifying and testing of large numbers of potential inhibitors. The effectiveness of this form of treatment may be considerably enhanced if innate immunity, particularly phagocytosis, can be stimulated at mucosal surfaces and systems need to be developed for delivery and maintenance of
therapeutically effective concentrations of inhibitors at mucosal surfaces.
AIM
Electron microscopy images of Helicobacter
pylori (left panel) and Candida albicans attaching to a human lymphocyte (right panel).
(H. pylori reproduced by kind permission of
Scientific American, SCIENCE &
MEDICINE; C. albicans contributed by A.
Cassone, Istituto Superiore di Sanita, Rome)
72
The main objectives of the project are:
To identify novel adhesion-blocking agents by screening of combinatorial carbohydrate and
antibody fragment libraries for molecules that bind to either defined adhesins or host receptors
for H. pylori, C. albicans or rotavirus. Receptor binding regions of microbial adhesins may also
be defined. Potential blocking molecules will subsequently be tested using in vitro adhesion
assays.
To produce adhesion-blocking agents for use in models of infection either by chemical synthesis or by expression in E.coli or yeast.
To optimize delivery and maintenance of effective concentrations of adhesion-blocking agents
at mucosal surfaces. Commensal bacteria that colonise mucosal surfaces and that have been
genetically engineered to produce surface bound or secreted forms of inhibitors will be investigated as a means of delivery.
To develop effective methods of treatment in models of infection using inhibitors singly or in
combination. Harnessing innate immune mechanisms to promote clearance of microbes will be
an important aspect of this therapy.
Acronym : ADRI
Keywords : microbial adhesion, combinatorial libraries, innate immunity,
Candida albicans, Helicobacter pylori, rotavirus, Streptococcus gordonii,
BabA, mannosylated protein MP65, antimicrobial
EXPECTED RESULTS
We expect to define novel inhibitors of microbial adhesion in each of the infections. These
may include antibody fragments, polypeptide/peptide adhesin analogues and synthetic carbohydrate analogues of receptors. Strains of commensal bacteria that are capable of producing inhibitors will be produced. Effective modes of treatment in relevant in vivo models of disease will be established as well as definition and enhancement of innate immune mechanisms of protection.
The results of this project should lead to the development of antimicrobial therapies for use
in human clinical trials. Major deliverables from this proposal that have potential for industrial exploitation and may be further developed with industrial partners are:
1 anti-adhesin monoclonal antibodies or fragments
2 adhesin analogues: synthetic peptides or recombinant polypeptides
3 receptor analogues: synthetic carbohydrates or antibody fragments
4 means of mucosal delivery with engineered live commensal bacteria
5 therapies for prevention of infection that have been tested in models of disease.
http://www.ucl.ac.uk/adri
Prof. Ivan Roitt
Department of Immunology
Windeyer Building
46 Cleveland St. - UK-London W1P 4JF
United Kingdom
T: +44 207 679 9360; F: +44 207 679 9400
Web-site: http://www.ucl.ac.uk
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Dr. Thomas Borén
Umeå Universitet
Department of Odontology
Umeå University
SE-901 85 Umeå - Sweden
T: +46 90 785 6036; F: +46 90 785 6129
Web-site: http://www.umu.se
Prof. Antonio Cassone
Department of Bacteriology & Medical
Mycology
Viale Regina Elena, 299 - IT-00161 Rome
Italy
T: +39 0649 387113; F: +39 0649 387112
Web-site: http://www.iss.it
Prof. Lennart Hammarström
Center for Oral Biology
Karolinska Institute, Novum
SE-141 57 Huddinge - Sweden
T: +46 8 608 91 15; F: +46 8 774 55 38
Web-site: http://www.ki.se
Dr. Charles Kelly
King’s College London
Department of Oral Medicine and Pathology
Floor 28 Guy’s Tower - GKT Dental Institute
UK-London SE1 9RT - United Kingdom
T: +44 20 7955 4966; F: +44 20 7955 4455
Web-site: http://www.kcl.ac.uk
Prof. Gianni Pozzi
Laboratory of Molecular Microbiology and
Biotechnology (L.A.M.M.B.)
Dipartimento di Biologia Molecolare
Policlinico Le Scotte
1S, Viale Bracci - IT-53100 Siena - Italy
T: +39 0577 233430; F: +39 0577 233334
Web-site: http://www.unisi.it
Dr. Ian M Tomlinson,
Diversys Limited - Compass House
Vision Park
Chivers Way, Histon - UK-Cambridge CB4 9AD
United Kingdom
T: +44 7770 608051; F: +44 1223 515799
Web-site: http://www.diversys.co.uk
Dr Colin Campion
Carbohydrate Synthesis Ltd.
Building 15, North Culham Estate
Culham Science & Engineering Centre
UK-Oxon. OX14 3GY - United Kingdom
T: +44 1865 407767; F: +44 1865 407985;
Dr. Marcel Vander Vaart,
Unilever Research Vlaardingen
Biotechnology Department
P.O. Box 114, - NL-3130 AC Vlaardingen
The Netherlands
T: +31 10 4605092; F: +31 10 4605383
Web-site: http://www.unilever.com
73
TREATMENT AND PREVENTION OF BACTERIAL
INFECTIONS BY ANTI-ADHESION COMPOUNDS
SUMMARY
To combat the growing number of antibiotic-resistant bacteria new therapeutic strategies are needed.
Our project aims to create novel therapeutics that can interfere with the adhesion of pathogenic bacteria to
carbohydrate ligands present on target tissues. For many bacteria this event is the first and crucial step that
leads to an infection. The molecules are evaluated in various adhesion assays and their applicability will be
evaluated with relevant animal model studies.
PROBLEM
The project addresses the important issue of increased resistance of pathogenic bacteria to
conventional antibiotics. Over-usage of conventional antibiotics is one of the factors that has
contributed to the current existence of an alarming number of resistant bacteria. To illustrate
the usage: 10.000 tons were used in the EU in 1997, 50% of which for human medicine.
Although there have been important advances in the availability of antiviral and anti-fungal
agents, no truly novel antibacterial drugs have been marketed in more than 10 years. Currently
around 160 antibiotics are available, which are based on a few basic chemical structures. There
is a realisation that alternative intervention strategies are needed. The present project comprises such an alternative strategy. Unlike classical antibiotics which kill by interfering with
some critical metabolic pathway specific to micro-organisms, anti-adhesion compounds as proposed here, do not kill the pathogens. Their use simply results in the failure of the pathogen
to find a suitable site for infection or it results in clearance of attached pathogens from the
mucosal surfaces. In such a scenario selection for resistance is unlikely.
AIM
Our objective is to create novel therapeutics that prevent infection by pathogenic bacteria. We
apply a new approach to develop compounds which bind tightly to adhesion proteins on the
bacterial surface. This prevents the binding of the bacteria to target tissues and thus the infection that would follow. This anti-adhesion strategy is also used by Nature as exemplified by the
oligosaccharides present in breast milk protecting newborn babies. Four types of pathogenic
bacteria, causing septicaemia, meningitis, gastric ulcers and urinary tract infections, are targeted.
EXPECTED RESULTS
The approach towards our goals is to make multivalent carbohydrate molecules that can prevent bacterial adhesion to target tissues. Many bacteria bind specifically to certain carbohydrate
structures. Multivalency is a design principle which is crucial in
our project. A multivalent system allows simultaneous binding to
multiple copies of the receptor and thus leads to the strong binding required for application of this method. We will achieve multivalency by linking the carbohydrates to small scaffold molecules,
dendrimers and polymers. The compounds will be tested against
several relevant pathogens in vitro and ultimately in vivo as well.
We expect to find several high-affinity compound based on the
multivalent presentation of the carbohydrates.
74
Acronym : POLYCARB
Starting date : 1st May 2002
Contract Type : Shared Cost
Keywords : Anti-adhesion, Carbohydrate binding, Multivalency,
Glycodendrimers, Glycopolymers
Results from the research efforts of the present project could lead to the identification of highly promising compounds which can inhibit adhesion of pathogenic bacteria and thus prevent
the infection that would follow. These properties make them potential therapeutics as a
stand-alone treatment or in combination with conventional antibiotics that can be used more
effective in such a scenario.
Dr. R. J Pieters
Utrecht Institute for Pharmaceutical Sciences
Department of Medicinal Chemistry
Utrecht University
Sorbonnelaan 16, NL-3584 CA Utrecht, The
Netherlands
T: (+31) 30.253 6944/4232; F: (+31) 30.253 6655
Email [email protected]
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PARTNERS:
Prof. J. Finne
Department of Medical Biochemistry
University of Turku
Kiinamyllynkatu 10, FIN-20520 Turku , Finland
T: (+358) 2 333 7240; F: (+358) 2 333 7229
E-mail [email protected]
Prof. J. Hacker
Institute for Molecular Biology of Infectious
Diseases
University of Würzburg
Röntgenring 11, D-97070 Würzburg, Germany
T: (+49) 931 312575; F: (+49) 931 312578
Prof. I. S. Roberts
1.800 Stopford Building
University of Manchester
Oxford Road, Manchester M13 9PT, UK
T: (+44) 161 275 5601; F: (+44) 161 275 5656
Dr. K. A. Krogfelt
Department of Gastrointestinal Infections
Statens Serum Institute
Artillerivej 5, 2300 Kopenhagen, Danmark
T: (+45) 326 83745; F: (+45) 326 83873
75
CONTROL OF LUNG INFECTION, ALLERGY
AND INFLAMMATION: ASSESSMENT OF THE
THERAPEUTIC POTENTIAL OF RECOMBINANT
FORMS OF THE LUNG SURFACTANT PROTEINS
SP-A AND SP-D
SUMMARY
The surfactant proteins A and D (SP-A and SP-D) occur naturally in the lung and play a role in the defence against
viral, bacterial and fungal infections and against inflammation induced by allergic reactions. At present, lung
surfactant preparations in clinical use do not contain SP-A or SP-D, although recombinant forms of both these
proteins can be produced. The addition of recombinant forms of SP-A and SP-D to existing surfactant
preparations, or by use on their own, may prove to be of therapeutic use in controlling lung inflammation seen in
a wide range of infections and allergies.
PROBLEM
Lung infection and inflammation is a growing problem within all countries of the EU,
and the infections are routinely treated with antibiotics. The pharmaceutical industry
is interested in the development of protein therapeutics, which can be used as alternatives to antibiotics. There is a relatively fragile protective barrier, the alveolar lining
layer, which controls the interaction between the atmosphere and the lung. This film,
known as lung surfactant, plays two important roles, prevention of lung collapse during respiration and provision of a first line of defence against the extremely varied
range of particles, allergens and microbes that are present in the environment. The
lung surfactant is a surface- active mixture of phospholipids and four main surfactant
proteins – SP-A, SP-B, SP-C and SP-D. The SP-B and SP-C proteins are small, highly
hydrophobic, polypeptides, which are strongly associated with the phospholipid portion of the surfactant, whereas SP-A and SP-D are large (approximately 600kDa) and
complex, disulphide-bonded, proteins of a more hydrophilic nature. They can bind, via
their lectin domains, to arrays of carbohydrate structures on the surfaces of pathogenic microbes and to glycosylated allergens, thus initiating defence against a range of viral,
fungal and bacterial lung infections and modulating allergic reactions. There is evidence of lowered levels of SP-A, and SP-D, in the lung surfactant of a growing number of types of infectionor allergy-mediated lung inflammation, which strengthens the case for testing the use of recombinant forms of these proteins as therapeutic alternatives to antibiotics.
AIM
The principal aim of this project is to test the effectiveness of recombinant forms of the human
lung surfactant proteins SP-A and SP-D, and of a fusion between a fragment of SP-D and the
Fab fragment of an antibody directed against the receptor for IgA on phagocytic cells, as therapeutic agents in a variety of conditions of lung infections and inflammation. To proceed to a
position where novel recombinant therapeutics can be considered as clinically useful additions
to existing artificial lung surfactant mixtures they must be fully characterised in terms of their
physical and biological functions. The functional work involves a wide range of in vitro and in
vivo assays, which will be carried out both in the presence and absence of added lipids/artificial surfactant. The in vivo assays will make use of mouse, rat and pig models of lung infection
and allergy with a variety of common lung pathogens and allergens. In addition, in order to fully
understand how the biological effects shown by the recombinant SP-A and SP-D are mediated
and controlled, studies on the putative receptors for these molecules will be carried out and
also the involvement of SP-A and SP-D in innate immunity at sites, other than the lungs, will be
examined.
EXPECTED RESULTS
1. The establishment of efficient procedures for the production of biologically active recombinant
forms (either full-length or fragments) of the human lung surfactant proteins SP-A and SP-D.
2. Full, physical and biochemical, characterisation of the various recombinant preparations of
SP-A and SP-D.
76
Acronym : lung SP-A and SP-D
Keywords : lung, surfactant, allergy, infection, inflammation, therapeutic agent
3. Use of a range of in vitro binding and cellular studies, to compare the properties of the
recombinant preparations with those of natural SP-A and SP-D preparations.
4. The in vivo testing of the recombinant forms of SP-A and SP-D in mouse, rat and pig models of lung inflammation, mediated by infection and allergy.
5. The assessment of the effects of lipid, and artificial surfactant, preparations on the properties of recombinant forms of SP-A and SP-D.
6. Assessment of the mechanisms of action, at the cellular and molecular levels, of natural
and recombinant forms SP-A and SP-D on triggering of immune cells.
The development of effective synthetic surfactant preparations, which contain phospholipids
and the recombinant forms of the hydrophobic peptides SP-B or SP-C, or synthetic peptides
modelled on the structures of SP-B or SP-C, is being vigorously carried out by several pharmaceutical companies. A next major step in the development of such surfactants could be the
addition of recombinant versions of SP-A and SP-D. Incidence and severity of respiratory distress syndrome is markedly reduced when the present artificial surfactant mixtures are used
prophylactically in infants with established disease. However, there is a significant percentage of the pre-term-treated infants (up to 40%) who grow up with chronic lung disease.
Therefore surfactant therapies, which include the use of natural types of anti-microbial agents
(such as the recombinant forms of SP-A and SP-D), could help in the prevention of chronic
lung disorder – thus providing both an increase in survival and a reduction in costs with
respect to pre-term baby healthcare in the Community. Other groups of patients (cystic fibrosis patients, those suffering from fungal infections of the lung, severe asthmatics), may also
benefit from the administration of preparations containing recombinant forms of SP-A and SPD. This therapeutic approach also addresses the growing problem of bacterial resistance to
existing antibiotics and the perhaps overuse of existing antibiotics. If this therapy can provide
an alternative to, or at least reduce the use of, antibiotics then this will make a valuable contribution to healthcare in the Community.
http://www.uni-konstanz.de/FUF/Bioinformatik/EUCollectins/
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Prof. Kenneth.B.M. Reid
MRC Immunochemistry Unit
Department of Biochemistry, University of Oxford
South Parks Road Oxford OX1 3QU UK
T: (+44) 1865 275353; F: (+44) 1865 275729
Prof. Cristina Casals Carro
Dto de Bioquimica y Biologica Molecular
Facultad de Ciencias Quimicas
Universidad Complutense 28040 Madrid
Spain
T: (+34) 91 394 4261; F: (+34) 91 394 4672
PARTNERS:
Prof.Uffe Holmskov
Immunology and Microbiology University of Odense
Winslovsparken 19 DK 500 Odense
Denmark
T: (+45) 6550 3775; F (+45) 6591 5267
Prof. Klaus P. Schaefer and
Dr.Wolfram Steinhilber
Byk Gulden Pharmaceuticals
Department of Molecular Biology
Byk-Gulden-Strasse 2
78467 Konstanz
Germany
T: (+49) 7531 8432 72/80; F: (+49) 7531 8433 60
[email protected]
Prof.Henk P. Haagsman
Protein & Meat Technology Department
TNO Nutrition and food Research Institute
Utrechtsweg 48 PO Box 360 3700 AJ Zeist
Netherlands
T: (+31) 30 694 4382; F: (+31) 30 235 2365
Dr. Coos Batenburg
Department of Biochemistry and Cell Biology
Faculty of Veterinary Medicine
PO Box 80176 3508 TD Utrecht
The Netherlands
T: (+31) 30 253 5381; F: (+31) 30 253 5492
77
DEVELOPMENT OF A PHOTODYNAMIC
TREATMENT TO ERADICATE AND CONTROL THE
CURRENT SPREAD OF INFECTIOUS ANTIBIOTIC
RESISTANT MICROORGANISMS IN MAN
SUMMARY
The DYNAMICRO study aims to develop a revolutionary new treatment, which will provide clinicians with an alternative to combat the major European healthcare threat posed by antibiotic resistant microbial pathogens including methicillin-resistant bacteria such as Staphylococcus aureus (MRSA). The innovative treatment involves
killing microbes by the combination of a light activated agent (Photosensitiser) and light, a system known as
Photo-Dynamic Therapy (PDT). The aim is to optimise these anti-bacterial drugs for potential use in:
(i) Treatment of infected wounds/ulcers; (ii) Treatment of skin infections (atopic eczema, impetigo); (iii) General
skin disinfection and of the nares to treat “carriers” of infection e.g. health care workers and (iv) Treatment of eye,
ear and dental infections.
Within the first 10 months the studies have identified a range of novel light activated drugs, which can totally
eradicate MRSA at low concentration and have no effect on human cells.
PROBLEM
The generic versatility of bacteria has proven to be so remarkable at the beginning of the 21st
century that it is a fact that multi-drug resistant bacteria threaten the closure to successful antibacterial chemotherapy. Resistance to all known classes of antibiotics is on the increase, rendering certain therapies ineffective. Clinicians are being forced to replace traditional antibiotic
therapies with more potent ones, which if in turn are overcome by bacterial resistance, could
result in the emergence of ‘untreatable’ infections, leading to deaths from infection on a scale
unknown since before the discovery of the first antibiotic penicillin in the 1920’s. A global
healthcare imperative for new anti-bacterials directed against MRSA and other multi-drug
resistant bacterial now exists. The DYNAMICRO drug series represents such a new approach,
one with already proven activity in vitro and likely to be defiant of bacterial efforts to develop
resistance.
AIM
The DYNAMICRO Programme seeks to develop a revolutionary new treatment
for the prevention/treatment of both drug-sensitive and multi-drug resistant
infections based on photodynamic therapy. The innovative treatment involves
killing microbes by the combination of a photoactive agent (photosensitiser)
and a light activated source, a system known as PhotoDynamic Therapy (PDT).
The initial aim of the research programme is to develop an optimised PDT
System for the treatment of bacterial skin infections and of infected
wounds/ulcers. Other applications are also being investigated and this exciting technology could be developed into a range of new medicines to fight infections instead of using antibiotics, which are increasingly impotent in stemming
the spread of multi-drug resistant bacteria e.g. MRSA. The potential benefit of
this technology is immense and could result in a saving of hundreds of millions
of euros per year for the European Community as well as providing a significant
product opportunity for the SMEs involved in the Programme.
EXPECTED RESULTS
Although the DYNAMICRO Programme is only in it’s first year, the consortium has already synthesised a range of novel light activated drugs that have demonstrated significant anti-bacterial properties at very low concentrations. Furthermore, these drugs have a “therapeutic window” whereby they are active in killing bacterial cells, including multi-drug resistant bacteria
(e.g. MRSA), whilst not damaging cultured human cells. Final studies will include tissue distribution and efficacy at treating models of infection.
78
Acronym : DYNAMICRO
EC contribution : 693,700 €
Starting date : 1 December 2001
Contract Type : CRAFT
Keywords : MRSA, Photo-Dynamic Therapy (PDT), Staphylococcus aureus,
antimicrobial, antibacterial, infection.
It is currently envisaged that the current series of novel light activated drugs would have
application for treatment of topical human infections and to replace ‘skin applied’ antibiotics.
This has been concluded from discussions with clinicians (dermatologists & microbiologists)
and the development of products for the treatment of dermal, ocular, dental and aural infections are predicted.
The novel light activated drugs would be administered topically, and initial development activity will focus on products for the treatment of topical infectious diseases. In fact such products can be considered as replacement products for current topical antibiotics, thus providing
clinicians with a much-desired alternative therapy.
The clinical need for the novel light activated drugs already exists in hospitals worldwide and
is growing fast. Many clinicians consider the continuing emergence of drug resistant bacteria
as the major potential threat to public health. Consequently there is a high level of interest in
new therapies that can eradicate bacteria in a way that avoids the risk of future resistance.
These novel light activated drugs hold this promise.
Therefore, these novel light-activated drugs offer the potential to significantly improve the
current means of treatment and of reducing potential future problems with antibiotic-resistant bacteria. If vancomycin resistant S. aureus does emerge, this technology may offer one
of only a few viable alternative treatments able to prevent topical infections and treat “healthcare” carriers of infection, a major problem in hospitals the world over.
Dr. William Rhys-Williams
Destiny Pharma Ltd
The Sussex Innovation Centre
Science Park Square
Falmer
Brighton BN1 9SB
United Kingdom
Tel.: +44 1273 704 440
Fax: +44 1273 704 499
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PARTNERS:
Dr Keith Brain
An-eX Analytical Services Ltd
Victoria House
250 Cowbridge Rd East
Cardiff CF5 1GZ
United Kingdom
Tel.: +44 2920 874 952
Fax: +44 2920 874 952
Mr Robert Zeller
Waldman Eclairage SA
Rue de l’Embranchement
Zone Industrielle
F-67117 Reichstett
France
Tel.: +49 7720 601477
Fax: +49 7720 601356
Dr Benoit Pugin
Solvias AG
Klybeckstrasse 191
Postfach
CH-4002 Basel
Switzerland
Tel.: +41 61 686 6335
Fax: +41 61 686 6311
79
ANTIMICROBIAL ENDOTOXIN NEUTRALISING
PEPTIDES TO COMBAT INFECTIOUS DISEASES
SUMMARY
Drug-resistant bacteria are reducing the effectiveness of antibiotics and cause high morbidity and economical
costs in Europe and world-wide. Application of classical antibiotics upon infection may actually increase bacterial
endotoxin release thus promoting septic endotoxic shock, a syndrome claiming hundreds of thousands of lives
every year only in Europe. We propose a new therapeutic approach based on the modification of host defence
peptides to confer them both antibiotic and anti-septic activity. Since these peptides act directly on bacterial
membranes, resistance is unlikely to occur. Our strategy will systematically evaluate the dependence of antimicrobial and antiseptic activity of modified peptides as a function of their tertiary structure and biophysical analysis of
peptide-membrane/endotoxin interaction. The project also includes evaluation of large-scale synthetic strategies
and preclinical studies of potential side effects of the candidate substances.
PROBLEM
There is a strong medical but also economical motivation for the development of novel antimicrobial drugs due to both the ever growing bacterial resistance to conventional antibiotics and
the high mortality caused by septic shock, which particularly affects the ageing population and
patients in intensive care units. When bacterial infection spreads through the body (sepsis),
there is an overload of bacterial debris to which the immune system responds by releasing proinflammatory and inflammatory cell mediators. Gram-negative bacteria, in particular, contain a
lipopolysaccharide (LPS) that is the most potent elicitor of this response known. When sepsis
develops, classical antibiotics have to be used with care since they may increase endotoxin
release. Therefore, new drugs, based on a different mechanism of action than existing antibiotics are urgently needed to combat the spread of antibiotic-resistant bacterial strains and to
overcome Gram-negative sepsis.
AIM
The objective is to design semi-synthetic peptides that simultaneously act as endotoxin antagonists and antimicrobial agents and that are not antigenic or toxic to humans. The strategy is
based on an innovative type of peptide modification, i.e. to insert hydrophobic components to
endogenous human cationic peptides to fine-tuning their binding/insertion into membranes
and interaction with bacterial endotoxin, and to increase their selectivity towards bacterial
membranes. By a multidisciplinary approach, we aim to improve our understanding of the peptides’ mechanism of action and the factors underlying selectivity and endotoxin neutralisation.
Dramatic effect of antimbiotic peptides
on the cell envelopes
of Gram-negative bacteria that burst
80
Acronym : ANEPID
Contract Type : Shared Cost
Keywords : antimicrobial peptides, lipopeptides, novel antibiotics, sepsis,
endotoxin neutralisation, bacterial membrane, membrane mimetics,
drug design, pre-clinical studies, SAR studies.
EXPECTED RESULTS
Formulation of the model for interaction between (lipo)peptides and lipopolysaccharide and
structural requirements for optimising the biological activity. Design, preparation and biological testing of peptide analogues as antimicrobial agents and endotoxin neutralisers. We
expect to increase the potency of endogenous peptide fragments by introducing the chemical
modifications, particularly lipophylic groups. Potential drug candidates will be selected based
on their biological activity and their side effects and strategies for large scale production, and
in vivo activity will be evaluated.
The most promising drug candidates will be extensively tested in pre-clinical studies and if
those trials prove satisfactory, exploitation concept will be formulated and partners will be
sought for next steps in drug development. Therefore the most important potential application will be as new drug to treat bacterial infections and particularly sepsis.
Karl Lohner
Institute of Biophysics and X-Ray
StructureResearch
Austrian Academy of Sciences
Schmiedlstr. 6 A 8042 Graz, Austria
T : (+43) 316 4120 323 ; F : (+43) 316 4120 390
Email : [email protected]
PARTNERS:
Sylvie Blondelle
Biochemistry/Microbiology Torrey Pines
Institute For Molecular Studies
3550 General Atomics Court San Diego CA 92121,
USA
T : (+1) 858 455 3843 ; F : (+1) 858 455 3804
Roman Jerala
Laboratory for molecular modelling and NMR
spectroscopy
National Institute of Chemistry
POB 3430 Hajdrihova 19 SLO 1000 Ljubljana,
Slovenia
T : (+386) 1 4760372 ; F : (+386) 1 4259244
Massimo Porro
BioSynthsrl
Zona Industriale Loc. Sentino I 53040 Siena, Italy
T : (+39) 0577 704685 ; F (+39) 0577 704688
Ignacio Moriyon
Departamento de Microbiología
Universidad de Navarra
Irunlarrea 1 PO Box 177 E 31008 Pamplona, Spain
T : (+34) 948 425600 ; F (+34) 948 425649
Klaus Brandenburg
Biophysics Division
Forschungszentrum Borstel Parkallee 10, D 23845
Borstel, Germany
T : (+49) 4537 188235 ; F : (+49) 4537 188632
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SCREENING ASSAYS FOR NEW BACTERIAL
INHIBITORS BASED ON TARGETS ACTIVE
IN SEPTATION
SUMMARY
Our goal is to provide assays and to screen for compounds able to inhibit bacterial proliferation. Blocking cell
division undermines a crucial stage in the development of infection, i.e. the propagation of the microbe. Therefore
we use as inhibitable targets molecules that are essential for the formation of the division septum.
PROBLEM
Very efficient mechanisms to effect division have evolved in microbes using a diverse set of
molecules among which only a handful are conserved in most pathogenic bacteria. We will
focus on essential and phylogenetically conserved bacterial proteins (i.e. FtsA and LdcA). In
addition to being themselves inhibitable targets, these proteins interact with themselves and
with other components of the septation machinery (septator), what will reveal potential targets
in other proteins.
AIM
Already existing and newly derived assays applied to the screening of libraries of natural and
synthetic compounds and “biased” libraries will select candidate compounds for their industrial development as leads for new antibiotics. Cell division proteins and one recently discovered cell wall biosynthetic protein are the proposed targets to build in vitro assays amenable to
HTS. The assays will be developped first at the labortatory scale by the academic participants.
The assays will be transferred to the industrial participant to implement the screening of large
libraries of compounds to select hits with antibacterial properties. Compounds with the desired
activities will be further developed into leads for their potential application in combating disease. The study of the biochemical activities, their three dimensional structure, and the interactions of both types of protein will supply key elements to apply structure activity correlation
for the chemical optimisation of leads.
EXPECTED RESULTS
We expect to obtain compounds (natural and synthetic) to inhibit bacterial proliferation by blocking cell division, a process crucial for infection. These compounds will form part of the new therapies required to
effectively combat infectious disease and to overcome the widespread
resistance to antibiotics common among pathogenic bacteria.
Credits to STUDIO MV
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Acronym : SANITAS
Starting date : March 1st 2001
Keywords : antibiotics / cell division / High Throughput Screening /
protein structure
The industrial Participant (Aventis Pharma), having the expertise and commitment to discover new antibacterials, will develop and market those selected compounds that fulfill the
needs for their clinical use as new antibiotics.
http://www.cnb.uam.es/~mvicente/SANITAS.html
Miguel Vicente
Centro Nacional de Biotecnología
CSIC Campus de Cantoblanco
28049 Madrid. Spain
T: (+34) 91 585 46 99; F: (+34) 91 585 45 06
Web-site: http://www.cnb.uam.es/~mvicente/
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PARTNERS:
Alfonso Valencia
Centro Nacional de Biotecnología
CSIC Campus de Cantoblanco
28049 Madrid. Spain
T: (+34) 91 585 45 70; F: (+34) 91 585 45 06
John Hodgson
Infectious Disease Group
Aventis Pharma
102 route de Noisy
F-93235 Romainville cedex. France
T: (+33) 14 991 46 49; F: (+33) 14 991 26 42
Jan Löwe
Laboratory of Molecular Biology
Medical Research Council
Hills Road, Cambridge CB2 2QH, UK
T: (+44) 01223 40 24 02; F: (+44) 01223 21 35 56
Web-site: http://www2.mrclmb.cam.ac.uk/groups/JYL/index.html
Joachim-Volker Höltje
Max-Planck-Institut für
Entwicklungsbiologie.
Spemanstraße 35, 772076 Tübingen, Germany
T: (+49) 70 71 601 412; F: (+49) 70 71 601 447
Web-site:
http://www.eb.tuebingen.mpg.de/dept2/home.ht
ml
Orietta Massidda
University of Cagliari
Via Palabanda 14, 09124 Cagliari, Italy
T: (+39) 070 675 84 85; F: (+39) 070 66 80 01
Jean-Marie Ruysschaert
LCPMI. Université Libre de Bruxelles
Bld. du Triomphe, Campus de la Plaine, (CP 206/2)
1050 Bruxelles, Belgium
T: (+32) 2 650 53 77; F: (+32) 2 650 51 13
83
RAPID ANTIBIOTIC SUSCEPTIBILITY TESTING
USING DIELECTROPHORESIS
SUMMARY
The objectives of the RASTUD project are: 1) Design and construction of a miniaturised integrated
dielectrophoresis antibiotic susceptibility analyser. 2) Reduction in traditional antibiotic susceptibility analysis
time from 16-24h to less than 1h. 3) Susceptibility profiles for organisms taken directly from clinical specimens
such as blood and urine within 3h. 4) Analysis sensitivities of between 103-104 bacteria/ml. 5) Statistical
appraisal of error rates compared to gold standard methods.
PROBLEM
Most hospital laboratories use a traditional system of agar diffusion assays or broth dilution
tests to determined antibiotic susceptibility, which require an incubation stage of 24 h or
longer. Diagnosis is often made, and an antibiotic treatment prescribed, before a clinician has
the benefit of laboratory tests to ensure specificity of treatment, often leading to inappropriate
prescription. Current rapid methods of assessing the antibiotic susceptibility of an organism
are limited, the majority of systems requiring pre-processing of organisms and still relying on
growth of bacteria.
AIM
Dielectrophoresis (DEP) is an electrical phenomenon, which can be performed at almost realtime due to the absence of a growth phase in the analyses. When cells are placed in a non-uniform electric field they can be induced to collect upon microelectrodes. The extent of collection
varies as a function of the electric field frequency and is determined by cell characteristics.
Changes in the surface chemistry or structure of a cell caused by antibiotic treatment leads to
a corresponding change in the dielectrophoretic frequency response. Preliminary work for
selected examples has demonstrated that dielectrophoresis can detect changes in collection
spectrum caused by treatment with antibiotics within minutes. The project will determine
whether differences in dielectrophoretic spectra occur with a wide range of bacterial species
following exposure to antibiotics and to assess the extent of discrimination between antibiotic
resistant and sensitive bacteria.
EXPECTED RESULTS
The system will be redesigned, miniaturised and a fully integrated prototype system will be produced as part of this research, to demonstrate the application of dielectrophoretic antibiotic
susceptibility determination within clinical environments.
84
Acronym : RASTUD
Keywords : Antibiotic susceptibility testing, antibiotic resistance,
dielectrophoresis, AC electrokinetics, electric fields, rapid, real-time,
biomedical, microsystems, microfabrication, microelectrodes,
equipment, miniaturisation, automation, prototype, clinical, public
health, point of care testing
This will provide market opportunities for rapid microbiological analytical techniques, rapid
antibiotic susceptibility testing and point of care testing systems and will enable a faster and
more informed prescription of appropriate antibiotics, reducing hospital costs and improving
public health.
Peter Salmon
Cell Analysis Ltd
The Old Stable
Farnham Lane, Farnham Royal
Slough, Berkshire SL2 3SE, ENGLAND
T: (+44) 1753 647229; F: (+44) 1753 647226
PARTNERS:
Dr Ralf Höper
Protron Mikrotechnik GmbH
Fehrenheitstrasse 1
D-28359 Bremen
Germany
Tel.: +49 421 223 4818
Fax: +49 432 223 4820
Ms Anne Bolmström
AB Biodisk
Dalvägen 10
S-16956 Solna
Sweden
Tel.: +46 8 730 0760
Fax: +46 8 831 8158
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Dr. Andrew Brown
University of York
PO Box 373
York YO10 5YW
United Kingdom
Tel.: +44 1904 432458
Fax: +44 1904 43433
Dr. Marcos Rodrigues
Sheffield Hallam University
Howard Street
Sheffield S1 1WB
United Kingdom
Tel.: +44 114 225 4486
Fax: +44 114 225 2060
Prof. Miguel Sancho
Universidad Complutense de Madrid
Avenida de Seneca 2
S-28040 Madrid
Spain
Tel.: +34 91 394 4388
Fax: +34 91 394 5196
Prof. Wolfgang Benecke
Institute for Microsensors, Actuators and
Systems (IMSAS)
University of Bremen
Kufsteinerstrasse 1
D-28359 Bremen
Germany
Tel.: +49 421 218 2712
Fax: +49 432 218 4259
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DIFFERENTIAL DIAGNOSIS OF INFECTIOUS
LUNG DISEASES
SUMMARY
In this CRAFT PulmInfect project, the consortium strives to develop platform technologies to enable differential
diagnosis of infectious lung diseases on various detection platforms.
PROBLEM
With the currently existing (single parameter) tests, infectious lung diseases are often difficult
to diagnose. This may be because only low amounts of infectious agents can be collected (e.g.
in small children), or because of the presence of multiple infectious agents simultaneously, or
because the infectious agent is a-typical and therefore not tested for. Moreover, a tumour may
be underlying the apparently infectious lung problems. All these factors often are resulting in
long diagnostic intervals and poor prognosis.
AIM
In the present project we aim to increase diagnostic sensitivity by a novel approach, detecting
simultaneously both the infectious agent(s) as well as the immune response(s) directed against
this agent. This approach will speed-up the diagnosis and increase the reliability. Thus, differential diagnosis will be aimed at by multi-parameter detection on various detection platforms.
EXPECTED RESULTS
Various prototype detection platforms for the differential diagnosis of infectious lung diseases
will be developed. Moreover, (basic) technologies implemented in the development of these
(diagnostic) tests may be implemented in other applications (outside the scope of this project).
86
Acronym : PulmInfect
Starting date : June 1st 2002
Keywords : multi-parameter diagnosis, infectious lung diseases,
antigens, antibodies, nucleic acids
The prototype(s) will be exploited via third parties that produce and distribute diagnostic
tests (e.g. diagnostic or pharmaceutical industries). The underlying technologies may be
exploited in other applications (both in research and in diagnostics).
Dr. C.J.T. Visser
KREATECH Biotechnology BV
P.O. Box 37078, 1030 AB Amsterdam, The
Netherlands
T: (+31) 20-6919181; F: (+31) 20 6963531
PARTNERS:
L. Verstappen
DIAMED BENELUX NV
Hertoginstraat 82, 2300 Turnhout, Belgium
T: (+32) 14470890; F: (+32) 14424957
J. Bensadon Belicha
VITRO SA
Apartado, 1286 F.D., 28080 Madrid, Spain
T: (+34) 91 3821620; F: (+34) 91 763668
Prof. Dr. H.A. Verbrugh
Erasmus University Medical Center
Rotterdam
Dept. Med. Microbiol. & Inf. Diseases
Dr. Molewaterplein 40, 3015 GD Rotterdam, The
Netherlands
T: (+31) 10 4633510; F: (+31) 10 4633875
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Dr. P.R. Klatser
Koninklijk Instituut voor de Tropen
KIT Biomedical Research
Meibergdreef 39, 1105 AZ Amsterdam, The
Netherlands
T: (+31) 20 5665440; F: (+31) 20 6971841
Prof. Dr. J. Anné
Katholieke Universiteit Leuven
Rega Institute
Dept. Microbiology and Immunology
Laboratory of Bacteriology
Minderbroedersstraat 10, 3000 Leuven, Belgium
T: (+32) 16 324067; F: (+32) 16 324198
Dr. L. Michiels
Limburgs Universitair Centrum
Biomedisch Onderzoeksinstituut (BIOMED)
Universitaire Campus, Gebouw A, 3590
Diepenbeek, Belgium
T: (+32) 11 268111; F: (+32) 11 268199
Prof. Dr. A. Costa García
Universidad de Oviedo
Departamento de Química, Física y Analítica
C/ Julián Clavería No 8, 33006 Oviedo, Spain
T: (+34) 985103488 -125; F: (+34) 985104040
Prof. Dr. H.J. Tanke
Leiden University Medical Center
Dept. Molecular Cell Biology
Wassenaarseweg 72, 2333 AL Leiden, The
Netherlands
T: (+31) 71 5276196; F: (+31) 71 5276180
87
DEVELOPMENT OF STRATEGIES FOR CONTROL
AND PREVENTION OF ANTIBIOTIC RESISTANCE
IN EUROPEAN HOSPITALS
SUMMARY
The project will gather human antibiotic consumption data, establish antibiotic resistance rates in targeted
bacterial pathogens, investigate current guidelines on antibiotic policies and infection control policies and
investigate standardisation of methods used to track human resistant pathogens. The required data are gathered
via a series of questionnaires sent out to European Microbiologists, starting at the beginning of 2002. Initially the
questionnaires are being sent out via ESCMID, but participation from all willing parties is encouraged. To ensure
participation, contact the project co-ordinator. The success of this study relies entirely on the willingness of
Microbiologists across Europe to participate in this study and to provide data. For the first time the variability of
the parameters detailed above will be explored across a common set of European hospitals. All of the assimilated
data will be modelled by a medical statistician and strategies associated with lower antibiotic resistance rates
will be identified
PROBLEM
Antibiotic resistance is an escalating problem, especially in hospitals. As yet, no Europe-wide
vehicles operate to assess the effectiveness of control measures to decrease the incidence of
antibiotic resistance. The overall objective of the project is to lay the foundations for a better
understanding of the emergence and epidemiology of antibiotic resistance in human
pathogens and to evaluate and harmonise strategies for prevention and control of antibiotic
resistant pathogens in European hospitals.
AIM
• To identify antibiotic policies associated with lower resistance rates.
• To identify infection control policies associated with low rates of Alert organisms.
• To develop a DNA typing database and data exchange format for tracking epidemic
antibiotic-resistant micro-organisms.
EXPECTED RESULTS
The project expects to confirm the suspected causal relationship between antibiotic consumption and resistance. It also expects to deliver a consensus set of recommendations to control
the emergence and spread of antibiotic resistance.
Multiply-resistant K. pnemoniae
from Aberdeen outbreak demonstrating
extended spectrum beta-lactamase activity
88
Acronym : ARPAC
EC contribution : 700 000 €
Keywords : antibiotic, consumption, resistance, infection control,
antibiotic policies, epidemiology, DNA fingerprinting, consensus
guidelines
Guidelines and strategies will be developed with a view to their incorporation into local hospitals. These will include optimum antibiotic and infection control policies as well as data
exchange strategies for molecular fingerprinting.
http://www.abdn.ac.uk/arpac/index
Dr. Ian M. Gould (Dr. Fiona M. MacKenzie)
Medical Microbiology
Aberdeen Royal Infirmary
Foresterhill, UK-Aberdeen AB25 2ZN, Scotland
T: (+44) 1224 554954; F. (+44) 1224 550632
PARTNERS:
Prof Marc J.Struelens
Service de Microbiologie
Université Libre de Bruxelles
Hopital Erasme & Unité d'Epidémiologie des
Maladies Infectieuses
Ecole de Santé Publique
808, route de Lennik, BE-1070 Bruxelles, Belgium
T: (+32) 2 555 45 19; F: (+32) 2 555 31 10
Prof. Dr. H. Goossens
University of Antwerp
Universiteitsplein 1, BE-2610 Wilrijk (Antwerp), Be
T: (+32) 3 821 37 89; F: (+32) 3 825 42 81
Dr Kevin Towner
Public Health Laboratory
University Hospital
Queen's Medical Centre
UK-Nottingham NG7 2UH, UK
T: (+44) 115 9709163; F: (+44) 115 9422190
Prof Jos van der Meer
General Internal Medicine
Univ. Hospital Nijmegen
P.O. Box 9101, NL-6500 HB Nijmegen, The
Netherlands
T: (+31) 24 3614763; F: (+31) 24 3541734
Prof. Vladimir Krcmery
St. Elisabeth Cancer Institute
University of Trnava
Heydukova 10, 812 50 Bratislava, Slovak Republic
T/F: (+42) 1 7 5292 4308
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Dr. Gunnar Kahlmeter
Clinical Microbiology
Central Hospital
SE-351 85 Vaxjo, Sweden
T: (+46) 470 587460; F: (+46) 470 587 455
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Prof. P.J. van den Broek
Department of Infectious Diseases
C5-P, PObox 9600, NL-2300 RC Leiden
The Netherlands
T: (+31) 071 5262290; F: (+31) 071 5266758
Dr. Peter Gerner-Smidt
5, Artillerivej, DK-2300 Copenhagen S
Denmark
T: +45 3268 3798; F: (+45) 3268 8238
Prof. Dr. Jordi Vila
Laboratori de Microbiologia
Hospital Clinic
Villarroel, 170, ES-08036 Barcelona, Spain
T: (+34) 93 2275522; F: (+34) 93 2275454
Dr. Giuseppe Cornaglia
Instituto di Microbiologica
Strada le Grazie 8, IT-37134 Verona, Italy
T: (+39) 045 8027196; F: (+39) 045 584606
Prof. Fernando Baquero
The University Hospital Ramóm y Cajal
Carretera de Colmenar km, ES-9100 Madrid, Spain
T: (+34) 91 336 83 30; F: (+34) 91 336 88 09
Dr Barry Cookson, Director
Laboratory of Hospital Infection
CPHL, PHLS
61 Colindale Avenue, UK-London NW11 5HT, UK
T: (+44) 020 8200 4400 ext 4221/4249; F: (+44)
020 8200 7449
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SCIENTIFIC EVALUATION ON THE USE OF
ANTIMICROBIAL AGENTS IN HUMAN THERAPY
SUMMARY
During a two year period, actions are being taken to harmonise the collection of antimicrobial consumption data
in all EU countries. Retrospective data for ambulatory care and hospitals are being collected for the period 19972001. A prospective data collection system, based on a validated register of available antibiotic products linked to
the correct ATC/DDD classification, will become effective in 2003. Standardised national data will be assembled in
a European database for international comparison of antibiotic use in relation to antibiotic resistance patterns
and socio-economic and general health parameters.
PROBLEM
There is an established, albeit complex, relationship between consumption of antimicrobials
and the prevalence of drug resistant bacteria. Although several antibiotic resistance surveillance programmes are operational at the EU level, a programme for the collection of data on the
consumption of antimicrobials is lacking. In most European countries, some information on the
consumption of antimicrobials at a national level exists. Large differences can be observed,
however, in the structure and the accessibility of these databases. Moreover, in order to compare antimicrobial consumption at the EU level, internationally applicable methods need to be
established including both a uniform classification system and a common unit of measurement.
AIM
This project aims to construct and maintain a European database on antimicrobial consumption
based on standardised and validated national data using the DDD (Defined Daily Dose)/ATC
(Anatomical Therapeutic Chemical) classification system. Quantitative consumption data will
be complemented with a database offering an inventory of projects focused on antibiotic consumption (published research as well as ongoing projects). The database will be accessible for
health authorities and scientists in order to link antibiotic consumption to resistance patterns
and to assess the impact of intervention strategies at the community and hospital level.
EXPECTED RESULTS
The main results of the pilot phase of the ESAC project will be the availability of an administrative database of retrospective data of antibiotic consumption of all European countries (19972001) and a continuous and comprehensive surveillance system of antibiotic consumption,
containing data from 2002 on.
http//www.esac.ua.ac.be
90
Acronym : ESAC (funded by DG SANCO)
(European Study on Antimicrobial Consumption)
Prof. Dr. Herman Goossens
Dept. of Microbiology University Hospital Antwerp
Wilrijkstraat 10 B 2650 Edegem-Antwerp BE
Tel +32 3 821 37 89 Fax +32 3 825 42 81
[email protected]
PARTNERS:
Dr. Erik Hendrickx Epidemiology Department
Institute for Public Health
J. Wytsmanstraat 14 B 1050 Brussel Belgium
Keywords : Antimicrobial consumption, antimicrobial resistance, ATC
(Anatomical Therapeutic Chemical) classification, DDD (Defined Daily
Dose), quality of use indicators.
Dr. Ludvik Stika
State Institute for Drug Control
Srobarova 48, 100 14 Praha 10, Czech Republic
Dr. Dominique Monnet
Dept. of Microbiological Research and
Development Statens Serum Institut
Artillerivej 5, DK 2300 Copenhagen S, Denmark
Annemette Anker Nielsen
Danish Medicines Agency
Frederikssundsvej 378, DK 2700 Bronshoi,Denmark
Dr. Robert Vander Stichele
Dept. of Microbiology
Universty of Antwerp Universiteitsplein 1,
B 2610 Wilrijk, Belgium
Dr. Pentti Huovinen
P.O.Box 57 SF 20521 Turku, Finland
Dr. Monique Elseviers
Dept. of Microbiology University of Antwerp
Universiteitsplein 1, B 2610 Wilrijk, Belgium
Pirkko Paakkari
National Agency for Medicines
P.O.Box 55 DF-00301 Helsinki, Finland
Matus Ferech
Prof. Dr. Didier Guillemot
Unité des Agents Antibactériens
Institut Pasteur
25-28 rue du Dr. Roux, F-75017 Paris, France
Monique Boets
Dr. Helmut Mittermayer Krankenhaus der
Elisabethinen Linz Institut für Hygiene,
Mikrobiologie und Tropenmedizin Fadingerstrasse
1, A-4010 Linz, Austria
Boyka Markova Microbiology Department
University Hospital "Alexandrovska
1 Georgy Soffiiski St, 1431 Sofia, Bulgaria
Dr. Borislav Borissov Bulgarian Drug Agency
26, Yanko Sakazov Blvd., 1504 Sofia, Bulgaria
Arjana Andrasevic Clinical Microbiology
University Hospital for Infectious Diseases
Mirogojska 8, 10 000 Zagreb, Croatia
Dr. Arne Rodloff,
Institut für Medikal Mikrobologie
Universität Leipzig
Liebigstrasse 24, D-04103 Leipzig, Germany
Dr. Helen Giamarellou
4th. Department of Internal Medicine
Sismanoglio Hospital
151 26 Maroussi Attikis, 11526 Athens, Greece
Dr. Anastasia Antoniadou
4th. Department of Internal Medicine
Sismanoglio Hospital
151 26 Maroussi Attikis, 11526 Athens, Greece
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Mr. Philippe Maugendre,
Agence Française de Sécurité Sanitaire des
Produits de Santé (AFSSAPS)
Blvd. Anatole France 143-147, F-93285
Saint-Denis Cedex, France
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Dr. Michael Borg Infection Control Unit
St Luke's Hospital - G'Mangia, MSD08 Malta
Hege Salvesen Blix
Norwegian Institute of Public Health
Postboks 4404 Nydalen, N-0403 Oslo, Norway
Dr. Waleria Hryniewicz
Chairman National Drug Institute
Chelmska 30/34, 00-725 Warsaw, Poland
Pawel Grzesiowski National Drug Institute
Dr. Luis Caldeira OMPS-Observatório do
Medicamento e Produtos de Saúde
Instituto Nacional da Farmacia e do Medicamento
(INFARMED) Parque de Saude de Lisboa-Av. Brasil,
53-Pav.24 1749-004 Lisboa Portugal
Irina Codita Cantacuzino Institute Romania
Dr. Leonid Stratchounski
Institute of Antimicrobial Chemotherapy
Smolensk State Medical Academy
P.O.Box 5, 214019 Smolensk, Russia
Viliam Foltan Faculty of Pharmacy UK
Katedra organizacie a riadenia farmacie
Kalinciakova 8, 832 32 Bratislava, Slovak Republic
Dr. Milan Cizman Department of Infectious
Diseases University Medical Centre Ljubliana
Japljeva 2, 1525 Ljubliana, Slovenia
Prof. Dr. Fernando Baquero
Serv. De Microbiologia Hospital Ramon y Cajal
National Institute of Health (INSALUD)
Carretera de Colmenar KM. 9 100, ES-28034
Madrid, Spain
Dr. Gabor Ternak Dept. of Infectiology
County Hospital Rákóczi út 2,.7623 Pécs, Hungary
Jose Campos Centro Nacional Microbiologia
Instituto Carlos III Ministry of Health
Cra. Pozuelo, ES-28222 Madrid, Spain
Dr. Karl G.Kristinsson
Landspitali - University Hospital
POBox 1465, 121 Reykjavik, Iceland
Prof. Dr. Otto Cars
Uppsala University Hospital
S-751 85 Uppsala, Sweden
Edmond Smyth Microbiology Department
Beaumont Hospital Beaumont, Dublin 9, Ireland
Dr. Christian Ruef
Division of Infectious Diseases and Hospital
Epidemiology HAL14C, University Hospital of
Zürich - CH-8091 Zürich, Switzerland
Dr. Guiseppe Cornaglia
Institute of Microbiology University of Verona
Strada le Grazie 8, I-37134 Verona, Italy
Sandra Berzina Clinical Microbiolog
P. Stradins Clinical Univrsity Hospital
Pilsonu str 13, LV-1002 Riga, Latvia
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Dr. Robert Hemmer
Service National des Maladies Infectieuses
Centre Hospitalier de Luxembourg
4 rue Barble L-1210 Luxembourg, Luxembourg
Dr. Rolanda Valinteliene
Head, Public Health Research Department
Institute of Hygiene
Didzioji 22, LT-2001 Vilnius, Lithuania
Dr. Robert Janknegt Maasland Ziekenhuis
P.O.Box 5500, 6130 MB Sittard, The Netherlands
Serhat Unal Department of Medicine
Hacettepe University-School of Medicine
06100 Ankara, Turkey
Prof. Dr. Peter Davey
Department of Clinical Pharmacology
Ninewells Hospital-Medicines Monitoring Unit
DD1 9SY Dundee, Scotland
91
TOWARDS CONTROLLING ANTIMICROBIAL USE
AND RESISTANCE IN LOW-INCOME COUNTRIES –
AN INTERVENTION STUDY IN LATIN AMERICA
SUMMARY
The project will describe antimicrobial use and resistance in healthy children in urban and remote rural
communities of Bolivia and Peru. Antimicrobial use will be analysed by means of household surveys with a
triangulation of methods. Antimicrobial resistance will be analysed in commensal flora using a robust resistance
screening method. Resistant bacteria will be investigated for genetic resistance mechanisms. Influencing factors
including policy and non-human use will also be analysed. To improve the situation a community intervention
package focusing on rational use of antimicrobials will be evaluated. It will be developed and implemented in
collaboration with the local public and private health services on the basis of the generated results enforced by
the national drug policy. The project will contribute to a more appropriate antimicrobial use and evidence based
antimicrobial policy.
PROBLEM
Antimicrobials represent one of the major interventions to reduce mortality in low-income countries (LIC), and in many of these countries
they constitute a substantial part of the overall health budget. The
effectiveness of these drugs is diminishing due to the emergence and
spread of bacterial resistance. Resistance to antimicrobials is now a
worldwide problem, which may be particularly serious in LIC where
resistance rates were found even higher than in industrialised countries, and where alternative
antimicrobials are often not available or too expensive.
Increased resistance rates are found not only among infectious pathogens, but also in the resident indigenous microbiota of healthy people. The high prevalence of antimicrobial resistance
observed in LIC is likely due to several factors, including both irrational use of antimicrobials
and unique environmental conditions, such as crowding and poor sanitation. Many factors contribute to irrational use of antimicrobials: lack of laboratory facilities to guide prescribing, uninformed self-medication due to the widespread availability of non-prescription antimicrobials,
and sub-optimal therapeutic regimes, due to lack of finances to complete treatment courses or
to the use of substandard drugs.
However, the recent findings of high prevalence of antimicrobial resistance in commensal bacteria from human population of a remote rural community of South America, which has very low
human antimicrobial consumption, raise the question about the role of other factors (e.g. nonhuman use of antimicrobials), favouring the emergence and spreading of antimicrobial resistance.
To reduce antimicrobial consumption, multi-faceted interventions are needed. They should
include education of providers and measures to improve the public’s knowledge about the risks
and benefits of antimicrobial therapy. Surveillance of antimicrobial resistance is of critical
importance both to provide information on the magnitude and trends in resistance and to monitor the effect of interventions. Development and strengthening of local laboratory and standardization of techniques and data interpretation are essential. Irrational use and bacterial
resistance to antimicrobials are major public health problems amplified in low-income countries.
AIM
The main objective is the development of a conceptual framework of the dynamics of antimicrobial use and resistance in healthy children in urban and remote rural areas of Bolivia and
Peru. According to the findings an intervention strategy involving local health services in
addressing the public will be developed and implemented. It will be evaluated on antimicrobial
use and bacterial resistance trends in the intervention group compared to the control group by
using household surveys and applying an innovative and robust resistance screening method
of the commensal microbiota. The project will contribute to the development of evidence based
antimicrobial policy at national and international levels.
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Acronym : ANTRES
Project number : ICA4 – 2001-10014
Keywords : antimicrobial use, bacterial resistance, screening method,
commensal microbiota, IEC intervention, low-income countries,
Escherichia coli, antimicrobials, Latin America, Bolivia, Peru
EXPECTED RESULTS
• Improvement of the general public’s understanding about the risks and benefits of antimicrobial therapy.
• Advanced knowledge of the interrelationship between human antimicrobial use and resistance epidemiology taking influencing factors into account.
• Monitoring strategies for antimicrobial use and antimicrobial resistance to support the governmental institutions involved in public health and drug policy making.
• Intervention package strategies documented feasible and effective for improving rational
use of antimicrobials in low-income settings.
The results will provide evidence-based improvements of the community health services related to antimicrobial use and resistance and propose the utilised simple screening method for
routine analysis of antimicrobial resistance with implications for national and international
surveillance and control.
www.unifi.it/infdis/antres
Prof Paradisi Franco
Area Critica Medico-Chirurgica
Università di Firenze
Clinica Malattie Infettive
Viale Morgagni 85
50124 Florence Italy
T: (+39) 055 4279482/4279431;
F: (+39) 055 4279480
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PARTNERS:
Prof Bonanni Paolo
Salute Pubblica
Università di Firenze
Viale Morgagni 48, 50134 Florence, Italy
T: (+39) 055 3262402; F: (+39) 055 3262436
Dr Lanza Van den Berghe Oscar
Accion Internacional por la Salud AIS Bolivia
Casilla Correo 568
Avda. Abel Iturralde 1178-Miraflores
La Paz, Bolivia
T: (+591) 2 222987; F: (+591) 2 222987
Prof Rossolini Gian Maria
Biologia Molecolare
Ospedale"Le Scotte"
Viale Bracci, 53100, Siena, Italy
T: (+39) 0577 23326/23327; F: (+39) 0577 233325
Dr Trigoso Christian
Instituto Nacional de Laboratorios de Salud
INLASA
Casella M-10019
Rafael Zubieta No. 1889-Miraflores, La Paz, Bolivia
T: (+591) 22 226670; F: (+591) 22 228254
Dr Falkenberg Torkel
Public Health Sciences
Division of International Health (IHCAR)
11645 Stockholm, Sweden
T: (+46) 8 51776487; F: (+46) 8 311590
Prof Gotuzzo Eduardo
Universidad Peruana Cayetano Heredia
Instituto de Medicina Tropical “Alexander von
Humboldt"
Lima, Peru
T: (+51) 14 823910; F: (+51) 14 823404
Dr Guillaumet Javier
Teko Guaraní
Casilla 20
Calle Sargento Macedo s/n, Camiri, Bolivia
T: (+591) 3 9522565; (+591) 3 9522580
Dr Llanos Zavalaga Luis Fernando
Instituto Nacional de Salud
Microbiology
Cápac Yupanqui 1400, Lima 11, Peru
T: (+) 51 14 712529; F: (+51) 14 717443
Dr Gamboa Barahona Herlan
Distrito de Salud Cordillera
Casilla 20
Calle Sargento Macedo s/n, Camiri, Bolivia
T: (+591) 3 9522782; F: (+591) 3 9522147
Dr Naupay Ruben
Referencial Laboratory of Loreto
Garcia Saenz 332, Iquitos–Loreto, Peru
T: (+51) 94 223369; T: (+51) 94 243518
Dr Rodriguez Ferrucci Hugo
Hospital Apoyo Yurimaguas
Calle Progreso 305-307, Yurimaguas–Loreto, Peru
T: (+51) 94 352135; F: (+51) 94 352135
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ANTIBIOTIC RESISTANCE SURVEILLANCE &
CONTROL IN THE MEDITERRANEAN REGION
SUMMARY
ARMed is composed of three sub-projects linking with and utilising comparable methodology such as, the
European Antimicrobial Resistance Surveillance System (EARSS), European Study on Antibiotic Consumption
(ESAC) and the HARMONY infection control project. Overlap will be avoided by focusing specifically on a group of
countries in the southern and eastern Mediterranean that have not been included in these programmes.
1. ARMed-EARSS will assemble a database on antibiotic resistance by undertaking a surveillance study in
Mediterranean partner countries through the collection of comparable antimicrobial susceptibility data utilising
a standardised protocol for Staphylococcus aureus, Streptococcus. pneumoniae, Enterococcus faecium and
faecalis and Escherichia coli strains isolated from routine blood cultures
2. ARMed-ESAC shall collect data on antibiotic consumption by an agreed standardised methodology
concentrating on hospital settings.
3. ARMED-HARMONY will evaluate current infection control policies and practices in the Mediterranean partner
countries through the use of structured questionnaires and finalise tools for policy design.
4. Data from the three sub-projects will be linked into an integrated report which will elucidate any relationships
between antimicrobial resistance, antibiotic consumption and infection control in the participating countries.
Dissemination of information will be achieved through a project website, international publication of articles in
medical journals and communication with government health officials and the national press.
5. A symposium in an international congress will be organised at the end of the project to present the results and
recommendations to the scientific community.
PROBLEM
EU Council Resolution 1999/C 195/01 "A strategy against the microbial threat" highlights
antibiotic resistance as a major challenges facing public health and identifies the need for surveillance of antibiotic resistance, adherence to infection control principles and promotion of
optimal antibiotic use. Although preliminary evidence indicates high resistance prevalence in
the Mediterranean, co-ordinated networks studying the problem are still, on the whole, absent
in much of the region. ARMed will link with current or recently finalised European studies and
extend them to non-participating southern and eastern Mediterranean partner countries. It will
study the epidemiology of antimicrobial resistance, antibiotic consumption trends, infection
control measures and disseminate results widely within the region.
AIM
Over a period of 4 years, ARMed aims to:
1. Initiate a network of experts on antibiotic resistance in the southern and eastern
Mediterranean region
2. Assemble a database on antibiotic resistance in selected medically relevant pathogens by
undertaking a surveillance study in the Mediterranean partner countries through the collection of comparable antimicrobial susceptibility data using a standardised protocol
3. Compare results from ARMed with similarly collected data from the European Mediterranean
countries by the EARSS network, creating a more complete picture of resistance in the region
4. Investigate antibiotic consumption patterns and infection control guidelines in use in the
participating Mediterranean countries with a view to highlight any specific cultural issues
particular to this region
5. Link the data from the three separate sub-projects to elucidate relationships between antimicrobial resistance, antibiotic consumption and infection control.
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Acronym : ARMed
Starting date : January, 1st 2003
Contract Type : Thematic Network
Keywords : Antimicrobial resistance, Infection Control,
Antibiotic consumption, Mediterranean.
EXPECTED RESULTS
1. Detailed antimicrobial resistance data for the most relevant pathogens collected in a standardised and uniform method will be made available for the first time and will (as a result
of EARSS and ARMed) cover the whole Mediterranean region.
2. The antibiotic consumption data, particularly at hospital level, should assist in the establishment of surveillance methods for antibiotic use within the participating Mediterranean
countries.
3. An overview of the current infection control infrastructure in the participating
Mediterranean partner countries will be elucidated. Guidelines and tools shall be specifically adopted for the characteristics of the Mediterranean region.
4. The final symposium will allow discussion on the issues concerning antibiotic resistance
within the Mediterranean countries amongst the scientific community. In addition the dissemination of information by the individual project participants at national level will
increase public awareness of the microbial threat and influence policy and decision makers
in the respective countries.
The challenge of the “Microbial Threat” has been recognised in the European Union over the
past years. The same challenge in the Mediterranean can only start to be addressed when validated data on the occurrence of antibiotic use and resistance is available to public health
planners within the countries concerned. Unless this data is collated, there is a real risk that
the populations of Mediterranean countries will do nothing, leaving this major public health
threat uncontrolled. ARMed will facilitate a concerted co-operation towards the identification
and control of antimicrobial drug resistance between the participating centres in the EuroMediterranean countries involved.
Dr Michael Angelo Borg
Infection Control Unit, St Luke's Hospital
St. Luke's Road, MSD 07, Guardamangia, Malta
T: (+356) 21235447; F: (+356) 21235447
PARTNERS:
Paul Schrijnemakers
Department of infectious Disease
Epidemiology
National Institute of Public Health and the
Environment
A. V. Leeuwenhoeklaan 9, 3720 BA Bithoven,
The Netherlands
T: (+31) 30 274 3486; F: (+31) 30 274 4409
Web-site: http//www.earss.rivm.nl
Prof Dr Herman Goossens
Laboratory of Microbiology
University of Antwerp - Universiteitsplein 1,
B-2610 Wilrijik-Antwerp, Belgium
T: (+32) 3 8213789; F: (+32) 3 8254281
Web-site: http://esac-www.uia.ac.be/esac/
Dr Berry Cookson
Laboratory of Hospital Infection
Central Public Health Laboratory
PHLS Headquarters
61, Colindale Avenue, NW9 5DF, London, UK
T: (+44) 0 20 8358 3299; (+44) 0 20 8200 7449
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Prof Saida Benredjeb
Laboratoire de Microbiologie
Hopital Charles Nicolle, Boulevard du 9 Avril, 1006
Tunis, Tunisia
T: (+216) 71564765; F: (+216) 71568744
Prof Dr Deniz Gur
Clinical Microbiology Lab
Hacettepe University -Sihhiye, 6100, Ankara, Turkey
T: (+90) 312 3105925; F: (+90) 312 3243284
Prof Ossama Rasslan
Infection Control Unit
Ain Shams Faculty of Medicine
Ramsis Ext. Street, Abbassia square, Cairo, Egypt
T: (+20) 2 4842562, (+20) 2 4842563;
F: (+20) 2 6846899
Prof Mohammed Benbachir
Laboratoire de Microbiologie
Faculte de Medecine - IbnRochd University Hospital
19 rue Tarik BNDU ZYAD, Casablanca, Morocco
T: (+212) 22269057; F: (+212) 22269057
Dr Despo Pieridou-Bagatzouni
Microbiology Departrment
Nicosia General Hospital - Cyprus Ministry of Health
10, Markou Drakou, 1449 Nikosia, Cyprus
T: (+357) 22 673945; F: (+357) 22 303471
Ziad Elnasser
Microbiology Laboratory
King Abdulla Teaching Hospital
Jordan University of Science and Technology
Pathology and Microbiology Dept
22110 Irbid, Jordan
T: (+962) 79544224; F: (+962) 27095070
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TREAT – A SYSTEM FOR BALANCING
ANTIBIOTIC TREATMENT AGAINST
DEVELOPMENT OF DRUG RESISTANCE
SUMMARY
The goal of the project is to develop a framework for the building and testing of medical decision support
systems. The framework is based on a novel technology for the construction of very large stochastic models.
The project will demonstrate the value of the proposed framework through its successful application in a specific
medical area, the antibiotic treatment of infections.
PROBLEM
There is a large and rising mortality (about 100.000 annual deaths in Europe) associated with
inappropriate antibiotic treatment of patients with severe infections. The inappropriate antibiotic treatment has resulted in increased bacterial resistance to antibiotics. Resistant bacterial
strains do not respect national borders and with the current level of travel, the problem of bacterial resistance cannot be solved at the national level.
AIM
1. The purpose of the project is to develop a novel framework for the construction and testing
of medical decision support systems and to demonstrate its soundness through a successful
application of the framework to a clinical problem of major importance. A successful application implies that the system a) can be integrated into the informational infrastructure of the
involved hospitals, b) is clinically acceptable from the user interaction point of view, and c)
can improve diagnosis or therapy in the medical area addressed.
2. The clinical problem chosen is the selection of antibiotic therapy for the treatment of severe
infections. A medical decision support system, TREAT, will be developed, that can help doctors reduce substantially the about 100,000 annual deaths in Europe associated with inappropriate antibiotic treatment of patients with severe infections, and help to curb the development of bacterial resistance to antibiotics.
3. TREAT will be tested to demonstrate that it can be integrated in the clinical and informational environment and that improvements in antibiotic therapy can be obtained in a large controlled multicentre clinical trial in geographic regions with widely differing patterns of antibiotic resistance.
EXPECTED RESULTS
By month 36, the clinical value and acceptability of TREAT will be demonstrated, TREAT will be
installed in hospitals in three different countries and a product marketing plan for two versions
of the system will be available. By month 48, the clinical trials of both systems are concluded
and strategic alliances for the world wide commercialisation of TREAT are identified.
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Acronym : TREAT
Project number : IST-1999-11459
Keywords : Decision support, medical information system, bacterial
resistance, antibiotics, causal probabilistic networks, bacterial infection, treatment, infections
The steps necessary to ensure that the results will be disseminated scientifically will be taken,
and TREAT will be made commercially available. Widespread clinical adoption of TREAT is
expected to lead to reduced mortality, improved quality of life and savings in the health care
system. The medical objective of the project is thus to develop and test a decision support
system, TREAT, that can help doctors reduce substantially the large and rising mortality associated with inappropriate antibiotic treatment of patients with severe infections, and help to
curb the development of bacterial resistance to antibiotics.
The results of the clinical testing are expected to show that:
1) TREAT can be integrated into the informational infrastructure of the involved hospitals.
2) TREAT is clinically acceptable from the user interaction point of view.
3) TREAT can give major reductions in mortality, compared to current clinical practice.
Based on these results, a plan for world-wide commercialisation of TREAT will be made, and
the results will be disseminated to open a path for the use of this novel technology in other
medical areas.
www.treat.dk/
Dr.Tech., Ph.D. Steen Andreassen
Center for Model-based Medical Decision
Support
Aalborg University
Fredrik Bajers Vej 7C? DK-9220 Aalborg, Denmark
T: (+45) 9635 8812; F: (+45) 9815 5816
Email: [email protected] www.treat.dk
PARTNERS:
General Manager Erling Henningsen
Judex A/S, Lyngvej 8, DK-9000
Aalborg, Denmark
T: (+45) 9818 6900; F: (+45) 9818 8019
www.judex.dk
Prof. Leonard Leibovici
Department of Medicine
Rabin Medical Center
Beilinson Campus, 49100 Petah-Tiqva, Israel
T: (+972) 3 937 6501, (+972) 3 9376505
Web-site: www.tau.ac.il
Prof., Dr. Franz Daschner
Institut für Umweltmedizin und
Krankenhaushygiene
Univeritaetsklinikum Freiburg
Hugstetter Strasse 55, D-79106 Freiburg, Germany
T: (+49) 761 270 5471; F: (+49) 761 270 5485
Web-site: www.ukl.uni-freiburg.de/iumwkra
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Prof., MD Henrik C. Schønheyder
Department of Clinical Microbiology
Aalborg Hospital
P.O. Box 365 Hobrovej 18-22, DK-9000
Aalborg, Denmark
T: (+45) 9932 3219; F: (+45) 932 3216
Web-site: www.nja.dk/aas
Prof., Ph.D. Roberto Cauda
Instituto Clinica Malattie Infettive
Università Cattolica del Sacro Cuore
Largo F. Vito n°1, I-00168 Rome, Italy
T: (+39) 06 30154945; F:(+39) 06 30154934
Email:
[email protected]
Web-site:www.rm.unicatt.it
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97
SELF-MEDICATION WITH ANTIBIOTICS AND
RESISTANCE LEVELS IN EUROPE
SUMMARY
Self-medication with antibiotics is an underestimated problem in combating antimicrobial resistance. The survey
undertaken in this study will provide information on the levels of self-medication in different countries of Europe,
explore underlying reasons and possible relationship with penicillin non-susceptibility of invasive isolates of
Streptococcus pneumoniae.
PROBLEM
Actual consumption of antibiotics by people may be the result of self-medication, using antibiotics bought directly over the counter (OTC) without a prescription, ‘leftover’ antibiotics from
earlier prescribed treatment courses, antibiotics ordered via the internet or other sources outside one’s own country. Data on the degree of OTC use in Europe are hardly available, nor of the
use of antibiotics from the home medicine cabinet. The few studies describing the situation in
Spain and in Greece, suggest a considerable OTC use. Campaigns to return unused medicine to
the pharmacy indicate the availability of large quantities of unused antibiotics in the medicine
cabinet at home. Studies in the US suggest that up to 25% of people have ‘leftovers’ of antibiotics at home many of whom intended to use them without consulting a doctor. Moreover, data
in the US suggest that antibiotics are frequently (26%) obtained without a prescription. Selfmedication may lead to inappropriate use, in particular to a more or less frequent use of low
dosages of antibiotics for minor ailments, for which there is no indication for antimicrobial therapy. Low dosage and long duration of therapy with antibiotics have been identified as risk factors for resistance to S. pneumoniae.
AIM
The project aims to
1.Assess the prevalence of self-medication, including both the consumption of OTC antibiotics
and leftovers from prescribed antibiotics
2. Assess beliefs and attitudes towards antibiotic use, as well as care-seeking behaviors and
the appropriateness of use of antibiotics for common diseases
3. Explore the possible relationship between penicillin non-susceptibility of invasive isolates of
Streptococcus pneumoniae and self-medication with antibiotics
EXPECTED RESULTS
• Quantification of levels of self-medication (OTC and use of ‘leftovers’ of antibiotics) in different European countries
• Final report: a final report will be prepared describing in detail the threat of self-medication
of antibiotics in at least 19 participating countries, and with recommendations on possible
control measures, and recommendations on the country-specific factors that have to be taken
into account when designing interventions.
• Feedback by Internet will be given in the form of summary tables. The achievement of these
objectives will provide information for use by national governments and European authorities
in formulating policies on antibiotic use.
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Acronym : SAR (funded by DG SANCO)
Start date : Octber 1st 2002
Keywords : self-medication, antibiotics, prevalence
The project provides essential information for policy decisions, for example on regulatory
measures. The study will be done in close collaboration with other EU projects on the threat
of antimicrobial resistance. The European Antimicrobial Resistance Surveillance System
(EARSS) and European Surveillance of Antimicrobial Consumption (ESAC) projects provide an
ideal platform where inappropriate antibiotic use can be studied in relation to a selection of
resistance and overall antibiotic consumption. Some of the Member States contemplate
broadening the number of medications (including antibiotics) that might change from prescription-only status to OTC status. The results of this study will provide country-specific
information about the risks and possibilities of such liberalization of prescription medication.
Results will be published in peer reviewed professional journals and disseminated through
the EARSS network, the ESAC network and the EURODURG network. They will be sent also to
WHO and EMEA in order to reach out to the international bodies.
http://www.earss.rivm.nl/
Prof. Flora Haaijer-Ruskamp
Clinical Pharmacology
Ant Deusinglaan 1, 9713 AV Groningen,
The Netherlands T: (+31) 50 3636858
PARTNERS:
Prof.dr Mittermayer
Medical Microbiology and Hygiene
Elisabethinen Hospital Linz
Fadingerstrasse 1, 4010 Linz, Austria
Prof. Legakis Microbiology
Medical School University of Athens
M Asias Str. 75, 11527 Athens, Greece
Dr. Codita Institutul Cantacuzino
Splaiul Independentei 103, Bucuresti sector 5,
70100 Bucharest,
Dr. Cornaglia Instituto di Microbiologia
Universita degli Studi di Verona
Strada Le Grazie 8, 37134 Verona, Italy
Dr. Gubina
Institute of Microbiology and Immunology
University of Ljubljana Zaloska 5, 1015 Ljubljana,
Slovenia - Email: [email protected]
Dr. Borg Health Division
St. Luke's Hospital MSD 08 G'Mangia, Malta
Dr. Campos Centro Nacional Microbiologia
Carretera Poluelo, 28220 Majadahonda, Madrid,
Spain Email: [email protected]
Prof.dr Goossens Medical Microbiology
University Hospital UIA
Wilrrijkstraat 10, 2650 Antwerpen, Belgium
Prof. Markova
Microbiology, Laboratory of Clin.
Microbiology Alexander University Hospital
Georgii Sofiiski Street 1, 431 Sofia, Bulgaria
Dr. Monnet
(AMTI) Division of Microbiology
Statens serum Institut
Artillerivei 5, 2300 Copenhagen, Denmark
Prof. Raz HaÉmek Medical Center
18101 Afula, Israel - Email: [email protected]
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Dr. Hemmer
National Service of Infectious Diseases
Centre Hospitalier de Luxembourg
Rue Barble, 1210 Luxembourg, Luxembourg
Dr. Hryniewicz
Sera and Vaccins Central Research Lab
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Dr. Naaber Clinical Microbiology
University of Tartu - Ravila 19, 50411 Tartu, Estonia
Prof. Stratchounski
Clinical Pharmacology and Antimicrobial
Chemotherapy - Smolensk State Medical Institute
PO box 5, Krupskaya 28, 214019 Smolensk, Russia
Dr. Langsadl Clinical Microbiology
National TB & Respiratory Diseases Institute
Krajinska 91-101, 82556Bratislava, Slovak Republic
Dr. Fuzi National Centre for Epidemiology
Budapest, Hungary - Email: [email protected]
Dr. Canica Unidade Resistente Antibiotics
Dr. Ricardo Jorge Instituto Nacional de Saude
Av Pedre Cruz , 1699 Lisboa, Portugal
Larissa Grigoryan Clinical Pharmacology
Ant Deusinglaan 1, 9713 AV Groningen, The
Netherlands T: (+31) 50 3636858
99
TV-FILM: “THE BATTLE AGAINST ANTIBIOTIC
RESISTANT BACTERIA”
SUMMARY
This project will result in the production of a television film on antibiotic resistance. The aim is to raise awareness
of this growing problem in the general public as one measure to reduce the current misuse of antibiotics.
PROBLEM
Antimicrobial resistance has become a threat to human health, causing increasing morbidity
and mortality. One major factor causing this problem is inappropriate usage of antibiotics.
These agents are widely used for respiratory tract infections such as common colds, which are
not caused by bacteria and where they have no effect. This misuse is partly driven by poor
knowledge about the nature of infection diseases and the role of antibiotics in the general population. Once established, antibiotic resistance can be transmitted among individuals and
across countries. The development has rapidly turned into an increasing amount of lethal cases
within Europe and we are now on the threshold to an explosive development of incurables.
Countermeasures need therefore to be internationally co-ordinated.
AIM
The aims of the project is to produce a television program on antibiotic resistance, its causes
and consequences which can be used as a tool to promote appropriate antibiotic usage in every
European country. This film will facilitate the understanding of the European as well as global
perspectives of antimicrobial resistance and increase the awareness of the problem and its consequences in the community. The team works closely together with renowned European scientists from the field.
EXPECTED RESULTS
In each country where the film is shown it shall be followed up local measures:
-TV talk shows with experts in connection with the broadcast of the film Articles in other media
100
Acronym : TV Film (funded by DG SANCO)
Start Date : 1st December 2002
Keywords : TV-film, public perception, antibiotic resistancew
The film will influence a broad audience wherever it is broadcast and they will in their turn
encourage scientists, doctors and pharmacists to influence inappropriate prescriptions of
antibiotics.
Anders Wahlmark
METER Film & Television
Sweden
WITH SCIENTIFIC EXPERT ADVICE FROM:
MD Pentti Huovinen
Antimicrobial Research Laboratory
PO Box 57, Kiinamyllynkatu 13, FIN-20520 Turku,
FINLAND
T: (+358) 2 251 9255; F: (+358) 2 251 9 254
MD JavierGarau
Dept of Medicin
Hospital Mutua de Terrassa
Terrassa, 508221 Barcelona, SPAIN
T: (+34) 93 736 50 50; F: (+34) 93 736 50 37
Helen Giamarellou
4th Department of International Medicine
Sismanoglion Hospital
1 Sismanoglio Str, 151 26 Athens, GREECE
T: (+30) 210 803 3817; F: (+30) 210 803 9543
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Herman Goossens
University Hospital Antwerp
Wibrujkstraat 1, B-2650 Edegem, BELGIUM
T: (+32) 3 821 37 89; F: (+32) 3 825 42 81
Helmut Mittermayer
Elisabethinen Hospital Linz
Fadinger Strasse 1, A-4010 Linz, AUSTRIA
T: (+43) 70 7676 3680; F: (+43) 70 7676 3686
Edmond Smyth
Microbiology Department
Beaumont Hospital
Beaumont, Dublin 9, IRELAND
T: (+353) 01 809 2646; F: (+353) 01 809 2871
Niels Frimodt-Möller
Department of Research and Development
5 Artillerivej, DK-2300 Copenhagen S, DENMARK
T: (+45) 3268 8145; F: (+45) 3268 3887
MD Otto Cars
Control
S-171 82 Solna, SWEDEN
T: (+46) 8 457 24 99; F: (+46) 8 31 36 10
Rober Hemmer
National Service of Infectious Disease
Centre Hospitalier de Luxemburg
L – 1210 LUXEMBURG, LUXEMBURG
T: (+352) 4411 3091; F: (+352) 4412 79
Didier Guillemot
Suite des Agents Antibacterieux
Institut Pasteur
25 – 28 Rue Du DR Roux, 750 15 Paris, FRANCE
T: (+33) 145 68 8174; F: (+33) 145 68 8319
Richard Wise
Dept of Medical Microbiology
City Hospital
Birmingham, ENGLAND
T: (+44) 0121 507 4255; F: (+44) 0121 551 7763
Karl G. Kristinsson
Dept. Of Nicrobiology
Landspital Universitet Hospital
S/ Hringbraut
101 Reykjavik, ICELAND
T: (+354) 560 19 52; F: (+354) 560 19 57
Giuseppe Cornaglia
Dip. Patologia
Universitá di Verona
Sttrada le Grazie 8, 39134 Verona, ITALY
T: (+39) 045 80 27 196; F: (+39) 045 58 46 06
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101
NETWORK FOR AUTOMATED BACTERIAL
STRAIN FINGERPRINTING IN EUROPE
SUMMARY
There is urgent need to monitor and control the alarming spread of antibiotic-resistant and pathogenic bacterial
strains in European member states. Strains need to be characterised by common standards to detect and track
their dissemination. The aim of the project is to create a network including leading European microbiology
laboratories for data exchange on bacterial molecular characterisation ("fingerprinting"), based on the only fully
automated technology, ribotyping. Standard procedures for data exchange and attribution of common codes to
identical bacterial strains from different countries will be defined, and a common database with on-line access
created. Broadened exploitation of this standard fingerprinting system will be ensured by developing
comparable standards for manual ribotyping procedures, and by assessment of data complementarity with other
powerful fingerprinting methods and definition of type correspondence between methods.
PROBLEM
photo credit: Chris Timmers
The emergence of new antimicrobial resistant bacterial strains is a natural process, which limits the use of any newly developed antibiotic. Moderation of antibiotic consumption must be
implemented, but it is unlikely to result, on its own, in a significant decrease in resistance rates,
due to the stability of resistance mechanisms and fitness of resistance strains even in the prolonged absence of antibiotics. Bacterial strains, which have acquired resistance mechanisms,
can indeed survive for long periods of time without selective pressure from antibiotics. These
strains can also become disseminated to new patients, hospitals and communities, often crossing borders to achieve international pandemic status. Therefore, antibiotic resistance will continue to increase if no measures are taken to control the dissemination of these resistant
strains.
To better identify these strains, several molecular methods providing a DNA profile of each bacterial strain have been developed. A number of such "fingerprinting" techniques are currently
used. However, European standardisation of the relevant procedures and data exchange
remains to be achieved.
This lack of co-ordination between clinical microbiology laboratories results in loss of information that is crucial for the rapid identification and control of the dissemination of virulent or
resistant clones, at the local as well as European scales. In addition, this situation limits the scientific knowledge necessary to policy-makers to take more efficient control measures against
infections, and has profound consequences on the risk of infection in European member states
as well as on public health costs.
AIM
The aim of the project is to create a network including leading European microbiology laboratories to exchange data and develop a common language for the characterisation of bacterial
strains, in order to monitor and better control infections caused by pathogenic and resistant
bacteria. The language will be based upon automated ribotyping of bacterial isolates using
RiboPrinter® equipment (Qualicon Europe Inc., Warwick, UK).
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Acronym : Gene
Contract Type : Concerted action
Keywords : Ribotyping, molecular epidemiology, microbial diversity,
identification, characterization, typing, antimicrobial drug
resistance, bacterial virulence, fingerprinting, standardization.
EXPECTED RESULTS
1 Development of data exchange procedures between the RiboPrinter users and construction
of a common library of ribotypes.
2 Standardisation of manual ribotyping to produce data comparable with RiboPrinter standards.
3 Determination of the correspondence between the digital codes for important strains
defined on the basis of ribotype, on the one hand, and other methods, on the other hand,
with the creation of a database containing ribotypes complemented with other molecular
typing data and clinically important characteristics.
It is hoped (and believed!) that the common database of ribotype fingerprints and its crossreferences to other typing techniques and databases will prove to be a useful resource for
epidemiological investigation of the distribution and prevalence of bacterial clones, as well
as for studies of the microbial strain diversity. The results will be available for use to all institutions using the ribotyping technology and other interested parties.
http://www.ewi.med.uu.nl/gene
Dr. Sylvain BRISSE
Uhnité biodiversité des Bactéris Pathogènes
Emergentes
Institut Pasteur
28 rue du Docteur Roux
75015 Paris
T: + 33 (0) 1 40 61 33 57
F: +33 (0) 1 45 68 88 37
Dr. F.J. Allerberger
Institute for Hygiene and Social Medicine
[email protected]
Dr. Martin Altwegg
University of Zürich
[email protected]
Dr. Vivian Fussing
[email protected]
Prof. Patrick A. D. Grimont
Institut Pasteur de Paris
[email protected]
Dr. Marc Lange
Institut Pasteur of Lille
[email protected]
Dr. Edoardo Carretto & Prof. Piero Marone
IRCCS Policlinico ”San Matteo”
[email protected]
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Dr. Panayotis Tassios &
Prof. Nicholas Legakis
University of Athens
[email protected]
Dr. Herminia de Lencastre
Universidade Nova de Lisboa
[email protected]
Dr. Alex Van Belkum
Erasmus University Medical Center
Rotterdam
[email protected]
Dr. Rob Willems & Dr. Herre Heersma
National Institute of Public Health and
Environmental Protection
[email protected]
Prof. Edouard Bingen
Hôpital Robert Debré
[email protected]
Caroline Durbin
Qualicon Europe Ltd.
[email protected]
Prof. Keith Klugman
The South African Institute for Medical
Research
[email protected]
Dr. Daniel Jonas
University Hospital Freiburg
[email protected]
Alexandr Nemec
[email protected]
Dr. Lenie Dijkshoorn
[email protected]
Prof. Marc Struelens
The Université Libre de Bruxelles
[email protected]
103
IMPACT OF MENINGOCOCCAL
EPIDEMIOLOGY AND POPULATION
BIOLOGY ON PUBLIC HEALTH IN EUROPE
SUMMARY
The project targets meningococcal disease, one of the most severe childhood infections in Europe, with incidence
rates of up to 50/100,000 for children aged 0-4 and mortality rates approaching 20%.
A pan-European infrastructure for the investigation and surveillance of meningococcal disease will be built up for
the analysis of the meningococcal population structure and the dynamics of the spread of the causative organism,
Neisseria meningitidis. The insights obtained will improve public health interventions.
PROBLEM
Meningococcal disease remains a major childhood infection in Europe, with an appreciable
number of cases in other age groups, notably young adults. There is currently no comprehensive childhood vaccine against this disease, the severity of which, combined with its rapid progression and non-specific symptoms, results in an unacceptable burden of childhood morbidity and mortality. In addition, the emergence of meningococci with reduced penicillin susceptibility is a major concern. The public health management policies and the development of effective vaccines are confounded by the epidemiology of meningococal disease, which is itself governed by the complex population biology of the causative organism.
AIM
An improved understanding of the spread of hyperinvasive and antibiotic resistant meningococci throughout Europe will be obtained by integrated epidemiological and population genetic studies that employ the latest molecular isolate characterisation techniques (MLST) and
electronic data transfer via the Internet. Strain collections from all parts of Europe, in combination with surveillance data, will be analysed for the rigorous definition of hyperinvasive strains.
This will help to determine the distribution of meningococcal lineages associated with disease.
The hypothesis that the spread of antibiotic resistant strains is linked to antibiotic usage, and
therefore different in different parts of Europe, will be investigated. Collections of isolates with
representative levels of antibiotic resistance throughout Europe will be made and isolates characterised by both MLST and nucleotide sequence analysis of the genes encoding antibiotic
resistance. Elucidation of the underlying resistance mechanisms and the dynamics of distribution of resistance genes among clonal lineages will be investigated with the intention of tracking and ultimately predicting the spread of antibiotic resistance in these organisms.
There is evidence that particular hyperinvasive lineages are also hypervirulent, that is are likely to cause severe meningococcal disease and death. This will be examined by characterising
by MLST the isolates obtained from large numbers of patients with known severity of meningococcal disease as assessed by a variety of clinical scoring paradigms. These data will permit the
establisment of rigorous definitions of hypervirulent meningococci and will also promote the
unification of clinical scores, with the potential to improve clinical management throughout.
EXPECTED RESULTS
An enhanced infrastructure for studies and surveillance of meningococcal disease will be created and molecular methods disseminated throughout the Community. Rigorous definitions of
disease-associated and antibiotic resistant meningococci, and an understanding of meningococcal population biology and the spread of meningococcal disease, will be generated. The
project will provide the infrastructure and the insights essential for harmonised and improved
public health and clinical management.
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Acronym: EU-MENNET
Starting date : September 1st, 2001
Keywords : Neisseria meningitidis – epidemiology –population biology
– multilocus sequence typing – antibiotic resistance
An important and novel element of exploitation and dissemination of the results of this project
will be the creation of websites, which will allow (i.) the real time data deposition into databases; (ii.) the distribution of regular summaries of the epidemiological and strain characterisation data; and (iii.) the dissemination of regular updates of the guidelines for the public
health and clincal management of meningococcal disease. This information will be of great utility to those public health institutions with responsibility for management of cases and outbreaks of meningococcal disease.
Since all of the national meningococcal reference laboratories in Europe are participants in the
consortium information on latest technologies and newest methods available for the detection
and typing of meningococci will automatically be made available Europe-wide. This guarantees
that, within a short period of time, unified methods will be applied in all reference laboratories
of the Community and that expertise and experience will be shared widely.
http://neisseria.org/nm/emgm/eumennet/
Prof. Matthias Frosch, MD
Institute of Hygiene and Microbiology
University of Wuerzburg
Josef-Schneider-Str. 2
DE-97080 Wuerzburg - Germany
T: +49 931 201-5160; F +49 931 201-3445
Web-site: http://www.hygiene.uni-wuerzburg.de/
PARTNERS:
Prof. Dominique A. Caugant
Department of Bacteriology
P.O. Box 4404 Torshov - NO-0403 Oslo - Norway
T: +47 22 04 2311; F +47 22 04 2518
Prof. Andrew Fox
Meningococcal Reference Unit
Manchester Public Health Laboratory
Withington Hospital
UK-Manchester M20 2LR - United Kingdom
T: +44 161 291 4631; F: +44 161 446 2180
Dr. Mary Ramsay
Immunisation Division
PHLS Communicable Disease Surveillance
Centre - Colindale
UK-London MW9 5EQ - United Kingdom
T: +44 208 200 6868; F: +44 208 200 7868
Sigrid Heuberger, PhD
National Reference Centre for Meningococci Bundesstaatliche bakt.- serologische
Untersuchungsanstalt
Beethovenstr. 6 - AT-8010 Graz - Austria
T: +43 316 321643; F: +43 316 388470
Paula Kriz, MD, PhD
NRL for Meningococcal Infections
Srobarova 48 - 100 42 Prague 10 -Czech Republic
T: +420 2 6708 2259; F: +420 2 6731 1454
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Prof. Simon Kroll FRCP
Department of Paediatrics,
Imperial College School of Medicine, St
Mary’s Hospital campu,
Norfolk Place, - UK-London W2 1PG - UK
T: +44 20 7886 6220; F: +44 20 7886 6284
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Dr. Martin Maiden
Wellcome Trust Centre for the Epidemiology
of Infectious Disease,
Department of Zoology, University of Oxford,
South Parks Road - UK-Oxford OX1 3FY - UK
T: +44 1865 271284; F: +44 1865 271284
Muhamed-Kheir Taha, MD, PhD
Unite des Neisseria & Centre National de
Reference des Meningocoque
Institut Pasteur
28 rue du Dr Roux - FR-75724 Paris cedex 15 - FR
Phone: +33 1 45 68 8438; Fax: +33 1 45 68 8338
Dr. Georgina Tzanakaki
National Meningococcal Reference
Laboratory
National School of Public Health
196, Alexandras Avenue - Athens
Greece
T: +30 1 646-5982 ; F: +30 1 643-2258
Julio A. Vázquez, PhD
Reference Laboratory for Meningococci
National Center for Microbiology
Ctra Majadahonda-Pozuelo, Km2
ES-28220 Majadahonda
Spain
T: +34 91 5097901; F: +34 91 5097966
Dr. Ulrich Vogel, MD
Institut fuer Hygiene und Mikrobiologie
Universitaet Wuerzburg
Josef-Schneider-Str. 2 - DE-97080 Wuerzburg
Germany
T: +49 931 201 3802; F: +49 931 201 3445
105
STRENGTHENING INTERNATIONAL
SALMONELLA SURVEILLANCE THROUGH
STRAIN TYPING AND DIFFERENTIATION
SUMMARY
The added value to surveillance of using molecular methods to genotypically subtype selected salmonellas,
including organisms characterised by the possession of multiple drug resistance, and the feasibility of capturing
molecular information on such organisms in real-time throughout the EU surveillance network will be studied.
PROBLEM
In recent years, clones of salmonella organisms with multiple drug resistance have caused
numerous outbreaks of infection in many countries throughout the EU. It is hoped that by genotypically characterising these strains in real-time by a variety of molecular methods and by
transferring such information electronically that outbreaks can be rapidly recognised and
appropriate intervention methods introduced and implemented.
AIM
There are five objectives: 1) To develop standard laboratory protocols for the genotyping of subtypes of the major salmonella serotypes and phage types. This will include clonal lines characterised by the possession of multiple antimicrobial drug resistance. 2) To create an on-line,
searchable database of genotypic information on these strains, 3) To establish an external quality assurance scheme for the laboratory methods used, 4) to genotype in real-time, a large sample of salmonella strains in several countries using selection criteria that maximise outbreak
recognition power, 5) To develop recommendations for incorporating genotyping into national
and international surveillance of salmonella strains.
EXPECTED RESULTS
Development and implementation of an international database for salmonellas, including
organisms with multiple drug resistance, based on their genotypic characteristics.
106
Acronym : SALM-GENE
Keywords : Salmonellas; genotyping; international databases;
multiple drug resistance
Outbreak control throughout the EU; rapid exchange of relevant genotypic data; the current
necessity of the exchange of strains to be significantly reduced.
Dr E J Threlfall
Public health Laboratory Service
Laboratory of Enteric Pathogens
61 Colindale Avenue - UK-London NW9 5HT
United Kingdom
T: +44 0 20 8200 4400 x 3144;
F: +44 0 20 905 9929
PARTNERS:
Noël Gill
Centre
61 Colindale Avenue - UK-London NW9 5EQ
United Kingdom
T: + 44 20 8200 6868 x 4462;
F: + 44 20 8200 7868
Ian Fisher
Centre
61 Colindale Avenue - UK-London NW9 5EQ - UK
T: + 44 20 8200 6868 x 4543;
F: + 44 20 8200 7868
Christian Berghold
Salmonella Zentrale
Bundesstaatliche - Bakteriologisch-serologische - Untersuchungsanstalt
Beethovenstraße 6 - AT-8010 Graz - Austria
T: + 43 316 321643; T: + 43 316 388470
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Peter Gerner-Smidt
Dept of Gastrointestinal Infections
Artillerivej 5 - DK-2300 Copenhagen - Denmark
T: + 45 3268 3798; F: +45 3268 8238
Anja Siitonen
Laboratory of Enteric Pathogens
Mannerheimintie 166 - FI-00300, Helsinki -Finland
T: +358 9 474 48245; F: +358 9 474 48238
Helmut Tschäpe
Robert Koch-Institut - Bereich Wernigerode
Burgstrasse 37 - Germany
DE-38843 Wernigerode/Harz
T: +49 3943 679 237; F: +49 3943 679 207
Ida Luzzi
Laboratory of Medical Bacteriology &
Mycology
Viale Regina Elena 299 - IT-00161 Rome - Italy
T: +3906 4990 2171; F: +3906 4938 7112
Wim Wannet
National Institute of Public Health and the
Environment
Diagn. Lab. For Infectious Diseases and
Perinatal screening
PO Box 1 NL-3720 BA, Bilthoven -The Netherlands
T: +31 30 274 2105; F: +31 30 274 4418
Giles Edwards
Scottish Salmonella Reference Laboratory,
Dept of Bacteriology - Stobhill Hospital
UK-Glasgow G21 3UW - United Kingdom
T: +44 141 201 3015; F: +44 141 558 5508
Miquel Usera
Laboratorio de Enterobacterias
Centro Nacional de Microbiologia
ES-28220 Majadahonda, Madrid - Spain
T: +34 91 509 7901; F: +34 91 509 7966
Patrick Grimont
Unite des Enterobacteries
Institut Pasteur
28 Rue du Docteur Roux
T: +331 456 88340; F: +331 456 88837
107
HUMAN ENTERIC PATHOGEN
SURVEILLANCE NETWORK
SUMMARY
Enter-net is an established and thriving EU-wide network for the laboratory-based surveillance of human
Salmonella and Verocytotoxin-producing Escherichia coli (VTEC) infections. By involving the microbiologist in
charge of the national reference laboratory and the epidemiologist responsible for national surveillance of these
organisms, the key professionals in every European Union (EU) country are participating. Data from all
20 countries are being collated every month to create international salmonella and VTEC databases. Outbreak
recognition and the efficiency of investigations in the EU has improved, and national surveillance has been
strengthened. Enter-net is a continuation of the Salm-Net surveillance network (1994-1997) which was also
funded by the European Commission and concentrated upon harmonisation of slamonella phage-typing and the
establishment of a timely international salmonella database. Enter-net is continuing to extend the efforts to
prevention of E.coli VTEC infections.
The hub also acts as the distribution point for all urgent enquiries on incidents and outbreaks of enteric
pathogens. Often these only affect individual countries, but international outbreaks have been identified by
conveying information on outbreaks between members of the network. When international outbreaks are
recognised their coordination is managed by the hub.
PROBLEM
The problem of widespread and increasing antimicrobial resistance has been highlighted by
many fora, including the European Commission. Strengthening surveillance is vital for monitoring the trends in resistance and for identifying the emergence of more resistant strains. To
achieve this, however, there has to be harmonisation of the results of antimicrobial tests.
Previously, it was thought that this would require all laboratories to use common standards and
methods, but an international study by the Enter-net laboratories demonstrated that this is not
the case, that the qualitative results from the reference laboratories are comparable, and this
will allow for the international surveillance of antimicrobial resistance. All of these harmonised
typing methods are underpinned by international quality assurance schemes.
AIM
Enter-net, like the majority of surveillance networks, relies on microbiological confirmation of
cases of salmonella and VTEC infections. The key aspect to this is ensuring that the microbiological methods are harmonised between the reference laboratories in Europe. Enter-net has
succeeded in this aim in that single phage-typing schemes for sub-typing of the two main
serovars causing human infections in Europe – S. enteritidis and S. typhimurium – are now
available for the European laboratories participating in the network.
ACHIEVED RESULTS
A significant public health benefit of Enter-net has been the recognition of international outbreaks of infection. This has allowed for the implementation of public health interventions to
prevent further cases occurring. There have been many international outbreaks recognised by
Enter-net, and the most recent example demonstrates that the dissemination of information on
unusual events can lead to timely interventions. Dissemination of information via Enter-net on
an outbreak of Salmonella Oranienburg originating in Germany led to associated cases being
identified in seven EU countries. Product withdrawals were implemented in two countries, and
in three others contaminated chocolate was impounded prior to being released to consumers.
108
Acronym :ENTER-NET
(funded by DG SANCO)
Keywords : Surveillance, Salmonella, VTEC, epidemiology, enteric infection
EXPECTED RESULTS
The network will continue to provide and improve recognition of outbreaks of Salmonella and
E. coli O157 involving more than one country and to facilitate more efficient and effective outbreak investigation. The quality of reference microbiological services in certain countries will
be improved through the distribution of materials and by participation in external quality
assurance. Using the network to begin international surveillance of campylobacter will be an
efficient use of resources and will provide the Commission with information which is essential
to address the issues raised by the very high incidence of this infection in many EU countries.
An important series of research projects is either already underway, or envisaged for the near
future, which, if successful, will eventually increase the surveillance power of the network to
detect outbreaks even earlier.
Quarterly summary epidemiological reports will be posted on the web site for general access.
Laboratory quality assurance reports will be distributed to each participant. Reports on outbreak investigations, significant trends, and research results, will be prepared for publication
in peer-reviewed scientific journals including Eurosurveillance. The Workshop report and an
Annual Report on the epidemiology of the major human enteric bacterial pathogens in the EU
will be circulated.
Authoritative and standardised information on trends in antibiotic resistance will be provided
to inform the need for further applications by the Commission of the precautionary principle
in relation to antibiotic usage and to evaluate actions already undertaken.
http://www.Enter-net.org.uk
http://www.phls.co.uk/inter/enter-net/menu.htm
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Dr. Noël Gill
Prof. Bill Reilly
Dr. Henry Smith
PHLS Communicable Disease
Surveillance Centre
61 Colindale Ave, London NW9 5EQ, UK
T: +44-20-8200-6868
MOB: +44-773-636-2507
F: +44-20-8200-7868
with the participation of 20 European countries
109
EUROPEAN ANTIMICROBIAL RESISTANCE
SURVEILLANCE SYSTEM
SUMMARY
The European Antimicrobial Resistance Surveillance System (EARSS), funded by DG SANCO of the European
Commission and coordinated by the Dutch National Institute for Public Health and the Environment (RIVM), is an
international network of national surveillance systems, which collects comparable and validated antimicrobial
susceptibility data for public health purposes. EARSS performs ongoing surveillance of antimicrobial susceptibility in Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis/faecium
causing invasive infections, and monitors variations of antimicrobial resistance in time and from place to place.
In December 2001, around 600 microbiological laboratories serving some 970 hospitals in 27 countries provided
susceptibility data on around 65 000 invasive isolates.
PROBLEM
Mean proportion (1999-2001) of Streptococcus
pneumoniae penicillin non-susceptibility (PNSP)
in invasive isolates reported per country. France
delivered aggregated data for the first two
quarters of 2001.
Antimicrobial resistance (AMR) is an emerging public health problem with local, national, and
international dimensions as described in ‘the Copenhagen Recommendations’. Antimicrobial
resistance is clearly an emerging problem. However, the precise impact of this problem is less
clear to the European and scientific community. Before being able to quantify the impact on
public health it is necessary to have more comparable surveillance data available. One of the
recommendations made at the EU Conference 'The Microbial Threat' in 1998 was that a
European surveillance system of antimicrobial resistance should be set up, therefore EARSS
has been established.
AIM
EARSS aims to obtain comparable and reliable antimicrobial resistance data of
main indicator pathogens in Europe to monitor antimicrobial resistance in time and
from place to place. EARSS also aims to assess risk factors for antimicrobial resistance and to enable policy makers and health care workers to monitor the impact
of their interventions.
EXPECTED RESULTS
For pathogens (Streptococcus pneumoniae, Staphylococcus aureus, Escherichia
coli, and Enterococcus faecalis/faecium) causing invasive infections, resistance
levels are available for important groups of antimicrobials from 27 European countries. In the EARSS annual report 2001, results are described in detail for all four
pathogens collected in 2001, as well as trends in penicillin non-susceptible S.
pneumoniae (PNSP) and methicillin resistant S. aureus (MRSA) during the period
1999-2001. Aggregated information is directly available to health care workers,
policy makers, and a wider public, at an interactive website
(http://www.earss.rivm.nl). From EARSS data, it can be concluded that proportions of antimicrobial resistance vary markedly between European countries, most
likely as a result of differences in hospital infection control and the consumption of
antibiotics
Mean proportion of Staphylococcus aureus
methicillin resistance (MRSA) in blood
isolates from the period 1999-2001.
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Acronym : EARSS
(funded by DG SANCO)
Start date : january 1st 2001
Keywords : surveillance, antimicrobial resistance, monitor trends,
invasive pathogens, public healh action
Policies to combat resistance should be tailored specific to country and hospital level. The
results as presented in the EARSS annual report 2001 emphasise the need to implement the
Council Recommendations on the Prudent Use of Antibiotics in Human Medicine. As laid down
in the Council Recommendations, it has recently been decided that multi-disciplinary organisations, called Intersectorial Coordinating Mechanisms (ICMs), will be established at the
national level. The ICMs will be responsible for information exchange and co-operation
between the parties involved at the national level. The ICMs are responsible for implementing
the Council’s recommendations and should consider the recommendations as formulated in
the EARSS Annual Report 2001. This applies particularly to countries with high proportions of
resistance for all bacterial species.
In order to get more insight into the causes and mechanisms of these striking differences, in
the near future several research initiatives will be set up in close cooperation with the EARSS
network. EARSS already cooperates closely with the project ‘European Surveillance on
Antimicrobial Consumption’(ESAC). EARSS and ESAC have recently started to collaborate with
the project ‘Self-medication of Antimicrobials and Resistance Levels in Europe’ (SAR), and
future cooperation is planned with the project ‘Antibiotic Resistance in Mediterranean countries’ (ARMed).
http://www.earss.rivm.nl
Paul Schrijnemakers
Institute for Public Health and the
Environment (RIVM)
Dept. of Infectious Disease Epidemiology
(CIE-pb 75) PO Box 1
3720 BA Bilthoven
the Netherlands
T: + 31 (0) 30 274 3486
F: + 31 (0) 30 274 4409
url: http://www.earss.rivm.nl
PARTNERS:
Austria (AT)
H. Mittermayer, W. Koller
Belgium (BE)
H. Goossens, E. Hendricxk
Bulgaria (BG)
B. Markova
Croatia (HR) S. Kalenic
A.Tambic Andrasevic
Czech Rep. (CZ)
P. Urbaskova
Denmark (DK)
D. Monnet
Estonia (EE)
P. Naaber
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Finland (FI)
P. Huovinen, O. Lyytikäinen
France (FR)
P. Courvalin, H. Aubry-Damon
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Germany (DE)
W. Witte, U. Buchholz
Greece (GR)
N. Legakis, G. Vatopoulos
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Hungary (HU)
M. Füzi
Ireland (IE)
O. Murphy, D. O' Flanagan
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Iceland (IS)
K. Kristinsson, H. Briem
Israel (IL)
R. Raz
Italy (IT)
G. Cornaglia, P. D'Ancona
Luxembourg (LU)
R. Hemmer
Malta (MT)
M. Borg
Netherlands (NL)
A. de Neeling, E. Tiemersma
Norway (NO)
A. Hoiby, E. Bjørløw
Poland (PL)
W. Hryniewicz
Portugal (PT)
M. Caniça, M. Paixao
Romania (RO)
I. Codita
Russia (RU)
L. Stratchounski
Slovenia (SI)
M. Gubina
Slovakia (SK)
L. Langsadl
Spain (ES)
F. Baquero, J. Campos
Sweden (SE)
B. Liljequist, O. Cars
United Kingdom (UK)
A. Johnson, M. Wale
111
EUROPEAN NETWORK OF NOSOCOMIAL
INFECTIONS (IMPLEMENTATION PHASE II)
SUMMARY
The general aim of the next phase of the HELICS collaboration is the creation of a robust and validated
surveillance system and establishment of a European Database on Nosocomial Infections to explore areas where
improvement in healthcare delivery are possible.
PROBLEM
Since the first Council of Europe recommendations in 1974, a series of initiatives (e.g. DANOP,
WHO.CARE, ESICM surveys, EURO.NIS and EPIC projects) highlighted the need of harmonisation of NI control policies in Europe. They led to the first HELICS project (HELICS I), which created the scientific conditions for a harmonised approach of surveillance in surgery and ICU.
Following this first consensus phase, pilot European comparative evaluations have been possible between surveillance networks in several countries. Prepared in the perspective of the decision 2119/98/EC, a report presented in 1999, to the DG SANCO (HELICS II), by the HELICS group
proposed a Global Strategy for the Implementation of a Network on Nosocomial Infections. This
approach associates surveillance, control, training and research, and can be done in a bottomup and consensual manner, in co-operation with the professional and scientific bodies, with an
organisation of ‘network of existing networks’ around meaningful targeted sub-projects. Such
a step-by-step development of the network permits a progressive involvement of this complex
field within a European perspective. The current HELICS activity comprises recommendations
on surveillance, control and training and is a continuation of this step-by-step approach.
AIM
The three main objectives of the current HELICS activities are:
• A descriptive objective: to establish large reference data sets to mirror national data in order
to guide preventive activities and stimulate complementary or intervention studies
• A methodological objective: to improve the quality and comparability of data and the capacity of data exchange between the existing European national or regional networks
• A research and evaluation objective: which would take advantage of these large European
database(s) to guide preventive activities and stimulate comparative analysis and applied
research
Two additional facilitating objectives will contribute to the success of the surveillance activities.
They concern the use of data for infection control and training of professionals:
• Use of data: to disseminate the European data, analyses, results, comments and technical
advice as well as recommendation for the control of specific risks which will come to light
through the European database.
• Training: to create a network of trained professionals sharing concepts related to surveillance
such as prioritisation, methods of collection, checking and validation, use of data and methods for analysis of European data.
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Acronym : HELICS
(funded by SANCO)
EXPECTED RESULTS
A further step in ‘harmonisation’ of the European surveillance policies and a new initiative to
reduce the occurrence of hospital infection and to improve the quality of patient care through
exchanges of experience between the national/regional surveillance centres. The European
Database will help mirroring national data in order to guide preventive activities and stimulate complementary studies and data exchange between existing European Networks.
Aggregated results will be disseminated to the scientific community and the general public by
the HELICS Internet page and the annual published report showing agreed comparative
analysis by countries, type of hospital/unit, type of patients or surgical interventions, level of
risk factors, etc. The results of patient-based analysis will be accessible in the same way and
participating members can make requests for complementary analyses.
http://helics.univ-lyon1.fr/
Prof. Jacques Fabry
Laboratoire d'Epidemiologie et de Santé
Publique
University Claude Bernard Lyon 1
8, Avenue Rockefeller, F-69373
Lyon Cedex 8, France
T: (+33) 4 78 77 70 31; F: (+33) 4 78 00 93 86
PARTNERS:
Dr Carl Suetens
ISP Louis Pasteur
Rue J. Wytsman 14, B-1050
Brussels, Belgium
T: (+32) 2 642 57 22; F: (+32) 2 642 54 10
Dr Petra Gastmeier
Freie Universität Berlin
Institüt für Hygiene
Campus Virchow Klinikum, D-13344 Berlin,
Germany
T: (+49) 30 450 61002; F: (+49) 30 450 61900
Dr Annette de Boer
Epidemiology RIVM
P.O. Box 1, NL-3720 Bilthoven, The Netherlands
T: (+31) 30 27 436 79/91; F: (+31) 30 274 44 09
Dr Barry Cookson
Lab. of Hospital Infection
Public Health Lab. Service
61, Colindale Avenue, NW9 5HT London, UK
T: (+44) 181 200 4400; F: (+44) 181 200 7449
Dr José Rossello Urgell
Sce Médecine Préventive
Hôpital Vall d'Hebron, 119-129 DRO 35, E-08035
Barcelona, Spain
T: (+34) 93 48 942 14/19; F: (+34) 93 489 41 11
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NOVEL APPROACHES
FOR THE CONTROL OF FUNGAL DISEASE
SUMMARY
This Consortium is studying Candida albicans, the major fungal pathogen in humans. We are exploiting the latest
molecular and genomic technologies to dissect host-fungus interactions and the key fungal virulence factors:
yeast-hypha morphogenesis, cell wall, phenotypic switching and secreted hydrolases. Our findings will be
relevant to the improvement of current antifungal therapies.
PROBLEM
Current antifungal therapies are less effective than antibacterial drugs, and the incidence of
drug-resistant Candida strains is increasing rapidly due to the common prophylactic use of antifungal drugs. Clearly new more effective antifungal therapies need to be developed. This
process will be enhanced significantly by an increased understanding of how the fungus thrives
in its human host and combats host defences.
AIM
1. To establish state-of-the-art technologies for the analysis of fungal virulence factors and
host-fungus interactions.
2. To create an integrated picture of the regulation of virulence factors during Candida
infections.
3. To provide advanced models that describe fungus-host interactions.
4. To exploit these findings for the development of novel antifungal therapies.
EXPECTED RESULTS
Tongue
Candida albicans penetrating the tougue
during an oral infection
Hypha
Invasive hypha of Candida albicans
114
1. Arrays for Candida albicans transcript profiling.
2. A publicly available relational database for C. albicans genomics.
[http://genolist.pasteur.fr/CandidaDB/]
3. A Consortium Internet Site for internal communication and external dissemination.
4. A novel high-throughput “interaction screen” for the identification of human and C.
albicans proteins involved in host-fungus interactions.
5. A set of well-defined C. albicans regulatory mutants for the analysis of virulence factors
and host fungal interactions.
6. A list of C. albicans genes that respond to specific regulatory pathways.
7. A list of C. albicans genes that respond to environmental changes relevant to fungal disease progression.
8. A coherent picture describing the expression of specific virulence factors during C. albicans
infections.
9. Well-defined molecular models describing C. albicans responses to host factors.
10. A set of molecular responses in human cells to C. albicans infections.
11. A list of novel potential antifungal targets identified during this project.
Acronym: Galar Fungail
Keywords : Fungal infections; Candida; virulence factors; morphogenesis;
cell wall; phenotypic switching; secreted aspartyl proteinases; transcript
profiling.
Galar Fungail data will provide a strong platform for the development of new antifungal therapies by pharmaceutical companies and for academic studies of C. albicans pathogenicity.
The data will be made publicly accessible on the Consortium Internet Site and published in
scientific journals.
Project web-site: http://www.pasteur.fr/recherche/unites/Galar_Fungail/
Prof. Alistair Brown
Molecular and Cell Biology
Institute of Medical Sciences
University of Aberdeen
Foresterhill, UK-Aberdeen AB25 2ZD, UK
T: (+44) 1224 273183; F: (+44) 1224 273144
Web-site: http://www.abdn.ac.uk/
• •
•
•
•
PARTNERS:
Prof. Claude Gaillardin
Laboratoire de Génetique Moléculaire et
Cellulaire, INRA
Rue de l’Université, 147, FR-75338 Paris Cedex 07
T: (+33) 1 30 81 54 52; F: (+33) 1 30 81 54 57
Prof. Joachim Ernst
Institut fuer Mikrobiologie
Universitaet Duesseldorf
Universitaetsstr. 1, Geg 26.12
DE-40225 Düsseldorf
Germany
T: (+49) 211 811 5176; F: (+49) 211 811 5176
Prof. Angel Dominguez
University of Salamanca
Dept. de Microbiologia y Genetica
Avda. campo Charro s/n, ES-37071 Salamanca
T: (+34) 923 294 677; F: (+34) 923 224 876
Dr. Jose Perez Martin
CSIC, Dept of Microbial Biotechnology
Centro Nacional de Biotecnologia
Campus de Cantoblanco-UAM, ES-28049 Madrid
T: (+34) 91 585 4704; F: (+34) 91 585 4506
Dr Frans Klis
Laboratory for Microbiology
Swammerdam Institute for Life Sciences
University of Amsterdam
Nieuwe Achtergracht 166
NL-1018 WV Amsterdam
T: (+31) 20 525 7834; F: (+31) 20 525 7056
Prof. Rafael Sentandreu
Universitat de València
Secció Departamental de Microbiología
Facultat de Farmacia
Avgda, Vicént Andrés Estellés s/n
ES-46100 Burjassot, València
T: (+34) 96 386 46 83; F: (+34) 96 386 46 83
Dr. Bernhard Hube,
Nachwuchsgruppe Pathogenitatsfaktoren bei
Pilzinfektionen,
Robert Koch-Institut, NG4, Nordufer 20,DE-13353
Berlin
T: (+49) 1888 754 2116; F: (+49) 30 4547 2328
Drs. Cornelia Kurischko & Frank Haenel
Hans-Knoell-Institut fuer NaturstoffForschung e.V.
Beutenbergstrasse 11, DE-07745 Jena
T: (+49) 3641 656683; F: (+49) 3641 656694
Dr. Christophe d'Enfert
Unité de Physiologie Cellulaire
Institut Pasteur, FR-75724 Paris Cedex 15,
T: (+33) 1 40 61 32 57; F: (+33) 1 45 68 87 90
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NOVEL APPROACHES TO COMBAT
MULTIDRUG RESISTANCE (MDR)
IN PATHOGENIC YEAST
SUMMARY
In view of the increasing threat of Candida infections, particularly to immuno-compromised patients (immunosuppressive therapy, AIDS), it is imperative to look for novel drugs with new targets. The experimental rationale is
to develop a screening system on a suitable yeast background for effectors which block an early regulatory step
in the acquired mechanism of the pathogen’s drug defence machinery. The novel and innovative aspect of this
approach is searching for inhibitors of the synthesis of regulators and of their newly identified target MDR genes
in favour of regaining the efficiency of already existing antifungals.
PROBLEM
Candida albicans accounts for more than 50% of fungal infections in immuno-compromised
individuals and thus, becomes a severe concern to clinicians. The search for novel drugs has
become urgent as the common Candida strains acquire resistance to antifungals (multidrug
resistance, MDR) and thus, severely hamper successful therapy. In this context leading experts
in the field have joined their efforts to combat MDR of C. albicans on the following complementary areas: 1) systematic analysis of the functional domains of the transcription regulators
and cell surface glycoproteins involved in MDR. 2) Search for new targets of MDR transcription
regulators by using micro-array DNA chip technology. 3) Development of a screening system, in
a suitable yeast background, for a block in an early regulatory step (including entry of the drug)
in the acquired mechanism of MDR.
AIM
The main objectives of the project are to understand the mechanisms underlying MDR in order
to define new antifungal targets and to develop a yeast based test system suitable for screen
of negative effectors of the regulation of MDR components. The basic idea of this approach is
to block an early regulatory step in the acquired mechanism of MDR of a pathogenic yeast. This
will be monitored by testing the expression of a gene, able to inhibit growth, set under control
of the promoter of a specific drug exporter, e.g. Pdr5p, Cdr1p, or of an enzyme related to protein glycosylation such as Pmt1p. This strain will only grow under conditions when the regulatory step is inhibited. The novel and innovative aspect of this approach in combating MDR of
human pathogens is to block the synthesis of regulators of MDR genes in favour of regaining
the efficiency of already existing antifungals. This approach emphasises the inhibition of regulatory components of the MDR defence machinery rather than looking for new antifungals, to
which organism will develop resistance in due course of time.
Potential effectors (regulatory inhibitors) will allow the growth of constructed yeast strains in
selective media. This positive growth selection marker is of significant advantage since it
excludes false positive signals by general metabolic inhibitors.
EXPECTED RESULTS
• Establishment of functional domains of MDR gene products (transcriptional regulators, drug
transporters)
• Revealing of the subset of genes up-regulated by drug treatment
• Identification of targets of MDR regulatory genes by DNA array analysis
• Establishment of the role of cell surface glycosylation in MDR
• Identification of glycosylation target genes of C. albicans
• Construction of C. albicans mutant strains lacking or over-expressing pmt genes
• Establishment of a yeast based screening system for inhibitors and novel components of the
MDR regulatory network in C. albicans
• The screening system will also have potential to reveal so far unknown cofactors of MDR transcription regulators of drug entry/export machinery, including cell surface glycosylation.
116
Acronym : Combating MDR in pathogens
Keywords : Multidrug resistance (MDR), antifungals, Candida albicans,
drug transport-ers, cell surface glycosylation, transcription factors,
DNA-array technology, MDR regulatory network, Saccharomyces
cerevisiae, screening system
Elaboration of novel strategies to combat multidrug resistance of animal and human
pathogens has a strong relevance to the quality of life and health aspects of the European
population. Development of novel approaches to combat these infections is, therefore, of fundamental interest for the population in Europe and elsewhere. The participating industrial
partner in collaboration with participating clinical partners will use the obtained results for
further clinical testing of Candida infection treatment.
The yeast based screening system has the potential to reveal new components of the MDR
regulatory cascade. Eventually, it may be exploited to search for pharmaceuticals related to
other diseases (tumour chemotherapy), thus offering a promising alternative to animal tests.
http://www.multi-drug-resistance.org
http://www.multi-drug-resistenz.de
http://www.botanik.uni-bonn.de/hoefer/index.html
CO-ORDINATOR:
Prof. Dr. Milan Höfer
Botanisches Institut der Rhein. Friedr.-WilhUniversität Bonn,
Kirschallee 1, 53115 Bonn, Germany
Tel.: +0049-228-735545; Fax: -735504
[email protected]
http://www.uni-bonn.de
PARTNERS:
Prof. Dr. Julius Subik
Comenius University
Dept. of Microbiology and Virology
Mlynska dolina B-2 - 84215 Bratislava - Slovakia
T: +421 2 60296631; F: +421 2 602064
Dr. Dominique Sanglard
Centre Unuversitaire Hospitalier Vaudois
Institute of Microbiology
Rue de Bugnon 44 - CH-10011 Lausanne
Switzerland
T: +41 21 3144083; F: +41 21 3144060
Prof. Dr. Rajendra Prasad
Jawaharlal Nehru University
School of Life Sciences
New Mehrauli Road - New Delhi 110067 - India
F: +91 11 6165886 or 91 11 6187338
or +91 11 6198234
• •
•
Prof. Dr. Claude Jacq
Ecole Normale Superiere
Laboratoire de Génétique Moléculaire
CNRS UMR 8541 - 46, rue d'Ulm
FR-75231 Paris Cedex 05
France
T: +33 1 44323570; F: +33 1 44323941
Prof. Dr. Joachim Ernst
Institut für Mikrobiologie der Heinrich-HeineUniversität
Universitätsstr. 1 - Gebäude 26.12.01
DE-40225 Düsseldorf - Germany
T/F: +49 211 8115176
Priv.Doz. Dr. Joachim Morschhäuser
Institut für Molekulare Infektionsbiologie der
Bayer
Jul.-Maxim.-Univ. Würzburg
Röntgenring 11 - DE-97070 Würzburg - Germany
T: +49 0931 312152; F: +49 0931 312578
•
117
EXPLOITING YEAST CELL WALL FOR
HIGH THROUGHPUT SCREENING OF
ANTIMICROBIAL AGENTS
SUMMARY
The main objective of this project is to exploit the molecular knowledge of yeast cell wall for High Throughput
Screening (HTS)of antimicrobial agents. To this end, the activity of a consortium of 10 laboratories (one company,
one SME and 7 public research institutes) will be to convert the molecular data on essential gene targets involved
in cell wall cross-linking and remodelling pathways into HTS and to use the powerful technologies in genomics
and proteomics and bio-informatics, to characterize the cell wall compensatory mechanism that is induced when
cell wall is weakened in order to identify new targets. This challenging research requires complementary
approaches and techniques in genomic, molecular biology, cell biology, proteomic, enzymology, underpinned by
the complete engagement of industrial companies for further technology transfer. The deliverables will be the
production of a number of assays for HTS of antimicrobial agents, and the expected goal is to find novel antifungal molecules acting specifically on the designed targets.
PROBLEM
Human Pathogenic candida albicans
forming germings tubes for
invagination in the body.
(Courtesy of Dr Neil Gow and
G. Gooday, Aberdeen University)
Plant pathogenic Magnaporthe grisea :
infecting rice leaves (the rice disease named
piriculariosis )
(Courtesy of Dr MH Lebrun and JL Zundel from
Aventis Crop Science, Lyon)
Fungi represent a serious medical problem, since the frequency of fungal diseases has
seriously increased during the last decade. They are also causative agents of food spoilage
with important economic losses. The pharmaceutical and agro-industry have been quite slow
in developing novel antifungals for at least two reasons: (i) an incomplete knowledge of the
mechanisms of pathogenicity and (ii) lack of well-characterised targets from which an assay
can be used for HTS of antimicrobial agents. Furthermore, the slow development is accentuated by the fact that a drug-discovery program (from the identification of the target to the production of the antifungal molecule) is labour intensive, very expensive and time consuming.
The extracellular matrix, also called the cell wall, is the ideal 'target' of anti-microbial molecules
since it is not present in mammalian cells and weakening this barrier causes immediate cell
lysis accompanied by cell death. The fungal cell wall is composed of three macromolecules: glucans, mannoproteins and chitin which are intertwined with each other in a modular structure.
Since the basic architecture of the wall is largely conserved among pathogenic and non-pathogenic fungi, it is the purpose of this project to use at first the yeast Saccharomyces cerevisiae
whose the genome sequence is known and publicly available as the model system in order to
speed up the progress of the work.
A second and strong argument to use S. cerevisiae as the model system comes from the recent
finding that most treatments which tend to weaken the cell wall activate a 'salvage pathway'
which leads to compensatory changes in the cell wall composition. The molecular and biochemical determinants of this so-called 'compensatory mechanism' which also occur in pathogenic fungi are still totally unknown. Therefore, the disposal of the whole S. cerevisiae genome
on filters (DNA arrays) combined with powerful proteomic and bioinformatic technologies will
provide a remarkable frame for the study of this mechanism. The characterisation of the compensatory mechanism represents the second major objective of this project. It is foreseen that
this study will provide novel potential drug-targets.
AIM
Overview of a high throughput
screening platform
(Courtesy of Dr L. Carrano from Biosearch Sa)
118
The scientific objectives of this proposal are (1) to characterise cross-linking and remodelling
pathways which are the basis for the modular structure of the cell wall in pathogen and nonpathogen fungi, using S. cerevisiae as the model system., and (2) to identify by an integrated
approach involving genomics, proteomics and bio-informatics the determinants of the cell wall
compensatory mechanism which is induced when the cell wall is weakened by various treatments (drugs, stress, etc).
The technological objective is to provide assays from selected cell wall targets for high through-
Acronym : EUROCELLWALL
Keywords : yeast, fungi, cell wall, targets, screening, genomic,
proteomic.
put screening. The socio-economic impact of the project is to speed up the search of novel
effective antifungal drugs. These objectives will be achieved by a strong commitment between
molecular and cellular identification of cell wall targets and their biochemical characterisation
to design assays allowing development of drug-discovery program.
EXPECTED RESULTS
1 Provide assays of different cell wall enzymes for high throughtput screening tests
2 Evaluate enzymes of the cross-linking and remodeling pathways as novel potential cell wall
targets
3 Identify new targets from genomic and proteomic analysis
4 Identify antifungal activities
It is hoped and believed that at least a couple of enzymes assays will go up to the complete
HTS development by our Industrial partners, and that genomic and proteomic approaches will
provide us many new hints to develop strategies to circumvent the very critical ‘compensatory
mechanism’
http://www.dkfz-heidelberg.de/funct_genome/index.html
(support for data on microarray)
http://www.insa-tlse.fr/gba/eurocellwall.htm (under construction)
Prof. Jean Marie Francois
Centre de Bioingenierie Gilbert Durand
Département de Génie Biochimique et
Alimentaire
Institut National Des Sciences Appliquées
FR-31077 Toulouse cedex 04 - France
T: + 33 5 6155 9492; F: +33 5 6155 9400
Web-site: http://www.insa-tlse.fr/gba/
Web-site: http://site.voila.fr/labojmf/index.html
PARTNERS:
Dr. Laura Popolo
Università degli Studi di Milano
Dipt Fisiologia e Biochemica generali
Via Celoria 26 - IT- 20133 Milan - Italy
Prof. Cesar Roncero
Departamento de Microbiologia
Edificio Departamental.H-219.
Avda Campo Charro s/n - ES- 37007 Salamanca
Spain
Dr J. Arroyo
Departamento de Microbiologia II
Facultad de Farmacia
Universidad Complutense
Dr L. Carrano
Microbial Technologies
BIOSEARCH Italia Spa
IT-21040 Gerenzano - Italy
•
•
•
Prof Graham GOODAY
Department of Molecular & Cell Biology
•
•
Prof. Neil Gow
Department of Molecular & Cell Biology
Prof Widmar Tanner &
Dr Sabine Strahl-Bolsinger
Lehrstuhl für Zell biologie und
Planzenphysiologie
Universität Regensburg
DE-93040 Regensburg - Germany
Prof Francisco Del Rey
Departamento de Microbiologica y Genetica
Instituto de Microbiologica Bioquimica
Universidad de Salamanca/CSIC
Campus Miguel de Unamuno - ES-37007
Salamanca - Spain
Dr. Joerg Hoheisel
Functional Genome Analysis
Deutsches Krebsforschungszentrum
Im Neuenheimer Feld 506
DE-69120 Heidelberg - Germany
Dr Jean Luc ZUNDEL
AVENTIS Cropscience SA
Centre de Recherches de La Dargoire
14-20 rue Pierre Baizet - FR-69263 Lyon - France
Dr Massimiliano Fenice
Dipartimento di Agrobiologia e Agrochimica,
University of Tuscia
Via San Camillo DeLellis, - IT-01100 Viterbo - Italy
119
NEW TOOLS TO INVESTIGATE
AND SUPPRESS HIV DRUG RESISTANCE
SUMMARY
New drugs will be developed by targeting new amino acids in the non-nucleoside binding pocket of HIV-1 reverse
transcriptase (RT) that are less prone to be mutated (i.e. W229, Y318 and N136/137), including the dimerisation
interphase of the heterodimeric enzyme (i.e. the 134SINNET139 stretch in the p51 subunit of HIV-1 RT).
Antimetabolites (i.e. purine nucleoside phosphorylase inhibitors) will be used in an attempt to modify the
resistance spectrum of NRTIs and NNRTIs and to select for attenuated mutant virus strains. Several HIV-1 and FIV
RTs in which one or several amino acids or amino acid blocks have been exchanged, and a RT FHIV [hybrid
between HIV and feline immunodeficiency virus (FIV) in which the RT gene of HIV-1 has been placed in the FIV
genetic background] will be constructed and developed to allow investigation of NNRTI drug resistance in an in
vivo (cat) model.
PROBLEM
Resistance to the currently available drugs is one of the major obstacles to achieve a long-term
suppression of HIV in drug-treated HIV-infected individuals. There is a direct correlation
between drug resistance and deterioration of the clinical status of the treated individuals.
Therefore, drug resistance development deserves careful attention in future treatment strategies.
AIM
New avenues will be explored to develop alternative approaches to more efficiently address
HIV drug resistance by developing new type of drugs and novel treatment modalities.
EXPECTED RESULTS
Crystal structure of the HIV-1 reverse
transcriptase complexed with a
non-nucleoside RT inhibitor.
120
The non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) may become the
cornerstone of future HIV treatment modalities when resistance can be adequately
suppressed or avoided. Therefore, this proposal is entirely focussed on this resistance issue. New (or modified) NNRTIs will be targeted and fine-tuned to those interaction points in the HIV-1 RT that are unable to mutate without heavily compromising
the integrity of the RT and the fitness of HIV-1. In addition, entirely new targets on HIV
RT (i.e. the interphase at the binding between both p66 and p51 subunits of the
enzyme) will be explored and characterized, and new drugs (either non-nucleoside
small molecules, or short peptides) will be designed and synthesized in an attempt
to disturb the action of HIV RT. The role of endogenous nucleotide pool ratios in the
HIV-infected cells on drug resistance development will be investigated, with the aim
to force the virus to use alternative (and less optimal) drug-resistance patterns upon
drug combination with antimetabolites in order to weaken the fitness and resistance
level of the virus mutants. Also carefully selected RNA mutagens will be investigated
on their potential role to hypermutate the RNA genome of HIV in a selective manner, causing
error catastrophy in HIV. Cells from different body compartments, including T-lymphocytes and
macrophages will be included in this study. In contrast with nucleoside RT inhibitors (NRTIs),
there is a gap between in vitro research and clinical investigation of the NNRTIs, due to the lack
of an appropriate animal model that can address important issues, in particular drug resistance, in a preclinical setting. Therefore, NNRTI-sensitive feline immunodeficiency virus (FIV) RTs
and FIVs (RT-FHIVs) will be designed, constructed and generated to allow the establishment of
a useful and representative small animal (cat) model for studying the NNRTI resistance patterns
in vivo.
Acronym : HIV resistance
Keywords : AIDS – human immunodeficiency virus (HIV) – feline immunodeficiency
virus (FIV) – reverse transcriptase – dimerisation – drug resistance – error catastrophy
– non-nucleoside reverse transcriptase inhibitors (NNRTIs) – nucleoside RT inhibitors
(NRTIs) – RT-FHIV – chemotherapy – antimetabolites.
Pharmaceutical and biotechnological companies will be involved in a later stage of the
research to develop the novel drugs and concepts to the clinic. HIV-infected individuals will
benefit from the outcome of the research project.
Prof. Jan Balzarini
Rega Institute for Medical Research
Minderbroedersstraat 10
B-3000 Leuven. Belgium.
T: (+32) 16 33.73.52; F: (+32) 16 33.73.40
PARTNERS:
Prof. M.-J. Camarasa
Instituto de Química Médica
Consejo Superior Investigaciones Cientificas
Juan de la Cierva 3, 28006 Madrid, Spain
T: (+34) 91 562.29.00; F: (+34) 91 564.48.53.
Dr. H. Egberink
Department of Infectious Diseases &
Immunology
Institute of Virology, Veterinary Faculty
Yalelaan 1, 3584 CL Utrecht, The Netherlands
T: (+31) 30-2532.485; F: (+31) 30 2536.723
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Prof. E. Gago
Department of Pharmacology
University of Alcada
28871 Alcade de Henares, Madrid, Spain
T: (+34) 91 854.514; F: (+34) 918 854.591
Prof. C.-F. Perno
Department of Experimental Medicine
University of Rome “Tor Vergata”
Via Montpellier 1, 00135 Rome, Italy
T: (+39) 06 7259.6553; F: (+39) 06 7259.6552
Dr. D. Stammers
Structural Biology Division
Wellcome Trust Centre for Human Genetics
University of Oxford, Roosevelt Drive, Oxford OX3
7BN, UK
T: (+44) 1865 287.565; F: (+44) 1865 287.547
Prof. B. Öberg
Medivir AB
Lunastigen 7, S-14144 Huddinge, Sweden
T: (+46) 8 6083.1116/48 8 728.6322; F: (+46) 8
6083.3199/46 8 331.399
Prof. A. Karlsson
Karolinska Institute
Division of Clinical Virology
F68, Huddinge University Hospital, S-141 86
Huddinge/Stockholm, Sweden
T: (+46) 8 5858.7932; F: (+46) 8-5858.7933
121
HIV REVERSE TRANSCRIPTASE INHIBITOR AND
ITS CONSEQUENCES FOR VIRAL VIRULENCE
SUMMARY
VIRULENCE will provide a strategy against a major health threat: occurrence of resistance against therapeutic
HIV/AIDS-measures. Reverse transcriptase inhibitors play an essential role HIV-treatment. RT variants with
reduced susceptibility to these inhibitors emerge even under potent combination therapies and may become less
virulent. Thus, emergence of RTI resistance negatively affects the inhibitor efficacy but may also result in virus
variants with reduced pathogenicity. VIRULENCE aims at elucidating clinical consequences of changes in viral fitness for optimal use of antiviral drugs. VIRULENCE is first to address this through development and validation of
improved fitness assays and their application on one or more existing well-defined longitudinal clinical cohorts.
The molecular mechanisms for viral fitness and resistance will be elucidated and new compounds active against
drug resistant HIV will be defined. This integrated approach will improve care of patients with limited therapeutic
option.
PROBLEM
VIRULENCE will provide a strategy against a major health threat: occurrence of resistance
against therapeutic HIV/AIDS-measures. The emergence of anti-viral drug resistance in HIV
infected individuals undergoing the commonly administered treatment with Highly Active
Antiretroviral Therapy (HAART), is a significant drawback of the therapy efficacy that aims for a
complete and sustained inhibition of viral replication. Failure to suppress viral replication is
demonstrated by increasing concentration of the virus detected in the plasma of these individuals. In drug-naive individuals starting HAART, a combination of three drugs targeting the viral
reverse transcriptase and protease enzymes, complete inhibition of viral replication is usually
achieved effectively. Escape from this initial HAART regimen, as observed by a rise in viral load,
generally occurs in approximately 10% of the patients under treatment per year. Subsequent
therapy changes usually result in a viral suppression for a significantly shorter period, due to
the rapid emergence of resistance to the administered drugs. Viruses expressing resistance to
several, if not all of the available drugs are frequently observed in patients that received and
failed several different HAART regimens. It is estimated that, resistance to two or more reverse
transcriptase inhibitors (RTIs) and or protease inhibitors (PIs) can be observed in approximately 50% of the patients under HAART therapy, with hardly any effective therapeutic options left.
Hence, in order to be able to find new drugs and improved corresponding therapies that can
fight HAART resistant HIV effectively it is essential to obtain a thorough understanding of the
resistance effects and involved mechanisms regarding HIV-virulence.
The VIRULENCE project will focus on investigating the reverse transcriptase. As described
above, reverse transcriptase variants expressing resistance emerge even under potent combination therapy. These variants in some cases express a reduced replication capacity (viral fitness) as a consequence of the resistance mutations selected, and as such become less virulent.
Thus, the emergence of resistance to RTIs negatively affects the efficacy of the drug to inhibit
viral replication, but on the other hand creates virus variants that are less virulent than the wildtype. The clinical consequences of changes in viral fitness are as yet unknown. Therefore, it is
of utmost importance to get a better understanding of the consequences of high level resistance on the pathogenesis and virulence of these viruses in vivo. Moreover it will provide a
sound basis for the screening (within this project) and future development of new drugs that
effectively inhibit the replication of these highly RTI-resistant HIV variants.
AIM
The main scientific and technological objectives of the VIRULENCE project are:
• To determine the clinical consequences of high-level resistance against HIV Reverse
Transcriptase inhibitors (RTIs) on viral virulence in treated individuals.
• To develop, standardise and validate diagnostic tests for the in vivo management and in vitro
determination of viral fitness.
• To clarify the underlying molecular mechanisms relating changes in drug susceptibility with
122
Acronym : VIRULENCE
Starting date : September, 1st 2002
Keywords : HIV-1, drug-resistance, reverse transcriptase, fitness,
replication capacity, virulence, pathogenicity.
viral virulence.
• To identify and test novel RT inhibitors against highly drug resistant HIV-variants.
• To create highly characterised reference materials available to the scientific community.
• To create a public accessible database on the consequences of drug resistance for viral fitness and in vivo virulence.
EXPECTED RESULTS
• Determined clinical impact of drug resistance on viral fitness and underlying molecular
causes.
• Standardised and validated diagnostic tests for patient management.
Public sample-bank of resistant variants suitable for the identification of novel inhibitors.
• Database summarising all aspects of HIV RT-inhibitor resistance and consequences for viral
virulence.
• New lead compounds active against resistant viruses.
Availability and implementation of new, and clinically validated diagnostic tests to determine
viral fitness. Rational understanding of the role of viral fitness in the management of patients
expressing treatment failure. Further exploration of identified lead compounds active against
RTI resistant viruses.
Dr. Rob SCHUURMAN
University Medical Center
Dept. of Virology (G04.614)
Heidelberglaan 100, NL-3584 CX UTRECHT
T: (+31) 30 2506526; Fax: (+31) 30 2505426
PARTNERS:
Dr. Bonaventura Clotet
Department of Retrovirology
Hospital Universitari Germans Trias I Pujol
Badalona
Barcelona, Spain
Dr. Dave Stammers
The Wellcome Trust Centre for Human
Genetics
Division of Structural Biology Chancellor
Masters and Scholars of the University of Oxford
Oxford, UK
Dr. Barbara Schmidt
National Reference Center for Retroviruses
Institute for Clinical and Molecular Virology
Erlangen, Germany
Prof. Dr. Jan Balzarini
Rega Institute for Medical Research
Laboratory of Virology and Chemotherapy
Katholieke Universiteit Leuven
Leuven, Belgium
Prof. Dr. Francois Clavel
IMEA
Hopital Bichat
Institut National de la Santé et de la Recherche
Médicale
Paris, France
• •
• •
•
•
•
Prof. Dr. Ben Berkhout
Department of Human Retrovirology
University of Amsterdam, Amsterdam
The Netherlands
Prof. Dr. Claudia Balotta
Institute of Infectious and Tropical Diseases
Luigi Sacco Hospital
University of Milan
Milan, Italy
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RATIONAL APPROACHES TOWARDS
UNDERSTANDING AND OVERCOMING
HIV PROTEASE INHIBITOR RESISTANCE
SUMMARY
The objective of this project is to determine and monitor development of resistance against human
immunodeficiency virus (HIV) Protease (PR) inhibitors in five European countries, to develop, standardise and
validate diagnostic tests, to analyse changes in viral fitness on resistance development and determine the
underlying molecular mechanisms, and to design and test novel PR inhibitors against drug resistant HIV variants.
Complex approaches involving genotypic and phenotypic characterisation of HIV variants as well as biochemical
and structural analysis of resistant enzymes are required to accurately monitor drug resistance and changes in
virulence, and to provide new inhibitors. The proposed integrated studies will lead to a complete database
covering all aspects of HIV PR inhibitor resistance in Europe.
PROBLEM
Infection with human immunodeficiency virus (HIV) has recently become the leading infectious
disease killer worldwide and antiretroviral drugs are currently the only way to combat AIDS.
Potent antivirals have been developed against HIV reverse transcriptase (RT) and protease (PR)
and combination therapy including protease inhibitors (PI) has dramatically reduced disease
progression in most patients. However, emergence of drug resistant variants has been
observed even for the most potent combination therapies. Therefore, development of drug
resistance and spread of resistant variants are serious impediments to control of HIV infection.
AIM
It is the aim of this collaborative project to analyse the development of drug resistance against
HIV PR inhibitors in an European multicenter study and to provide the rational basis for understanding resistance development and the corresponding changes in viral fitness on a molecular level. To this end, seven closely collaborating groups from five European countries with longstanding experience concerning biochemical, structural and virological aspects of HIV PR and
its inhibitors will join forces in an interdisciplinary approach, also involving hospitals and policlinics treating AIDS patients.
EXPECTED RESULTS
Some expected deliverables of this project are a molecular understanding of resistance evolution and viral fitness, diagnostic assays for standardised and validated resistance monitoring,
novel PR inhibitors with broad activity profiles against resistant PR-species and possibly
reduced side effects, and an integrated database of all aspects of PI resistance development in
Europe.
124
Acronym : HIV PR inhibitors
Starting date : December 2001
Keywords : AIDS, drug resistance, protease inhibitor,
human immunodeficiency virus, viral fitness, inhibitor design,
combination therapy
Results on emerging HIV resistance patterns as well as phenotypic, biochemical and structural characterisation of the respective PR variants will be widely disseminated and rapidly
made publicly available. Several aims of the collaborative project are distinctly applied in
nature. Our plans for potential commercial exploitation of the results from the proposed work
include a prominent role for European SME companies. Contacts with large traditional pharmaceutical industry are also established.
PROJECT COORDINATOR:
Prof. Dr. Hans-Georg Kraeusslich
Abteilung Virologie
Universitaetsklinikum Heidelberg
DE-69120, Heidelberg - Germany
T: +49 6221 56 5001; F: +49 6221 56 5003
PARTNERS:
Dr. Joao Goncalves
Faculdade de Farmacia
Centro de Patogénese Molecular
Av. das Forcas Armadas - PT-1649-019 Lisboa
Portugal
T: +351 21 7934212; F: +351 21 7934212
Prof. Dr. Rolf Hilgenfeld
Institute of Molecular Biotechnology
Beutenbergstr. 11 - DE-07745 Jena - Germany
T: +49 3641 656061; F: +49 3641 656062
Dr. Jan Konvalinka
Institute of Organic Chemistry and
Biochemistry
Academy of Sciences of the Czech Republic
Flemingovo n. 2 - CZ-166 10 Praha 6
Czech Republic
T: +420 2 20183 218;F: +420 2 24310 090
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Dr. Vladimir Kral
Prague Technical University
Institute of Chemical Technology
Technická 5
CZ 16610 Prague 6 - Czech republic
T: +420 2 2435 4060; F: +420 2 311 2828
Dr. Fabrizio Mammano
INSERM U-552 Recherche Antiviale
IMEA/INSERM Hopital Bichat C. Bernard
46, rue H. Huchard - FR-75018 Paris
France
T: +33 1 40256368; F: +33 1 40256370
Dr. Monique Nijhuis
University Medical Center Utrecht
Eijkman-Winkler Institute,
Department of Virology
Heidelberglaan 100
NL-3508 GA Utrecht
The Netherlands
T: +31 302506526; F: +31 302505426
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125
STRATEGY TO CONTROL SPREAD
OF HIV DRUG RESISTANCE
SUMMARY
The proportion of newly HIV infected patients that is resistant to antiretroviral drugs, is increasing throughout
Europe. If this spread of resistance is not stopped, then a situation can occur in which no effective drugs will be
available for newly infected patients. Within the SPREAD-project clinicians, virologists and epidemiologists from
16 European countries are developing a strategy to control the transmission of resistant HIV.
PROBLEM
The mortality in the EU caused by AIDS has decreased dramatically since the introduction of
highly active antiretroviral drugs. In contrast, the number of new HIV infections has not
decreased in EU countries. Furthermore, it is now painfully apparent that resistance to antiretroviral drugs not only emerges in treated patients, but that resistant viruses can also be
transmitted to newly infected patients. At the moment, there is confusion about the true incidence of transmission of drug resistant HIV in Europe. Several factors are responsible for this
confusion. First, there is a lack of representative sampling within the different risk groups and
the different countries. Second, there is no consensus on the definitions for resistance. Third,
there is a great variety in the performance of laboratories applying resistance assays. Currently,
most laboratory techniques underestimate the presence of drug resistance mutations. Despite
of these limitations it is expected that approximately one fifth of all new HIV patients are infected with drug resistant strains.
The spread of resistance is particularly worrisome since treatment may be severely compromised in patients infected with drug resistant HIV. Transmission of drug resistance can also
have serious implications for professionals that have accidentally been exposed to HIV. These
persons receive post exposure prophylaxis (PEP) consisting of a combination of highly active
antiretroviral drugs. There is a risk that PEP will not be beneficial to resistant viruses. It is very
important to identify which groups in society are more likely to spread resistance, so prevention campaigns can be directly targeted towards these.
Subtype B is the most common subtype circulating in Europe. However, subtype B accounts for
only a small proportion of HIV isolates worldwide. In Europe non-B subtypes have been increasingly identified. Drug resistance profiles have been characterized almost exclusively for subtype B. It is not known what mutation patterns emerge in non-B subtypes.
AIM
The main objective of SPREAD is to develop and implement a European surveillance program
that collects comparable and reliable data on the transmission of HIV drug resistant viruses.
This objective will be met by:
• Prospective inclusion of antiretroviral naïve HIV patients across Europe using a standardized
representative sampling strategies.
• Monitoring of the performance of the resistance assays of the participating laboratories by a
quality control programme.
• Assessment of transmitted drug resistance using a scientific consensus model.
SPREAD will establish its main objective based on several aims:
• Determine the proportion of new HIV patients in Europe that has been infected with resistant
virus.
• Identify risk factors for transmission of resistance.
• Study the relation of transmission of resistance with both antiretroviral consumption and with
national treatment guidelines.
• Identification of mutant HIV-strains, especially in non-B subtypes. These strains will be used
to study changes in virulence, identify novel genetic resistance pathways and can be used to
improve the diagnosis of drug resistance.
• Development of a mathematical model, based on the epidemiological, clinical and virological
results, on the spread of drug resistant virus.
126
Acronym : SPREAD
Keywords : HIV, drug-resistance, surveillance, transmission, Europe,
prevention
EXPECTED RESULTS
SPREAD will be the first study that systematically collects information on the spread of transmission of drug resistance in HIV across Europe. The following results are expected:
• Overall incidence of the transmission of drug resistant virus both throughout Europe and
per country
• Risk factors for transmission of drug resistance
• Relation between the incidence of transmission and antiretroviral consumption and/or local
treatment guidelines
• Establishment of a European database of circulating HIV-drug resistant strains
• Construction of a mathematical model that will be pivotal in understanding the processes
that drive the evolution and spread of drug resistant virus.
The results of SPREAD will help to reduce, or even stop, the spread of resistant virus. Results
will be of use to physicians, patients, public health organisations, and the pharmaceutical
industry.
The benefits for physicians and patients:
• Guidelines concerning testing of newly infected patients for drug resistance
• Information that can help avoiding ineffective drugs after transmission of drug resistant HIV
• Guidelines for PEP
The benefit for public health organisations:
• Identification of groups in society to whom prevention campaigns should be directed
The benefit for the pharmaceutical industry:
• Information on HIV-drug resistance that can be used for development of drugs with better
resistance profiles.
www.spread-europe.org
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Dr. Charles A.B. Boucher, MD, PhD
University Medical Center Utrecht
Department of Virology (G04.614)
Heidelberglaan 100, NL-3584 CX UTRECHT
T: (+31) 30 2506526; F: (+31) 30 2505426
E-mail: [email protected] / [email protected]
Dr. Balotta
PARTNERS:
Dr Puchhammer-Stockl
Dr. Holm-Hansen
Prof. Dr. Vandamme
Dr. Nielsen
Dr. Salminen
Prof. Dr. Fleckenstein
•
Prof. Dr. Hatzakis
Dr. Schmit
Dr. Boucher/Dr. Wensing
Dr. Horban
Dr. Camacho
Dr. Clotet
Dr. Albert
Prof. Dr. Loveday
Prof. Dr. Hall
127
THE EUROSIDA NETWORK 2000-2004.
CLINICAL AND VIROLOGICAL OUTCOME OF
EUROPEAN PATIENTS INFECTED WITH HIV
SUMMARY
The primary objective of the EuroSIDA study is to collect demographic, clinical, therapeutic and laboratory data
on patients with HIV infection to determine the long-term virological and clinical outcome for patients from
Europe. Furthermore, to ensure that this knowledge is analysed, discussed and presented within the scientific
community to assist physicians and public health officials.
PROBLEM
Despite recent improvement in prognosis of HIV-infected patients from across
Europe due to use of powerful combination antiretroviral therapies, challenges
persist in relation to durability of responses and new co-morbidities. These challenges mainly concern development of resistance to existing treatments and coping with adverse drug reactions. In addition there is a dramatic progression in the
HIV epidemic in the Eastern European countries requiring development of treatment strategies in this setting also.
AIM
To continue a long-term, prospective collection of clinical, laboratory and therapeutic data as well as plasma on a large cohort of consecutive HIV infected
patients from across Europe. This in order to assess the factors associated with
the clinical, immunological and virological course of HIV infection, including effect
and toxicity of antiretroviral agents and other therapeutic interventions. Further
continue to provide and develop a surveillance system to describe temporal
changes and regional differences in the clinical course of HIV. These include: i) the overall pattern of HIV-related diseases and death and the emergence of new diseases, including drugrelated diseases; ii) the prevalence of patients with severe immunosuppression and high viral
replication; iii) the virological response to initial antiretroviral therapy.
EXPECTED RESULTS
• Long term prospective follow-up on a total of 10,900 unselected patients from 70 clinical centres in 26 European countries, providing 40,500 person-years of follow-up.
• The EuroSIDA main database will serve as a tool to study the course and evolution of chronic HIV infection in patients from across Europe in the future.
• The study will collect extensive information on adverse events including risk factors for and
the occurrence of cardiovascular disease events.
• In 1999, Poland, the Czech Republic and Hungary entered EuroSIDA.
Additional recruitment from the Newly Associated States in the eastern part of
Europe; Slovakia, Estonia, Latvia, Lithuania, Romania and Ukraine, will continue in 2002 to 2004.
• A central plasma repository of 27,000 samples will allow laboratory analyses
related to HIV resistance and HIV sub-typing.
128
Acronym : EURO-SIDA 2000-2004
Contract Type : Thematic Network
Keywords : HIV, AIDS, observational study, cohort, virology,
clinical outcome
The results are widely disseminated by publications in peer-reviewed journals and are used
by health authorities to formulate treatment guidelines. Findings related to the objectives listed above provide important knowledge for decision making in clinical practice in the care of
HIV patients.
http://www.eurosida.org
Dr. Jens D. Lundgren
Hvidovre University Hospital,
Dept. Of Infectious Diseases
144, Kettegaard Alle 30, DK- 2650 Hvidovre,
Denmark
T: (+45) 36323015;
F: (+45) 36473340
Web-site: www.eurosida.org
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and 72 clinical centres in 26 countries
in Europe
Latvia L Viksna
Argentina M Losso
Luxembourg R Hemmer
Austria N Vetter
Netherlands P Reiss
Belgium N Clumeck
Norway J Bruun
Czech Republic L Machala
Poland B Knysz
Denmark J Nielsen
Portugal F Antunes
Estonia K Zilmer
Romania D Duiculescu
France C Katlama
Slovakia M Mokrá
Germany M Dietrich
Spain J González-Lahoz
Greece J Kosmidis
Sweden A Blaxhult
Hungary D Banhegyi
Switzerland B Ledergerber
Ireland F Mulcahy
Ukraine N Chentsova
Israel I Yust
United Kingdom S Barton
Lithuania S Chaplinskas
Italy S Vella, A Chiesi
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129
EMERGING VARIANTS OF HEPATITIS B VIRUS:
NEW TOOLS FOR EPIDEMIOLOGICAL SURVEY,
DIAGNOSIS OF INFECTION, AND MONITORING
OF DRUG RESISTANCE
SUMMARY
Clinical centres from different part of Europe (France, Germany, Italy, United Kingdom, Poland) will select clinically
relevant samples. A retrospective and prospective assessment of Hepatitis B virus (HBV) carriers will be carried
out at different phases of chronic infection, including HBeAg positive and negative patients, cirrhotic patients
with or without hepatocellular carcinoma, patients with liver transplantation, as well as carriers who have
undergone antiviral therapy (interferon and nucleoside analogues). Variant genomes from clinical specimens will
be detected using sequencing as well as novel methodologies. Denaturating gradient gel electrophoresis,
heteroduplex mobility assay, allele specific PCR, DNA chip and DNA-designed variant specific immunoassays will
be evaluated and the best technology(ies) will be selected.Quantitation of variant viruses based on real time PCR
will be used to correlate the dynamics of viral replication with the outcome of disease. Replication competence,
pathogenicity and ability to escape to antiviral or immune pressure of defined variants will be determined by
established and newly developed protocols for transfection of
cloned genomes in eukaryotic cell lines. Delivery of HBV genomes
by adenovirus vectors in tupaias will be used as a novel system to
analyse the biology and pathobiology of variants in cultured
primary hepatocytes and in vivo. Genotypic and phenotypic results
will be correlated with the clinical status of patients to identify
clinically and biologically relevant mutations. The best strategies for
the diagnosis and monitoring of mutant HBV infection, including
antiviral therapy, will be defined.
PROBLEM
Chronic hepatitis B virus infection remains a major public health problem worldwide. New advances have been made recently in the field of vaccine prophylaxis
but also with antiviral therapy. However, due to its genome variability, HBV has
the capacity to escape spontaneaous, interferon alpha, and vaccine induced
immune response, as well as to specific polymerase inhibitors. Few information
is available on the epidemiology of these emerging variants as well as on their
clinical and therapeutical impact. It is therefore mandatory to gain more insight
in the molecular biology of these escape mutants, and to design new assays for
the detection of these mutants.
AIM
The objectives of our project regarding HBV genome variability will be to : 1) identify the clinical situations that favour emergence of vaccine-escape and drugresistant variants and evaluate their prevalence and spread in Europe, 2) characterise the genome of the variants, 3) study the pathobiology of the variants, 4)
develop new animal and in vitro infection models, 5) develop new molecular
assays for the diagnosis of variant infection and define the most accurate strategy to detect HBV infection and monitor its treatment, 6) make available recommendations and new diagnostic tools to the scientific and medical community.
130
Acronym : HEPBVAR
Keywords : Hepatitis B virus, genome variability, variants, genotype,
phenotype, cell culture, animal models, diagnosis, antivirals
EXPECTED RESULTS
The following milestones are expected : (1)a database of molecular epidemiology data useful
to develop new vaccines and drug formulations and to adapt screening assays for relevant
variants, (2) development and marketing of new diagnostic tools for the monitoring of HBV
infected patients, (3) knowledge on the molecular biology of HBV variants useful to improve
vaccination and treatment strategies, (4) development of new experimental models to validate treatments, (5) dissemination of the results in the scientific and medical community.
Results will be disseminated to the scientific and medical community, after filling patent applications when required. New experimental systems for the study of HBV replication and its
mutants may be set up, which in turn may have therapeutical implications for the evaluation
of new inhibitors on the mutants that are resistant to currently available drugs. New diagnostic assays specific of these variants will be designed and may be used routinely in clinical
practice for the monitoring of antiviral therapy of HBV infection.
CO-ORDINATOR:
Pr. Fabien Zoulim
INSERM Unit 271
151 cours Albert Thomas - FR-69003 Lyon - FR
T: +33 4 72 68 19 70; F: +33 4 72 68 19 71
PARTNERS:
Dr Chong-Gee Teo
Central and Public Health Laboratory
Hepatitis and Retrovirus Laboratory, PHLS
61 Colindale Avenue - UK- London NW9 5HT
United Kingdom
T: +44 181 200 44 00; F: +44 181 200 15 69
Prof. Michael Nassal
University Hospital Freiburg
Dept. of Internal Medicine II,
Molecular Biology
Hugstetter Str. 55 - DE-79106 Freiburg
Germany
T/F: +49 761 270 3507
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Pr. Feruccio Bonino
UO Epatologia (UOE) Azienda Ospedaliera
Universitaria Pisana
Via Paradisa, 2 Cisenalo - IT-54124 Pisa - Italy
T: +39 337 221762; F: 39 050 995 457
Dr Guy Vernet
BioMerieux
Chemin de l’Orme
FR-69280 Marcy l’Etoile - France
T: +33 4 78 87 24 08; F: +33 4 78 87 53 40
Pr Anna Podhajska
Department of Biotechnology
Intercollegiate Faculty of Biotechnology
Kladki 24 - 80 822 Gdansk - Poland
T/F: +48 58 3012 807
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131
ANALYSIS OF THE MOLECULAR MECHANISMS
OF HEPATITIS C VIRUS (HCV) RESISTANCE TO
ANTIVIRAL THERAPY
SUMMARY
The Hepatitis C Virus (HCV) infection affects about 300 million individuals worldwide and at least 5 millions within the EC. Despite recent improvements of therapy, eradication of HCV is not obtained in around 50% of treated
patients. This proposal aims to combine a wide functional genomics approach and post-genomic profiling
techniques to explore in detail the molecular mechanisms of HCV persistence under interferon therapy.
The project will identify new targets for antiviral therapy and contribute to the control of HCV infection.
PROBLEM
The Hepatitis C Virus (HCV) is a major human pathogen and around 300 million individuals are
chronically infected worldwide. In Europe, the prevalence of anti-HCV positivity ranges from 1
to 4-5 % and at least 5 million subjects are infected. Major features of HCV infection are a high
risk of chronicity (60-80%) and of developing severe liver lesions (cirrhosis and hepatocellular
carcinoma (HCC)). Our current understanding of the natural history of HCV chronic infection and
the epidemiological and biological impact of both overt and occult HBV co-infection in HCV
chronic hepatitis patients allows to anticipate a further important rise in HCC incidence over the
next two decades in Europe. HCV also triggers a polyclonal lymphoid cell proliferation and has
been implicated in malignant lymphoproliferative disorders.
Treatment of chronic HCV infection is based on interferon-a which, in combination with ribavirine, can eradicate the virus in around 30-40% of the treated patients. Pegilated-interferons,
which are given to the patient once a week and whose pharmacokinects properties garantee
costant steady state levels, has further ameliorated the results to about 50% of the patients.
Still, HCV eradication can be reached only in about 40% of patients infected with HCV type 1. It
is also debated whether or not interferon therapy might reduce, independently of its direct
antiviral effect, the risk of HCC development among patients with HCV-related cirrhosis. Several
studies have suggested that, in addition to the interplay between genetic variability and host
immune response, some HCV proteins (namely the HCV core, E2 and NS5A) might directly modulate interferon and other signal transduction pathways, which control IFN responses.
AIM
The scientific/technological objective of this project is to elucidate the mechanisms of HCV
resistance to the antiviral therapy.
Specific objectives of the project are:
1. To perform a comprehensive in vivo analysis of the HCV sequences encoding for proteins
(core, E2 and NS5A), implicated in the modulation of cellular signalling, in patients with different profiles of response to therapy.
2. To develop several new research tools to investigate the biological activities of such in vivo
HCV mutants, using in vitro and in vivo experimental approaches.
3. To analyze in detail the impact of HCV protein expression on : a) the major transduction networks controlling the cellular response to inteferon ; b) on the kinetics of ISGF-3 complexes
recruitment at the DNA level and on the composition of ISGF-3 containing DNA-bound complexes ; c) on the repertoire of IFN-responsive genes activated in vivo.
To accomplish these tasks a combination of biochemistry, cell biology and molecular biology
techniques will be coupled with innovative approaches, including the very powerful chromatin
immuno-precipitation (ChIP) technique and gene expression profiling using laser-based
microdissection and microarrays screening.
132
Acronym : HEPAC-RESIST
Keywords : Hepatitis C Virus; Antiviral therapies; Interferon;
HCV quasispecies; HCV replicons; Signal transduction; Transcription;
Chromatin Immunoprecipitation; Laser Microdissection; Microarrays.
EXPECTED RESULTS
The project will elucidate: a) how HCV proteins, namely core, E2 and NS5A, as well as the HCV
polyprotein itself, modulate major interferon-dependent cellular signalling pathways; b) how
viral genomic variability impact on the interplay between the virus and the host interferondependent innate immune response. To define host gene expression profiles that predict the
outcome of interferon-based therapies will improve our instruments for nosological and prognostic evaluation of HCV infection, a leading cause of morbidity and mortality in Europe.
The expected results will provide to both biomedical industry and physicians new instruments
for optimised managements of HCV chronic hepatitis in terms of prediction of outcomes and
tailoring of treatment to the single patient.
Prof. Massimo Levrero
Fondazione A. Cesalpino
University of Rome La Sapienza
Laboratory of Gene Expression
Policlinico Umberto I
Viale del Policlinico 155, 00161 Roma, Italy
T: (+39) 06 446 85 29; F: (+39) 06 4940594
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PARTNERS:
Dr. Dina Kremsdorf
INSERM U370
156, rue de Vaugirard, 75730 Paris Cedex 15, France
T: (+33) 01 40 61 56 44; F: (+33) 01 40 61 55 81
Dr. Czeslaw Wychowski
UPR 2511 CNRS
Institut Pasteur de Lille
Institut de Biologie de Lille
1, rue du Pr Calmette, 59021 Lille Cedex, France
T: (+33) 03 20 87 11 60; F: (+33) 03 20 87 10 19
Dr. Claude Auriault
UMR 8527 CNRS
Institut Pasteur de Lille
Institut de Biologie de Lille
1, rue du Pr Calmette, 59021 Lille Cedex, France
T: (+33) 03 20 87 11 60; F: (+33) 03 20 87 10 19
Prof. Gilles Duverlie
UMR 8527 UPJV
Universitè Jules Verne Picardie
Faculté des Sciences
3, rue de Louvels, 80036 Amiens Cx1, France
T: (+33) 03 22 82 79 17; F: (+33) 03 22 45 56 50
Dr. Claude Auriault
UMR 8527 Lille 2
Universitè de Droit et Sante Lille 2
42, rue Paul Duez, 59800 Lille Cedex, France
T: (+33) 03 22 82 79 17; (+33) 03 22 45 56 50
Dr. Illka Julkunen
Laboratory of Molecular and Viral
Immunology
Mannerheimintie 166 FIN-00300 Helsinki, Finland
T: (+358) 9 47 44 83 72; F: (+358) 9 47 44 83 55
Prof. Graham Foster
Department of Medicine
St Mary's Hospital Medical School
Queen Elizabeth the Quenn Mother Wing, London
W2 1NY, UK
T: (+44) 171 725 16 06; F: (+44) 171 724 93 69
Prof. Stefan Zeuzem
Internal Medicine II
Saarland University Hospital
Saarland University
Kirrberger Str. 66421 Homburg/Saar, Germany
T: (+49) 0 6841 16 23201; F: (+49) 0 6841 16 23267
Dr. Mariano Esteban
28049 Madrid Spain
T: (+34) 91 585 4503; F: (+34) 91 585 45 06
133
ADHESIVE INTERACTIONS IN MALARIA:
NEW TARGETS FOR INTERVENTION
SUMMARY
The high mortality associated with malaria is due to the adhesive properties of P. falciparum –infected
erythrocytes, which sequester in the deep vascular bed resulting in damage to vital organs. The goal of this
research project is to develop the technological and scientific base that will permit the rational design of
anti-adhesive drugs for the treatment of severe malaria. Chemotherapeutics that disrupt and prevent adhesion
of parasitized erythrocytes will be of immense clinical value as such an adjuvant treatment would substantially
improve microcirculation, reverse tissue hypoxia, remove the proinflammatory stimulus from endothelial cells
and bring the parasitized erythrocytes back into circulation where they are more accessible to drug killing.
PROBLEM
Erythrocytes infected with
Plasmodium falciparum.
photo credit: Alister Craig
Malaria is a major health problem not only in traditional endemic areas but increasingly at a
global level. Once sharp weapons in the campaign against this infectious disease have lost
their edge. Chemotherapeutics now frequently fail due to resistance mechanisms, and insecticides, once widely used to eradicate the malaria vectors, are now disqualified because of environmental considerations. In order to control the spread of this infectious disease a comprehensive and co-ordinated approach is required which would include new generations of antimalarial drugs with novel modes of action for both therapeutic and prophylactic use.
The morbidity and mortality inflicted by tropical malaria is associated with the adhesive properties of erythrocytes infected with the malarial parasite Plasmodium falciparum. By adhering
to the endothelial lining of venular capillaries the parasite evades elimination by the reticuloendothelial system of the host spleen. Sequestration of infected erythrocytes severely compromises the host, resulting in damage to vital organs including the brain, lung and kidney.
Clinical protection against malaria is associated with the acquisition of immunity to different
antigenic forms of the parasite-encoded adhesion molecule. Thus, inhibition of adhesive interactions is a valid target for therapeutic intervention. The feasibility of this concept has been
demonstrated in preliminary experiments where adhesion was reversed both in vitro and in vivo
upon the addition of certain glycans.
AIM
The main objective of this research proposal is to develop the technological and scientific base
that will permit the rational design of anti-adhesive drugs for the treatment of severe malaria.
A prerequisite to the successful development of anti-adhesion drugs for the treatment of severe
malaria is detailed knowledge of the molecular basis of adhesion phenomena in malaria,
including the role host and parasite-encoded factors play in this pathogenic process. Therefore,
this consortium incorporates a strong basic research program aimed at the elucidation of the
mechanisms underpinning adhesion of P. falciparum infected erythrocytes to host cells at the
molecular level. Information gained will be fed back to our rational drug design program. The
specific aims are:
• create parasite mutants that display various defects in adhesion properties;
• analyze the mechanism of differential expression of adhesins;
• examine the behavior of infected erythrocytes regarding their capability to bind to chondroitin-4-sulfate (CSA) and intercellular adhesion molecule 1 (ICAM-1), which both have been
associated with disease, using adhesion under shear flow that mimics more closely the conditions in vivo.
• study the parasite binding characteristics to endothelia derived from the microvasculature of
different target organs, to understand the contribution of contextual and differential expression of endothelial receptors to cytoadherence;
• identify novel host cell receptors involved in adhesion of parasitized erythrocytes to critical
target organs of sequestration;
• examine the possibility of a cross-talk between the endothelial cell and the parasitized erythrocyte upon binding.
• screen a large library of various glycans for their ability to disrupt adhesion of parasitized ery134
Acronym : ADMALI
Keywords : malaria, Plasmodium falciparum, anti-adhesive drugs
throcytes to human endothelial cells and CHO cells expressing defined human endothelial
cell surface receptors;
• glycans demonstrating anti-adhesion activity will be enrolled in a rational drug design program for optimization of their activity;
• all active substances will be tested for unwanted site effects, such as interference with
coagulation;
• promising substances will be tested for activity in a Saimiri monkey malaria model system;
• obtain the three dimensional structure of the conserved head structure of PfEMP-1.
EXPECTED RESULTS
1 A better understanding of the adhesive interactions between P. falciparum-infected erythrocytes and host cells at the molecular level;
2 Identification of new targets for both chemotherapeutic intervention;
3 Development of a novel class of antimalarial drugs that prevent adhesion of P. falciparuminfected erythrocytes.
It is hoped that our research and development will lead to a novel class of antimalarial drug.
The close collaboration with our industrial partner ensures that the scientific knowledge
gained will be rapidly transferred into new commercial products.
Prof. Dr. Michael Lanzer
Abteilung für Parasitologie
Hygiene Institut
Im Neuenheimer Feld 324 - DE-69120 Heidelberg
Germany
T: +49 6221 567845; F: +49 6221 564643
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PARTNERS
Dr. Alister Craig
Liverpool School of Tropical Medicine
Pembroke Place
UK-Liverpool L3 5QA - United Kingdom
T: +44 151 708 9393;
F: +44 151 708 1702 ( or +44 151 708 9007 )
Dr. Jürg Gysin
Université de la Méditerranée Aix Marseille
II
Unité de Parasitologie Expérimentale
Faculté de Médecine
Bd. Jean Moulin, 27 - FR-13385 Marseille - France
T: +33 4 91324633; F: +33 4 91324643
Dr. Artur Scherf
Institut Pasteur
Department of Immunology
25, rue du Dr. Roux - FR-75724 Paris - France
T: +331 4568 8616; F: +331 4561 3185
Dr. Daniel Vitt
4SC GmbH
Am Klopferspitz 19
DE-82152 Martinsried - Germany
T: +49 89 7007610; F: +49 89 70076329
Dr. Mats Wahlgren
Microbiology and Tumor Center
Karolinska Institute
Nobels väg 16 - SE-171 77 Stockholm - Sweden
T: +46 8 7287277;F: +46 8 310525
Dr. Percy Knolle
Zentrum für Molekulare Biologie Heidelberg
Im Neuenheimer Feld 282 - DE-69120 Heidelberg
Germany
T: +49 6221 546815; F: +49 6221 545893
135
MALARIAL CHEMOTHERAPY TARGETING
PLASMODIAL PHOSPHOLIPID
BIOSYNTHESIS: IMPLEMENTATION OF
A PRODRUG STRATEGY FOR ORALLY
ACTIVE COMPOUNDS
SUMMARY
The objective of this project is to develop a promising antimalarial drug that targets membrane biogenesis of the
erythrocytic stage of malaria parasites. Phospholipid synthesis of the parasite, but not the host, is specifically
blocked, and multiresistant malaria is susceptible to the compounds.
To date no resistance has been found. This innovative development of new chemical weapons against malaria,
capitalises on groundbreaking original work, and brings together a unique partnership of resources and
expertise. The work we propose here has real prospects of delivering first-line, original antimalarial drugs as
cheap replacements for chloroquine in areas of resistance.
PROBLEM
The impact of malaria on world health is enormous. The global threat of multidrug resistant
malaria makes imperative rapid clinical assessment of promising new therapeutics. In fact, no
current compound provides protection against malaria in all regions of the world. Drug development efforts must aim at obtaining compounds that work through new independent mechanisms of action that are structurally unrelated to existing antimalarial agents.
AIM
A recently discovered property of the new
antimalarials is a selective accumulation in
the P. falciparum-infected erythrocytes.
The mechanisms mediating this accumulation
and the intracellular localisation will be
studied during this programme
(A, Apicoplast, DV, digestive vacuole, M,
mitochondria, N, nucleus)
This programme concerns a new chemotherapy for P. falciparum and P. vivax malaria
that targets membrane biogenesis of the erythrocytic stage of the parasite. The most
promising drug interference is blockage of the choline transporter, a rate-limiting step
in phosphatidycholine synthesis and an essential step for intraerythrocytic growth of
the malaria parasite. Here we will focus on the following major points : 1- synthesis of
prodrugs to obviate the weak oral absorption of bis-cationic compounds. The concept
has been validated. This includes optimisation of two series, which satisfied criteria
for transfer to preclinical studies, and assessment of two other new delivery systems.
Oral absorption and physical properties of the compounds will be maximised. 2Characterisation of the interaction of the compounds with the infected erythrocyte
(we have recently shown that compounds are specifically accumulated at least 100 –
fold in the infected erythrocytes). This requires the synthesis of radio- and fluorescent
labelled compound derivatives of both bioprecursors and drugs. 3- Identification, isolation and
characterisation of the intrinsically-efficient drug target as a prerequisite to intracellular characterisation and a more rationale design of potent specific effectors. 4- Studies on the regulation of phospholipid metabolism and mechanisms involved in possible acquisition of resistance. Unique characteristics of serine-based lipid metabolism should provide further targets
for development of new therapeutic agents. 5- Thorough studies of biological and pharmacological activities including chemosensitivity of multiresistant malaria, therapeutic index after
oral formulations in mouse and monkeys, and toxic evaluation of lead compounds ; 6Determination of pharmacokinetics and toxic properties in animals and their metabolisation for
one compound under GMP and GLP procedures (as part of preclinical studies).
EXPECTED RESULTS
Synthesis and optimisation of 3 validated prodrug systems, their physico-chemical characterisation, thorough evaluation of antimalarial activities, toxicity and pharmacokinetics in animals,
and setting up predevelopment studies; Complementary research will define the molecular
mechanisms of drug activity and will more fully characterise parasite serine lipid metabolism.
This is an attractive pathway for drug development that is absent in mammalian cells and that
offers considerable potential for medium-term pharmacological interventions
136
Acronym : NOVEL ANTIMALARIALS
Keywords : Phospholipid metabolism, Anti-phospholipid,
Malaria chemotherapy, Development Pharmacology, Phospholipid,
Inhibitors, Plasmodium falciparum, Plasmodium vivax, Babesia,
The major aim is to develop orally prescribable antimalarial compounds (third generation) to
the preclinical stage. This will promote pharmacological and industrial application, have a
clear positive economic and social impact, will benefit the European citizen and people from
developing countries. Specific new therapeutics and patents are also anticipated.
Dr. Henri VIAL
UMR CNRS 5539
Dynamique Moléculaire des Interactions
Membranaires,
Département Biologie et Santé, Université
Montpellier II, CC 107
Place Eugène Bataillon, - FR-34095 Montpellier
Cédex 05 - France
T: +33 04 67 14 37 45;F: +33 04 67 14 42 86
• •
•
PARTNERS:
Dr. Michèle Calas
Amino acids
Peptides and Proteins Laboratory
University Montpellier II, cc22
Place Eugène Bataillon - FR-34095 Montpellier
Cedex 05 - France
T : +33 4 67 14 38 17 ; F : +33 4 67 14 48 66
Steve Ward
Pharmacology Research Labs
70 Pembroke Place Floor 1, Block H
UK-Liverpool L69 3GE
United Kingdom
T: +44 0151 794 8219; F: +44 0151 794 8217
Dr. Françoise Bressolle
Laboratoire de Pharmacocinétique Clinique
15 Avenue Charles Flahault
FR-34060 Montpellier Cedex 2 - France
T: +33 04 67 54 80 75; F: +33 04 67 54 80 75
Dr. Socrates Herrera
Fundacion Centro de Primates
Departamento de Microbiologia
Calle 4 b. N° 36-00AA 25574 - Facultad de Salud
Universidad del Valle - Cali
Colombia
T: +57 25 58 1946 ; F: +57 25 58 1061
Dr.Alan Thomas
Biomedical Research Centre
Postbox 3306 - Lange Kleiweg 139
NL-2280 GH Rijswijk - The Netherlands
T: +31 15 84 25 38; F: +31 15 284 39 86
137
GENETIC ANALYSIS OF THE CHLOROQUINE
DRUG RESISTANCE AND THE ACCELERATEDRESISTANCE-TO-MULTIPLE-DRUGS
PHENOTYPES IN THE HUMAN MALARIAL
PARASITE PLASMODIUM FALCIPARUM
SUMMARY
Chemotherapeutics against malaria now frequently fail in the field due to widespread resistance mechanisms.
We aim to do a genetic cross to identify genetic determinants of high level chloroquine drug resistance and the
accelerated-resistance-to-multiple-drugs (ARMD) phenotype. The study will provide new insight into the genetic
basis of chloroquine resistance and the ARMD phenotype in P. falciparum. A detailed knowledge of the molecular
mechanisms underpinning chloroquine resistance and the ARMD phenotype may provide new opportunities for
rational drug development.
PROBLEM
Physicians and medical support staff in many developing countries are engaged in a losing battle against malaria, an infectious disease that is endemic throughout most of Africa, Southeast
Asia and Latin America, causing an estimated 300-500 million clinical cases and 1-3 million
deaths annually. Options to control the spread of malaria are running out. Chloroquine, the first
line antimalarial for more than thirty years, now frequently fails in the field due to widespread
resistance. Alternative drugs that are as safe and affordable as chloroquine, and which could
replace chloroquine as the mainstay antimalarial, are not yet available. Unfortunately, chloroquine is not the only drug compromised by resistance mechanisms. Parasite strains have
emerged that are resistant to every common drug available, including chloroquine, quinine,
mefloquine, pyrimethamine, cycloguanil and sulfadoxin. To make matters worse, these multidrug resistant strains acquire resistances to new antimalarial compounds 1000 times more frequently than do wild-type clones. This finding suggests that multi-drug resistant strains are predisposed to acquiring resistance to novel drugs, a phenotype called accelerated resistance to
multiple drugs (ARMD). The mechanism responsible for the ARMD phenotype is not known,
despite its important medical and public health implications. The ARMD phenotype, when not
repressed, will inevitably undermine all future efforts to control malaria using intervention
strategies based on chemotherapy. The mechanism underpinning chloroquine resistance is
only partially understood, although a detailed understanding may provide new opportunities
for therapeutic interventions. Here we propose a genetic cross to study both chloroquine
resistance and the ARMD phenotype.
138
Acronym : MALCROS
Keywords : malaria, chloroquine resistance, genetic cross, multi-drug
resistance, genome scanning
AIM
The aim of the project is to identify candidate genes mediating chloroquine drug resistance
and the ARMD phenotype in the human malarial parasite P. falciparum.
EXPECTED RESULTS
We expect to identify genes that are altered in malarial parasites revealing chloroquine resistance and the ARMD phenotype. This will provide a better understanding of the molecular
mechanisms underpinning these two pathogenic phenotypes, which in turn, may open up
new opportunities for drug intervention.
The results may provide new insight into drug targets. As such the project may be of interest
to the pharmaceutical industry.
Prof. Dr. Michael Lanzer
Universitätsklinikum Heidelberg
Hygiene-Institut, Abt. Parasitologie
D-69120 Heidelberg, Germany
T: (+49) 6221 567845; F: (+49) 6221 564643
PARTNERS:
Dr. Alan Thomas
Foundation Biomedical Primate Research
Center (BPRC)
Lange Kleiweg 139, NL-2280 GH Rijswijk,
Netherlands
T: (+31) 15 2842538; F: (+31) 15 2843986
Prof. Dr. Dominique Mazier
Institut National de la Santé et de la
Recherche Médicale
184 Rue du Faubourg St-Antoine, Hôpital StAntoine, F-75571 Paris
France
T: (+33) 1 40 77 97 37; F: (+33) 1 45 83 88 58
Prof. Dr. Robert Sauerwein
Stichting Katholieke Universiteit
Geert Grooteplein 10, NL-6500 HB Nijmegen
Netherlands
T: (+31) 24 3614306; F: (+31) 24 3540216
•
•
•
Prof. Dr. Socrates Herrera
Fundacion Centro de Primates
Instituto de Inmunologia
Calle 4B # 36 – 00, Cali, Colombia
T: (+57) 2 558 3937; F: (+57) 2 557 0449
139
DEVELOPMENT OF A MALARIA RESISTANCE
DNA CHIP AS A PUBLIC HEALTH TOOL FOR THE
MANAGEMENT OF PLASMODIUM FALCIPARUM
MALARIA DRUG RESISTANCE
SUMMARY
This project aims at evaluating a newly developed DNA micro array for drug resistance monitoring in affected
areas. Results will allow to capture dynamics of drug resistance and to forecast its development by establishing a
community based Genetic Resistance Index, providing tools for rational and evidence based decisions on optimal
use of drugs. We will assess the relationship between in vivo drug sensitivity and in vitro susceptibility to several
anti-malarials and prevalence of multiple SNPs providing correlates for clinical resistance. Sequencing will
identify additional informative codons.
PROBLEM
Genetic diversity of malaria parasites is a major obstacle to develop rational control strategies
against this poverty related disease. Increase of health threatening drug resistance must be
monitored and prevented to protect introduced drugs.
AIM
1. to develop a sensitive, standardized tool for large-scale molecular epidemiological studies
on P. falciparum drug resistance based on a DNA Chip detecting SNPs.
2. to further document nucleotide polymorphism of genes associated with drug resistance in
field samples and develop novel markers.
3. to establish a Genetic Resistance Index (GRI) in several malaria endemic sites all over the
world.
4. to co-ordinate efforts of European molecular epidemiologists working in different field sites
to achieve a worldwide multi-locus survey of genes involved in resistance against major antimalarial drugs.
5. to transfer innovative, high throughput technology, and provide training to malaria endemic
countries to establish efficient tools for drug policy decision making.
EXPECTED RESULTS
During the initial phase of the project, we will develop our prototype SNP analysis chip into an
applicable tool. This includes the establishment of robust SOPs, inclusion of and verification of
newly identified SNP sites, as well as evaluation against currently used SNP analysis methods.
In parallel, health center and community surveys in the respective field sites will be set up by
field teams. Furthermore, studies on resistance markers for artemisinin derivatives and large
scale sequencing on known resistance markers will be conducted from the beginning of the
project onwards. As soon as health center and community surveys have been set up, in vitro
drug sensitivity studies will be performed. Upon conducting the first community surveys, samples will be prepared according to standard methods for molecular genotyping and analysed
using the produced SNP micro array, but also will be tested against RFLP based SNP analysis.
Also during this process, technology transfer will take place to two selected field sites with
appropriate infrastructure. Optimization and addition of new marker SNPs to the micro array
system will be ongoing throughout the process. Quality control and further system validation
will be performed by repetition of approximately 10% of samples for SNP micro array analysis,
but also for comparative analysis using classical SNP analysis methods.
During the analysis process, we will develop software (together with the bioinformatics units of
STI, IP, and CMDT). All data will be entered electronically into a database and upon first closure
(after the first round of surveys), the proposed genetic resistance index will be calculated and
a predictive model will be established and tested.
140
Acronym : RESMALCHIP
Keywords : DNA chips, malaria, drug resistance
Results should allow to forecast the development of malarial drug resistance through the
establishment of a community based Genetic Resistance Index, providing tools for rational
and evidence based decisions on optimal use of antimalarial drugs.
Prof. Virgílio E. do Rosário,
Centro de Malária e Outras Doenças Tropicais
Instituto de Higiene e Medicina Tropical
Rua da Junqueira, 96, P-1349-008, Lisbon, Portugal,
PARTNERS:
Dr Odile Mercereau-Puijalon
Institut Pasteur
25 rue du Dr ROUX, 75724 Paris Cedex 15, France
Dr. Hans-Peter Beck, Dr. Ingrid Felger
Swiss Tropical Institute
Socinstrasse 57, CH 4051 Basel, Switzerland
Prof. Anders Bjorkman
Karolinska Institutet, dept of Medicine
Unit of Infectious Diseases Karolinska Hospital
Stockolm, Sweden
•
•
• •
141
MILTEFOSINE FOR LEISHMANIASIS:
MOLECULAR BASIS OF MECHANISMS OF
ACTION, RESISTANCE AND COMBINATION
THERAPY
SUMMARY
This project was started to provide a comprehensive, complete and useful overview of the experiments concerning
the effects of natural anti-microbial compounds in food. To create this overview, a team of knowledge engineers
and microbiological experts worked closely together. Microbiological knowledge was extracted from literature,
and stored in a database. This database was the basis of the web-based prototype, through which the expert
knowledge can be accessed.
PROBLEM
It has been possible to generate miltefosine-resistant Leishmania relatively easily in the laboratory. Resistant parasites show cross-resistance with other antileishmanial drugs. The project
aims to uncover the mechanism (s) of resistance and design strategies to avoid this problem.
AIM
to define the molecular basis of mechanisms of action and resistance of Leishmania donovani
and to define new drug targets
to define the structure – activity relationship of miltefosine and phospholipid derivatives
against Leishmania using both extracellular promastigotes, including the identification of a target/receptor, and against amastigotes, including the determination of intracellular accumulation to the low pH phagosomal vacuole.
to examine the importance of in vitro and in vivo resistance to miltefosine developed in experimental models and to develop a strategy to avoid and/or overcome resistance through the use
of drug combinations, drug formulations and improved knowledge of pharmacokinetics in
infected models.
Miltefosine
EXPECTED RESULTS
To define the mechanism (s) of resistance of Leishmania to miltefosine and to propose drug
combinations that ensure that this problem does not arise in the clinic.
To identify new analogues of miltefosine with greater anti-leishmanial selectivity.
142
Acronym : MILTLEISH
Keywords : miltefosine leishmaniasis Leishmania donovani
resistance
The results will be used by both the pharmaceutical company that manufactures miltefosine
and by those, for example WHO, who organise clinical trials for antileishmanial drugs.
Dr Simon L. Croft
Department of Infectious and Tropical
Diseases,
London School of Hygiene and Tropical Medicine,
Keppel Street, London WC1E 7HT, UK.
Tel: +44 (0)20 7927 2345
fax: +44 (0)20 7323 5687
www.lshtm.ac.uk
•
•
•
PARTNERS:
Dr Francisco Gamarro
Consejo Superior de Investigaciones
Cientificas
c/Ventanilla 11
18001 Granada, Spain.
Tel: +34 958 805185
fax: +34 958 203323
Dr Philippe Loiseau
UPRES-EA 398, Biologie et Controle des
Organismes Parasites
Faculty of Pharmacy
University of Paris XI
Rue Jean-Baptiste Clement 3
92290 Chatenay-Malabry France
Tel: +33 1 46835553
fax: +33 1 46835557
Dr Oliver Kayser
Freie Universitat Berlin
Institute for Pharmacy
Department of Pharmaceutical Technology,
Biopharmacy & Biotechnology
Kelchstrasse 31,
Tel: +49 30 838 50689
Fax: +49 30 838 50616
Dr Daniel Perrissoud
Research Administration, Europeptides,
9 Avenue du Marais
95100 Argenteuil, France
Tel: +33 1 34 34 31 68
fax: +33 1 39 47 46 87
Dr Luis Rivas
Consejo Superior de Investigaciones
Cientificas
Velazquez 144
28006 Madrid , Spain
Tel: +34 1 5611800
fax: +34 1 5627518
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143
MOLECULAR TOOLS FOR MONITORING
EMERGENCE AND SPREADING OF DRUG
RESISTANCE AMONG NATURAL POPULATIONS
OF LEISHMANIA
SUMMARY
Leishmanias is a severe health problem in DCs and Europe. Pentavalent antimonials (SbV) will remain the first
line drug for the forthcoming years. Control is being challenged by increased SbV resistance. Our aim is to design
molecular tools for detecting emergence and spreading of drug resistance in visceral and mucocutaneous
leishmaniases. To achieve this, we will obtain samples from SbV susceptible and refractory patients (Nepal,
Bolivia, Peru), identify molecular modifications associated with SbV resistance in field isolates, transfer this
knowledge into molecular tools for drug resistance diagnosis, and define the population structure of sensitive
and resistance isolates for understanding the epidemiological dynamics of drug resistance.
PROBLEM
Leishmaniasis is serious health problem in developing countries and is emerging in Southern
Europe. Pentavalent antimonials (SbV) are the first line drug and because of their low cost, will
remain as such for the forthcoming years. Disease control is being challenged by increased SbV
resistance (up to 10% of the cases). The genetic bases of such resistance in natural parasite
populations needs to be tackled.
AIM
We aim to design molecular tools for gathering reliable information about the emergence and
spreading of drug resistance in the most severe forms of leishmaniases. To achieve this we will:
- obtain a representative collection of biopsies and parasite isolates from patients susceptible
or refractory to SbV treatment in areas endemic for anthroponotic visceral and zoonotic mucocutaneous Leishmaniases.
- identify genomic and functional modifications associated with SbV resistance in field isolates.
- develop molecular tools to diagnose drug resistance.
- identify the population structure of sensitive and drug resistant parasites for understanding
the epidemiological dynamics of drug resistance.
EXPECTED RESULTS
A collection of naturally resistant parasites, knowledge on natural mechanisms generating SbV
resistance and its transmission, PCR tools for preventing therapeutic failure and guidelines for
their use in epidemiological surveillance.
144
Acronym : LEISHNATDRUG-R
Project number : : ICA4-2001-10076
Keywords : Leishmania, surveillance
The users of our results are most likely
1. clinicians and researchers (molecular tools),
2. reference centres, Ministry of Health, surveillance networks (tools and guidelines), and
3. drug companies and researchers (collection of naturally resistant Lesihmania isolates).
Dr Jean-Claude Dujardin
Dept. Parasitology
Laboratory of Protozoology
Prins Leopold Institute for Tropical Medicine
Nationalestraat 155, B-2000 Antwerp, Belgium
T: (+32) 3 247 6358; F: (+32) 3 247 6362
PARTNERS:
B.P. Koirala Institute of Health Sciences
Dharan, Nepal
T: (+977) 2521017; F: (+977) 2520251
Centro Universitario de Medicina Tropical,
Iibismed
Universidad Mayor de San Simon
Cochabamba, Bolivia
Department of Infectious and Tropical
Diseases
London School of Hygiene and Tropical Medicine
Keppel Street WC1E 7HT, London, UK
T: (+44) 20 7636 8636
Laboratorio de Biologia de Tripanosomatidos
Instituto de Medicina Tropical Alexander von
Humboldt
Universidad Peruanan Cayetano Heredia
Lima, Peru
•
•
145
PRAZIQUANTEL RESISTANCE IN AFRICAN
SCHISTOSOMIASIS
SUMMARY
Praziquantel is the drug of choice for schistosomiasis, a disease affecting 200 million people, but recent field and
laboratory studies suggest that praziquantel resistant schistosomes have appeared. We will carry out parallel
studies in different laboratories to definitely ascertain the existence and the degree of drug resistance both in
the schistosomes that have been previously identified as resistant and in new isolates that we shall obtain from
different endemic areas. Using a combination of in vivo and in vitro approaches, we aim to produce a reliable
laboratory protocol as well as field-applicable tests for the diagnosis of praziquantel resistance. The biological
fitness and the genetics of resistant parasites will be defined, as these are crucial determinants of their potential
to spread in the population. The efficacy of alternative drugs will be determined as a measure to face possible
praziquantel failures.
PROBLEM
The drug of choice for the cure of schistosomiasis (a parasitic infection of 200 million people)
is praziquantel. Several reports have appeared indicating the development of resistance to this
drug.
AIM
To determine the existence and the magnitude of refractoriness to the major antischistosomal
drug, praziquantel, of the S. mansoni isolates that have been obtained so far from various
sources.
To isolate new parasite strains from uncured patients and assess their sensitivity to the drug.
To ascertain whether praziquantel resistance exists for S. haematobium as well.
To determine the extent of drug refractoriness during the long prepatent period of S. haematobium.
To suggest a suitable protocol for the determination of praziquantel resistance using both in
vivo and in vitro approaches.
To determine the biological fitness of resistant isolates and the mode of genetic transmission
of the trait, in order to evaluate their chances of spreading in the population.
To perform biochemical and molecular investigations aimed at the elucidation of the mechanism of praziquantel action and possibly leading to the development of field applicable diagnostic tests to detect praziquantel resistance.
To assess the efficacy of alternative drugs against praziquantel resistant parasites.
EXPECTED RESULTS
This project should provide a clear answer as to the existence and the extent of praziquantel
resistance in S. mansoni and in S. haematobium. The proposed studies will provide authenticated sensitive and resistant schistosome strains to be used as reference standards, as well as
optimised protocols for the assessment of resistance. A rational evaluation of the real danger
of the spreading of resistance in the population will be possible, based on the data to be collected on biological fitness and mode of inheritance of resistant schistosomes.
146
Acronym : SCHISTODRUG
Keywords : Schistosomiasis, praziquantel, drug resistance
Field applicable diagnostic assays of resistance should become available. The suitability of
alternative drugs will be known, a safety measure in case of potential situations of serious
praziquantel failure.
Dr Donato Cioli
Institute of Cell Biology
National Research Council
32 Via Ramarini, 00016 Monterotondo (RM), Italy
T: (+39) 0690091355; F: (+39) 0690091259
PARTNERS:
Prof Michael Doenhoff
University of Wales
Bangor, Gwynedd LL57 2UW, Bangor, UK
T: (+44) 1 248 3823; (+44) 1 248 371644
Prof Sanaa Botros
Pharmacology
Theodor Bilharz Research Institute
Warrak El-Hadar Imbaba, 12411 Giza, Egypt
T: (+20) 2 5720355; (+20) 2 5406656
Dr Amadou Mbaye
Region Medicale St Louis
BP 519, Saint Louis, Senegal
T: (+221) 961 13 88; F: (+221) 961 15 73
Dr Louis Albert Tchuem Tchuenté
Centre Schisto & Parasitology
University of Yaounde
P.O.Box 7244, Yaounde, Cameroon
T: (+237) 2210183; F: (+237) 221 50 77
•
•
Dr Dirk Engels
CDS/CPE/PVC
World Health Organization
1211 Geneva, Switzerland
T; (+41) 22 791.3824; F: (+41) 22 791.4869
•
•
147
PRAZIQUANTEL: ITS CENTRAL ROLE IN THE
CHEMOTHERAPY OF SCHISTOSOME INFECTION
SUMMARY
This project is focused on praziquantel usage and will involve a group of scientists from disease endemic countries (with a major focus on Africa). They will be linked with a group of scientists from European Member States
who share their common interest in researching the clinical, biological, epidemiological, pharmacological and
socio-economic aspects of praziquantel use for the control of schistosome infection and disease.
The project aims at providing methodological tools to deal with any outbreak of drug resistant parasites and will
develop strategies that, if implemented, will reduce the likelihood of emergence of drug resistant parasites.
PROBLEM
Praziquantel is the drug of choice for treatment of schistosomiasis and is the main tool of virtually all national and international schistosomiasis control programmes. Poor quality drugs or
drugs with lowered levels of active ingredients are factors that could lead to spurious report of
drug resistance but also, through decreased efficacy, lead to the emergence of parasites with
reduced drug sensitivity. This project aims at coordinating research on optimal use of praziquantel, in particular taking into account the problem of drug resistance.
AIM
A. To maintain a network of active partners already engaged in research on schistosomiasis, contributing available data on past, but more importantly present and
planned praziquantel use;
B. To establish 'best' practice for praziquantel use;
C. To define a set of requirements for classification of schistosome parasites as
'resistant' and support the development of reference centres;
D. To promote detailed study of schistosome strains arising from resistance monitoring;
E. To establish a defined resistant strain(s) that can be used for further investigations of the basis for praziquantel resistance;
F. To formulate policies to assist national health authorities to implement effective
control programmes using praziquantel;
G. To promote research and development of alternative drugs.
EXPECTED RESULTS
This project will allow an international concertation addressing clinical, biological, epidemiological, pharmacological and socioeconomic aspects of praziquantel, with a view to extending
its useful life-span as the prime chemotherapeutic for schistosomiasis. Praziguantel use will be
examined in the context of the use of chemotherapy for the treatment and control of schistosomiasis. Partners in the Concerted Action will be responsible for maintaining a watching brief
in their respective fields of expertise. Four annual expert committee meetings (2003-2006) will
provide a forum for the exchange of the latest research findings. Practical implications for praziquantel use will be explored. Summary reports will be distributed to the international community. The meetings will facilitate interaction with international agencies and pharmaceutical
companies. New strategies will be established for praziquantel use in the control of infection
and disease. New guidelines and protocols will be field-tested by the partners, many of whom
are in countries where vast quantities of praziquantel are being used for treatment or control.
Praziquantel-resistant parasites will be isolated and made available to the international scientific community. Samples of praziquantel will be collected to allow monitoring of drug quality.
The capacity to test praziquantel quality is an important role of this project.
148
Acronym : PRAZIQUANTEL
EC contribution : 558 000 €
Keywords : praziquantel, schistosomiasis
The concerted action will provide rational answers to questions related to the use of praziquantel in order to arm national health authorities and international agencies with appropriate information to ensure implementation of effective control programmes using praziquantel.
Prof. Paul Hagan
Division of Infection & Immunity
Institute of Biomedical and Life Sciences
University Avenue, Joseph Black Building, G12 8QQ
Glasgow, UK
T: (+44) 1413 302545; F: (+44) 1413 304600
PARTNERS:
Dr Donato Cioli
Istituto di Biologia Cellulare
National Research Council of Italy
via E. Ramarini 32, I-00016 Monerotondo, Italy
Prof. Bruno Gryseels
Helminthology Institute of Tropical Medicine
Prince Leopold
Nationalestraat 155, B-2000 Antwerp, Belgium
T: (+32) 3 247 6200; (+32) 3 237 6731
Dr Dirk Engels
Parasitic Diseases and Vector Control
Department of Communicable Diseases Control
Prevention and Eradication
World Health Organization
Avenua Appia 20, CH-1211 Geneva 27, Switzerland
T: (+41) 22 791 2111; F: (+41) 22 791 3111
Dr Moussa Sacko
Service de Parasitologie, Laboratoire Bamako
Coura
Institut National de Recherches en Sante Publique
P.O. Box 1771 Bamako, Mali
Dr Davy Koech
Centre for Biotechnology Research and
Development
Kenya Medical Research Institute
Mbaghati Road, Nairobi, Kenya
Dr Amadou Mbaye
Lutte contre les Bilharzioses
Unite de Recherche Operationnelle et Scientifique
Ministere de la Sante Publique et Action Sociale
Rue Thevenot 26, Saint-Louis, Senegal
Prof. Ezzat Hassan
Medical Research Institute
165 El Horreya, EG-21561 Alexandria, Egypt
Prof. Christopher Charles Appleton
Biology Division
School of Life & Environmental Sciences
University of Natal
George Campbell Building ZA-4041 Durban,
South Africa
Dr Maged Al-Sherbiny
Egyptian Reference Diagnostic Centre
Ministry of Health
Wezaret El Zerra 51, Agouza EG-12311 El Doqqi
Egypt +20 2 3362639
Dr Alan Fenwick
Schistosomiasis Vaccine Development
ProjectVacsera, 51 Wezaret El Zeraa Street, Agouza,
Cairo, Egypt
T: (+20) 2 3370786
Prof. SoadMokhtar
Pharmacology
Theodor Bilharz Research Institute
El-Nile Street, Imbaba, Warrak El-Hadar
EG-12411 Giza Egypt
T: (+20) 2 5409670
•
Mr Jonas Armand Kuissu
Centre for Schistosomiasis and Parasitology
Yaounde Cameroon
T: (+237) 2210183
•
•
Dr Mike Doenhoff
University of Wales – Bangor
Deiniol Road LL57 2UW, Bangor-Gwynedd, UK
•
Prof. Suad M. Sulaiman
Tropical Medicine Research Institute
El Qasser St., SD-11111 Khartoum, Sudan
T: (+249) 11 781 845
•
•
Mr Abdel Aziz Barkia
Service des Maladies Parasitaires
Direction de L'epidemiologie
et de Lutte Contre les maladies
Ministere de la Sante
Rue Ibn Al Haitam 14, Agdal, Rabat, Morocco
Prof. Rashida Barakat
Departement of Tropical Health
University of Alexandria,
High Institute of Public Health
El Horreya Avenue 165, EG-21599 Alexandria,
Egypt
Prof. Magdi Ismail
Parasitology Department
University of Zagazig,
Faculty of Medicine
Sahrkia governorate, EG-44111 Zaqazil,
El Egypt
T: (+20) 5 5234 5969
Dr Narcis Kabatereine
Vector Control Division
Ministry of Health of Uganda
P.O. Box 1661 15 Bombo Road, Kampala, Uganda
T: (+256) 41 251927
Mrs Rachel Fleetwood
Department of Clinical Veterinary Science
University of Bristol
Langford House, BS40 5DU
Bristol, UK
University of Zimbabwe
Mount Pleasant, ZW-30 Harare, Zimbabwe
149
ANTIMICROBIAL RESISTANCE TRANSFER
FROM AND BETWEEN GRAM-POSITIVE
BACTERIA OF THE DIGESTIVE TRACT AND
CONSEQUENCES FOR VIRULENCE
SUMMARY
Horizontal gene transfer is one of the main mechanism facilitating the spread of antibiotic resistance. There is a
gap in our knowledge of the extent and rate of prokaryotic gene flux as well as barriers for the expression and
maintenance of transferred genes in new hosts. This project focuses on the transfer of glycopeptide, macrolide,
and tetracycline resistance determinants between Gram-positive bacteria from digestive tract samples from
animals and human and food. The study in vitro and in vivo of: (i) the transferability and the stability of resistance
determinants; (ii) the link of resistance with virulence; (iii) the detection and characterisation of new resistance
determinants; (iv) the ability of food-ingested bacteria to pick up and retransfer resistance determinants will
further our knowledge as to how to control the spread of resistance and virulence markers.
PROBLEM
The intestinal tract provides favourable conditions for gene transfer between the resident bacteria. Dissemination of antibiotic resistance genes and virulence determinants between and
within bacteria of the digestive tract and food-ingested bacteria by horizontal gene transfer has
been documented. However, our understanding of the extent of prokaryotic gene flux between
and within these reservoirs and barriers for their transfer and expression is limited. Increased
knowledge of the transmissibility and stability of resistance/virulence markers and a better
understanding of the transfer mechanisms involved will help to predict the outcome of different strategies for controlling the spread of resistance and virulence markers.
AIM
There are four overall objectives:
to analyse the transferability of resistance genes and linked virulence determinants in Grampositive bacteria from digestive tract and food in vitro and in vivo;
to examine the stability of resistance genes in the absence of antibiotic selection in vitro and in
vivo;
to detect new antibiotic resistance genes from environmental and mammalian digestive tract
samples and determine if they are contained within mobile genetic elements ;
to investigate the ability of food-ingested bacteria, generally regarded as safe, to pick up resistance determinants and transfer them to human digestive microflora in vivo.
The analysis will focus on glycopeptide, macrolide and tetracycline resistance determinants.
EXPECTED RESULTS
The main results will be:
• Characterisation of genetic elements involved in glycoprotein, mucolide and
tetracycline resistance in strains of the digestive tract from various origins.
• Assessment of their stability, transferability and links with virulence. Role of
antibiotic selection.
• Detection and characterization of new genes conferring resistance to these
antibiotics and antimicrobial peptides.
• Assessment and analysis of horizontal transfer of these antimicrobial resistance
determinants in both directions between pathogenic bacteria and commensal
anaerobes of the digestive flora.
• Analysis of transfer of these antibiotic resistance markers to food ingested bacteria specifically lactic acid bacteria generally regarded as safe.
• Better understanding of prokaryotic gene flux in the digestive tract.
150
Acronym : ARTRADI
Project number : QLK2– 2002 - 00843
Keywords : Antimicrobial resistance genes, transferability, stability,
virulence, intestinal microbiota, food ingested bacteria
At the end of the project, the results will contribute to improving our understanding of genetic exchanges leading to the spread of both antibiotic resistance and virulence factors. The
partners will be in a position to make recommendations that will allow the implementation of
policies aimed at decreasing the risk of transfer of antibiotic resistance genes to potentially
pathogenic bacteria.
• Macroarray (macrolide and tetracycline resistance genes) to be used for the screening of
resistance genes from different environments.
• Bacterial artificial chromosome (BAC) libraries of all the different antibiotic resistance genes
present in the different European environments. These gene libraries will represent a
resource that can be screened for the production of biologically useful substances.
• Guidelines for the control of antibiotic resistance gene transfer in the digestive tract.
• Safety issues related to consumption of antibiotics.
http://www.microfun.u-psud.fr/microfun.
Prof. Anne COLLIGNON
Université Paris Sud
Unité Ecosystème Microbien Digestif et Santé
Rue jean Baptiste Clément
F-92296 Châtenay-Malabry Cedex
France
T: (+33) 1 46835529; F: (+33) 1 46835537
Web-site: http://www.u-psud.fr
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PARTNERS:
Prof. Carla PRUZZO
Universita degli Studi di Ancona
Istituto di Microbiologia
via Ranieri- Monte d'Ago
I-60131 Ancona
Italy
T: (+39) 0712204697; F: (+39) 0712204693
Dr Karen SCOTT
Rowett Research Institute
Gut Microbiology and Immunology Division
Greenburn Road, Bucksburn,
Aberdeen AB21 9SB - UK
T: (+44) 1224 712751; F: (+44) 1224 716687
Dr Peter MULLANY
Eastman Dental Institute
25 Gray's Inn Road
London WC1X8LD - UK
T: (+44) 20 79062253; F: (+44) 20 79061127
Dr Arnfinn SUNDSFJORD
University of Tromsoe
Department of Microbiology and Virology
IMB
Breivika
N-9037 Tromsoe - Norway
T: (+47) 77646202; F: (+47) 77645350
Prof. Paola MASTRANTONIO
Laboratorio di Batteriologia e Mycologia Medica
Viale Regina Elena 299
I-00161 Roma
Italy
T: (+39) 06 49902335; F:(+39) 06 49387112
Dr Maria SAARELA
VTT Biotechnology
Tietotie 2, Espoo
FIN-02044 VTT
Finland
T: (+358) 9 4564466; F: (+358) 9 4552103
Dr Gérard CORTHIER
Unité Ecologie et Physiologie du Système
Digestif
INRA-CRJJ
Domaine de Vilvert
F-78352 Jouy en Josas
France
T: (+33) 1 34652067; F: (+33) 1 34652462
Web-site: http://www.inra.fr/ENG/INRA
OVERVIEW/overview.html
151
ANTIBIOTIC RESISTANCE IN BACTERIA
OF ANIMAL ORIGIN – II
SUMMARY
This concerted action will create a network of national veterinary reference laboratories in Europe and establish
a surveillance system for monitoring the occurrence and emergence of antibiotic resistance among bacteria from
food animals. It involves 19 laboratories in 18 European countries. An external quality control for the capability
of laboratories to perform susceptibility testing of bacteria correctly will be performed. Different bacterial strains
with known susceptibility patterns will be sent four times each year to the different laboratories for testing and
the results entered into a central database. Each year the data generated in the individual laboratories will be
collected centrally and a report on the occurrence of antibiotic resistance among the different bacterial species
isolated from food animals will be generated and published.
PROBLEM
The development of antimicrobial resistance among bacteria from food animals is considered a
major public health problem in the European Union. Knowledge of the occurrence of antimicrobial resistance in the different countries is requirements to do something about the problem.
There is currently no central collection of susceptibility data from the different veterinary diagnostic laboratories in Europe. In addition, there is a lack of standardisation of susceptibility
testing methods between the different laboratories.
AIM
The main objective of this concerted action is to:
Improve the quality of the data obtained with the monitoring of antimicrobial resistance among
food animals in the European Union and utilise the results already available.
The specific objectives are to:
1. Create a network of national veterinary reference laboratories in the EU Member States.
2. Harmonise the susceptibility testing of bacteria from food animals in European veterinary
reference laboratories.
3. Collect and evaluate the susceptibility data from these laboratories.
4. Make comparable results available for the public and decision-makers.
EXPECTED RESULTS
1. An external quality assurance system for all national veterinary reference laboratories in the EU countries ensuring harmonisation of the susceptibility testing
performed.
2. A network of these laboratories.
3. Comparable and quality controlled information on the resistance situation
among food animals in the different EU countries will be known.
152
Acronym : ARBAO-II
Keywords : Antibiotic resistance, susceptibility testing, monitoring,
bacteria, animals
The ability to point out new and emerging problems with antimicrobial resistance at and early
stage could assist in performing successful interventions resulting in a higher food safety.
A reduction of the prevalence of resistant bacteria in food of animal origin will be a benefit for
the export of high safety food from the EU countries. Furthermore, and reduction of the occurrence of antimicrobial resistant bacteria will also have beneficial consequences for the
European citizen because of reduced mortality and morbidity.
Frank Aarestrup
Danish Veterinary Institute
Bulowsvej 27, DK-1790 Copenhagen V, Denmark
T: (+45) 35 30 01 00; F: (+45) 35 30 01 20
PARTNERS:
Pascal Sanders
French Agency for Food Safety (FAFS)
La Haute Mache, Javené, 35133 Fougeres, France
T: (+33) 1 49 77 13 50; F: (+33) 1 49 77 90 05
Dik Mevius
Institute for Animal Science and Health
(ID-DLO)
PO Box 65, 8200 AB, Lelystad, Netherlands
T: (+31) 320 23 82 38; F: (+31) 320 23 80 50
Christopher Teale
The Veterinary Laboratory Agency
Shrewsbury Regional Laboratory
Kendal Road, Harlescott, SY1 4HD, Shrewsbury, UK
T: (+44) 1932 35 76 93; F: (+44) 1932 35 72 14
Christian Berghold
National Referenzzentrale für Salmonellen
Beethovenstrasse 6, A-8010 Graz, Austria
T: (+43) 316 32 16 43; F: (+43) 316 38 84 70
Patrick Butaye
Veterinary and Agrochemical Research
Centre
Groeselenberg 89, 1180 Brussels, Belgium
T: (+32) 2 379 06 00; F: (+32) 2 379 04 01
Andrzej Hoszowski
National Veterinary Research Institute
Partyzantow 57, 24-100 Pulawy, Poland
T: (+48) 81 886 32 70; F: (+48) 81 886 25 95
Tuula Honkanen-Buzalski
National Veterinary and Food Research
Institute
PO Box 45, Hameentie 57, 00581 Helsinki, Finland
T: (+358) 9 39 31 600; F: (+358) 9 39 31 811
Email:
[email protected]
0
Alice Amado
Laboratorio National de Investigacáo
Veterinaria
Estrada de Benfica 701, 1549-011 Lisboa, Portugal
T: (+351) 21 711 52 98; F: (+351) 21 711 53 83
Reiner Helmuth
Federal Institute for Helth Protection of
Consumers and Veterinary Medicine
Postfach 330013, Diedersdorferweg 1, 14191 Berlin,
Germany
T: (+49) 30 84 12 22 33; F: (+49) 30 84 12 29 53
Miguel Moreno
Complutense University of Madrid
Ciudad Universitaria, 28040 Madrid, Spain
T: (+34) 91 394 35 44; F: (+34) 91 394 63 82
John Egan
Central Veterinary Research Laboratory
Abbotstown, Castleknock, 15 Dublin, Ireland
T: (+353) 1 60 72 619; F: (+353) 1 82 13 010
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Hilde Kruse
National Veterinary Institute
PO Box 8156 Dep, Ullevaalsveien 68, 0033 Oslo,
Norway
T: (+47) 22 96 46 10; F: (+47) 22 46 00 34
Luca Busani
Lab. Medicina Veterinaria
Viale Regina Elena 299, 00161 Roma, Italy
T: (+39) 06 49 902 691
Antonio Battisti
Istituto Zooprofilattico Sperimentale delle
Regioni Lazio e Toscana
Via Appia Nuova 1411, 00178 Roma, Italy
T: (+39) 06 790 991; F: (+39) 06 793 407 24
Anders Franklin
National Veterinary Institute
Travv. 12A, 75189 Upsala, Sweden
T: (+46) 18 674 000; F: (+46) 18 309 162
Katharina Stärk
Bundesamt für Veterinärwesen
Schwarzenburgstrasse 161, 3003 Bern, Switzerland
T: (+41) 31 323 95 44; F: (+41) 31 323 95 43
Maria Passiotou
Veterinary Laboratory of Chalkis
PO Box 250, 34100 Chalkis, Greece
T: (+30) 2220 10 42 521; F: (+30) 2220 10 41 421
Alda Vizbule
State Veterinary Medicine Diagnostic Cente
of Food and Veterinary Service
Lejupes 3, 1076 Riga, Latvia
T: (+371) 76 20 526; F: (+371) 76 20 434
153
PATHOLOGY AND ECOLOGY OF THE GENUS
CLOSTRIDIUM IN HUMANS, ANIMALS, AND
FOODSTUFFS: IDENTIFICATION, EPIDEMIOLOGY
AND PROPHYLAXIS
SUMMARY
The overall aim of this concerted action is to exchange across Europe the scientific knowledge and technical
know-how about the genus Clostridium to create a standardised basis for new studies and improved techniques in
all aspects concerning this bacterial genus. The project workplan includes the organisation of four workshops, the
edition of congress proceedings, scientific and technical booklets and the creation of a website.
PROBLEM
The Clostridia are a group of ubiquitous, spore-forming anaerobic bacteria. The genus comprises many species. Although some species have been studied in detail, the genus is generally not well characterised. Some of the species are pathogenic for humans and/or animals
among which those causing tetanus, botulism, enteritis, gas gangrene are the best known.
Clostridial diseases and infections are often under-diagnosed because the isolation and identification of the clostridial species can be uneasy and sometimes inaccurate. Moreover, identification of clostridial species relies, at least in part, on the demonstration of the production in
vitro of a specific toxin, or on the demonstration of the presence of their encoding genes, some
of which can be lost in vitro. The epidemiology of the diseases has been barely investigated,
because of the lack of standardised techniques for the diagnosis and identification of the
Clostridia. This standardisation across Europe and the presence of reference laboratories is a
requirement to perform valuable epidemiological studies. The mechanisms by which many
Clostridia cause disease are still not well understood. The toxins produced by the bacteria seem
to be often involved in the pathogenesis of the diseases. A knowledge of the structures and
modes of action of these toxins has already allowed their therapeutic use in some non-clostridial clinical conditions. However, some of the toxins still need to be further characterized. These
toxins also form the basis for vaccines, powerful tools in medicine, and more effective vaccines
should be developed with the help of genetic manipulation technologies. Our increased knowledge of the molecular genetics and cellular physiology of the bacteria will in turn allow
improved methods for culturing or for impairing the growth of the bacteria for diagnostic and
vaccine production purposes.
AIM
This concerted action aims to gather the scientific and technical know-how of several leading
European laboratories in the field of the genus Clostridium. Collaboration between human medicine, veterinary medicine, microbiology and food microbiology departments should make possible standardisation of techniques and protocols for epidemiological, bacteriological and
genetic studies as well as development of new vaccines. Reviewing and updating the current
knowledge on the genus Clostridium is the main purpose of this project.
EXPECTED RESULTS
Information in all the fields concerning the genus Clostridium (ecology and pathogenesis,
molecular genetics and taxonomy, virulence factors and vaccination, classical and molecular
identification and epidemiology, antibiotic resistance, food safety and spoilage, sporulation)
will be disseminated in various forms:
1. Direct exchange of information in the four meetings
2. Extended exchange of information through scientific booklets summarizing the topics of the
meetings
3. Collection of protocols and technical information in technical booklets
4. Website for permanent and easy exchange of information
154
The proceedings of the workshops and the scientific booklets will be disseminated to all partners and external participants, to veterinary and medical practitioners and routine diagnostic
laboratories, to food microbiologists, and to private companies and stock breeders, to some
Acronym : : Genus Clostridium
Keywords : Clostridium, taxonomy, toxins, pathology, molecular genetics,
vaccines, food safety, diagnosis, sporulation, epidemiology.
extent. The technical booklets will be disseminated to researchers, diagnosticians and laboratory personnel, working with Clostridium sp.
It is also the aim of the Concerted Action to disseminate the newly acquired knowledge to non
scientific communities, such as stock breeders in the veal, beef, porcine, and poultry sectors
and such as meat supermarkets, during their national and regional meetings and via seminars
organized by pharmaceutical companies.
Jacques MAINIL Université de Liège
Faculté de Médecine Vétérinaire – Bactériologie
Bât B43a - Sart Tilman, 4000 Liège, Belgium
T: (+32) 4 366 40 50; F: (+32) 4 366 41 22
PARTNERS:
Georges DAUBE
Piet DEPREZ
Joachim FREY
Frédéric CARLIN
Patrick FACH
Kriston SARRIS
Michel POPOFF
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Sinikka PELKONEN
Klaus AKTORIES
Ingo JUST
Erko STACKEBRANDT
George NYCHAS
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Benaouda KADRA
Cesare MONTECUCCO
Per E. GRANUM
Magne KALDHUSDAL
Bjorn ENGSTROM
Vladimir KMET
Maria Grazia MENOZZI
Michael PECK
Joan SMYTH
David TAYLOR
Richard TITBALL
Michel DELMEE
Alex VAN BELKUM
Karl PEDERSEN
Marina CERQUETTI
155
RAPID ANTIBIOTIC DETECTION FOR ILLEGAL
AND UNLICENSED SUBSTANCES IN ANIMAL
FEEDINGSTUFFS
SUMMARY
In the EU, human health concerns have resulted in the antibiotic growth promoters avoparcin, spiramycin, tylosin,
virginiamycin and zinc bacitracin being banned from use in animal feedingstuffs. Existing "Community methods
of analysis" for feedingstuffs are microbiologically based, non-specific, susceptible to feed component interference and unsuitable for effective policing of the ban. This project aims to develop new screening and confirmatory
tests. This twin-track approach will facilitate the effective implementation of EU policy.
PROBLEM
In relation to antimicrobial resistance, this project will play an important role in developing the
tools required to ensure that animal feedingstuffs do not contain antibiotics that may be harmful to the European consumer.
AIM
The project has five objectives: (1) The development of antisera to be employed in immunoassays for each of the growth-promoters; (2) The development, validation and interlaboratory collaborative testing of compound-specific chemical assays for determination of each of the
antibiotics; (3) The development of prototype immunoassay test kits for each antibiotic; (4) Full
validation of the prototype immunoassay test kit performance and (5) End-user testing of prototype immunoassay test kits and dissemination of chemical confirmatory methods.
EXPECTED RESULTS
Screening immunoassay dipstick tests will be developed. Using the novel idea of antiidiotype
monoclonal antisera as surrogate ligands, carbon labelled monoclonal antisera, raised to each
drug, will be employed in a competitive assay format resulting in robust, reliable and highly
specific assays, less susceptible to interference than previous immuno- or microbiological
assays. Mass spectrometric confirmatory techniques will also be developed, allowing
unequivocal identification/quantification of each antibiotic. Collaborative testing will establish
their usefulness as definitive EU methods of analysis.
156
Acronym : Feedstuffs-RADIUS
Keywords : animal, feedingstuffs, banned antibiotics, performance enhancers,
dipsticks, immunoassays, LC-MS
Successful execution of the project’s objectives will enable (a) the rapid identification and
semi-quantification of the banned feed additives in animal feedingstuffs using the screening
immunoassays produced and (b) confirmation of the identity of the banned additives by a
multi-residue mass spectrometric method. The latter method will be made available to testing laboratories free of charge in ISO format. The screening methods will eventually be commercialised by the industrial partner in the consortium. It is hoped that the resulting kits will
be suitable for ‘field’ use and will enable rapid decisions to be made about the quality/safety of animal feedingstuffs in situ (e.g. at ports, feed mills etc).
Dr John McEvoy
Department of Veterinary Science
Queen's University of Belfast
Stoney Road, Stormont BT4 3SD Belfast, UK
T: (+44) 0 28 90525606; F: (+44) 0 28 90525754
Web-site: www.afsni.ac.uk
PARTNERS:
Dr Fortüne Kohen
Department of Biological Regulation
Weizmann Institute of Science
P.O. Box 26, 2 Hertzl Street, 76100 Rehovot,
ISRAEL
T: (+972) 8 9342763 / 9343995; F: (+972) 8
9344165
Dr Martin Salden
Euro-Diagnostica BV
P.O. Box 5005, Beijerinckweg 18, NL-6802 EA
Arnhem
The NETHERLANDS
T: (+31) 263 630364; F: (+31) 263 645111
Prof Carlos van Peteghem
Laboratory of Food Analysis
University of Ghent
Harelbekestraat 72, B-9000
Ghent, BELGIUM
T: (+32) 9264 8134; F: (+32) 9264 8199
Dr Robert Schilt
Department of Residue Analysis
TNO
PO Box 360, Utrechtsweg 48, 3700 AJ Zeist, The
NETHERLANDS
T: (+31) 30694 4840; F: (+31) 30696 2513
Mr Jan Wichers
Section Ind. & Specialty BioChem
ATO
P.O. Box 17 Bornsesteeg 59, NL-6700 AA
Wageningen, The NETHERLANDS
T: (+31) 317 475330 / 478526; F: (+31) 317 475347
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DEFINING AND VALIDATING GUT HEALTH
CRITERIA IN YOUNG PIG, BASED ON DIGESTIVE
PHYSIOLOGY, MICROBIOLOGY AND MUCOSAL
IMMUNOLOGY INVESTIGATIONS FOR TESTING
ALTERNATIVE STRATEGIES TO IN-FEED ANTIBIOTICS
SUMMARY
Health degradation of pigs at weaning in intensive production systems is expected after the recent ban on in-feed
antibiotics and metals by the EU. Many alternatives with claimed effectiveness have invaded the market prior to
being fully evaluated. Our objective is to provide scientific basis for understanding the functional disturbances of
the gut which favour enteric infections. We propose to use the disturbed weaned pig as a model for defining the
major criteria for gut health. We will measure the changes occurring in gut physiology and digestion, mucosal
integrity and function, bacterial populations, and intestinal immunity, using the most modern techniques.
The ability of selected novel feed additives and rearing strategies to maintain or restore gut health will be
assessed in large pig trials using the most pertinent defined health criteria. This will contribute to set up a
uniform approach to testing alternatives at the EU level and will assist socio-economical partners in making
well-informed decisions.
PROBLEM
The HEALTHYPIGUT project is aimed at understanding the mechanisms of gut disorders and
preventing them through the use of alternatives substances to in-feed antibiotics.
AIM
The major objective of this project is to provide scientific basis for understanding the disturbances of animal gut that ultimately favour enteric infections, in order to define relevant gut
health criteria. It will be met by addressing four subsidiary objectives: 1) to characterise the
major changes in digestive physiology, microbiology and immunology occurring before clinical
diarrhoea; this will be done using models of disturbed gut in the newly-weaned pig; 2) to generate in vitro systems (microbial cultures, and tissular and cellular techniques) as tools for
future screening of novel alternatives; these systems will also be used for investigating the
mechanisms underlying the gut disorders; 3) to validate in vivo the defined gut health criteria
through testing the effect of selected new feed additives and rearing strategies in pig production trials; and 4) to foresee the economic effects of a generalised antimicrobials ban in the EU.
All this is crucial if knowledge-based strategies are to be developed by feed industries and
farmers, in agreement with consumers’ expectations and decision-makers.
EXPECTED RESULTS
The project will identify: 1) the most relevant criteria for gut health in piglets, including parameters of digestive physiology and digestion, microbiology and immunology;
2) the appropriate methodologies and needs for objective, multidisciplinary, assessment of gut health; 3) guidelines for objective assessment of novel alternatives to infeed antibiotics and metals; and 4) the consequences of adopting such alternatives
on animal health, performance, environment, market balance and social welfare.
158
Acronym : Healthypigut
Starting date :: October 1st 2001
Keywords : piglet, gut disorders, weaning, digestion, in-feed-antibiotics,
digestive physiology, digestive microbiology, mucosal immunology
Future application include a better understanding of the mechanisms of gut disorders, serving for more objective evaluation of the effectiveness and modes of action of new alternatives
to in-feed antibiotics. End users: feed industry and farmers, policy makers.
http://www.rennes.inra.fr/healthypigut/
DR LALLES Jean-Paul
UMRVP, INRA
65, rue de St-Brieuc, 35042 Rennes cedex, France
T: (+33) 2 23 48 53 52; F: (+33) 2 23 48 53 70
Web-site: http://www.inra.fr/
PARTNERS:
DR SEVE Bernard
UMRVP, INRA
35590
Saint-Gilles, France
T: (+33) 2 23 48 50 48; F: (+33) 2 23 48 50 80
DR MROZ Zdzislaw
research branch
ID-DLO
P.O Box 65, Runderweg 2, 8200 AB Lelystad, The
Netherlands
T: (+31) 320 238 238; F: (+31) 320 238 050
Web-site: http://www.id.wageningen-ur.nl/
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DR AKKERMANS Antoon
Laboratory of microbiology
Wageningen University
Hesselink Van Suchtelenweg 4, 6703 CT
T: (+31) 317 483 486; F: (+31) 317 483 829
Web-site: http://www.ftns.wau.nl/
PR STOKES Chris
Division of molecular and cellular biology
University of Bristol
Langford house
langford BS 18 7DT Bristol, UK
T: (+44) 117 928 9243; F: (+44) 117 928 9505
Web-site: http://www.bris.ac.uk/
DR ROTHKOETTER Hermann-Josef
Hannover Medical School
30623
Hannover, Germany
T: (+49) 511 532 2900; F: (+49) 511 532 2948
Wew-site: http://www.mh-hannover.de/
DR OSWALD Isabelle
laboratoire de pharmacologie et toxicologie
INRA
BP3, 180, chemin de tournefeuille, 31931
Toulouse cedex 9, France
T: (+33) 5 61 28 54 80; F: (+33) 5 61 28 53 10
DR MENGHERI Elena
INRAN
Via Ardeatina 546, 00179 Roma, Italy
T: (+39) 6 5032412; F: (+39) 6 5031592
Web-site: http://inn.ingrm.it/
DR DIRKZWAGER Annemarie
De Schothorst
Meerkoetenweg 26, 8218 NA Lelystad, The
Netherlands
T: (+31) 320 252294; F: (+31) 320 255030
Web-site: http://www.schothorst.nl/
PR SOUFFRANT Wolfgang
Department of Nutritional physiology "Oskar
Kellner"
FBN
Wilhem-Stahl-Allee 2, D-18196 Dummersdorf,
Germany
T: (+49) 382 08 68 666; F: (+49) 382 08 68 652
Web-site: http://www.fbn-dummerstorf.de/
DR DUPRAZ Pierre
Unité d'economie et sociologie rurale
INRA
rue Adolphe Bobierre, 35011 Rennes cedex, France
T: (+33) 2 23 48 56 06; F: (+33° 2 23 48 53 80
PR BOSI Paolo
DIPROVAL
University of Bologna
Via Rosselli 107, 42100 Reggio Emilia, Italy
T: (+39) 0522 290522; F: (+39) 0522 2905523
Web-site: http://www.stpa.unibo.it/
159
SPORE PROBIOTICS: AN ALTERNATIVE
TO ANTIBIOTICS IN ANIMAL HUSBANDRY
SUMMARY
This project aims at investigating how bacterial spores could be used as probiotic agents in order to reduce the
need for antibiotics against pathogenic E. coli in poultry.
PROBLEM
The overuse of antibiotics in the farming industry has profound public health implications. Of
most concern is the proliferation of drug-resistant bacteria and the transfer of these bacteria
from farm animals to humans.
AIM
Recently, Denmark has made the landmark decision of banning anti-microbials as growth promoters in pigs. This decision reinforces several key actions in the 5th framework programme to
'find antibiotic alternatives' and 'reduce their use in animal husbandry'. One option is the use
of probiotics or competitive exclusion (CE) agents. We have recently discovered that bacterial
spores are effective CE agents and can suppress the colonisation of E. coli pathogens in poultry. We have assembled a group of three spore researchers and two commercial partners, coordinated by a leading Agricultural Research facility.
EXPECTED RESULTS
We will determine how spores work as CE agents/probiotics and commercially exploit them.
160
Acronym : SPOREBIOTICS
Keywords : probiotics, antibiotics, CE agents
Spores are remarkably robust and could be mixed with animal feed.
Prof. Martin Woodward
Vetenirary Laboratories Agency
Department of Bacterial Diseases
Department for Environment, Food and Rural Affairs
Woodham Lane, New Haw, KT15 3NB
Addlestone, UK
T: (+44) 1932 357582
PARTNERS:
Prof. Antonio Barletta
Dipartimento Di Fisiologia Generale Ed
Ambientale
Universita Degli Studi Di Napoli Federico Ii
Via Mezzocannone 8, I-80134 Napoli, Italy
Dr José Monteiro
Instituto de Biologia Experimental e
Tecnologica
Quinta do Marques, Apartado 12, P-2780-901
Oeiras, Portugal
Mr Graeme Robinson
School of Biological Scienses
Royal Holloway and Bedford New College
Egham Hill, TW20 0EX Egham, Surrey, UK
Prof. Manuel Nunes da Ponte
Instituto de Tecnologia Quimica e Biologica
PO Box 127, Avenida da Republica, Estacao
Agronomica Nacional,
P-2784-505Oeiras, Portugal
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Prof. Aldo Olivieri
Consorzio Farmaceutico e Biotecnologico
Bioprogress
Bioprogress SPA
via Paduni, 240, I-03012 Anagni, Italy
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161
DEVELOPMENT OF A COMPETITIVE
EXCLUSION PRODUCT FOR POULTRY
MEETING THE REGULATORY REQUIREMENTS
IN THE EUROPEAN UNION
SUMMARY
The main objective of this project is the development of a competitive exclusion (CE) product, which should
replace the use of antibiotics as growth promoters in the industrial production of poultry. Using this product,
an economic breeding of poultry can be maintained and the expansion of antibiotic resistance genes to
human-pathogens can be restricted as well. The CE-product will consist of various bacterial strains derived from
the gut of healthy chicken. These bacteria should prevent chicks from infection with Salmonella and
Campylobacter during their first days of life. To guarantee the safety of the final product, intensive investigations
concerning the properties of the isolated microorganisms, such as the ability of transferring antibiotic resistance
genes, have to be carried out. The final composition has to correspond with the regulations for feed additives
in the EU. Further it is aimed to develop a diagnostic kit based on molecular biological methods for the
determination of certain antibiotic resistance genes in bacteria.
PROBLEM
The animal production in the EU faces a very difficult situation due to the ban of some feed
antibiotics, the foreseeable total ban within the near future and the understandable consumer
objections to their intensive use. These developments lead to a situation with a demand for
alternatives. On the one hand, to control the problems associated with increased occurrence of
infectious disease due to the ban of antibiotics (e.g. performance losses in animal husbandry,
food borne disease in humans, increased use of therapeutic antibiotics…) and on the other
hand, to meet the requirements in the European Community to guarantee safety in animal production (Commission Directive 94/40) and safe animal derived products, free of contaminants
(e.g. antibiotics, hormones, toxins…). Especially the poultry industry deals with this problem,
particularly concerning the transfer of pathogens (Salmonella, Campylobacter, E. coli) from
chickens to humans.
Commercially produced poultry lack the natural contact between chicks and mother hens, thus
providing them the natural infection stress and triggering of their immune system. As a consequence, day old chicks do not establish a microflora very quickly after hatching, thus being very
susceptible to pathogen colonisation, with special reference to Salmonella. Competitive exclusion is a promising method to protect chicks from infections with pathogenic bacteria.
Inoculation of day old chicks with undefined microflora derived from adult chicken is an effective method to protect them effectively from pathogen invasion. Using undefined CE to increase
the colonisation resistance of chicks against Salmonella has been proven in literature successfully.
AIM
The intended project will focus on the development of a defined CE-product, which in contrast
to non-defined products available in non-European markets meets the requirements for registration in the European Union. It is also planned to develop a diagnostic test kit for the determination of certain antibiotic resistance genes.
The defined CE-product developed in the course of this project will be used as feed additive in
poultry production. It can be used instead of antibiotic growth promoters to prevent poultry
from contamination with Salmonella, Campylobacter and E. coli. The diagnostic test kits will be
validated and commercialised.
162
Acronym : CEX
Keywords : Antibiotics, growth promoters, competitive exclusion,
antibiotic resistance, diagnostic test kits,
EXPECTED RESULTS
• Replacement of antibiotics in animal nutrition
• Standard operating procedure (SOP), which describes the lab process for growth of
bacteria in detail
• SOP for the production process to be able to obtain products for stability investigations and
feeding trials
• Improved stability of bacteria against humidity, temperature and pH
• Simple method for quantification of strains in fermentation, product and feed
• Method for determining bacteria resistant against antibiotics
• Test system to quantify Salmonella in feed and meat
• Suppression of pathogenic bacteria by using CE-products in comparison to control groups;
validation of test kits
Dr. Gerd Schatzmayr
R&D Department, Biomin
Industriestrasse 31A-3130 Herzogenburg, Austria
T: (+43) 2782 803; F: (+43) 2782 82330-500
Web-site: http://www.biomin.net
PARTNERS:
Dr. Roberto Kron-Morelli
Agrifutur srl
Via Campagnole 8, I-25020 Alfianello, Italy
T: (+39) 030 9934776; F: (+39) 030 9934777
Dr. Michel Van der Rest
Research and Development
Microscreen BV
L.J. Zielstraweg 1, NL-9713 GX Groningen,
Netherlands
T: (+31) 50 3166787; F: (+31) 50 3166797
Web-site: http://www.microscreen.com
Dr. Michael Hutzinger
Inovia
Durisolstrasse 7, A-4600 Wels, Austria
T: (+43) 7242 65810515; F: (+43) 7242 65810516
Web-site: http://www.inovia.at
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Eng. Josif Pazuric
SC Nutrientul
Campului Nr. 1, Ro-3720 Palota Pihor, Romania
T: (+40) 59 4161825947;F: (+40) 59 415695
Dr. Andreas Loibner
Department of Environmental Biotechnology
IFA-Tulln
Konrad Lorenzstrasse 20, A-3430 Tulln, Austria
T: (+43) 2272 66280 553; F: (+43) 2272 66280-503
Web-site: http://www.ifa-tulln.ac.at
Prof. Laszlo Babinsky
Department of Animal Nutrition
University of Kaposvar
Guba S. utca 40, H-7400 Kaposvar, Hungary
T: (+36) 82 412285; F: (+36) 82 313562
Web-site: http://www.kaposvar.pate.hu
Prof. George Zervas
Faculty of Animal Science
Agricultural University of Athens
Iera Odos 75, EL-11855 Athens, Greece
T: (+30) 210 5294411; F: (+30) 210 5294413
Web-site: http://www.aua.gr
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163
MULTI-RESIDUE DETERMINATION OF
(FLOURO)QUINOLONE ANTIBIOTICS IN FOODSTUFFS FROM ANIMAL ORIGIN BY LC-MS-MS
SUMMARY
The European Union has established safe Maximum Residue Limits (MRLs) for residues of veterinary drugs
in animal tissues entering the human food chain to safeguard human health The establishment of MRLs in the EU
is governed by Council Regulation 2377/90/EEC. Therefore, the development of highly performing analytical
techniques, such as liquid chromatography hyphenated to mass spectrometry (LC-MS-MS) detection for the
determination of antibiotics in food matrices, is requested for the implementation of the EC directives.
A complete procedure has been developed and validated by the Joint Research Centre (JRC), in collaboration with
the University of Liège, for the identification and the quantification of 11 (fluoro)quinolones simultaneously in
pig kidney.
PROBLEM
Since the fifties, the use of antibiotics in human and veterinarian medicine as well as in industrial farming has dramatically increased, leading to the appearance of antimicrobial resistances. Nowadays, bacterial infections in farm animals have to be treated with the most recently
developed drugs such as (fluoro)quinolones. On the other hand, the widespread use of antibiotics in agriculture has resulted in the potential presence of these compounds in consumed
foodstuffs such as milk, meat and eggs having therefore important consequences on public
health. Indeed, parallel resistances have been observed in hospitals where the same classes of
antibiotics are used.
AIM
To face the problem, the European Union has established Maximum Residue Limits
(MRL) for residues of veterinary drugs in animal tissues entering the human food
chain (Council Regulation 2377/90/EEC). But there is still a lack of analytical methods for the quantification of recent antibiotics such as the (fluoro)quinolones in
foodstuffs and for their monitoring as requested by the Council Directive 96/23/EC.
Taking into account the variety of samples to be investigated (liver, kidney, meat,
milk, eggs, etc) and the low detection limits required, liquid chromatography (LC)
coupled with tandem mass spectrometry (MS-MS) represents a very powerful analytical technique combining high selectivity, detection sensitivity and rapidity of
analysis.
ACHIEVED RESULTS
Recently, a complete procedure based on an LC-MS-MS method has been developed by JRC, in
collaboration with the University of Liège, for the identification and the quantification of 11 (fluoro)quinolones simultaneously in pig kidney. This recent class of antibiotics is one of the most
frequently used in industrial farming. It shows an expended spectrum that includes the Gram
positive bacteria as well as the Gram negative ones. They are indicated in the treatment of urinary, digestive and pulmonary diseases. The studied compounds are: danofloxacine,
cinoxacine, ciprofloxacine, enoxacine , enrofloxacine, flumequine, marbofloxacine, nalidixic
acid, norfloxacine, ofloxacine and oxolinic acid.
Pig kidney samples, controlled as negative with regards to antibiotics, were obtained from the
Belgian Veterinary Inspection. They were used as blank samples and spiked with standard solutions of (fluoro)quinolones of various concentrations. The analytical method involves an off-line
sample preparation by solid phase extraction followed by the chromatographic separation of
the compounds and their on-line detection in MS-MS. The use of a solid phase extraction as a
sample preparation technique not only allows the clean-up of the matrix but also in this case a
factor 4 of preconcentration for the studied compounds before analysis. An electrospray ionisation interface was selected for the LC-MS-MS coupling allowing a smooth ionisation of the
compounds. The use of tandem mass spectrometry implies the selective detection of each compound in a complex matrix by following one of its particular fragmentation reactions.
164
Acronym : Antibiotics project LC-MS-MS analysis
Contract Type : JRC
Keywords : LC-MS-MS analysis, (flouro)quinolone antibiotics, maximum residue limits
The LC-MS-MS method has then been validated at five different concentration levels in pig
kidney samples in terms of selectivity, linearity, accuracy, repeatability and estimated intermediate precision.
Of particular interest are the limits of detection (LODs) and quantification (LOQs) of the
method which were estimated to 10 and 35ng.g-1, respectively, what enables the single run
quantification of the 11 (fluoro)quinolones at a 4-fold lower concentration level than the
Maximum Residue Limit required.
EXPECTED RESULTS:
In order to support the consumer health protection policies, the LC-MS-MS analysis of antibiotics in animal tissues will very soon have to include other classes of compounds such as
macrolides, among which tylosin and spiramycin have been banned by the Commission for
animal husbandry (Council Regulation 2821/98/EC).
POTENTIAL APPLICATIONS:
An important aspect that could be studied is the control of antibiotics in the environment, i.e.
water. In this case, the matrix will be simpler but will probably contain trace levels of compounds. If so, bigger size sampling combined with a sample preparation method involving a
pre-concentration step will allow sensitive detection by LC-MS-MS analysis.
A. Rodriguez,
G. Van Vyncht,
B. Toussaint,
G. Bordin
EC-JRC-IRMM, Retieseweg,
B-2440 Geel, Belgium
e-mail : [email protected]
PARTNERS:
G. Maghuin-Rogister,
A. Janosi
Laboratory of Analysis of Foodstuffs from
Animal Origin,
Faculty of Veterinary Medicine,
University of Liège, B-4000 Liège, Belgium
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165
CARTRIDGES WITH MOLECULARLY
IMPRINTED RECOGNITION ELEMENTS
FOR ANTIBIOTIC RESIDUES MONITORING
IN MILK
SUMMARY
Bacteria exposed excessively to antibiotics may develop resistance. To ensure our future capacity in fighting
infectious diseases it is therefore of vital importance to decrease the spread of antibiotics globally. Antibiotics
are commonly used in veterinary medicine. Simple and cheap methods for their monitoring in milk and meat are
therefore needed and required by regulatory authorities. In this project, a portable microfabricated plug-in
cartridge for flow analysis of beta-lactam antibiotic residues in food will be developed. Robust synthetic polymers
molecularly imprinted with beta-lactams will serve as the recognition elements using fluorescent detection.
The device will be compared to existing methods for the analysis of beta-lactam antibiotics and will be critically
evaluated in field tests.
PROBLEM
The needs to evaluate the safety of selected veterinary drugs residues used in food-producing
animals, define acceptable daily intakes for humans, and recommend maximum residue limits
has been recognized world-wide by various public authorities. Until now, the analysis or detection of these substances is most commonly performed using microbiological methods and
physical-chemical HPLC methods in the post-screening phase. There are disadvantages to that
approach as these tests can only be performed in a well-equipped laboratory. Aside from the
fact that this approach is costly, there is a tendency to bring the "laboratory to the samples",
that is to identify critical points in the production chain and perform the control directly at these
points: e.g. production chain management, hazard analysis critical control points. To make that
a feasible approach, simple and cheap devices for on-site monitoring of critical parameters are
needed. A number of rapid tests with detection sensitivities for most b-lactam antibiotics in the
low ppb (mg/kg) range, are already commercially available for screening residues in various
biological fluids such as milk, plasma, and urine. These tests however cannot discriminate
among different beta-lactam drugs. Alternative confirmatory methods with
equivalent or better detection sensitivity are therefore needed, to confirm
the identity or identities of the specific beta-lactams eliciting presumptive
positive responses and to determine their residual concentrations.
AIM
To meet the need of alternative confirmatory methods and
practical instrumentation for on-site monitoring and discrimination of beta-lactam residues in milk, the main objective of
this project is to develop and optimize a plug-in detection cartridge supporting a molecularly imprinted polymer assay. This
cartridge will consist of a microfabricated column accommodating an optical detection window. Molecularly imprinted
polymers (MIPs) in the form of beads will be used as packing
materials and recognition elements. Analyte binding will be
detected by fluorescence. Different assay formats, labels, and
optical detection schemes will be evaluated. The best candidates will be optimized to meet the required assay specificity
and sensitivity. Participation of a national reference laboratory in the project will enable field trials of the developed cartridge in real experimental conditions and validation of the analytical method.
166
Acronym : CREAM
Keywords : analysis, antibiotic residues in milk, b-lactams,
fluorescent labels, portable microsystem, molecular imprinting
EXPECTED RESULTS
The concept of a cartridge supporting a molecularly imprinted polymer (MIP-cartridge) will be
demonstrated. State-of-the-art replication techniques in plastics will ultimately be utilised to
produce cheap and disposable cartridges. A portable instrumentation will be developed to
enable screening tests to be performed at the farm and processing dairy with MIP-cartridge
prototypes. Field trials will assess the validity of this analytical system.
This novel analytical instrumentation will be designed and optimised to allow handling by
non-chemists in non-laboratory settings. The approach relies on robust molecularly imprinted polymers as the recognition element and has therefore obvious advantages over conventional immunological and biochemical methods in terms of stability (mechanical, chemical,
and thermal). The detection of beta-lactam antibiotics in milk has been chosen as a model
system to demonstrate the general principle of the system. It is anticipated that the approach
can be applied also to other substances needed to be easily monitored in non-laboratory settings.
http://www-samlab.unine.ch/activities/projects/cream/cream.htm
Dr. Maria Kempe
Biomedical Polymer Technology Group
Dep. of Cell and Molecular Biology
Biomedical Centre, B10
University of Lund
SE-221 84 Lund - Sweden
Phone: +46 46 2220857; Fax: +46 46 2221410
Web-site: www.lu.se
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PARTNERS:
Dr. Giovanni C. Fiaccabrino
Institute of Microtechnology
University of Neuchâtel
Rue Jaquez-Droz 1 - P.O. Box 3
CH-2007 Neuchâtel - Switzerland
Phone: +41 32 7205644; Fax: +41 32 7205711
Web-site: http://www-samlab.unine.ch/
Dr. Martin French
Kalibrant Ltd.
2 Oakwood Drive
Loughborough Park - Loughborough
UK-Leicestershire LE11 3 NH - United Kingdom
Phone: +44 1509 631706; Fax: +44 1509 631739
Web-site: www.kalibrant.com
Dr. Johannes A. van Rhijn
RIKILT-DLO
Bornsesteeg 45
P.O. Box 230
NL-6700 AE Wageningen - The Netherlands
Phone: +31 317 475597; Fax: +31 317 417 717
Web-site: www.rikilt.dlo.nl
Dr. Maria C. Moreno-Bondi
Department of Analytical Chemistry
Chemistry Faculty
Complutense University
Phone: +34 91 3944196; Fax: +34 91 3944329
Web-site: http://www.ucm.es/
167
BIOSAFETY EVALUATION OF PROBIOTIC
LACTIC ACID BACTERIA USED FOR HUMAN
CONSUMPTION
SUMMARY
Probiotic bacteria, mainly lactic acid bacteria (LAB) (e.g. lactobacilli, lactococci, enterococci, and bifidobacteria)
have been considered safe for human consumption. However, recent reports of clinical infection, the spread of
antibiotic resistance genes, and development of new and/or modified probiotic LAB strains, have caused concern
of safety. This project aims to assess the biosafety of LAB. Isolates from healthy humans and immunocompromised patients, commercially available and new probiotic LAB will be studied. After polyphasic taxonomic
identification, their biosafety will be assessed by:
(1) the detection of antibiotic resistance and horizontal transfer of antibiotic resistance genes;
(2) the detection of known and new virulence properties;
(3) evaluation of immunological adverse effects of LAB; and
(4) the survival, colonisation, and genetic stability of probiotic LAB in the human gut.
The project will result in recommendations for biosafety and biosafety testing of LAB.
PROBLEM
In recent years, interest has renewed in health promotion and disease prevention by the incorporation of probiotic bacteria into foods to counteract harmful bacteria in the intestinal tract.
Most of these bacteria are part of fermented dairy products, but some are sold in the form of
powders, capsules or tablets as nutritional dietary supplements, or as biotherapeutic agents.
The bacteria used as probiotics usually belong to the genera Lactobacillus, Lactococcus,
Bifidobacterium and Enterococcus. This group of genera is also called lactic acid bacteria (LAB)
because of similar physiological and biochemical properties and the sharing of a common ecological niche: the gastro-intestinal tract. These probiotic LAB have a long history of safe use,
but recent clinical reports associate LAB with human infection in healthy and immunocompromised subjects, and there is evidence that these organisms play an important role in the spread
of antibiotic resistance genes.
AIM
The aim of the project is the safe use of probiotic Lactic Acid Bacteria (LAB) (e.g. lactobacilli,
lactococci, enterococci, bifidobacteria) for human consumption, by proposing: (1) criteria, standards, guidelines, and regulations; (2) procedures and standardised methodologies of premarketing biosafety testing and post-marketing surveillance.
EXPECTED RESULTS
Culture collection and database of probiotic and other LAB.
Standardised methodologies to detect antibiotic resistance in LAB.
Investigation of (potential) virulence properties in LAB, and their association with clinical disease and results obtained in rabbit and rat endocarditis models.
Potential immunological adverse effects of LAB.
Genetic stability and colonisation of probiotic LAB in the human gastro-intestinal gut.
Recommendations for biosafety evaluation of probiotic LAB.
168
Acronym : PROSAFE
Keywords : biosafety, probiotics, lactic acid bacteria
The progressive increase in bacterial antibiotic resistance has stimulated scientists to look for
other strategies to combat spread and effects of microbial resistance. Probiotics may provide
an alternative to antibiotics, and the safe use of probiotics might reduce the use of antibiotics
and thus the selective pressure.
The final result of the present project proposal will be a classification of potential probiotic
strains with respect to safety. It will allow the discrimination of strains that may cause problems and strains that can safely be used as a food additive, even for people with a disease.
The end user groups of these results are: academia, general food industry and starter culture
producers, consumers
http://www.proeuhealth.vtt.fi
Prof. Dr. H. Goossens
University Hospital Antwerp
Wilrijkstraat 10
2650 Edegem
Belgium
T.: 0032 3 821 37 89 ; F.: 0032 3 825 42 81
Prof. Dr. Ir. Jean Swings
University of Ghent
K.L. Ledeganckstraat 35
9000 Ghent
Belgium
T: 0032 9 264 51 16
F: 0032 9 264 50 92
Prof. Dr. Wolfgang Witte
Wernigerode Branch of the Robert Koch
Institute
Burgstrasse 37
38855 Wernigerode
Germany
T: 0049 39 43 679 246
F: 0049 39 43 679 207
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Prof. Dr. Marie-Bénédicte Romond
Université de Lille 2
3, Rue du Pr. Laguesse
BP 83
59006 Lille Cedex
France
T: 0033 320 964 037
F: 0033 320 964 037
Dr. Philippe Moreillon
Beaujon hôpital
100, Bld. Général Leclercq
92110 Clichy
T: 0033 1 40 875 227
F: 0033 1 40 875 495
Dr. Anne Mensink
NUMICO Research B.V.
Bosrandweg 20
P.O. box 7005
6700 CA Wageningen
The Netherlands
T: 0031 317 467 805
F: 0031 317 466 500
Prof. Dr. Emmanuel Wiertz
P.O. box 9600
2300 RC Leiden
The Netherlands
T: 31 71 526 39 31
F: 31 71 524 81 48
169
ANTIMICROBIALS ONLINE
SUMMARY
This project was started to provide a comprehensive, complete and useful overview of the experiments concerning
the effects of natural anti-microbial compounds in food. To create this overview, a team of knowledge engineers
and microbiological experts worked closely together. Microbiological knowledge was extracted from literature,
and stored in a database. This database was the basis of the web-based prototype, through which the expert
knowledge can be accessed.
PROBLEM
Food safety, quality, convenience and environmental awareness are predominant issues in the
modern food industry. Manufacturers have to produce a wide range of minimally processed
foods that are safe to eat and have long shelf lives, compatible with the infrequent shopping
habits of the modern consumer. On the other hand, the same consumer is increasingly reluctant to accept artificial, synthetic food preservatives. This has re-awakened interest of the
food industry to exploit some of the antimicrobial compounds known to exist in nature.
However, the scientific literature on the subject of natural antimicrobial systems has now
become so extensive that a cursory search is insufficient to assess the practical potential of
any single antimicrobial compound in foods. Furthermore, there is a need to distinguish
between antimicrobial properties demonstrated in laboratory systems and those in real foods
where interactions can have a profound effect on activity. The burgeoning research literature
and the explosive expansion in unverified information available on the Internet have together contributed to information obscurity for many food professionals. Consequently, much of
the existing knowledge generated in research laboratories is not being transferred to industrial practice. Moreover, no platform exists for collecting and structuring new knowledge.
AIM
The main aim of this project is to provide structured information on the properties and application of natural antimicrobial compounds through a web-based expert knowledge system.
EXPECTED RESULTS
A web-based expert knowledge system for storing and exchanging
knowledge on natural antimicrobial systems for use as preservatives in
foods, initially accommodating four compounds.
Notable features will include:
• Information on chemical structure, physical properties, stability, biological activity in vitro,
model systems and in foods, dependency of activity on environmental factors, interactions
with other food preservatives and food components, mode of action, production statistics,
toxicity and regulatory status.
• Full reference details for each primary paper from which data extracted.
• Simple searching options to enable the identification of compounds by source, common
name, chemical name and/or a combination of all of these
• Protocol (proposed standard) for sharing information on antimicrobials and other compounds.
170
Acronym : A/OL
Contract Type : Accompanying measure
Keywords : antimicrobial database
The results will be presented and searchable via a web-based user-interface. A prototype of
this interface can be found on the web-site www.ato.dlo.nl/AOLKnowledge/index.asp
The overview of the applicability of anti-microbial compounds in various food types will help
food industry and food professionals to design new and reformulate old food products in line
with changing consumer demands without compromising safety and quality. Researchers in
the field of food science will be able to search for gaps in the knowledge of food safety and
they will thus be able to perform research in this area in an efficient way.
Beneficiaries will be:
• The food industry.
• Food professionals in retailing, government and academia.
• Research scientists in industrial and academic R&D (by identifying gaps in knowledge).
• Research funding bodies (by identifying research needs).
• Regulatory authorities including the future European Food Standards Authority.
• Consumers (by widening the availability of minimally processed food products and, if this
study is successful and the system is expanded as suggested above, by providing easy
access to user-friendly information on food preservatives and processes).
www.ato.dlo.nl/aol/index.asp for general information on the project.
http://www.ato.dlo.nl/aolKnowledge/index.asp for a view of the prototype.
Prof. Dr. Jan Top
Production & Control Systems
ATO
P.O.Box 17, Bornsesteeg 59, 6700 AA
T: (+31) 7 475314; F: (+31) 7 475347
Web-site: www.ato.dlo.nl
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171
SAFETY EVALUATION OF HORIZONTAL GENE
TRANSFER FROM GENETICALLY MODIFIED
ORGANISMS TO THE MICROFLORA OF THE FOOD
CHAIN AND HUMAN GUT
SUMMARY
The project addresses horizontal gene transfer (HGT) of marker genes from genetically modified (GM) food crops
and derived products to bacteria in the food chain. Frequencies of HGT as well as the impact of HGT will be
determined for a quantitative risk assessment of GM crops. In addition food models and models of the
gastrointestinal tract will be evaluated for analysing DNA integrity and HGT.
PROBLEM
Antimicrobial resistance is a serious problem challenging the clinical efficacy of antibiotics.
Antibiotic resistance is spreading due to various mechanisms of which the use of antibiotics is
certainly the most important one. An additional spread of antibiotic resistance may be caused
by the application of antibiotic resistance markers as selectable markers for the genetic modification of food crops. Such markers may become introduced into bacteria of the microflora of
the food chain including the gastrointestinal (GI) tract of man via HGT.
AIM
The aim of the project is to provide a quantitative risk evaluation based on scientific knowledge
and evidence concerning the frequency and consequence of HGT of marker genes from GM food
crops to bacteria present in the food chain and human gut. An additional aim is the development and validation of model systems to study HGT.
EXPECTED RESULTS
Food and GI tract model systems will be established to study the integrity of DNA and HGT.
Transfer of marker genes from GM crops to bacteria in the food chain can only take place via
transformation events. Subsequent transfer of acquired marker genes from transformed bacteria may be accomplished via transformation and conjugation events. Data on both transfer
mechanisms are envisaged results of the project. Prerequisite for transformation is the presence of biologically functional DNA as well as the development of competence for transformation in the recipient bacterium. Data on the integrity of DNA and competence development in
food and GI tract model systems will be collected. Since transfer of marker genes were anticipated to be extremely rare events, sensitive detection procedures base on PCR and cell sorting
systems will also be a result of the project. A quantitative risk assessment will be based on the
data collected in the model systems in combination with exposure assessment and the consequence (hazard) of gene transfer. The spread of antibiotic resistance genes in nature that are
used for GM crop development will be estimated together with the nature of the resistance
gene in relation to the therapeutic significance of the antibiotic to assess the consequence of
gene transfer.
172
Acronym : GMOBILITY
Starting date : March 1st 2000
Keywords : genetically modified organisms, food chain, horizontal
gene transfer, antibiotic resistance genes, safety assessment,
gastrointestinal tract, intestinal microflora
The GI tract model systems can be used for analysing DNA degradation. In a broader perspective, the GI tract model systems can be used to analyse the digestion of food products
including the analysis of the availability of micro- and macro-nutrients. The models were
shown applicable for analysing conjugative transfer of antibiotic resistance genes between
bacteria. The models can also be used to analyse the fate of the microflora in the presence of
antibiotic residues. An essential part of the research can therefore be implemented for further
studies on the spread of antibiotic resistance and the impact of antimicrobials on the composition of the intestinal microflora and intestinal health. The risk assessment can be implemented in policy making. The results are particularly useful for the scientific community as
well as governmental bodies.
www.entransfood.com/RTDprojects/GMOBILITY/gmobility.html
Dr. Jos van der Vossen
Risk Management and Microbiology
TNO Nutrition and Food Research Institute
PO BOX 360, Utrechtseweg 48, 3700 AJ
Zeist, The Netherlands
T: (+31) 30 6944720; F: (+31) 30 6944901
Web-site: www.voeding.tno.nl
PARTNERS:
Dr. Jos van der Vossen
Risk Management and Microbiology
TNO Nutrition and Food Research Institute
PO BOX 360, Utrechtseweg 48, 3700 AJ
Zeist, The Netherlands
T: (+31) 30 6944720; F: (+31) 30 6944901
Dr. Henk Aarts
RIKILT-DLO
PO BOX 230, Bornsesteeg 45, 6700 AE
T: (+31) 317 475604; F: (+31) 317 417717
Web-site: www.rikilt.wageningen-ur.nl
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Prof. Dr. Walter Hammes
Institute of Food Technology
University of Hohenheim
Garbenstrasse 28, 70599 Stuttgart, Germany
T: (+49) 711 4592305; F: (+49) 711 4594199
Web-site: www.uni-hohenheim.de
Dr. Gerard Corthier
UEPSD-Fonctions des Bacteries Intestinales
Institut National de Recherches Agronomiques
Batiment 440-CRJ-INRA, 78350
Jouy-en-Josas, France
T: (+33) 1 34652467; F: (+33) 1 34652462
Web-site: www.inra.fr
Dr. Birgit Nørrung
Division of Microbiological Safety
Danish Veterinary and Food Administration
Mørkhøj Bygade 19, 2860 Søborg, Denmark
T: (+45) 33 95 61 80; F: (+45) 33 95 66 98
Web-site: www.vfd.dk
Dr. Alexander Haslberger
Institute for Microbiology and Genetics
University Vienna
Dr. Bohrgasse 9, A-1030 Vienna, Austria
T: (+43) 1 31991484 ext 203; F: (+43) 1 319914884
ext 234
Web-site: www.univie.ac.at
Dr. Margaret Davies
Molecular Science Departement
TNO BIBRA Interantional Ltd
Woodmansterne Road, Carshalton SM5 4DS
Surrey, UK
T: (+44) 20 8652 1025; F: (+44) 20 8661 7029
173
AN ECOLOGICALLY SAFE SELECTION SYSTEM
FOR TRANSGENIC CROPS BASED ON MODIFIED
PLANT-TUBULIN GENES
SUMMARY
A selection system is proposed for plant transformation that is exclusively based on genetic information already
present in the host plant and that does not require antibiotic resistance genes. Driving force for this approach is
the public concern about a potential spread of antibiotic resistance genes in soil and intestinal bacteria possibly
accentuated by the agricultural use of transgenic crops. The project is based upon truncated tubulins lacking the
binding site for carbamates. The homologous tubulin promotor within a DNA fragment drives the expression of
this truncated tubulin devoid from exogenous sequences (minimal expression unit). The suitability of this
selection system is demonstrated for rice as a model system for Graminean crops.
PROBLEM
There is a potential risk for spread of antibiotic resistance genes in soil and intestinal bacteria
as a result of use of antibiotic genes as markers, also giving rise to major public concern.
AIM
1. Production of a selection marker cassette (tubulin promotor, coding sequence coding for
alpha-tubulin conferring resistance to carbamate herbicides, and full-length beta-tubulin).
2. Transformation of rice with this construct and establishment of a selection and transformation protocol based on carbamates.
3. Cotransformation of the selection marker cassette with rice phytochrome as model test and
assays for expression and functionality of the transgene.
4. Use of “clean DNA” technology to avoid extraneous backbone and vector sequences.
The tasks will include:
A. Adaptation of molecular tools
1. Coding sequences and promotors for different rice alpha- and beta-tubulins
2. Designed variants of rice alpha-tubulin with truncated or modified C-terminus
3. Tandem vector made of truncated rice TUBA1, rice beta-tubulin, and different promotors
(CaMV-35S for moderate, rice CDPK for low expression and the homologous rice TUBA/B promotor)
B. Homologous transfection into rice embryos
1. Tests for conferred carbamate resistance
2. Correlation of resistance levels to expression level of the construct and adjustment of the
selection regime
3. Check for potential changes in cold resistance
C. Cotransfection with rice phytochrome as candidate gene
1. Transformation by the clean-DNA technology
2. Tests for phytochrome expression and functionality in transformed calli
3. Tests for stability of carbamate resistance and phytochrome overexpression during development and propagation of transgenic plants. Checking for potential negative effects of tubulin expression on plant development.
174
Acronym : EcoTub
Starting date : 1 October 2000
Keywords : plant transformation, tubulin
EXPECTED RESULTS
1. Transformation cassettes: homologous tubulin promotor driving balanced expression of
tubulin dimers that confer carbamate resistance.
2. Carbamate-resistant rice plants that do not contain any DNA sequences of foreign origin.
3. A selection protocol based on carbamate and possibly cold resistance.
4. Rice plants that overexpress phytochrome and are carbamate-resistant and transformation
vectors for phytochrome over-expression.
5. Data on trait stability, regulation of the tubulin system and potential side effects of the
selection marker.
A novel plant transformation system based on genetic information already present in the host
plant.
PD Dr Peter Nick
Institut für Biologie II
Albert-Ludwigs-Universität Freiburg
D-79104 Freiburg, Germany.
T: (+49) 761 203 2646/2669
F: (+49) 761 203 2612
PARTNERS:
Dr Diego Breviario
Istituto Biosintesi Vegetali
Centro Nazionale di Ricercha
I-20133 Milano, Italy
T: (+39) 02 2369 9441
F: (+39) 02 2369 9411
Dr Paul Christou
John Innes Centre
Norwich Research Park
NR4 7UH, Norwich
UK
T: (+44) 1603452 571
F: (+44) 1603 456 8442
•
•
•
175
SEA LICE RESISTANCE TO
CHEMOTHERAPEUTANTS: DIAGNOSIS,
MECHANISMS, DYNAMICS AND CONTROL
SUMMARY
Throughout Northern Europe, the salmon louse, Lepeophtheirus salmonis, seriously affects the marine phase
of Atlantic salmon production. The development of sustainable methods of pest management has been unable
to keep pace with the intensification of production, leading to excessive and very fragile reliance on very few
chemotherapeutants, a situation that may promote development of resistance in the parasites.
PROBLEM
Resistance against organophosphates has been demonstrated in sea lice in important
salmon producing countries, and resistance against pyrethroids and avermectins
seems to be emerging. Most reports of clinical failures of control agents are though of
an anecdotal nature. Unless they are verified as cases of resistance using validated
diagnostic methods, control actions are difficult. Possible actions should be based on
knowledge of the underlying mechanism in order to be effective. A thorough knowledge
of the dispersion of the genetic material between salmon farms is vital for the assessment of the risk posed by resistance development in sea lice. The main objective of this
project is, by a multidisciplinary effort involving scientists and aquaculture industry in
Norway, UK, Ireland and Canada, to develop strategies for identification and control of
resistance development in sea lice.
Adult female sea lice (Lepeophtheirus
salmonis) on a salmon
AIM
The aims of the project are:
1) to develop biochemical and toxicological methods (bioassays) for early detection of reduced
sensitivity to the pyrethroids cypermethrin and deltamethrin, the organophosphate azamethiphos, the avermectin emamectin and the chitin synthesis inhibitor teflubenzuron,
2) to study the underlying mechanisms using biochemical and molecular biology techniques,
3) to monitor sensitivity against the available chemotherapeutants in Norway, Scotland, Ireland
and eastern Canada,
4) to study the dispersal of genetic material between individual farms under different control
strategies by developing and applying microsatellite PCR-methods
5) to propose control strategies for handling resistance problems based on the knowledge generated through the project.
Evaluation of bioassay results
EXPECTED RESULTS
The project is expected to result in:
1) validated protocols for the diagnosis of resistance in sea lice,
2) a thorough description of underlying mechanisms (e.g. altered detoxification, target
site mutations),
3) an overview of the sensitivity towards the available control agents in Norway,
Scotland, Ireland and eastern Canada,
4) a genetic population structure analysis on lice from adjacent salmon farms within
fjords (Norway), in defined single bay management schemes (Scotland and Ireland),
and in a location with unusual hydrogeography (Canada).
176
Acronym : SEARCH
Keywords : Sea lice, resistance, pyrethroids, organophosphates,
avermectins, teflubenzuron, bioassays, mechanisms, gene-flow
The basic diagnostic protocols will be established in each of the participating countries, and
will provide the industry and the authorities with knowledge-based tools to handle cases
where resistance has developed. The project will help to establish strategies that minimize
the risk of eroding the agent’s effect, and with minimal consequences for the industry and the
environment.
http://www.iacr.bbsrc.ac.uk/pie/search-EU/
Dr Tor E. Horsberg
The Norwegian School of Veterinary Science
P.O. Box 8146, Dep.N-0033 Oslo, Norway
T: (+47) 2 296 4983; F: (+47) 2 296 4752
PARTNERS:
Dr R Powell
National Unversity of Ireland
Galway, Ireland
Dr Greg Devine
IACR-Rothamsted Experimental Station
UK
Mr Paal Haldorsen
Hydro Seafood AS
Hundsnes, Norway
Mr Andrew Grant
Marine Harvest Scotland Ltd.
Fort WilliamUK
•
•
Mr John Burka
University of Prince Edward island
Charlottetown, Canada
•
177
IMPROVED PROCEDURES FOR FLATFISH
LARVAL REARING THROUGH THE USE OF
PROBIOTIC BACTERIA
SUMMARY
This project provides a new and systematic approach to improving the rearing conditions of larval turbot and halibut by using biological, non-antibiotic dependent measures of disease control. Bacteria, which have the potential
to inhibit the proliferation of damaging bacteria during the early rearing stages of turbot larvae, will be identified.
This will be beneficial by improving health and disease resistance of the larvae as well as in reducing the use of
antibiotics in larval rearing. The bacteria will be tested for their ability to inhibit known larval pathogens in laboratory scale trials before testing the applicability in commercial hatcheries. The results will be transferable to
many other larval rearing systems in aquaculture.
PROBLEM
Strategies to control bacterial growth (particularly the growth of unwanted bacteria)
in intensive rearing of marine fish larvae have frequently resorted to the prophylactic use of antibiotics, with the attendant problems which arrive when antibiotic
resistance develops in bacteria. This problem is not limited to the larval culture system as resistance can often be transferred to micro-organisms in other environments. Subsequently such resistance may spread to bacteria causing disease in
humans. We aim to reduce the reliance on antibiotics by developing methods that
can positively regulate the microbial flora of flatfish by other means.
Inhibition plate assay
AIM
The project aims to identify promising bacteria that can be used to regulate the bacterial
microflora during rearing of larval turbot and thereby improve larval survival and health. After
development in the laboratory the effectiveness of this approach will be tested on an industrial scale. Large numbers of bacterial isolates from a range of sources will be provided which are
suitable for use as probiotics of larval flatfish. These will be screened using in vitro tests to
identify those suitable for use as probiotics, and promising candidates as probiotics will be
tested for their effects on larvae and in disease control during larval rearing on a small scale.
Methods for delivery of such bacteria to larvae will be optimised and the most promising candidate probiotics will finally be tested on an industrial scale.
It is intended to transfer the
science-based knowledge and understanding of bacterial interactions with marine fish larvae
to practical use in industrial-scale production sites for turbot and other species of fish.
EXPECTED RESULTS
The project is expected to identify a large number of bacteria that could potentially be used in aquaculture to improve larval rearing. The mechanisms by which
bacteria cause losses in larval turbot rearing as well as the mechanisms which
potential probiotics inhibit their actions will be determined and this fundamental
knowledge will be important for developing future strategy in this area. The route
by which probiotic bacteria need to be delivered to larval rearing systems for optimum protective effect will be determined and this requires study of their stability,
efficiency of delivery via water or live food prey organisms, levels required in the
rearing system and frequency of application. The methods investigated in laboratory scale trials will be chosen so that they can be applied to commercial scale
rearing in due course.
Mette turbot
178
Acronym : PROBE
Project number : Q5RS-2000-31457
EC contribution : 1270000 €
Keywords : aquaculture, probiotics, antibiotics, turbot,
larval rearing, bacteria
The expected benefits to the industry should include improved health, disease resistance and
welfare of fish; enhanced survival and better reproducibility between batches of fish; reduced
use of antibiotics and, consequently, reduced environmental impact; rapid transfer of laboratory findings to commercial scale culture; the results gained will allow industry to become
more competitive and potentially lead to significant expansion and economic growth of the
flatfish aquaculture industry; application of the work to other larval rearing systems;
increased food safety through minimising the use of antibiotics in the food chain.
http://www.gla.ac.uk/probe
Dr Harry Birkbeck
Institute of Biomedical and Life Sciences
Joseph Black Building
University Avenue
Glasgow G12 8QQ
Scotland
T: (+44) 141 330 5843, F: (+44) 141 330 4600
Web-site: www.gla.ac.uk
•
•
•
•
PARTNERS:
Dr Øivind Bergh
Department of Aquaculture
Institute of Marine Health
PO Box 1870
Nordnes
N-5187 Bergen
Norway
T: (+47) 55236370; F: (+47) 55236379
Web-site: www.imr.no
Prof. Lone Gram
Department of Seafood Research
Danish Institute for Fisheries Research
Danish Technical University
Building 221
DK-2800 Lyngby
Denmark
T: (+45) 45252586; F: (+45) 45884774
Web-site: www.dfu.min.dk
Dr Jorunn Skjermo
Fisheries and Aquaculture
SINTEF
Brattoera
Gryta 2
N-7465 Trondheim
Norway
T: (+47) 73596367; F: (+47) 73596363
Web-site: www.sintef.no
Mrs Ana Riaza
Research and Development
Stolt Sea Farms SA
Lira
E-15292 Carnota, La Coruna
Spain
T: (+34) 981837501; F: (+34) 981761031
Web-site: www.stoltseafarm.com
Dr Miquel Planas
Instituto de Investigaciones Marinas
Consejo Superior de Investigaciones Cientificas
Eduardo Cabello 6
E-36208 Vigo
Spain
T: (+34) 986214457; F: (+34) 986292762
Web-site: www.csic.es
179
HAZARD ANALYSIS OF ANTIMICROBIAL
RESISTANCE ASSOCIATED WITH ASIAN
AQUACULTURAL ENVIRONMENTS
SUMMARY
Aquaculture is a mainstay of South East Asian economies, providing high quality food for local consumption and
export. The EU is an important market for South East Asian aquaculture products. Much of the industry has relied
on antibiotics to control disease, and this project is concerned with assessing the risk to the farmer and to the
consumer posed by potential antibiotic resistance in bacteria in the farmed species and in their environments.
It also aims to develop and promulgate control protocols to minimise those risks.
PROBLEM
Aquaculture, like other forms of animal agriculture, has hitherto relied heavily on antibiotics to
control infectious diseases caused by bacteria. The industry in South East Asia is very diverse.
It comprises both small-scale single household operations that are often part of integrated systems, and extremely large and intensive operations that are part of large corporations. Bacterial
diseases are a major problem in aquatic species, and especially so in the kinds of intensive
farming systems that have developed. The nature of the species being farmed, high density
stocking rates, and the nature of the farming environment, all compound to make the control of
these diseases difficult. Not surprisingly, wide scale antibiotic usage has underpinned disease
control measures, mostly through administration in feeds. Inevitably, there is a level of environmental contamination with the antibiotics. This poses a risk that resistance to antibiotics
will develop in environmental organisms, as well as in the endogenous bacterial population of
the farmed species, and in their pathogens. Antibiotic resistant bacteria in turn constitute a
direct threat to farmers, consumers and other forms of livestock that are often farmed in association with aquatic species, through potential transfer of resistance to human and animal
pathogens. It also threatens the viability of export markets that are increasingly sensitive to
food safety concerns. The recent rejection by the EU of some Asian aquaculture products containing chloramphenicol, is a case in point.
AIM
Assessment of the extent of antibiotic resistance in the aquaculture environment in South East
Asia.
• Identification of the resistance genes involved.
• Assessment of the potential for transfer of antibiotic resistance from the aquaculture environment to possible human pathogens.
• Identification of critical control points (CCP) where south East Asian fish farmers can apply
monitoring systems to prevent or eliminate antibiotic resistance.
• Dissemination of control protocols among the farming and disease control communities in
SEA.
EXPECTED RESULTS
• Standard Operating Procedures for sampling, isolation and antibiotic susceptibility testing
• A collection of antibiotic resistant bacterial isolates derived from aquaculture sites of economic importance in Malaysia, Thailand and Vietnam.
• Reliable identification and classification of antibiotic-resistant freshwater and marine isolates.
• Full taxonomic description of any new bacterial taxa.
• Resistance to antimicrobials determined for bacterial populations from different compartments of South East Asian aquaculture ecosystems.
• Quantitative data on antimicrobial susceptibility of a heterogeneous bacterial collection.
• Plasmid profiles of antibiotic-resistant isolates originating from diverse geographical and
aquaculture environments.
• Distribution of resistance genes among a sub-set of antibiotic resistant plasmid-harbouring
bacterial isolates.
180
Acronym : Asiaresist
Keywords : antibiotic, chloramphenicol, resistance, aquaculture,
Asia, gene, hazard analysis, HACCP.
• Assessment of transferability of resistance factors determined for bacterial strains isolated
from the aquaculture ecosystem.
• Definition of Critical Control Points for monitoring transferable antimicrobial resistance at
aquaculture sites, which can form the basis of Hazard Analysis Critical Control Point
(HACCP) systems applicable to aquaculture.
• A core public data base dealing with monitoring of antibiotics in aquaculture environments
On completion of this project, agencies in South East Asia responsible for environmental monitoring, farming regulation and food safety, will be better informed about antibiotic resistance
in aquaculture environments, and better placed to advise on, and, where appropriate, to
implement control procedures. The consumer will have greater assurance that food originating on South East Asian fish and prawn farms will not carry antibiotic resistant bacteria that
could pose a threat to health. Consequently, the international markets for the products of
South East Asian aquaculture will be strengthened, to the benefit of both the producers and
the consumers of potentially very high value foods.
http://www.dist.unige.it/asiaresist
Prof. Alan Teale
Institute of Aquaculture
University of Stirling
Stirling FK9 4LA, UK
T: (+44) 1786 467872; F: (+44) 1786 472133
Web-site: www.stir.ac.uk
•
PARTNERS:
Prof. Jean Swings
Ghent University
Ghent University,
K.L. Ledeganckstraat 35, Gent, B-9000 Belgium
T: (+32) 9 2645116; (+32) 9 2645092
Web-site: http://lmg.rug.ac.be/
Dr. Thanh Phuong Nguyen
Department of Applied Aquaculture and
Fisheries Biology
College of Aquaculture and Fisheries,
Cantho University
Campus 2, Cantho University
3-2 Street, Cantho, Vietnam
T: (+84) 71 830931; F: (+84) 71 830247
Web-site:
http://www.ctu.edu.vn/colleges/aquaculture/
•
•
Prof. Mohamed Shariff
Faculty of Veterinary Medicine
University Putra Malaysia
43400 Serdang, Selangor, Malaysia
T: (+60) 3 89468288; F: (+60) 3 89488246
Web-site: http://www.upm.edu.my
Dr Supranee Chinabut
Department of Fisheries
Aquatic Animal Health Research Institute
Kasetsart University Campus, Jatujak,
Phaholyothin Rd. Jatujak 10900 Bangkok, Thailand
T: (+66) 2 5796803; F: (+66) 2 561 3993
Web-site: http://www.fisheries.go.th/
Dr Stefania Bertone
Software Development Division
R.I.L.A.B. s.r.l. Research and Informatics for the
Environmental and Biomedical Laboratory
Via Guerrazzi 24/12B, 16146 Genova, Italy
T: (+39) 10 3626606; F: (+39) 10 3626606
Web-site: http://www.rilab.it
Prof. Dr. Carmelina Mauro Ruggiero
Giacomini
DIST – Medinfo
University of Genova
Via Opera Pia 13, 16415 Genova, Italy
T: (+39) 10 3536546; F: (+39) 10 3532154
Web-site: http://www.medinfo.dist.unige.it
181
RESIST ON RESISTANCE: MOBILISING
THE RESEARCH EFFORTS FOR COMBATING
MULTI-RESISTANCE AGAINST ANTIBIOTICS
SUMMARY
This project addresses the urgent need to create favorable conditions for making scientific breakthroughs in the
combat against multi-resistance against antibiotics and the development and implementation of a European
policy aimed against the resistance problem. The objectives are to identify the different ongoing and foreseen
European research activities and investigate modalities for implementing an integrated and co-ordinate a
European program multi-resistance against antibiotics.
PROBLEM
There is a growing awareness in the scientific and public health community that multi-resistance against antibiotics is an increasing threat. Bacteria resistant to a large variety of antibiotics are responsible for large proportion of infections worldwide. This pandemic is caused by
the inappropriate use of antibiotics in human and veterinary medicine, and often by the lack of
hygienic standards in hospitals and other health care facilities. For example, an unacceptable
15% of patients in European hospitals are infected with multi-drug resistant infections.
Patients may even die as a result of infections caused by (multi-) resistant strains. This amount
will dramatically increase if no appropriate strategies are developed. However, this requires
scientific breakthroughs and another approach to organizing joint research efforts.
There are a number of research efforts focused on combating multi-resistance but apparently
these are not sufficient to make a breakthrough in the combat against multi-resistance: there
is an urgent need to mobilize and co-ordinate research efforts and concentrate them in large
integrated research programs.
The emergence of new resistant clones is a natural process that limits the use of any newly
developed antibiotic. Moderation of antibiotic consumption must be implemented, but it is
unlikely to result, on its own, to a significant decrease in resistance rates, due to the stability
of resistance mechanism even in the prolonged absence of antibiotics.
Bacterial strains, which have acquired resistance mechanisms, can indeed survive for large
periods of time without selective pressure from antibiotics. The spread of these strains should
have serious focus. These strains can also become disseminated to new patients, hospitals
and communities, often crossing borders to achieve pandemic status. Therefore, antibiotic
resistance will continue to increase if no measures are taken to control the dissemination of
these resistant microorganisms.
AIM
The main objective is to investigate the feasibility of a joint European effort on anti-bacterial
resistance. The overall goal is to evaluate whether combining and organizing the research
efforts in the field of multi-resistance against antibiotics is feasible and to describe the critical
conditions for an effective and purposive European approach. The objectives can be translated into objectives focusing on scientific and technological aspects and specific objectives
focusing on the favorable conditions for these scientific break-throughs.
EXPECTED RESULTS
ROR results in a Knowledge Survey on which institutions in Europe are involved, how a combined and organized approach of European research efforts can be created. Moreover, the creation of a favorable environment which paves the way for further elaboration, concrete establishment and implementation of a future structure which is able to make strong steps forward.
182
Acronym : ROR
Contract Type : Accompanying Measure
Keywords : Multi-resistance, antibiotics, mobilization, surveillance,
hospital hygiene, antibiotic consumption
- Better standard of care;
- Development of new antimicrobial agents.
Prof. Dr. Jan Verhoef
Eijkman-Wonkler Institute, UMC Utrecht
Postbus 85500
Heidelberglaan 100
3584 CX
T: +31-30-250.7625;
F: +31-30-254.1770
PARTNERS:
Prof. Dr Jos Van der Meer
Dept. of Internal Medicine
University Med. Centre St. Radboud
P.O. Box 9101
Geert Grooteplein 8
6500 HB
Nijmegen
Netherlands
T: +31-24-361.8819; F: +31-24-354.1734
Dr David Livermore
Antibiotic Resistance Monitoring and
Reference Laboatory
PHLS Central Public Health Laboratory
61 Colindale Avenue, NW9 5HT
London, UK
T: (+44) 20 8200.4400; F: (+44) 20 8358.3292
Prof. Dr. Richard Wise
City Hospital NHS Trust
Dudley Road B18 7QH Birmingham, UK
T: (+44) 121 507.4255; F: (+44) 121 551.7763
• •
• ••
•
•
Prof. Dr. Waleria Hryniewicz
Sera and Vaccines Central Research
Laboratory
30/34 Chelmska str., 00 - 725 Warsaw, Poland
T: (+48) 22 841.3367; F: (+48) 22 841.2949
Prof. Dr. Jiri Schindler
Charles University
3rd Faculty of Medicine
Ruská 87, CZ 100 00 Prague, Czech Republic
T: (+420) 2 671.6253; F: (+420) 2 6716.2516
Dr. Alkiviadis Vatopoulos
Dept. of Hygiene & Epidemiology
Athens Univ. Medical School
75 Asias street, GR-115 27 Athens (Goudi), Greece
T: (+30) 210 777.1165; F: (+30) 210 770.4225
Dr Eva Bán
Microbiology
Szent Laszlo Hospital
Gyáli út 5-7, 1097 Budapest, Hungary
T: (+36) 1 215.8515; F: (+36) 1 215.6501
Priv.Doz. Dr. Uwe Frank
Inst. Für Umweltmedizin und
Krankenhaushygiene
Universitätsklinikum Freiburg
Hugstetter Strasse 55, D - 79106 Freiburg,
Germany
T: (+49) 761 270.5481; F: (+49) 761 270.5486
183
European Commission
EUR 20495 — Antimicrobial resistance research
Luxembourg: Office for Official Publications of the European Communities
2003 — 183 pp. — 21,0 x 29,7 cm
ISBN 92-894-4454-1
15
KI-NA-20-495-EN-C

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