characterization of escherichia coli strains

CHARACTERIZATION
OF
ESCHERICHIA
COLI
STRAINS
AND
SALMONELLA ENTERICA SEROVARS ISOLATED IN GALLUS GALLUS AND
THEIR ANTIMICROBIAL SUSCEPTIBILITY
BY
WESONGA STEPHEN MAKOKHA
REG, NO. I56/5747/03
A THESIS SUBMITTED TO THE SCHOOL OF PURE AND APPLIED
SCIENCES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
AWARD OF THE DEGREE OF MASTER OF SCIENCE INFECTIOUS
DISEASES DIAGNOSIS OF KENYATTA UNIVERSITY
SEPTEMBER 2010
ii
DECLARATION
This thesis is my original work and has not been presented for a degree in any other
university or any award.
Wesonga Stephen Makokha
Signature:
Date:
We confirm that the candidate under our supervision carried out the work reported in this
thesis.
Prof. Geoffrey M. Muluvi
Department of Biochemistry and Biotechnology,
Kenyatta University,
Box 43844,
Nairobi.
Signature:
Date:
Prof. Paul .O. Okemo
Department of Plant and Microbial Sciences,
Kenyatta University,
Box 43844,
Nairobi.
Signature:
Date:
iii
DEDICATION
To
My family especially…
Mum Susan Makokha Kimomoi and Late Dad Nelson Makokha Kimomoi who gave me
the inspiration to eke on. To my wife Doreen Asha Makokha and daughter Alisaah Grace
Nambuye for the spiritual support. My brothers and sisters for encouragement. My friends
for the company and being there during the time of need.
To all, may the Almighty reward you abundantly.
To Pastor Rod Parsely…
“This is the generation that should seek God first!
Don’t just dream!
……………Live it.”
Thanks to Jehovah for the gift of life to do his will!
Amen!!!
iv
ACKNOWLEDGEMENT
I would like to express my heart-felt appreciation to my supervisors, Prof. Geoffrey
Muluvi and Prof. Paul. O. Okemo, for their tireless support, contribution, encouragement
and inspiration.
I also wish to thank other members of Kenyatta University, Prof. E. Njagi, Dr. G. Orinda,
Dr. Dan Masiga, and Dr. F. Muli; other teaching and technical staff of Biochemistry and
Biotechnology; and fellow students for the ample atmosphere you accorded me during
my work.
I am also grateful to my friends and colleagues Dr. Mark Wamalwa, Dr. Omondi
Omuomo, Peter Maloba Agira, Sifuna Antony, Allan Jalemba, Stella Kitavi, Jonathan
Mateba and many others for their support and encouragement.
Finally, I wish to appreciate the technical support and advice I received from Dr. Samuel
Kariuki of the Center for Microbiology Research (CMR) and Dr. Oundo both of KEMRI.
v
TABLE OF CONTENTS
Contents
Declaration
Dedication
Acknowledgement
Table of Contents
List of Tables
List of Figures
Abbreviations and Acronyms
Abstract
CHAPTER ONE: INTRODUCTION
1.1 Background
1.2 Problem statement
1.3 Justification
1.4 Research Hypothesis
1.5 Research Question
1.6 Objective
1.6.1 Specific objectives
1.7 Significance
CHAPTER TWO: LITURATURE REVIEW
2.1 Poultry industry (white meat trends) in Kenya
2.2 Nutritive value of indigenous chicken
2.3 Vitamin B6 for Cardiovascular Health
2.4 Food borne disease challenge
2.5 Salmonella enterica serovars
2.5.1 Salmonella characteristics, nomenclature and habitat
2.5.2 Salmonella isolation, manifestation and pathogenesis
2.5.3 Control of Salmonellosis
2.5.4 Epidemiology of Salmonella
2.6 Escherichia coli strains
2.6.1 E. coli characteristics, nomenclature and habitat
2.6.2 E. coli pathogenesis
2.6.3Uropathogenic E. coli and neonatal meningitis
2.6.4 Diarrhoeagenic Escherichia coli
2.6.4.1 Enterotoxigenic E. coli (ETEC)
2.6.4.2 Enteropathogenic E. coli (EPEC)
2.6.4.3 Enterohemorrhagic E. coli (EHEC)
2.6.4.4 Enteroinvasive E. coli (EIEC) and Enteroaggregative E. coli (EAggEC)
2.6.5 Epidemiology of E. coli
2.6.6 Control of Escherichia coli
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2.7 Drug resistance challenge
2.8 Bacterial resistance to antibiotics
2.9 The basis of microbial resistance to antibiotics
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CHAPTER THREE: MATERIALS AND METHODS
3.1 Sample source
3.2 Specimen collection
3.3 Sample size
3.4 Bacterial isolation and characterization
3.4.1 Escherichia coli strains
3.4.2 Salmonella enterica serovars
3.5 Biochemical tests for E. coli and Salmonella enterica
3.6 Serological test
3.7 Antimicrobial susceptibility testing
3.7.1 Escherichia coli strains and Salmonella enterica serovars
3.8 Plasmid DNA isolation
3.8.1. Escherichia coli and Salmonella enterica serovars
3.9 In-vitro conjugation experiment
3.10 Data analysis
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CHAPTER FOUR: RESULTS AND DISCUSSIONS
4.1Bacterial isolation and characterization
4.2 Serological tests
4.2.1 Serology of Salmonella enterica
4.2.2 Serology of Escherichia coli
4.3 Antimicrobial Susceptibility Testing
4.3.1 Escherichia coli strains
4.3.2 Salmonella typhimurium
4.4 Co-infection strains
4.5 Plasmid profiles
4.6 Discussions
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CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusions
5.2 Recommendations
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REFERENCES
68
APPEDINCES
Appendix I: Reagents
Appendix II: Table 12: Biochemical identification test kit (API20E)
Appendix III: Antimicrobial regiment/zone diameters in mm of bacterial strains
Appendix IV: Table 13: Zone diameter interpretative standards for MIC
83
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vii
LIST OF TABLES
Table 1: The distribution of enteric pathogens in the four districts samples
40
Table 2: District cross-tabulation of bacterial strains
41
Table 3: Pearson Correlation of strains
42
Table 4: Serotype of Salmonella enterica
43
Table 5: Serotypes of E. coli strains
43
Table 6: Antimicrobial sensitivity response (zone diameters) of E. coli and
Salmonella typhimurium case summaries
45
Table 7: Antimicrobial resistance patterns of E. coli strains
51
Table 8: Antimicrobial resistance patterns of Salmonella typhimurium strains
53
Table 9: Co-infection strains susceptibility
57
Table 10: Co-infection strains antimicrobial resistance patterns
57
Table 11: Strains, antimicrobial resistance pattern, number of plasmids and
plasmid sizes
59
Table 12: Biochemical identification test kit (API20E)
84
Table 13: Zone Diameter Interpretative Standards and equivalent MIC
86
viii
LIST OF FIGURES
Figure 1: Caged indigenous chicken at the slaughter house/market outlet
8
Figure 2: Processing of indigenous chicken in the slaughter house/market outlet
9
Figure 3: District percentage resistance of E. coli
48
Figure 4: District percentage susceptibility of E. coli
49
Figure 5: District percentage intermediate of E. coli
50
Figure 6: District percentage resistance of Salmonella typhimurium
54
Figure 7: District percentage susceptibility of Salmonella typhimurium
55
Figure 8: District percentage intermediate of Salmonella typhimurium
56
Figure 9: Gel electrophoresis of Escherichia coli and Salmonella typhimurium
Plasmid DNA
58
ix
ABBREVIATIONS AND ACRONYMS
MDR
Multidrug resistant
FAO
Food and Agriculture Organization
WHO
World Health Organization
FBDs
Food borne diseases
DNA
Deoxyribonucleic acid
USDA
United States Development Agency
API 20E
Analytical Profile index
ANOVA
Analysis of variance
NCCLS
National Committee for Clinical Laboratory Standards
XLD
Xylose Lysine decarboxylase agar
SDS
Sodium dodecyl sulphonate
CDC
The Center for Disease Control and Prevention
H2 S
Hydrogen sulphide
LPS
Lipopolysaccharide
HE
Hektoen enteric
HIV
Human immunodeficiency virus
STEC
Shiga toxin–producing E. coli
A/A
Acid/acid
Ak/A
Alkaline/Acid
KB
Kilobase
BBB
Blood-brain barrier
x
ETEC
Enterotoxigenic Escherichia coli
EPEC
Enteropathogenic E. coli
EHEC
Enterohemorrhagic E. coli
EIEC
Enteroinvasive E. coli
EAEC
Enteroaggregative E. coli
DAEC
Diffusely adherent E. coli
LT
Heat-labile enterotoxin
ST
Heat stable toxin
RTFs
Resistance transfer factors
NCCLS
National Committee for Clinical Laboratory Standards
FDA
Food and Drug Administration
E. coli
Escherichia coli
MIC
Minimum Inhibitory Concentration
Spp
Species
ATCC
American Type Culture Collection
GIT
Gastrointestinal tract
MOLFD
Ministry of Livestock, Fisheries and Development
Amp
Ampicillin
Aug
Augumentin
Cot
Cotrimoxazole
Chl
Chloramphenicol
Crx
Cefuroxime
xi
Gen
Gentamicin
Kan
Kanamycin
Nal
Nalidixic acid
Nor
Norfloxacin
Tet
Tetracycline
xii
ABSTRACT
Indigenous chicken production in most African countries including Kenya is traditionally
based on free feed resources available in the surrounding environment. There is a high
risk of zoonosis that could be an important source of enteric pathogens transmissible to
humans. The apparently healthy chicken, like other food animals shed enteric pathogens,
notably Salmonella spp. and Escherichia coli that are associated with antimicrobial
resistance. The purpose of this study was to characterize and investigate antimicrobial
resistance of Escherichia coli and Salmonella enterica isolated from indigenous chicken
rectal swabs in a leading slaughterhouse cum market outlet in Nairobi, Kenya. Seventy E.
coli strains showed resistance phenotypes to one, two or more antibiotics. The most
common antimicrobial resistance pattern was the single resistance pattern to Tetracycline
(21.43%), followed by Ampicillin, Cotrimoxazole and Tetracycline (14%), Augumentin,
Ampicillin, Cotrimoxazole and Tetracycline (4.29%), Augumentin, Ampicillin,
Cotrimoxazole, Tetracycline, Kanamycin and Chloramphenicol (2.86%), Ampicillin,
Cotrimoxazole, Tetracycline, Chloramphenicol, Cotrimoxazole and Tetracycline
(2.86%); and Cefuroxime, Ampicillin, Cotrimoxazole, Tetracycline, Chloramphenicol,
Cefuroxime, Ampicillin, Cotrimoxazole, Chloramphenicol, Ampicillin, Cotrimoxazole,
Augumentin and Ampicillin (1.43%) respectively. The highest rate of resistance was
against Tet (55.7%), followed by Cot (40%). Third in line of resistance was Amp
32.86%, followed by Aug (11.43%), low or moderate resistance was against Chl (8.57%),
Kan (4.29%), and Crx (2.86%) (P<0.0002). Salmonella typhimurium recovered displayed
single resistance pattern to Tet (16.67%), Gen Cot Tet (8.33%), Amp Cot Tet (8.33%),
Aug Amp Cot Tet (8.33%) and Amp Cot Tet Chl (16.67%). The highest resistance was
against Tet (58.3%), Cot (41.7%), Amp (33.3%), Chl (16.7%), Aug and Gen (8.3%)
respectively (P<0.0001). Conclusion: Routine surveillance at slaughter/market outlets for
Escherichia coli and Salmonella enterica Typhimurium should be done to identify
infected flocks as a procedure for food safety and security program.
1
CHAPTER ONE
INTRODUCTION
1.1 Background
Food animals harbour food borne pathogens and act as a source of contamination, which
is of paramount importance in the spread of Salmonella and Escherichia coli in humans
(Van den Bogaard et al., 2001; White et al., 2001). The shedding of pathogens by
apparently asymptomatic healthy animals is increasing concern as a source, and
distribution of food borne diseases (FBDs) (Bayleyegn et al., 2003; Dufrenne et al.,
2001; Van den Bogaard et al., 2001). The process of evisceration during slaughter of
food animals is regarded as one of the most important sources of carcass and organ
contamination with pathogens (Van den Bogaard et al., 2001).
Food items such as poultry products are regarded as the common source of food borne
Salmonellosis and E. coli (Oosterom, 1991; Bebora et al., 1994). An increase in the
consumption of poultry meat and eggs has led to an increase in the number of food borne
illnesses attributed to Salmonella and E. coli strains in many countries (Jianghong et al.,
2002). Salmonella and Escherichia coli strains are recognized as human food borne
pathogens that cause diarrhoea, gastroenteritis, septicaemia, hemorrhagic colitis,
hemolytic uremic syndrome, thrombotic thrombocytopenic purpura; and in Escherichia
coli strains may fulminate to kidney dysfunction (Doyle, 1999; Wolfgang et al., 2001;
Cheesbrough et al., 1997; Graham et al., 2000; Machiel et al., 1997).
The strains are among pathogens which have been demonstrated to have by far the
greatest impact, through mortality and morbidity (Kotula and Pandya, 1995; Kariuki et
2
al., 1999; Bebora et al., 1994; Dufrenne et al., 2001; Daly et al., 2000; Beery et al., 1985;
Schoeni et al., 1994). WHO estimates 2.1 million deaths from diarrhoea worldwide that
are mainly caused by contaminated food including poultry (FAO/WHO, 2002). As such,
the contamination and proliferation of these pathogens in poultry products (foods) are a
great concern to poultry production, food safety and public health.
Current therapy for infections due to Salmonella and E. coli strains depends primarily on
the use of antibiotics effective against the pathogens (White et al., 2002, Kariuki et al.,
2000; Bebora et al., 1994). This strategy has allowed the decrease in shedding of
Salmonella and E. coli strains in poultry (Van den Bogaard et al., 2001). However, in the
past decade or so, it has become apparent that reliance on disease control is becoming
increasingly difficult for a number of reasons, among them the growing antimicrobial
resistance to widely used antibiotics such as ampicillin, co-trimoxazole, chloramphenicol,
streptomycin and tetracycline (Kariuki et al., 2000; Bebora et al., 1994; White et al.,
2002). In Kenya, for example, multi-drug resistance to antibiotics has been reported in
several strains of Salmonella enterica serovar Typhimurium and E. coli (Kariuki et al.,
2000; Bebora et al., 1994, Kariuki et al., 1999).
In Nigeria, Morocco, Saudi Arabia, and Northern India, chicken has been described as an
important source of antimicrobial resistance in humans (Van den Bogaard et al., 2001).
Recently, Bass et al., 1999 described a high incidence of integrons encoding MDR
among chicken isolates as part of Transposon Tn21. Integron gene sequence are thought
to be a primary source of resistance genes and thus suspected to serve as reservoirs of
antimicrobial resistance genes within microbial populations of enteric bacteria including
E. coli and Salmonella enterica serovars (Roe et al., 2002). The aadA gene is highly
3
conserved among shiga toxin-produncing and avian clinical E. coli isolates respectively
(Zhao et al., 2001; Bass et al., 1999). Sulphonamide resistance is marker for the
presences of class 1 integrons, a novel group of mobile genetic elements, which play an
important role in the dissemination of antimicrobial resistance. Integrons contain
interchangible gene cassettes linked to other structural features including the
sulphonamide resistance gene (sul 1), and may be present on plasmids (Cormican et al.,
2002).
Consequently, resistance to antibiotics in strains of Salmonella and E. coli is being
viewed as a potential threat to the public health and long-term viability of poultry in
Kenya, thus necessitating the development of innovative management strategies. The
present study proposes to evaluate (or investigate) whether there are antimicrobial
resistant strains of Salmonella enterica and E. coli in rectal swabs from indigenous
Gallus gallus (chicken) sold in a leading slaughterhouse/market outlet in Nairobi-Kenya.
A clear understanding of patterns of antimicrobial resistance and diversity is important in
management/control of resistance to drugs and developing effective therapeutic
approaches to infections due to E. coli and Salmonella strains in poultry and will
contribute to improved poultry production for sustainable food security and income
generation among poultry keepers in the country.
4
1.2 Problem statement
There is concern that some antibiotics are rapidly losing their effectiveness as resistance
is spreading within and between different bacteria. Antibiotics are the "miracle drugs"
that are extensively used for the treatment and prevention of infectious diseases in
humans and as well as in food-producing livestock, poultry. Antibiotics have greatly
enhanced human life expectancy, reduced mortality, improved quality of life and almost
won the war against many infectious diseases (Mohamed et al., 2001).
However, despite advances in medical science, infections due to Salmonella and E. coli
strains remain the most important food borne diseases (FBDs) of human (Jianghong et
al., 2002). Moreover, a majority of FBDs are associated with the consumption of
contaminated poultry meat and eggs. Though many approaches have been employed to
counter these infections both in human and poultry, application of effective antibiotics
(therapy) is the main control strategy. However, recently therapy has not been efficient
due to the widespread emergence of drug resistance to Salmonella and E. coli isolates in
human (Kariuki et al., 1999; Cormican et al., 2002; Kariuki et al., 2000).
1.3 Justification
The indigenous Gallus gallus play a major role as a source of the increasing white meat
demand with an annual production estimate of 20,000 metric tonnes (Malanga, 2008).
However, the shedding of Salmonella and E. coli pathogens that cause high morbidity
and mortality by apparently asymptomatic healthy indigenous chicken is of concern as a
source, distribution of food borne diseases (FBDs) and emergence of antibiotic resistance
(Bayleyegn et al., 2003; Dufrenne et al., 2001; Van den Bogaard et al., 2001) and creates
5
a reservoir of resistant microorganisms in the environment that could infect humans
through the food chain (Mohamed et al., 2001).
The zoonoses have become a matter of public concern necessitating the need for more
attention in this area. Hence, the characterization of antimicrobial resistance patterns in
isolates of Salmonella and E. coli strains is important in strategic surveillance, which is
an essential necessity to curb outbreaks, control, and initiate counter measures for
antimicrobial
resistant
bacteria
from
indigenous
chicken
in
leading
slaughterhouse/market outlet in Nairobi-Kenya. These will shed light on the level of
bacterial antibiotic resistance, leading to a more effective way of controlling spread,
distribution and infections associated with the pathogens in food chain. Thus, the net
effect will be increased consumer confidence, a healthy breed, and compliance with the
global need of food security.
1.4 Research Hypothesis
Indigenous Gallus gallus (chicken) are a major source of Salmonella and E. coli borne
diseases associated with different levels of resistance to antibiotics.
1.5 Research Questions
Do indigenous Gallus gallus (chicken) in leading slaughterhouse/market outlet in
Nairobi-Kenya contain Salmonella and E. coli isolates which are resistant to antibiotics?
Are the enteric pathogens sensitive or resistant to antimicrobial agents?
Do the enteric pathogens (Salmonella and E. coli) in indigenous chicken contain
plasmids?
6
1.6 Objective
The objective of this study was to characterize and investigate antimicrobial resistance of
E. coli and Salmonella strains isolated from indigenous Gallus gallus (chicken) in a
leading slaughterhouse/market outlet in Nairobi-Kenya.
1.6.1 Specific objectives
1. To isolate and identify E. coli strains and Salmonella serovars from indigenous chicken
rectal swabs.
2. To investigate resistance to antibiotics of the isolates.
3. To isolate plasmids from E. coli strains and Salmonella serovars.
1.7 Significance
The scientific data generated is envisaged to provide an understanding of antimicrobial
resistance patterns and diversity, among E. coli and Salmonella strains from indigenous
Gallus gallus with a view of improving on intervention strategies for management and
control of the spread, distribution and infections associated with the strains. This will lead
to an opportunity of healthy breed, improved nutritional values, food safety, increased
consumption, and alleviate suffering by reducing disease burden thus enhancing
livelihood.
7
CHAPTER TWO
LITERATURE REVIEW
2.1 Poultry industry (white meat trends) in Kenya
Poultry diseases are recognized as a major constraint to increasing poultry production in
developing countries (Gueye, 1999). Among them, Salmonella and E. coli strains have
been demonstrated to have by far the greatest impact, through mortality, morbidity and
the prevention of use of genetically improved breeds that provide an opportunity for a
transition from subsistence to a market-oriented production (Kariuki et al., 1999; Bebora
et al., 1994). Salmonella and Escherichia coli strains are also recognised human food
borne pathogens (Cheesbrough et al., 1997; Graham et al., 2000).
Poultry keeping is one of the most popular livestock enterprises in Kenya due to its low
capital space requirements. Kenya has an estimated 37.3 million birds (MOLFD, 2007).
Of these, free-ranging indigenous birds comprised 84.1% (31.4 million), 8.4% were
layers (3.1 million), 5.7% (2.1 million) were broilers while other poultry species (ducks,
turkeys, pigeons, ostriches, guinea fowls and quails) accounted for 1.8% (0.7 million)
(FAO, 2007). Every household keeps at least 5-20 local breeds scavenging in open (kept
under free-range conditions) (Kiptarus, 2005; Gueye, 1998; Njue, 2002). Indigenous
chicken genotypes include the Rhode Island Red, Light Sussex, New Hampshire Red,
Black Australorps, white leghorns, Plymouth Rock, barred Rock and buff Rock (Figure
1) (FAO, 2007). The main genotypes of commercial layers are Isa Brown and Ross,
while commercial broiler genotypes include Arbor Acres, Hybro, Cobb (United
Kingdom) and Hypeco (Holland) (FAO, 2007).
8
Figure 1: Caged indigenous chicken at the slaughter house/market outlet in NairobiKenya
The mean annual estimated poultry meat Production is 20,000 metric tonnes valued at
Ksh 131 million (Malanga, 2008). The United States Development Agency (USDA)
predicts poultry consumption will rise from 100.2 pounds per person in 2003 to 108.9
pounds per person by 2013 (FAO, 1988). The Food and Agriculture Organization (FAO)
findings show that there is a global shift from red meat to white meat consumption and
forecast the demand to reach 45% of the total 19 million tonnes of meat utility by 2005 in
Africa, of which 70% is anticipated to be sourced from Poultry (FAO, 1988). Apart from
increased quantitative production of animal protein in rural households, chicken meat
provides protein of a higher biological value than that of red meat (Figure 2) (Aichi,
1998).
9
Figure 2: Processing of indigenous chicken in the slaughter house/market outlet in
Nairobi-Kenya
Indigenous chicken has many values in the contemplary society ranging from social,
cultural and religious importance throughout the continent and Kenya (Guèye, 2002;
Njue, 2002). Chicken is a very popular food in this country as well as other parts of the
world (Kiptarus, 2005; Aichi, 1998). Thus chicken is a good source of protein with all the
essential amino acids, contains fat that is less saturated than beef fat, vitamin B-complex
that are involved as cofactors in energy metabolism throughout the body (Mitch et al.,
2004).
2.2 Nutritive value of indigenous chicken
Chicken is a good source of the trace mineral for example selenium that is of
fundamental importance to human health (Wen et al., 1997). Our bodies use selenium to
10
produce glutathione peroxidase, which is important for cancer protection. Glutathione
peroxidase is one of the body's most powerful antioxidant enzymes, used in the liver to
detoxify a wide range of potentially harmful molecules (Lapenna et al., 1998). When
levels of glutathione peroxidase are too low, these toxic molecules are not disarmed and
wreak havoc on any cells with which they come in contact, damaging their cellular DNA
and promoting the development of cancer cells. However, Selenium has been shown to
induce DNA repair and synthesis in damaged cells, to inhibit the proliferation of cancer
cells, and to induce their apoptosis, the self-destruct ion sequence the body uses to
eliminate worn out or abnormal cells (Clark et al., 1996; Hocman, 1988). Selenium is
also an essential component of several major metabolic pathways, including thyroid
hormone metabolism, and immune function (Broome et al., 2004).
2.3 Vitamin B6 for Cardiovascular Health
Chicken contains vitamin B6 that plays a pivotal role in many biological functions
through methylation process which involves addition of methyl groups to other molecules
such as proteins, enzymes, chemicals, DNA, or amino acids like homocysteine (a toxic
amino acid). When levels of B6 are inadequate, the availability of methyl groups decline
leading to accumulation of homocysteine that damages blood vessel walls thus
considered a significant risk factor for cardiovascular disease (Jacobsen, 1998, Hirsch
and Pia De la Maza, 2002, Michelle et al., 2001, Schnyder and Roffi, 2002).
2.4 Food borne disease challenge
Today there is an increasing concern over food borne pathogens spreading from farm
animals to human populations. Epidemiological data have demonstrated that a significant
source of drug-resistant food borne infections in humans is the acquisition of resistant
11
bacteria originating from animals (Angulo et al., 1998, Holmberg et al., 1984). This
source of infection has been demonstrated through several different types of food borne
disease follow-up investigations, including laboratory surveillance, molecular subtyping,
and outbreak investigations (Tacket et al., 1985; Holmberg et al., 1984).
More studies have confirmed that using antimicrobial drugs in poultry increases the risk
of selecting for resistant food borne pathogens, and that these pathogens can then be
transferred to humans through direct contact with either contaminated food or animals
(Van den Bogaard et al., 2001; White et al., 2001). Due to the lack of alternative
strategies, most attempts to control gastrointestinal tract microflora in chickens have so
far relied on the use of broad-spectrum antibiotics (Apajalahti et al., 2004).
However, the recent and widening concern over disseminating antibiotic resistance genes
has led to bans on the prophylactic use of many antibiotics in a number of countries
(Apajalahti et al., 2004). In indigenous chicken, the diet and the environment affect the
microbial status of the gastrointestinal tract. Dirty litter and other animal management
parameters affect microbial composition of the chicken gastrointestinal tract by providing
a continuous source of bacteria through ingestion (Apajalahti et al., 2004).
Resistant strains from the gut readily soil poultry carcasses during slaughter and as such
result in poultry meat to often be contaminated with multiresistant strains (Van den
Bogaard et al., 2001). Thus raw retail chicken meats are potential vehicles for
transmitting food borne diseases (Zhao et al., 2001). Additionally, these retail chicken
meats are often associated with direct hand-to-mouth exposure to enteric pathogens and
cross-contamination of the kitchen environment and ready-to-eat foods (Zhao et al.,
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2001). Many infections are transmitted through food and cause illness ranging from mild
gastroenteritis to severe illness requiring hospitalization (Pinner et al., 2003). The task of
providing accurate information on trends in specific food borne pathogens capable of
causing syndromes is at the hands of researchers (Pinner et al., 2003).
Salmonella enterica serovars and E. coli are prominent food pathogens. Factors
influencing the occurrence of food borne illnesses are complex and include human
population increase, poverty, changing life-styles-including more adventurous eating,
more convenience foods, less time devoted to food preparation; ever-evolving
technologies for food production, processing, distribution, and emergence of newly
recognized microbial pathogens (Jianghong et al., 2002).
2.5 Salmonella enterica serovars
Salmonellosis is one of the most common and widely distributed food borne diseases
(WHO, 2005). It constitutes a major public health burden and represents a significant cost
in many countries. Millions of human cases are reported worldwide every year and the
disease results in thousands of deaths (WHO, 2005). Salmonella infections are mainly
asymptomatic in poultry, but are associated with widespread human illness from this
source (Saeed et al., 1999). Therefore, there is continuing interest in finding ways of
preventing flock infection and hence contamination of poultry products with Salmonella
(Saeed et al., 1999). Pullorum disease, (S. pullorum) and fowl typhoid (S. gallinarum) are
two classic and distinctive diseases of poultry that have received considerable attention
because of their economic impacts (Snoeyenbos, 1994).
13
Salmonella enterica-associated gastroenteritis is an important food borne human disease
(Cormican et al., 2002). Most serotypes are capable of infecting a variety of animal
species, including humans. There is considerable variation with time and geographical
location in serotypes commonly associated with human Salmonellosis notably S. enterica
serovar Typhimurium and S. enterica serovar Enteritidis (Cormican et al., 2002),
serotype Typhimurium is responsible for various disease manifestations, usually in the
form of mild gastroenteritis with low mortality, but it can cause septicemia with high
mortality (Salvatore et al., 2004).
The level of contamination of chicken and chicken products with pathogens associated
with gastroenteritis such as Salmonella spp. is significantly increasing in many countries
(Dufrenne et al., 2001). For example Salmonella serotypes were isolated from 22.0% of
broiler flocks, and from 15.3% of the layer flocks in The Netherlands (Dufrenne et al.,
2001). The Center for Disease Control and Prevention (CDC) estimates that there are 1.4
million cases with about 582 annual deaths related to Salmonellosis in the USA
(Wolfgang et al., 2001) and 12.5 million episodes globally (Gopinath et al., 1995).
In Denmark, the incidence of human Salmonellosis increased rapidly in the second half
of the 1980s because of the spread of Salmonella in broiler chickens, layer hens and
swines (Wegener et al., 2003). About 65% of domestically acquired Salmonella
enteritidis infections in Denmark was linked to Danish layer hens phage PT6 and PT8;
and the highly resistant phage were associated with imported broiler chickens and poultry
products (Molbak et al., 2002). The most important cause of Salmonellosis has been
attributed to broiler chickens and layer hens (Wegener et al., 2003).
14
In Kenya, Salmonella typhimurium and S. enteritidis account for a higher percentage of
human isolates (Oundo et al., 2000, Kariuki et al., 2000), and correlates with cases in the
USA where 24% of human Salmonellosis and 19% of animal isolates are due to
Salmonella typhimurium (Bender et al., 2001).
2.5.1 Salmonella characteristics, nomenclature and habitat
Salmonella is a Gram-negative facultative anaerobic rod-shaped bacterium in the family
of Enterobacteriaceae, also known as enteric bacteria. Salmonella is a motile bacterium
with the exception of S. gallinarum and S. pullorum and they are all nonsporeforming.
There is a widespread occurrence of Salmonellosis in animals, especially poultry (FDA,
1998).
There are over 2500 serotypes, of Salmonella (WHO, 2005). Different strains of
Salmonellae have been identified, and these are placed into groupings called serovars on
the basis of their antigens (Snoeyenbos, 1994). The latest nomenclature, which reflects
recent advances in taxonomy (Popoff, 2001), in the genus Salmonella consists of only
two species: S. enterica and S. bongori (Le Minor and Popoff, 1987; Popoff et al., 1994;
Cooper, 1994). Salmonella enterica is divided into six subspecies, which are
distinguishable by certain biochemical characteristics (Brenner et al., 2000; Farmer et al.,
1985). Strains of Salmonella are classified into serovars on the basis of extensive
diversity of lipopolysaccharide (LPS) antigens (O) and flagellar protein antigens (H) in
accordance with the Kauffmann–White scheme. Approximately 2500 serovars are
recognized (Popoff et al., 1994) with the number constantly increasing. The most
common serovars that cause infections in humans and food animals belong to subspecies
enterica.
15
Salmonellae have a wide range of hosts (Hohmann, 2001). Although primarily intestinal
bacteria of animals and birds, Salmonellae are widespread in the environment and
commonly found in farm effluents, human sewage and in any material subject to faecal
contamination and are transmitted to humans by contaminated foods of animal origin
(Tauni and Österlund, 2000; Refsum et al., 2002; Hohmann, 2001). Some serovars show
remarkable host specifity for instance Salmonella typhi and Salmonella gallinarium are
strictly found in humans and birds respectively (Jorgensen, 2001; Boyd and Hartl, 1997).
Wild animals are healthy carriers of a broad range of salmonella serotypes (Hudson et al.,
2000; Refsum et al., 2002). Furthermore, most European countries suggest that
Salmonella has established a reservoir in the wild birds (Kapperud et al., 1998) and
hedgehogs (Erinaceus europeus) (Handeland et al., 2002; Jorgensen, 2001).
Epidemiological and bacteriological evidence indicate that these animals may transmit
the infection to human (Tauni and Österlund, 2000; Handeland et al., 2002) or to
livestock (Humphrey and Bygrave, 1988).
2.5.2 Salmonella isolation, manifestation and pathogenesis of infections
The most commonly used media selective for Salmonella are Salmonella-Shigella (SS)
agar, bismuth sulfite agar, Hektoen enteric (HE) medium, brilliant green agar, xyloselysine-deoxycholate (XLD), and MacConkey agar. All these media contain both selective
and differential ingredients (Edwards and Ewing, 1972).
Salmonella organisms are aetiological agents of diarrhoeal and systemic infections in
humans, most commonly as secondary contaminants of food originating from the
environment, or as a consequence of septicaemia in food animals (EU, 1992). Onset of
the illness is usually 6 - 48 h. The infective dose is 15–20 cells; which depends upon age
16
and health of host, and strain differences among the members of the genus. Acute
symptoms include nausea, vomiting, abdominal cramps, diarrhea, fever, and headache,
which may last for 1 to 2 days or may be prolonged. Chronic consequences include
arthritic symptoms that may follow 3 - 4 weeks after onset of acute symptoms (FDA,
1998).
The infections are caused by Salmonella serovars (e.g., Typhimurium). About 12-24
hours following ingestion of contaminated food (containing a sufficient number of
Salmonella), symptoms appear (diarrhea, vomiting, fever) and may last 2-5 days usually
before spontaneous cure. Salmonella infections vary with the serovar, the strain, the
infectious dose, the nature of the contaminated food, and the host status (Gulig, 1996;
Aabo et al., 2000). Salmonella pathogenesis is initiated by oral ingestion and penetration
into the intestinal epithelium; induce degeneration of enterocyte microvilli causing
profuse macropinocytosis, which leads to the internalization of bacteria (Goosney et al.,
1999; Gulig, 1996).
2.5.3 Control of Salmonellosis
Salmonella enterica remains one of the most important food borne pathogens of humans
and is often acquired through consumption of infected poultry meat or eggs. Control of
Salmonella infections in chicken is therefore an important public health issue (Beal et al.,
2004). Three types of typhoid vaccines are currently available for use: (1) an oral liveattenuated vaccine, (2) a parenteral heat-phenol-inactivated vaccine, (3) a newly
developed capsular polysaccharide vaccine for parenteral use, a fourth vaccine, and an
acetone-inactivated parenteral vaccine are available only to the armed forces in USA
(Beal et al., 2004).
17
Hazards from Salmonella can be prevented by heating food sufficiently to kill the
bacteria, holding chilled food below 4.4 ºC, preventing post-cooking cross contamination
and prohibiting people who are ill or are carriers of Salmonella from working in food
operations (Ward et al., 1997). Salmonella surveillance and control of poultry industry at
slaughter should be done to identify infected flocks as regulatory procedures for food
safety and security program (Smith et al., 1989; Nielsen et al., 1995; Veling et al., 2002).
Indiscriminate distribution and use of antibiotics should be discouraged. Disease
prevention should be practical at bird feeding stations; the public should be educated to
maintain clean feeders and to remove spilled and soiled feed from the area under the
feeder. Feeders occasionally should be disinfected with a 1:10 ratio of household bleach
and water as part of the disease-prevention program. In the event of a die-off from
Salmonellosis, more rigorous disinfection of feeding stations is necessary and station use
should be discontinued temporarily.
Other potential point sources of infection include garbage, sewage wastewater, and
wastewater discharges from livestock and human operations should be monitored for
example a 1995 outbreak of S. enteriditis in California poultry was traced to sewage
treatment plant wastewater, which entered a stream that bordered the poultry farm;
contamination of feral cats and wildlife of the water of the stream was thought to be the
source of entry of S. enteriditis in the poultry (Snoeyenbos, 1994).
2.5.4 Epidemiology of Salmonella
Salmonellosis is one of the most common and widely distributed food borne diseases. It
constitutes a major public health burden and represents a significant cost in many
18
countries (WHO, 2005). Millions of human cases are reported worldwide every year and
the disease results in thousands of deaths (WHO, 2005). In addition to acquiring infection
from contaminated food, human cases have also occurred where individuals have had
contact with infected animals, including domestic animals (WHO, 2005).
In Africa, nontyphoidal salmonellae are the most common cause of bloodstream
infections in children younger than five years (WHO, 2005) and in recent series of HIVinfected African adults in who isolates of up to 35% are obtained (Hohmann, 2001;
Kariuki et al., 2005). Nontyphoidal Salmonella are important food borne pathogens that
cause gastroenteritis, bacteremia, and subsequent focal infection. These bacteria are
especially problematic (cause opportunistic infections) in a wide variety of
immunocompromised
individuals,
including
patients
with
malignancy,
human
immunodeficiency virus (Hohmann, 2001; Kariuki et al., 2005), or diabetes, and those
receiving corticosteroid therapy or treatment with other immunotherapy agents.
Endovascular infection and deep bone or visceral abscesses are important complications
that may be difficult to treat (Hohmann, 2001).
While meningitis caused by nontyphoidal salmonellae is uncommon in economically
developed countries, it is more frequent in tropical countries, particularly in children
younger than six months, and thus associated with higher case-fatality rates than
meningitis caused by other bacteria (Hogne et al., 2004). At Haydom Lutheran Hospital,
a rural hospital in northern Tanzania, clinicians noted an extraordinarily high case-fatality
rate (>60%) from pediatric meningitis in the period January 1998 to April 2000 (Hogne et
al., 2004). Plasmid-borne antibiotic resistance is very frequent among Salmonella strains
involved in pediatric epidemics (e.g., Typhimurium, Panama, Wien, and Infantis).
19
Resistance to ampicillin, streptomycin, kanamycin, chloramphenicol, tetracycline, and
sulfonamides is commonly observed compounding the problems (WHO, 2005).
During the last decade, antibiotic resistance and multiresistance of Salmonella spp. have
increased a great deal due to increased indiscriminate use of antibiotics in the treatment
of humans and animals; and the addition of growth-promoting antibiotics to the food of
breeding animals (White et al., 2002; WHO, 2005). Strains of Salmonella which are
resistant to a range of antimicrobials, including first-choice agents for the treatment of
humans, have emerged and are threatening to become a serious public health concern
(Holmberg et al., 1984).
Salmonella enterica associated gastroenteritis is an important FBD throughout the world
(Cormican et al., 2002), the Center for Disease Control and Prevention (CDC) estimates
that there are 1.4 million cases with about 582 annual deaths related to Salmonellosis in
the USA (Wolfgang et al., 2001) and 12.5 million episodes globally (Gopinath et al.,
1995). In Kenya, Salmonella typhimurium and S. enteritidis account for a higher
percentage of human isolates (Oundo et al., 2000; Kariuki et al., 2000), and correlates
with cases in the USA where 24% of human Salmonellosis and 19% of animal isolates
are due to Salmonella typhimurium (Bender et al., 2001).
2.6 Escherichia coli strains
Escherichia coli is one of the normal bacterial floras of the gastrointestinal tract of
poultry and humans (Barnes et al., 1997; Bonten et al., 1990; Conway and Macfarlane,
1995). Ten to fifteen percent of the intestinal coliforms in chicken are of pathogenic
serotypes (Barnes et al., 1997). Colibacillosis is a common systemic infection caused by
20
E. coli in poultry. The disease causes considerable economic damage to poultry
production worldwide (Margie and Lawrence, 1999). Significant increase in appearance
of drug-resistant strains of E. coli isolated from poultry has complicated the problem
(Geornaras et al., 2001).
In humans, these strains are the foremost cause of urinary tract infections (Falagas and
Gorbach, 1995), as well as a major cause of neonatal meningitis (Klein et al., 1986),
nosocomial septicemia, and surgical site infections (Thielman and Guerrant, 1999).
Infection with Shiga toxin–producing E. coli (STEC) may also result in complications
including thrombocytopenic purpura, severe hemorrhagic colitis, and hemolytic uremic
syndrome (Griffin, 1995). While therapeutic options vary depending on the type of
infection, antimicrobials including trimethoprim-sulfamethoxazole, fluoroquinolones, and
third-generation cephalosporins are generally recommended for treating infections caused
by E. coli other than STEC (Thielman and Guerrant, 1999; Paton and Paton, 1998).
2.6.1 E. coli characteristics, nomenclature and habitat
E. coli are straight rods, aerobes and facultative anaerobes; ferment most sugars
producing gas but do not produce H2S on TSI agar slants (A/A with gas). They are indole
positive, methyl red positive, Voges Proskaur negative, simmon’s citrate negative,
catalase positive and urease negative (Soomro et al., 2002; Farmer et al., 1985).
Escherichia coli is a commensal of the lower gastrointestinal tract of mammals (Hartl and
Dykhuizen, 1984; Selander et al., 1987). According to the modified Kauffman scheme,
E. coli serotaxonomy is based on their antigenicity O (somatic), H (flagellar), and K
(capsular) surface antigen profiles. A total of 170 different O antigens, each defining a
21
serogroup, are recognized currently. The presence of K antigens was determined
originally by means of bacterial agglutination tests: an E. coli strain that was
inagglutinable by O antiserum but became agglutinable when the culture was heated, thus
considered having a K antigen. A specific combination of O and H antigens defines the
serotype of an isolate (Nataro and Kaper, 1998).
2.6.2 E. coli pathogenesis
E. coli is responsible for three types of infections in humans: urinary tract infections
(UTI), neonatal meningitis, and intestinal diseases (gastroenteritis). These three diseases
depend on a specific array of pathogenic (virulence) determinants (Falagas and Gorbach,
1995; Nataro and Kaper, 1998).
2.6.3 Uropathogenic E. coli and Neonatal meningitis
The pathogen causes 90% of the urinary tract infections (UTI) in anatomically normal,
unobstructed urinary tracts (Falagas and Gorbach, 1995; Betsy et al., 2002). The severity
range from asymtomatic through bacteriuria, cystitis and pyelonephritis associated with
O groups O1, O2, O4, O6, O7, O18 and O75; and K antigens K1, K2, K3, K5, K12 and
K13 (Garcia-Mart, 1996). The extra intestinal pathogenic Escherichia coli (ExPEC)
strains (Andrew et al., 2004) colonize from the faeces or perineal region and ascend the
urinary tract to the bladder. Bladder infections are 14-times more common in females
than males by virtue of the shortened urethra. The organisms are propelled into the
bladder from the periurethral region during sexual intercourse. With the aid of specific
adhesins they are able to colonize the bladder (Falagas and Gorbach, 1995).
22
Escherichia coli is the most common causative agent of gram-negative neonatal bacterial
meningitis and sepsis. The rates of mortality, morbidity and neurologic sequelae remain
high despite advances in intensive care (Korczak et al., 2005). Neonatal colonization
often results from maternal transmission during delivery (Teng et al., 2005). More than
half of the survivors develop long-term neurological sequelae, including seizure
disorders, hydrocephalus, physical disability, developmental delay, and hearing loss. Most
infections occur in the first month of life with a frequency of 0.22 to 2.66 per 1,000 live
births worldwide (Korczak et al., 2005).
Eighty percent of E. coli strains involved synthesize K-1 capsular antigens (K-1 is only
present 20-40% of the time in intestinal isolates). E. coli strains invade the blood stream
of infants from the nasopharynx or GI tract and are carried to the meninges (Klein et al.,
1986). The two major pathophysiological steps in E. coli neonatal meningitis consist of
bacteremia with intravascular growth and passage of bacteria across the blood-brain
barrier (BBB) (Klein et al., 1986). Also in other unique cases, E. coli has been implicated
with fulminating neonatal sepsis and meningitis due to wound infection following
circumcision of newborn male offspring after delivery (Scurlock and Pemberton, 1977).
2.6.4 Diarrhoeagenic Escherichia coli
Escherichia coli is one of the predominant species of facultative anaerobes in the human
gut and usually harmless to the host; however, a group of pathogenic E. coli has emerged
that causes diarrheal (intestinal) disease in humans referred to as diarrheagenic E. coli
(Nataro and Kaper, 1998) or commonly as pathogenic E. coli. These groups are classified
based on their unique virulence factors and analysis for pathogenic E. coli often requires
that the isolates be first identified as E. coli before testing for virulence markers. The
23
pathogenic groups includes enterotoxigenic E. coli (ETEC), enteropathogenic E. coli
(EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC),
enteroaggregative E. coli (EAEC), and diffusely adherent E. coli (DAEC) (Rodrigues et
al., 2002; Levine, 1987; Nataro and Kaper, 1998).
2.6.4.1 Enterotoxigenic E. coli (ETEC)
ETEC is recognized as the causative agent of travelers' diarrhea and illness is
characterized by watery diarrhea with little or no fever. ETEC infections have been
implicated in sporadic waterborne outbreaks and raw vegetables. Pathogenesis of ETEC
is due to the production of enterotoxins the genes for which may occur on the same or
separate plasmids for example a heat-labile enterotoxin (LT), a heat stable toxin (ST),
resistant to boiling for 30 min. There are several variants of ST, of which ST1a or STp is
found in E. coli isolated from both humans and animals, while ST1b or STh is
predominant in human isolates only. The infective dose of ETEC for adults has been
estimated to be at least 108 cells; but the young, the elderly and the infirm may be
susceptible to lower levels (Nataro and Kaper, 1998).
2.6.4.2 Enteropathogenic E. coli (EPEC)
EPEC is an important category of diarrheagenic E. coli that has been linked to infant
diarrhea in developing countries affecting infants under one year of age, with the highest
prevalence occurring in those under six months of age (Gomes et al., 1991, RobinsBrowne, 1987; Nataro and Kaper, 1998; Jav et al., 2004). Studies in Brazil, Mexico, and
South Africa have shown that 30 to 40% of infant diarrhea can be attributed to EPEC that
causes a profuse watery diarrheal disease (Nataro and Kaper, 1998). EPEC outbreaks
have been linked to the consumption of contaminated drinking water as well as some
24
poultry meat products. Pathogenesis of EPEC involves intimin protein (encoded by eae
gene) that causes attachment and effacing lesions (Hicks et al., 1998); but it also involves
a plasmid-encoded protein referred to as EPEC adherence factor (EAF) that enables
localized adherence of bacteria to intestinal cells (Tobe et al., 1999).
2.6.4.3 Enterohemorrhagic E. coli (EHEC)
EHEC are recognized as the primary cause of hemorrhagic colitis (HC) or bloody
diarrhea, which can progress to the potentially fatal hemolytic uremic syndrome (HUS)
and thrombotic thrombocytopenic purpura (Alexandre et al., 2001). EHEC are typified by
the production of verotoxin or Shiga toxins (Stx). Although Stx1 and Stx2 are most often
implicated in human illness, several variants of Stx2 exist. Of these, O157:H7 is the
prototypic EHEC and most often implicated in illness worldwide (CDC, 1993; Griffin
and Tauxe, 1991; Karmali, 1989; Nataro and Kaper, 1998). EHEC infections are mostly
food or water borne, thus the strains have been described as important and emergent foodborne pathogens (Alexandre et al., 2001).
2.6.4.4 Enteroinvasive E. coli (EIEC) and Enteroaggregative E. coli (EAggEC)
EIEC causes an invasive, dysenteric form of diarrhea in humans (Jav et al., 2004).
Pathogenicity of EIEC is primarily due its ability to invade and destroy colonic tissue
(Nataro and Kaper, 1998; Jav et al., 2004).
The distinguishing feature of EAggEC strains is their ability to attach to tissue culture
cells in an aggregative manner. These strains are associated with persistent diarrhea in
young children (Jav et al., 2004; Nataro and Kaper, 1998). They resemble ETEC strains
25
in that the bacteria adhere to the intestinal mucosa and cause non-bloody diarrhea without
invading or causing inflammation (Levine, 1987; Nataro and Kaper, 1998).
2.6.5 Epidemiology of E. coli
Numerous incidents of fatal food borne diseases (FBDs) associated with pathogenic E.
coli strains have been reported over a wide geographic distribution in Canada, United
Kingdom, China, Argentina, Japan (Anonymous, 1995), Swaziland (Isaäcson et al.,
1993), Malawi (Paquet et al., 1993); Kenya (Sang et al., 1996; Kariuki et al., 1999),
Central African Republic (Germani et al., 1997), Cameroon (Germani et al., 1998),
Nigeria (Olorunshola et al., 2000) and Ivory Coast (Dadie et al., 2000). For example, E.
coli O157:H7 strains were isolated from 12 of 33 chicken samples in Seattle (Samedpour
and Liston, 1994). Similarly, in Kenya, four E. coli isolates from each child and chicken
living in close contact in Thika and Kiambu on small-scale farm gave identical gel
electrophoresis pattern indicating an association (Kariuki et al., 1999).
The impact is enormous, for instance, CDC estimates 20,000 illnesses and 250 deaths
each year in the USA with 30 separate outbreaks in 1994 with the latest data indicating
62,000 illnesses, 1,800 hospitalization and 52 deaths per year (Gregory et al., 1996). This
is attributed to the fact that E. coli strains can survive and multiply when stored between
0 ºC, 6 ºC and 12 ºC; and in dry foods with a wide range of water activity and pH values
(Samedpour and Liston, 1994).
2.6.6 Control of Escherichia coli
A biofilm-based vaccine for the dreaded E. coli, a bacterial disease that kills over 10 per
cent of chicken has been developed by a researcher Dr. Gowda in India and tested by
26
Prof G Krishnappa, director of the Institute of Animal Health and Veterinary Biologicals
on 70,000 chickens at two farms in Devanahalli and three farms in Mangalore
(Balakrishnan, 2004). The new drug may give protection to humans too in the food chain
(Balakrishnan, 2004).
2.7 Drug resistance challenge
Many food borne pathogens have developed resistance to antimicrobial agents during the
last decade including Salmonella and Escherichia coli strains (White et al., 2002). The
pathogens are an emerging problem in the community as well as nosocomial settings
(Schrag et al., 2002). Multidrug-resistant (MDR), Salmonella enterica serovar
Typhimurium phage type DT104 is currently the second most prevalent in England,
Wales, United States and Canada of which outbreaks have been reported in poultry (Boyd
et al., 2001).
There is a rapid international dissemination and excess mortality associated with drug
resistance in zoonotic Salmonella serovars (Helms et al., 2002). The increase in
antimicrobial resistance reported for human pathogens and the forecast by some
commentators that a post antibiotic era is on the horizon (Cohen, 1992) have greatly
stimulated research into the origins of antimicrobial resistance. Bacteria usually develop
their genes for drug resistance from plasmids (called resistance transfer factors, or RTFs).
Such bacteria are able to spread drug resistance to other strains and species during genetic
exchange processes (Cohen, 1992).
Salmonella typhimurium with reduced antimicrobial susceptibility associated with high
mortality in children less than 5 years (Graham et al., 2000; Lee et al., 2000; Kumar et
27
al., 1995) have been isolated in Africa and Kenya (Oundo et al., 2000; Kariuki et al.,
2000) of which the grossly affected antibiotics include ampicillin, co-trimoxazole,
streptomycin, chloramphenicol, and tetracycline. Salmonella isolates with reduced
fluoroquinolone susceptibility are on the increase (Hakanen et al., 2001) and ceftriaxoneresistant Salmonella isolates, which produce plasmid mediated AmpC- type  -Lactamase
such as CMY-2 are increasing globally as reported recently in the USA (Shiraki et al.,
2004).
Escherichia coli isolates with multiple-antibiotic-resistant phenotypes, involving coresistance to four or more unrelated families of antibiotics have been isolated globally
(Yolanda et al., 2004; Shiraki et al., 2004). In Kenya, thirty-seven strains of E. coli
recovered from cases of septicaemia in chicken showed resistance to the common cheap
antibiotics
notably trimethoprim-sulphamethoxazole (100%), kanamycin (13.5%) and
gentamycin (2.7%) (Bebora et al., 1994).
However, in other related studies, E. coli producing CTX-M-2 -Lactamase with reduced
susceptibility to cephalosporins for example Ceftiofur and Penicillinases such as TEM-1
and TEM-2 has been identified in poultry in Japan (Shiraki et al., 2004). The presence
and proliferation of clinical isolates of Escherichia coli strains producing IMP-1 type
metalo--Lactamase in Japan, which shows resistance to carbapenems and cephamycins,
that are third generation antibiotics, has become a clinical concern. Recently a global
threat emerged because certain antimicrobial resistant bacteria such as vancomycinresistant enterococci emerged in animals (Shiraki et al., 2004).
28
2.8 Bacterial resistance to antibiotics
The emergence and dissemination of antimicrobial resistance in pathogenic bacteria has
become a serious concern worldwide since this limits the therapeutic options for
treatment of infection (WHO, 2005; Cohen, 2000; White et al., 2001; Miller et al., 2002).
This has been attributed to selective pressure favoring antimicrobial-resistant phenotypes
whenever antimicrobials are used, in treating disease in clinical medicine, preventing
disease and promoting growth in animal husbandry (Cohen, 2000; Salvatore et al., 2004).
Antibiotic resistance also arise from a number of mechanisms involving mutation in genes
encoding drug targets or systems that affect drug accumulation defined as endogenous
resistance distinguished from the exogenous resistance mechanisms that are typically
mediated by the acquisition of plasmids and transposons (Miller et al., 2002). In
Enterobacteriaceae the most common mechanism of antibiotic-resistance genes assemble
is through gene-capture by integrons (Bissonnette and Roy, 1992; Hall and Collis, 1995;
Huovinen et al., 1995). Pathogenic or commensal bacteria in animals might acquire
antimicrobial resistance genes and either directly infect humans with the zoonotic
pathogens or transmit their resistance genes to human pathogens or human commensals
(Witte, 1998).
2.9 The basis of microbial resistance to antibiotics
Bacteria may be inherently resistant to an antibiotic (Ibezim, 2005). For example, a gramnegative bacterium may have some gene that is responsible for resistance to antibiotic; an
outer membrane that establishes a permeability barrier against the antibiotic; lack a
transport system for the antibiotic; target or reaction that is hit by the antibiotic (Ibezim,
2005). Bacteria can acquire (develop) resistance to antibiotics, for example bacterial
29
populations previously sensitive to antibiotics become resistant. This type of resistance
results from changes in the bacterial genome. Acquired resistance is driven by two genetic
processes in bacteria: (1) mutation and selection (vertical evolution); (2) exchange of genes
between strains and species (horizontal evolution) (Ibezim, 2005).
Vertical evolution involves principles of natural selection (Salvatore et al., 2004).
Spontaneous mutation in bacterial chromosome transfers resistance to a member of the
bacterial population. Selective pressure of antibiotics allow resistant mutant to grow and
flourish (Salvatore et al., 2004). Development of antibiotic resistance may involve point
mutations which are usually random (Davies, 1994; Lacey, 1984). The incidence of
mutations among isolates of pathogenic Escherichia coli and Salmonella enterica is over
1 percent (Eugene et al., 1996). Intragenic recombination in Escherichia coli and
Salmonella enterica may result in mosaic genes which express proteins that have new
phenotypes that help the organism to survive (Maiden, 1998).
Escherichia coli and Salmonella enterica may develop antibiotic resistance due to
transposons (transposable elements) which are small regions of DNA that can move from
one place to another in the genome playing a role in the evolution of antibiotic resistance
as well as providing another method of genetic exchange (McDonald, 1993; Lawrence et
al., 1992). Transposons are also relevant for the dissemination of antibiotic resistance
genes, either by integration in transferable plasmids or by direct conjugation and further
integration in the bacterial chromosome (McDonald, 1993; Lawrence et al., 1992).
Transposable elements that contain genes in the central region are called transposons.
This central sequence may contain resistance to one or more antibiotics, for example
making multiple antibiotic resistances probable. Transposons make multiple antibiotic
30
resistances very likely; this mechanism provides an easy and efficient way for the transfer
of resistance to several antibiotics to be spread at one time (McDonald, 1993; Lawrence
et al., 1992).
However, horizontal evolution involves the acquisition of genes for resistance from
another organism in nature through conjugation, transduction and transformation
(Guiney, 1984). Conjugation involves direct cell-to-cell contact of two bacterial cells
using sex pili with subsequent transfer of DNA (Guiney, 1984). Transduction occurs
when a bacteriophage carries chromosomal DNA or plasmids from one species to another
(Lacey, 1984). While transformation involves acquisition of DNA directly from the
environment, having been released from another cell. Recombinants (new genotypes)
may occur when plasmids are transferred between mating bacteria. Since bacteria usually
develop their genes for drug resistance on plasmids (resistance transfer factors or RTFs),
they are able to spread drug resistance to other strains and species during genetic
exchange processes (Maiden, 1998).
31
CHAPTER THREE
MATERIALS AND METHODS
3.1 Sample source
Chicken samples were sourced from indigenous chicken at Kariakor a leading
slaughterhouse/market outlet in Nairobi, Kenya. The source of each individual chicken
was identified using the Veterinary officer’s daily records and interviewing of farmers.
The indigenous chickens were from four districts namely, Murang’a, Bomet, Kitui and
Kericho which were the main supplier of indigenous chicken.
3.2 Specimen collection
Saline-moistened sterile cotton-tipped wooden applicators were used to collect rectal
swabs from chicken into sterile buffered peptone water (BPW) in universal bottles stored
in a cool box and delivered to the laboratory within 2 hours of collection. Each specimen
was cultured on respective isolation media to isolate the bacteria.
3.3 Sample size
The sample size was calculated according to Lwanga and Lameshow (1991).
N=Z² 1-aP (1-P)/d²
N=sample size
Z=standard normal deviation=1.96
a=level of significance of 5%
P=Prevalence of condition under study=0.2
32
d=Precision of study=0.05
N=1.96²x1-0.05x0.2 (1-0.2)/0.05²
=102 samples.
Prevalence for Salmonella enterica (20%) and E. coli (12.9%) were used to determine
sample size. Thus a minimum of 102 samples were to be collected, hence in the study
104 samples were collected with each of the four districts Murang’a, Bomet, Kitui and
Kericho accounting for 26 samples (Kariuki et al., 2005; Kariuki et al., 1999).
3.4 Bacterial isolation and characterization
3.4.1 Escherichia coli strains
Each of the specimens was streak plated onto MacConkey agar (Oxoid) and incubated at
37 oC overnight. Colonies suspected to be E. coli were isolated from the MacConkey agar
plates and identified using biochemical tests, confirmed by API 20E strips (Himedia
KB2). The isolates were stored at –70 oC in microvials for further analysis (Kariuki et al.,
1999).
3.4.2 Salmonella enterica serovars
The rectal swabs were inoculated onto Xylose Lysine decarboxylase agar (XLD). Cotton
tipped swabs were used to spread rectal swabs on a plate. Using flamed-sterilized wire
loop, the inoculum was streaked onto four quadrants of the plates with flaming after each
quadrant has been streaked, to obtain discrete colonies after overnight incubation at 37
°C. The inoculated plates and bottles were incubated aerobically at 37 ºC for between 1824 h. The XLD agar plates were removed from the incubator and examined for non-
33
lactose fermenting black centered colonies with clear edges and identified using
biochemical tests. Confirmation of Salmonella strains was done using API 20E strips and
stored in 15% glycerol at –80 ºC.
3.5 Biochemical tests for E. coli and Salmonella enterica
Gram stain
A pure colony was spread and fixed on the slide by drying using a Bunsen burner flame.
The slide was allowed to cool, and then flooded with crystal violet solution for 30 sec,
followed with Grams iodine solution for 1 min, followed by draining excess iodine by
decolorizing using acetone for at least 10 sec and then washed with water. Counter
staining was done using Basic fuchsin and allowed to stand for 30 seconds. This was
followed by washing the slide and dried in the air. The slide was observed under light
microscopy at X40. Short rods that stained red / pink were considered gram negative.
Indole production
Two to five pure colonies were inoculated using a sterile wire loop in 2 ml of peptone
water in bijous bottles and incubated overnight at 35 oC. 0.5 ml of Kovac’s reagent was
added and examined after 1minute. Presence of rose red colour on upper layer was
considered positive (+), while absence of rose red or pale colour was considered negative
(-).
Voges-Proskauer (VP)
In each bijous bottle, 2.5ml of Methyl red-Voges Proskauer broth was added and
inoculated with pure colonies of test organisms. The bijous bottles were then incubated at
35 oC for 48 h, followed by addition of 0.6 ml or 6 drops of VP reagent A (α-naphthanol
34
solution), then 0.2ml (2 drops) of VP reagent B (40% KOH). The bijous bottles were
shaken and allowed to stand for 15 minutes. Pink red colour (reddish pink) of the broth
culture in the bijous bottles was considered positive (+), while colourless (pale) were
considered negative (-)
Methyl red test
Five millilitres of Methyl red-Voges Proskauer broth was distributed in bijous bottles and
inoculated with pure colonies of test organisms. The bijous bottles were incubated at 35
o
C for 48 h, followed by addition of 0.5ml or 5 drops of methyl red and observed for
colour change. The bijous bottles with red colour were considered positive (+), while
those which developed yellow colour were considered negative (-).
Simmons Citrate
Simmons Citrate agar slants in bijous bottles were stabbed using a sterile wire loop and
incubated for 48h at 35 oC. Positive (+) growth for example citrate utilization produce an
alkaline reaction and the medium change colour from green to blue, while no colour
change (no citrate utilization) was considered negative (-).
Triple Sugar Iron Agar
TSI slopes with a butt of about 1 inch (3.5cm and 2.5cm) were inoculated by stabbing the
butt and carefully streaking of slant using a sterile inoculating needle after slightly
touching the center of a discrete colony on selective media. The tubes were incubated
overnight at 35 oC. Production of acid (yellow) slant and acid (yellow) butt, gas, without
production of H2S (blackening of agar) was considered positive for E. coli. While an
alkaline (red) slant and yellow butt (acid), gas, with or without H 2S gas (blackening
35
tubes) was considered positive for Salmonella enterica.
Urease test
Two millilitres of Urea broth base (Oxoid) in bijous bottles were inoculated with single
colonies of organism and incubated for 5-6 h at 37 oC in a water bath. Two controls were
used, a negative control containing Urea broth base only and positive control containing
Proteus aureus standard organism. All bijous bottles in which colour changed to pink were
considered positive (+), while those that had no colour change were considered negative.
Confirmation by API 20E
Two millilitres of bacterial suspension of one single colony of the isolate was prepared in
Muellar Hinton broth and incubated overnight at 37 oC. The kits were opened aseptically,
and 50l of bacterial suspension added to each compartment and incubated at 37 oC for
18-24 h. The results were interpreted as positive (colour change of medium) or negative
(no colour) in accordance with HilMViC Biochemical identification test kit KB001
(Appendix II).
3.6 Serological test
Serological tests were performed on sterile glass slides. Using a sterile wire loop, a
portion of growth from an overnight culture on TSI was suspended in normal saline on a
slide and then mixed with a drop of serum using the wire loop. The slide was rocked to
ensure uniformity. The slide was then observed under X40 on a microscope for
agglutination. Controls containing standard E. coli ATCC 25922 and standard Salmonella
typhimurium were run concurrently. The slides with agglutination for both polyvalent and
36
monovalent antisera were considered positive and negative without agglutination
respectively.
3.7 Antimicrobial susceptibility testing
3.7.1 Escherichia coli strains and Salmonella enterica serovars
All isolates were routinely tested by the single-disk diffusion method. Mueller Hinton
Agar was prepared according to the manufacturer's instructions (Oxoid). With a sterile
wire loop, the tops of five isolated colonies of similar morphological type were
transferred to a tube containing 5 ml of Mueller Hinton broth medium. The broth was
incubated at 35 °C until its turbidity exceeded that of the 0.5 McFarland standard
(Appendix I). Within 15 minutes of adjusting the density of the inoculums using sterile
distilled water, a sterile cotton swab on a wooden applicator stick were used to streak the
dried surface of Mueller-Hinton plates in three different planes. The inoculated plates
were allowed to remain on a flat surface for 3 to 5 minutes for absorption of excess
moisture, and then antimicrobial disks, Augumentin (Aug) 30μg; Ampicillin (Amp)
10μg; Cefuroxime (Crx) 30μg; Norfloxacin (Nor) 10μg; Chloramphenicol (Chl) 30μg;
Gentimicin (Gen) 10μg; Kanamycin (Kan) 30μg; Nalidixic acid (Nal) 30μg; Tetracycline
(Tet) 30μg; Cotrimoxazole (Cot) 25μg were applied. After 16 to 18 h incubation, each
plate was examined, and the diameters of the complete inhibition zones noted and
measured using vernier calipers. The diameters of the zones of inhibition were interpreted
by referring to the table, which represent the NCCLS subcommittee’s recommendation
(NCCLS 2002). Controls for antibiotic potency were done using E. coli ATCC 25922 for
which the MIC of the antibiotics is known.
37
3.8 Plasmid DNA isolation
3.8.1. Escherichia coli and Salmonella enterica serovars
The organisms were grown in 4ml of Luria-Bertani broth containing appropriate
antibiotic in a loosely capped universal bottle and incubated at 37 °C for 18 - 24 h. Then
2 ml of culture were pipetted into Eppendorf tubes and spinned at 12,000 rpm for 5
minutes in a micro centrifuge. The medium was removed by aspiration, leaving the
bacterial pellets dry. Plasmid DNA was then isolated using the Birnboim and Doly
(1979), and Ish-Horowicz and Burke (1981) modified protocol. The bacterial pellets
obtained were resuspended in 100 μl of ice-cold Solution I (Appendix I) by vigorous
vortexing to disperse bacterial pellets. Then 200 μl of freshly prepared Solution II
(Appendix I) was added and the contents mixed by inverting the Eppendorf tubes rapidly
five times. The Eppendorf tubes were then stored on ice briefly followed by addition of
150 μl of ice-cold Solution III (Appendix I), vortexed gently in an inverted position for
10 seconds and stored on ice for 5 minutes. The Eppendorf tubes were centrifuged at
12,000 rpm for 5 minutes in micro centrifuge, and then transferred the supernatant to
fresh Eppendorf tubes followed by addition of an equal volume of phenol: chloroform
(1:1) mixture. The Eppendorf tubes were vortexed and centrifuged at 12,000 rpm for 2
minutes in micro centrifuge. The supernatant was then transferred to fresh Eppendorf
tubes followed by precipitation of double-stranded DNA with 2 volumes of ethanol at
room temperature. The mixture was allowed to stand for 2 minutes at room temperature,
and then centrifuged at 12,000 rpm for 5 minutes in micro centrifuge. The supernatant
was removed by gentle aspiration and the Eppendorf tubes allowed to stand in an inverted
position to drain away the fluid. The pellets were rinsed with 1 ml of 70% ethanol,
38
drained by gentle aspiration and allowed to dry in air for 10 minutes. The pellets were
then redissolved in 50μl of Tris EDTA (pH 8.0). Plasmid DNA was separated by mixing
25μl of plasmid DNA with 3μl of electrophoretic dye (0.07% bromophenol blue, 0.7%
sodium dodecyl sulphate and 33% of glycerol in distilled water) and electrophoresised
using 1 % agarose for 1h at 100 volts in a standard electrophoretic system. The plasmid
bands were strained with aqueous ethidium bromide (1µg/ml) for 10 minutes and then
washed continuously in tap water for 10 minutes. The plasmid sizes were determined by
co-electrophoresis with plasmids of known sizes for E. coli strains V517. The bands were
then visualized on an ultraviolet transilluminator and photographed with a Polaroid
instant camera.
3.9 In-vitro Conjugation experiment
Escherichia coli strains that were resistant to Ampicillin and susceptible to Nalidixic acid
were used for conjugation studies which were carried out as described by Yamamoto and
Yokota (1983). E. coli strains from indigenous chicken were the donor bacteria and
recipient was E. coli K12 Nalidixic acid resistant which had been grown on nutrient agar
overnight and sub-cultured in 3ml of tryptic soy broth in bijou bottles incubated at 37 oC
for 3h in a rotating incubator to attain the logarithmic phase. The log phase cultures of
donor and recipient bacteria were mixed in a ratio of 1:10 by transferring 1ml of culture
into 9ml fresh broth. Two milliliter of donor and recipient were mixed in duplicate sets.
One set was incubated at 37oC and the other at room temperature overnight. The sets
were then centrifuged at 13000rpm for 1 minute in a refrigerated micro-centrifuge using
1.5ml tubes to obtain pellets which were washed with sterile phosphate buffered saline.
The mixture was further centrifuged at 13000rpm for 1 minute and washed using sterile
39
phosphate buffered saline, which was then aspirated to remove the supernatant. The cells
were streak plated using a sterile wire loop on MacConkey agar containing 30μg/ml
Ampicillin and 30μg/ml Nalidixic acid to obtain transconjugants. Two controls were
used, MacConkey agar containing 30μg/ml Ampicillin and MacConkey agar containing
30μg/ml Nalidixic acid. The transconjugants were tested for susceptibility to the
antibiotics previously used for isolates.
3.10 Data analysis
The isolation and identification of organisms were entered as plus (+) for presence and
negative (-) for absence of organisms. Response to antibiotics was recorded as either
Susceptible (S), Intermediate (I), or Resistant (R). Plasmid fingerprinting was entered as
bands. All the data was entered into the computer and subsequently analysed using SPSS
11.5 (2000) and MS Excel 2000 package for windows at confidence interval of 95% to
determine significance. The overall trends were computed using descriptive and
quantitative analysis. These were further subjected to analysis of variance (ANOVA).
40
CHAPTER FOUR
RESULTS AND DISCUSSION
4.1 Bacterial isolation and characterization
During this study, 104 samples were collected from indigenous chicken (Gallus gallus)
rectal swabs. The samples collected were from four districts namely Bomet, Kericho,
Kitui and Murang’a that supply indigenous chicken to the leading slaughterhouse/market
outlet. The distribution of samples collected with/ without pathogens (Salmonella
typhimurium and E. coli) was as follows; Murang’a district had the highest number of 22
out of 26 samples (84.6%) with pathogens while 4 samples (15.4%) were free from the
pathogens; Bomet and Kitui districts followed with 21 out of 26 samples (80.8%) with
pathogens, while 5 samples (19.2%) were free from the pathogens; finally Kericho
district had 19 out of 26 samples (73.1%) with pathogens, while 7 samples (26.9%) were
free from pathogens (Table 1).
Table 1: The distribution of enteric pathogens (Salmonella typhimurium and E. coli)
/percentages in the four district samples
District
Samples collected
Totals
With pathogens
Without pathogens
Murang’a
22 (21.15%)
4 (3.85%)
26 (25%)
Bomet
21 (20.19%)
5 (4.81%)
26 (25%)
Kitui
21 (20.19%)
5 (4.81%)
26 (25%)
Kericho
19 (18.27%)
7 (6.73%)
26 (25%)
Grand Total
83 (79.8%)
21 (20.2%)
104 (100%)
41
The cumulative proportion of samples with Salmonella typhimurium and E. coli was 83
(79.8%), while 21 (20.2%) were free from the pathogens (Table 1). The eighty-three
samples with pathogens had strains of Salmonella typhimurium at a frequency of 15.66%
(13 out of 83) and E. coli 84.34% (70 out of 83) respectively. The district cross tabulation
of bacterial strains from indigenous chicken showed Murang’a district had the highest
number of E. coli strains (19), followed by Kitui (18), Bomet (17), and Kericho (16)
(Table 2). On the other hand, Bomet district had the highest number of Salmonella
typhimurium strains of 4, while Kericho, Murang’a and Kitui had 3 strains each
respectively (Table 2).
Table 2: District cross-tabulation of bacterial strains from indigenous chicken rectal
swabs
District
Strains
Totals
E. coli
S. typhimurium
Murang’a
19
3
22
Kitui
18
3
21
Bomet
17
4
21
Kericho
16
3
19
Grand total
70
13
83
On the other hand Bomet district had the highest strains of Salmonella typhimurium (4),
while Kericho, Murang’a and Kitui had 3 strains each respectively (Table 2). Pearson
Correlation of strains (Table 3) showed significant correlation of strains from all district
samples since the values were positive tending towards one.
42
Table 3: Pearson correlation of strains
District
Bomet
Kericho
Kitui
Murang’a
Pearson ChiSquare
Likelihood
Ratio
N of Valid
Cases
Pearson ChiSquare
Likelihood
Ratio
N of Valid
Cases
Pearson ChiSquare
Likelihood
Ratio
N of Valid
Cases
Pearson ChiSquare
Likelihood
Ratio
N of Valid
Cases
Value
Df
Sig. (2-sided)
21.000
20
.397
20.450
20
.430
19.000
18
.392
16.574
18
.553
21.000
20
.397
17.225
20
.638
44.000
42
.387
21.345
42
.997
21
19
21
22
N=sample number, DF=difference, Sig=Significance.
4.2 Serological tests
4.2.1 Serology of Salmonella enterica
The thirteen strains of Salmonella enterica were serotyped using Polyvalent-O group AG; Salmonella-O group C1 factor 6, 7; Salmonella-O factor 4; Salmonella- H factor d;
and Salmonella-O factor 1,9,12. Agglutination was observed with Polyvalent-O group AG and Salmonella-O factor 4 in all the strains Thus Salmonella enterica serovar
43
Typhimurium was the only serovar serotyped in the four district samples collected (Table
4).
Table 4: Serotype of Salmonella enterica
Serotype
Strains
Totals
Salmonella
Bomet
Murang’a
Kitui
Kericho
typhimurium
4
3
3
3
13
4.2.2 Serology of Escherichia coli
The seventy strains of E. coli were serotyped using Polyvalent 1: 020, 025, 063, 0153 and
0167; polyvalent 2: 044, 055, 0125, 0126, 0146 and 0166; polyvalent 3: 06, 027, 078,
0148, and 0159; polyvalent 4: 08, 015, 020, 063, and 0115 respectively. Two serotypes of
E. coli were present, enterotoxigenic E. coli (53) and enteropathogenic E. coli (17). The
highest number of enterotoxigenic E. coli were isolated from Bomet district (16),
followed by Murang’a (14), Kitui (13) and Kericho (10); while enteropathogenic E. coli
were as follows; Kericho lead with 6 strains, followed by Murang’a and Kitui (5); and
Bomet (1) respectively (Table 5).
Table 5: Serotypes of E. coli strains
Serotype
ETEC
EPEC
Bomet
16
1
Strains
Kericho
Murang’a
10
14
6
5
Kitui
13
5
Total
53
17
ETEC=Enterotoxigenic Escherichia coli, EPEC=Enteropathogenic Escherichia coli.
44
4.3 Antimicrobial Susceptibility Testing
Antimicrobial susceptibility test results with resistance to two antibiotics, or more, were
considered as multiple resistances. Among all the strains, 60.98% were sensitive to all the
antimicrobial agents used, while 39.02% were resistant to at least one or more
antimicrobial agents tested.
In the study, various antimicrobial agents were tested against Escherichia coli and
Salmonella enterica Typhimurium which included Augumentin, Cefuroxime, Nalidixic
acid, Norfloxacin, Ampicillin, Gentamicin, Cotrimoxazole, Tetracycline, Kanamycin, and
Chloramphenicol. The antimicrobial sensitivity response (zone diameters in mm) for each
antimicrobial agent is indicated in Appendix III and case summary (Table 6).
45
Table 6: Antimicrobial sensitivity response (zone diameters) of Escherichia coli and
Salmonella enterica Typhimurium case summaries
Antimic
Strains
N
Mean
Std.
Dev
Std.
Error
Aug
S. typhim
E. coli
Totals
S. typhim
E. coli
Totals
S. typhim
E. coli
Totals
S. typhim
E. coli
Totals
S. typhim
E. coli
Totals
S. typhim
E. coli
Totals
S. typhim
E. coli
Totals
S. typhim
E .coli
Totals
S. typhim
E. coli
Totals
S. typhim
E. coli
Totals
13
70
83
13
70
83
13
70
83
13
70
83
13
70
83
13
70
83
13
70
83
13
70
83
13
70
83
13
70
83
21.69
18.96
19.38
20.54
18.92
19.17
22.92
21.27
21.52
27.69
25.17
25.56
15.31
13.90
14.12
16.92
17.77
17.64
16.38
14.63
14.90
10.54
9.94
10.04
17.54
17.21
17.26
19.54
23.20
22.63
4.553
4.142
4.296
2.817
3.609
3.532
1.115
1.473
1.540
3.449
2.757
2.995
10.858
9.776
9.895
5.392
1.750
2.624
13.890
12.249
12.443
10.162
10.418
10.321
1.898
3.902
3.657
8.762
7.594
7.843
1.263
.492
.469
.781
.428
.385
.309
.175
.168
.957
.327
.327
3.011
1.160
1.080
1.496
.208
.286
3.852
1.454
1.358
2.818
1.236
1.126
.526
.463
.399
2.430
.901
.856
Crx
Nal
Nor
Amp
Gent
Cot
Tet
Kan
Chl
95% confidence
interval for mean
L limit U
Limit
18.94
24.44
17.98
19.94
18.45
20.31
18.84
22.24
18.06
19.77
18.40
19.93
22.25
23.60
20.92
21.62
21.19
21.86
25.61
29.78
24.52
25.82
24.91
26.21
8.75
21.87
11.59
16.22
11.97
16.27
13.66
20.18
17.36
18.19
17.07
18.21
7.99
24.78
11.73
17.53
12.20
17.61
4.40
16.68
7.48
12.41
7.80
12.28
16.39
18.69
16.29
18.13
16.47
18.06
14.24
24.83
21.40
24.99
20.93
24.33
Min
mm
Max
Mm
13
0
0
15
0
0
21
18
18
22
20
20
0
0
0
0
13
0
0
0
0
0
0
0
14
0
0
0
0
0
29
25
29
24
24
24
25
25
25
32
31
32
28
25
28
22
22
22
33
29
33
20
23
23
20
21
21
26
30
30
Antimic=Antimicrobial agents; Aug=Augumentin; Crx=Cefuroxime; Nal=Nalidixic acid;
Nor=Norfloxacin; Amp=Ampicillin; Gent=Gentamicin; Cot=Cotrimoxazole; Tet=Tetracycline;
Kan=Kanamycin; Chloramphenicol; N=number of isolates; S. typhim=Salmonella typhimurium;
E. coli= Escherichia coli; std=standard; L=Lower; U=Upper; Min=Minimum zone diameter;
Max=Maximum zone diameter, Dev=Deviation, mean of zone diameters.
Resistance, susceptible and intermediate categories were achieved using the published
zone diameter interpretive NCCLS data (Appendix IV: Table 13).
46
4.3.1 Escherichia coli strains
Seventy E. coli strains showed different drug resistance profiles in this study (Table 7).
The strains displayed resistance phenotypes to one, two or more antibiotics. Monovalent
resistance to antibiotics was observed in 18 strains; divalent resistance was displayed by
4 strains, trivalent resistance was observed in 10 strains, tetravalent resistance was found
in 6 strains, pentavalent resistance was seen in 3 strains, and hexavalent resistance was
found in 2 strains.
Thus multiple resistances were observed in 25 strains. The most common antimicrobial
resistance pattern of these strains was the single resistance pattern to Tet (21.43%),
followed by Amp Cot Tet (14%), Aug Amp Cot Tet and Cot (4.29%), Aug Amp Cot Tet
Kan Chl (2.86%), Amp Cot Tet Chl, Cot Tet (2.86%), and Crx Amp Cot Tet, Crx Amp
Cot Tet Chl, Amp Cot, Aug Amp, Aug Amp Cot Tet Chl, Aug Amp Cot Tet Kan (1.43%)
respectively.
Single resistance to tetracycline was present in 15 out of 18 strains, which exhibited a
monovalent resistance pattern, and in all other patterns with exception of only two
Murang’a strains which exhibited resistance pattern of (Aug, Amp) and (Amp, Cot)
(Table 7). Thus, the highest rate of resistance was against Tet (55.7%), followed by Cot
(40%) which too had single resistance of 3 out of 18 strains, and showed presence in all
other combination patterns excluding the Aug, Amp in Bomet strains. Third in line of
resistance was Amp 32.86%, which occurred only in combination patterns with exception
of two (Cot, Tet) in Murang’a strains. Aug (11.43%), Chl (8.57%), Kan (4.29%), and Crx
(2.86%) followed respectively.
47
E. coli strains displayed high percentage of resistance to test antibiotics and thus multiple
drug resistance was observed in the strains. Tetracycline resistance was the highest and
common in all district strains. Bomet strains (17.1%) were the leading, followed by
Murang’a strains (15.7%), Kitui strains (12.9%) and finally Kericho strains (8.6%).
Cotrimoxazole resistance was second, the highest being in Bomet strains (15.7%), Kitui
and Murang’a (10%), while Kericho (2.9%).
Kanamycin resistance was only in Kitui (2.9%) and Bomet (1.43%). Escherichia coli
resistance to Augumentin (5.7%) was only found in strains from Bomet and Kitui district,
Cefuroxime (2.9%) resistance in Bomet strains, Ampicillin resistance (17.1%) was
highest in Bomet strains, followed by Kitui strains (7.1%), Murang’a strains (5.7%) and
Kericho (2.9%). Chloramphenicol resistance was highest in Kitui strains (4.3%),
followed by Bomet (2.9%) and Kericho (1.43%). There was no resistance to Norfloxacin,
Nalidixic acid and Gentamicin. Thus, the three antibiotics are the drugs of choice for E.
coli strains from indigenous chicken (Figure 3).
48
Figure 3: District percentage resistance of E. coli to test antimicrobial agent
18
16
14
%R
12
bomet
10
kitui
8
kericho
6
Murang’a
4
2
0
aug
crx
nal
nor
amp
gen
cot
tet
kan
chl
Antimicrobial agents
Aug=Augumentin,
Crx=Cefuroxime,
Nal=Nalidixic
acid,
Nor=Norfloxacin,
Amp=Ampicilin, Gen=Gentimicin, Cot=Cotrimoxazole, Tet=Tetracycline,
Kan=Kanamycin, Chl=Chloramphenicol, % R= percentage resistance.
Murang’a had the highest fully susceptible strains (27.1%) to Nal, Nor, Gen, and Chl;
followed with Nal and Nor (25.7%) in Kitui strains; Gen (24.3%) in Bomet and Kitui
strains; Aug (24.3%) in Muran’ga strains; Aug, Nal, Nor, Gen (22.9%) in Kericho
strains; Chl (21.4%) in Bomet, Kitui and Kericho strains; Kan, Amp (20%) in Kericho,
Kan (20%) in Murang’a and Aug (20%) in Kitui strains; Amp and Cot (18.6%) in Kitui
and Kericho; Cot (17.1%) in Murang’a strains; Aug, Cot, Kan (15.7%) in Bomet and
Kitui; Tet (14.3%) in Kericho; Tet (12.9%) in Kitui Tet (11.4%) in Murang’a; Tet and
Amp (7.1%) in Bomet strains; and the least being Crx (1.43%) in Kitui and Murang’a
strains respectively (Fig 4).
49
Figure 4: District percentage susceptibility of E. coli
30
25
20
bomet
kitui
15
%S
kericho
Murang’a
10
5
0
aug
crx
nal
nor
amp
gen
cot
tet
kan
chl
Antimicrobial agents
Aug=Augumentin,
Crx=Cefuroxime,
Nal=Nalidixic
acid,
Nor=Norfloxacin,
Amp=Ampicillin, Gen=Gentimicin, Cot=Cotrimoxazole, Tet=Tetracycline,
Kan=Kanamycin, Chl=Chloramphenicol, % S= percentage susceptibility.
However, Escherichia coli strains showed the highest percentage intermediates in all the
four district strains to Cefuroxime with Murang’a (25.7%) leading followed by Bomet
(24.4%), Kitui (24.3%) and Kericho (22.9%). Kanamycin (10%) in Bomet was the
highest; Kitui and Murang’a (7.1%) followed and least Kericho (2.9%). Aug 2.9% in
Bomet and Murang’a was next, and finally Cot (1.43%) in Kitui strains respectively (Fig.
5).
50
Figure 5: District percentage intermediate of E. coli
30
25
20
bomet
kitui
15
%I
kericho
Murang’a
10
5
0
aug
crx
nal
nor
amp gen
cot
tet
kan
chl
Antimicrobial agents
Aug=Augumentin,
Crx=Cefuroxime,
Nal=Nalidixic
acid,
Nor=Norfloxacin,
Amp=Ampicilin, Gen=Gentimicin, Cot=Cotrimoxazole, Tet=Tetracycline,
Kan=Kanamycin, Chl=Chloramphenicol, % I= percentage intermediate.
The distribution of resistance patterns to test antimicrobial agents for E. coli in Bomet
isolates (14 out of 43) was the highest in the four district samples studied. Out of the
fourteen strains, twelve exhibited multiple drug resistance patterns while the two
displayed single drug resistance pattern (Table 7). Of the 12 strains which displayed
multiple drug resistance patterns; the three drug resistance pattern of Amp Cot Tet (5 out
of 14) was predominant, followed with the four resistance pattern of Aug Amp Cot Tet
(2 out of 14), Amp Cot Tet Chl and Crx Amp Cot Tet (1 out of 14) each; the five
resistance drug pattern of Aug Amp Cot Tet Kan (1 out of 14) and Crx Amp Cot Tet Chl
(1 out of 14), and finally the two resistance drug pattern of Aug Amp (1 out of 14).
51
Single resistance pattern (Tet) was observed in two Bomet strains which was the least
comparable to other district strains (Table 7).
Table 7: Antimicrobial resistance patterns of E. coli strains
Antimicrobial
resisted
One
Bomet
2
Two
1
Strains of E. coli
Murang’a
Kitui
5
5
1
2
Totals
Kericho
3
1
Three
5
Four
2
2
4
1
1
1
Five
3
1
2
1
1
1
1
1
1
1
1
Six
Grant Total
15
3
1
1
2
10
2
14
12
12
2
5
Aug=Augumentin,
Amp=Ampicillin,
Cot=Cotrimoxazole,
Chl=Chloramphenicol, Crx=Cefuroxime, Kan=Kanamycin.
Resistance
patterns
Tet
Cot
Aug Amp
Amp Cot
Cot Tet
Amp Cot
Tet
Aug Amp
Cot Tet
Amp Cot
Tet Chl
Crx Amp
Cot Tet
Aug Amp
Cot Tet Kan
Aug Amp
Cot Tet Chl
Crx Amp
Cot Tet Chl
Aug Amp
Cot Tet Kan
Chl
43
Tet=Tetracycline,
Murang’a and Kitui had the same tally (12 out of 43) each. Both had five strains
displaying the single drug resistance patterns to tetracycline. On the hand, Kitui had 2
strains while Murang’a had one which exhibited single drug resistance to Cotrimoxazole.
The two district strains varied slightly when it came to multiple drug resistance. Whereas
in Murang’a four displayed a three drug resistance pattern of Amp Cot Tet; Kitui had one
52
strain which displayed a four drug resistance pattern (Aug Amp Cot Tet) and the other
displayed a five drug resistance pattern (Aug Amp Cot Tet Chl). However, both
Murang’a and Kitui had strains which displayed a two drug resistance pattern. Murang’a
had two strains (Cot Tet), while Kitui had one (Amp Cot).
In contrast, Kericho had 5 out of 43 strains with three displaying a single drug resistance
pattern (Tet). While multiple drug resistance patterns were observed in two strains as
follows; one which displayed a three drug resistance pattern (Amp Cot Tet) and the other
a four drug resistance pattern (Amp Cot Tet Chl) (Table 7).
4.3.2 Salmonella typhimurium
Thirteen Salmonella strains were recovered from 13 samples of indigenous chicken rectal
swabs (12.75 %) (Table 1). The highest resistance was against Tet (58.3%), followed by
Cot (41.7%), Amp (33.3%), Chl (16.7%), Aug and Gen (8.3%) respectively (Table 8). Of
the five Salmonella typhimurium recovered, single resistance pattern was to Tet in the
Bomet strains. The tetravalent pattern Amp Cot Tet Chl occurred in one Kericho and
Murang’a strains, while Aug Amp Cot Tet (Kericho), Gen Cot Tet (Bomet), and Amp
Cot Tet (Kitui) were observed in one strains each respectively (Table 8).
53
Table 8: Antimicrobial resistance patterns of Salmonella typhimurium strains
Antimicrobial
resisted
One
Three
Strains of Salmonella typhimurium
Bomet Murang’a
Kitui
Kericho
2
1
2
1
1
Four
Grand total
1
1
3
Totals
1
1
1
2
1
1
2
7
Resistan
ce
patterns
Tet
Gen Cot
Tet
Amp Cot
Tet
Amp Cot
Tet Chl
Aug Amp
Cot Tet
Aug=Augumentin,
Amp=Ampicillin,
Cot=Cotrimoxazole,
Tet=Tetracycline,
Chl=Chloramphenicol, Crx=Cefuroxime, Kan=Kanamycin, Gen=Gentamicin.
Bomet had the highest tally of strains (3 out of 7) which exhibited varied drug resistance
pattern. Single drug resistance pattern (Tet) was only observed in two Bomet strains,
while one displayed a three drug resistance pattern (Gen Cot Tet) (Table 8). In Kericho
strains (2 out of 7), both displayed a four drug resistance pattern (Amp Cot Tet Chl and
Aug Amp Cot Tet). Murang’a and Kitui had the same tally of one each multiple drug
resistance. The Kitui strains displayed a three drug resistance pattern (Amp Cot Tet),
while the Murang’a strains exhibited a four drug resistance pattern (Amp Cot Tet Chl)
(Table 8).
Among
multidrug-resistant
strains,
resistance
to
Gentamicin,
Cotrimoxazole,
Tetracycline, Ampicillin, Chloramphenicol, and Augumentin was most often observed
(Table 8). All strains of Salmonella typhimurium were susceptible to Norfloxacin, and
Nalidixic acid.
54
The highest resistance of Salmonella typhimurium was against tetracycline with strains
from Bomet district leading 23.1%, followed by Kericho strains (15.4%), Kitui and
Murang’a strains (7.7%) respectively. Cotrimoxazole resistance was highest in Kericho
strains (15.4%), with Bomet, Murang’a and Kitui strains (7.7%). Ampicillin resistance
was observed in three district strains with Kericho strains leading with 15.4%, while both
Kitui and Murang’a strains had 7.7%. Chloramphenicol resistance was only in two
district strains of Kericho and Murang’a (7.7%). Resistance to Gentamicin was only in
Bomet strains (7.7%); Augumentin (7.7%) was only observed in Kericho strains. All
Salmonella typhimurium strains showed no resistance to Norfloxacin, Nalidixic acid,
Cefuroxime and Kanamycin (Fig 6).
Figure 6: District percentage resistance of Salmonella typhimurium to test antimicrobial
agent
25
20
bomet
15
%R
kitui
kericho
10
Murang’a
5
0
aug
crx
nal
nor
amp
gen
cot
tet
kan
chl
Antimicrobial agents
Aug=Augumentin,
Crx=Cefuroxime,
Nal=Nalidixic
acid,
Nor=Norfloxacin,
Amp=Ampicilin, Gen=Gentimicin, Cot=Cotrimoxazole, Tet=Tetracycline,
Kan=Kanamycin, Chl=Chloramphenicol, % R= percentage resistance.
55
The highest proportion of susceptible Salmonella typhimurium strains (30.8%) was
observed for Aug, Nal, Nor, Amp, Kan and Chl in Bomet strains; this was followed by
Gen (23.1%) in all the four district strains, Nal and Nor (23.1%) in Kitui, Kericho and
Murang’a strains; and finally Chl (23.1%) in Kitui strains (Fig 7). Aug (23%) in
Murang’a strains was the second highest, the least being Kitui and Kericho strains (15.4
%); which was also similar to Chl (15.4%) in Kericho and Murang’a strains. In contrast
to Amp, Cot (15.4%) in Kitui and Murang’a strains, were second highest. Tet (15.4%) in
Murang’a and Kitui strains was the highest amongst all the four district strains. Crx
(7.7%) in Kitui, Kericho and Murang’a; Amp, Cot, Kan (7.7%) in Kericho; Kan (7.7%)
in Murang’a and Kericho: finally Tet (7.7%) in Bomet and Kericho were the least (Fig 7).
Figure 7: District percentage susceptibility of Salmonella typhimurium to test
antimicrobial agent
35
30
25
bomet
20
kitui
15
kericho
%S
Murang’a
10
5
0
aug
crx
nal
nor
amp
gen
cot
tet
kan
chl
Antimicrobial agents
Aug=Augumentin,
Crx=Cefuroxime,
Nal=Nalidixic
acid,
Nor=Norfloxacin,
Amp=Ampicilin, Gen=Gentimicin, Cot=Cotrimoxazole, Tet=Tetracycline,
Kan=Kanamycin, Chl=Chloramphenicol, % S= percentage susceptibility.
56
Among the intermediates observed, Cefuroxime lead all the test antimicrobials used in
the study, the highest being 30.8% from Bomet strains, followed by Kitui, Kericho and
Murang’a (15.4%) respectively. Kanamycin followed subsequently with 15.4% in
Kericho and Murang’a; and 7.7% in Kitui strains. Aug (7.7%) was least in Kitui strains
(Fig. 8).
Figure 8: District percentage intermediate of Salmonella typhimurium to test antimicrobial
agent
35
30
25
bomet
%I
20
kitui
15
kericho
Murang’a
10
5
0
aug
crx
nal
nor
amp gen
cot
tet
kan
chl
Antimicrobial agents
Aug=Augumentin,
Crx=Cefuroxime,
Nal=Nalidixic
acid,
Nor=Norfloxacin,
Amp=Ampicilin, Gen=Gentimicin, Cot=Cotrimoxazole, Tet=Tetracycline,
Kan=Kanamycin, Chl=Chloramphenicol, % I= percentage intermediate.
4.4 Co-infection strains
Out of the total 83 strains of Escherichia coli (70) and Salmonella typhimurium (13)
isolated from indigenous chicken rectal swab, 4 samples (9.8%) had both E. coli and
Salmonella enterica Typhimurium (Table 9).
57
Table 9: Co-infection strains antimicrobial susceptibility
Co-infection
Sample source
Strains
Bomet
Murang’a
Kitui
Kericho
E. coli
1(R)
1(S)
1(S)
1(S)
Salmonella
1(R)
1(S)
1(S)
1(S)
2
2
2
2
typhimurium
Total
R=Resistant, S=Susceptible.
All the co-strains were susceptible to test antibiotics used except for the Bomet strains
which exhibited resistance. The two strains were both resistant to tetracycline. Salmonella
typhimurium strain displayed single resistance pattern to Tet, while Escherichia coli
exhibited a tetravalent pattern of Crx Amp Cot Tet (Table 10).
Table 10: Co-infection strains antimicrobial resistance patterns
Source
Bomet
Strain
E. coli
S. typhimurium
Resistance patterns
Crx Amp Cot Tet
Tet
Crx=Cefuroxime, Amp=Ampicillin, Cot=Cotrimoxazole, Tet=Tetracycline.
4.5 Plasmid profiles
In this study, plasmids were found in 19 (44.2%) of resistant Escherichia coli strains,
while 24 (55.8%) were plasmidless. On the other hand 5 (71.4%) of resistant Salmonella
typhimurium strains had plasmids, while 2 (28.6%) were plasmidless (Fig 9). The 3.0-and
5.6-kb plasmids that were associated with Ampicillin resistance in E. coli were not
transferable.
58
Figure 9: Gel electrophoresis of Escherichia coli and Salmonella typhimurium plasmid
DNA
MW(kb)
7.2______________
5.6______________
3.9______________
3.0______________
2.7______________
1
2
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Lane 1:Marker V517, 2:S64Mu, 3:E43Ki, 4:S82Ke, 5:S19Bo, 6:E50Mu, 7:E79Ke,
8:E8Bo, 9:E11Bo, 10:E17Bo, 11:S46Ki, 12:Marker V517, 13:E39Ki, 14:E27Ki,
15:E83Ke, 16: E24Ki, 17:E10Bo, 18:E44Ki, 19:E14Bo, 20:E19Bo, 21:S81Ke,
22:E72Mu, 23:E23Bo, 24:E74Mu, 25:E28Ki, 26:E43Ki
Marker V517= E. coli V517, S64Mu=Salmonella typhimurium strain 64 of Murang’a,
E43Ki=E. coli strain 43 of Kitui, S82Ke=Salmonella typhimurium strain 82 of Kericho,
S19Bo=Salmonella typhimurium strain 19 Bomet, E50Mu=E. coli strain 50 of Murang’a,
E79Ke=E. coli strain 79 of Kericho, E8Bo=E. coli strain 8 of Bomet, E11Bo=E. coli
strain 11 of Bomet, E17Bo=E. coli strain 17 of Bomet, S46Ki=Salmonella typhimurium
strain 46 of Kitui, E39Ki=E. coli strain 39 of Kitui, E27Ki=E. coli strain 27 of Kitui,
E83Ke=E. coli strain 83 of Kericho, E24Ki=E. coli strain 24 of Kitui, E10Bo=E. coli
strain 10 of Bomet, E44Ki=E. coli strain 44 of Kitui, E14Bo=E. coli strain 14 of Bomet,
E19Bo=E. coli strain 19 of Bomet, S81Ke=Salmonella typhimurium strain 81 of Kericho,
E72Mu=E. coli strain 72 of Murang’a, E23Bo=E. coli strain 23 of Bomet, E74Mu=E.
coli strain 74 of Murang’a, E28Ki=E. coli strain 28 of Kitui, and E43Ki=E. coli strain
43 of Kitui.
The 5.6-kb plasmid was present in all strains examined. E28Ki, E72Mu, E24Ki, E43Ki,
E44Ki, E50Mu, E83Ke, E8Bo, E17Bo, E11Bo, E10Bo, E74Mu, contained only the 5.6-kb
plasmid indicating relatedness, likewise to S19Bo, S82Ke and S64Mu strains. The 5.6- and
59
7.2-kb plasmids were present in strains E43Ki, E79Ke and E39Ki; 2.7- and 5.6-kb were
present in strains E27Ki and S46Ki; 3.0- and 5.6-kb were present only in strains E14Bo and
E19Bo; 3.9, - 5.6- and 7.2- kb were only present in one strain S81Ke. The 3.0-kb plasmid
was present only in strains E14Bo and E19Bo that exhibited resistance to Cefuroxime
(Table 11).
Table 11: Strains, antimicrobial resistance pattern, number of plasmids and plasmid sizes
Plasmid sizes kb
2.7, 5.6
3.0, 5.6
3.9, 5.6, 7.2
5.6
5.6, 7.2
Strains codes
Resistance pattern
E27Ki,
Aug Amp Cot Tet Chl
S46Ki, E23Bo
Amp Cot Tet
E14Bo
Crx Amp Cot Tet Chl
E19Bo
Crx Amp Cot Tet
S81Ke
Aug Amp Cot Tet
S19Bo, E8Bo, E83Ke, E10Bo
Tet
E43Ki,E44Ki
Cot
E50Mu
Cot Tet
E17Bo, E72Mu,
Amp Cot Tet
S82Ke, S64Mu
Amp Cot Tet Chl
E11Bo, E74Mu
Aug Amp Cot Tet
E24Ki, E28Ki
Aug Amp Cot Tet Kan Chl
E43Ki
E79Ke
E39Ki
Cot
Amp Cot Tet
Aug Amp Cot Tet
Aug=Augumentin,
Crx=Cefuroxime,
Amp=Ampicilin,
Cot=Cotrimoxazole,
Tet=Tetracycline, Kan=Kanamycin, Chl=Chloramphenicol, Mu=Murang’a, Ke=Kericho,
Ki=Kitui, Bo=Bomet, E27Ki=E. coli strain 27 of Kitui, S46Ki=Salmonella typhimurium
strain 46 of Kitui, E14Bo=E. coli strain 14 of Bomet, E19Bo=E. coli strain 19 of Bomet,
S81Ke=Salmonella typhimurium strain 81 of Kericho, S19Bo=Salmonella typhimurium
strain 19 Bomet, E8Bo=E. coli strain 8 of Bomet, E83Ke=E. coli strain 83 of Kericho,
E44Ki=E. coli strain 44 of Kitui, E50Mu=E. coli strain 50 of Murang’a, E17Bo=E. coli
strain 17 of Bomet, E72Mu=E. coli strain 72 of Murang’a, E23Bo=E. coli strain 23 of
Bomet, S82Ke=Salmonella typhimurium strain 82 of Kericho, S64Mu=Salmonella
typhimurium strain 64 of Murang’a, E11Bo=E. coli strain 11 of Bomet, E24Ki=E. coli
strain 24 of Kitui, E28Ki=E. coli strain 28 of Kitui, E43Ki=E. coli strain 43 of Kitui,
E79Ke=E. coli strain 79 of Kericho, E39Ki=E. coli strain 39 of Kitui, E74Mu=E. coli
strain 74 of Murang’a, E10Bo=E. coli strain 10 of Bomet.
60
4.6 Discussion
The study shows appearance of multidrug enteric pathogens (Salmonella typhimurium
and E. coli) in apparently healthy indigenous chicken sold in the slaughterhouse cum
market outlet in Nairobi, Kenya. The microbes may easily contaminate chicken carcasses
during removal of gastrointestinal content (Van den Boorgand et al., 2004), leading to a
possibility of cross contamination directly from raw chicken or indirectly via
contaminated surfaces or niches. The result of this study showed 79.8% occurrence of E.
coli and Salmonella typhimurium in collected samples. Correspondingly, furthering the
significant appearance of drug resistant strains of E. coli (P<0.0002) and Salmonella
typhimurium (P<0.0001) in indigenous chicken.
Indigenous chicken poke and scratch anything they find in the quest for food, thus they
may pick up pathogens and other materials such as drug residues from the environment.
This is in line with findings done by Appanjalati et al (2004). At the same time
indigenous chicken (Gallus gallus) are reared together with other monogatric animals
(Guèye, 2002; Appanjalati et al., 2004), this may lead to zoonosis. For example βlactamase-produncing E. coli associated with drug resistance was isolated from a dog
with recurrent urinary tract infections (Shiraki et al., 2004).
In a previous study, thirty-seven strains of E. coli recovered from cases of septicaemia in
chicken in Kenya showed resistance to Trimethoprim-Sulphamethoxazole (100%),
Kanamycin (13.5%) and Gentamycin (2.7%) (Bebora et al., 1994). In another study, high
resistance rate were observed in the chicken E. coli strains as Tetracycline (99.1%),
Cotrimoxazole (92.2%), Gentamicin (89.7%), Ampicillin (88.7%) and Chloramphenicol
(57.0%) in Saudi Arabia (Al-Ghamdi et al., 1999).
61
Avian E. coli from faeces has also been shown to display high multidrug resistance of
86.5% to one or more antibiotics (Robab et al., 2003) in Iran and in Spain up to 67% to
Cotrimoxazole and Fluoroquinolones (Al-Ghamdi et al., 1999). It is now evident in this
study that there is an upward trend in reference to the numbers of antibiotic drugs to
which E. coli strains and Salmonella typhimurium are challenging.
The isolation of trivalent, tetravalent, pentavalent, and hexavalent R-type in the E. coli
and; the monovalent, trivalent and tetravalent in Salmonella typhimurium, in indigenous
chicken has raised concern. Of particular, resistance to Augumentin as displayed in the
resistance patterns as follows (Aug Amp), (Aug Amp Cot Tet), (Aug Amp Cot Tet Kan),
(Aug Amp Cot Tet Chl), (Aug Amp Cot Tet Kan Chl) of E. coli strains (Table 7) and
Salmonella enterica Typhimurium (Aug Amp Cot Tet) (Table 8), the drug of first choice
for extra intestinal and serious intestinal infections in adults, may reduce the efficacy of
early empirical treatment, the consequence being treatment failure.
In Kenya the unregulated over-the-counter sale of these antibiotics due to self-treatment
of suspected infection in humans, and to a lesser extent for use in animals without
prescription contribute to emergence and rapid dissemination of resistance (Kariuki et al.,
2005). This has exacerbated the problem of controlling microbes in a disease setting and
has caused a resurgence of bacterial diseases. The high level of resistance to Tetracycline,
Cotrimoxazole and Ampicillin is of concern due to possible cross-resistance with
antibiotics used in human medicine, poultry and other food-producing animals.
Resistance to Tetracycline, Ampicillin and Cotrimoxazole was noted in Bomet strains of
E. coli and Salmonella typhimurium, which were followed by Kericho district Salmonella
62
typhimurium; Murang’a and Kitui district E. coli and least were Kericho E. coli strains
(figure 3 and 6). However, the district variations were not significant.
The 3-kb plasmid size isolated during this study in two E. coli strains E14Bo and E19Bo
(Figure 9 and Table 11) may be associated with resistance to (Crx). Cefuroxime is
widely used in the treatment of certain human infections, bovine mastitis, feline and
canine upper respiratory tract infections (Molla et al., 2003). This could be explained
from the view that indigenous poultry are usually raised together with other domestic
animals (e.g. monogastric species such as pigs and rabbits, small and large ruminants)
and in some cases with fish (Guèye, 2002). The E14Bo and E19Bo were isolated from
Bomet district samples with intense domestic animals. Perhaps previously acquired
resistance to Cefuroxime in E. coli while in a mammalian host, and then this resistant
strain somehow found a way into a poultry environment and was transferred to the
poultry GIT (Molla et al., 2003).
In Kenya, Salmonella typhimurium is predominantly isolated in adults with Salmonellae
bacteraemia without diarrhoeal disease (Kariuki et al., 2005).
Salmonella enterica
Typhimurium presents as diarrhoeal disease acquired as food poisoning with several
foods being implicated as transmitting vehicles of salmonellosis to human as poultry,
beef, pork, eggs, milk, vegetables, fresh fruits and juices in the food chain (Kariuki et al.,
2005; Gomez et al., 1997).
In this study, the presence of S. enterica serotype typhimurium in indigenous chicken
demonstrates the potential for food contamination during handling and processing. The
prevalence of multidrug resistant Salmonella typhimurium in indigenous chicken retail
63
outlet reflects a reservoir (carriage of the organism) of resistance in poultry that can be
transmitted to humans.
All strains of Salmonella typhimurium were not resistant to Norfloxacin, Kanamycin,
Nalidixic acid and the third generation Cephalosporin, Cefuroxime (Figure 6). In Zahraei
et al (2005), Salmonella strains, showed resistance to Kanamycin (34.6%), Tet, Amp,
Trim, and Nal (20.7%). Most of the later antibiotics are commonly employed in Kenya
both in the public health and veterinary practices in control of bacterial infections.
At first glance, the low prevalence rate in indigenous chicken of co-infection of
Salmonella typhimurium and E. coli (9.8%) is an indicator of possible differences in the
source of infection. However, bacteria communicate with each other through autoinducers. The signaling systems termed quorum sensing were described as mechanisms
through which bacteria regulate gene expression via cell density. The quorum sensing
functions are varied and reflect the needs of a particular species of bacteria to inhabit a
given niche (Nicola and Vanessa, 2006). Thus, the quorum sensing functions could have
contributed to the low co-infection levels in indigenous chicken.
The findings in co-infections of either being resistant as in the Bomet strains or
susceptible (Table 9), highlights the possibility of exposure to the various antimicrobial
agents. However, the resistance patterns in Bomet co-infection strains were different and
thus introduce a variation to how resistance may have occurred (Table 10). Carlson et al
(2001) described that multidrug resistant Salmonella typhimurium could arise as a result
of an inducer from other pathogenic bacteria such as E. coli that produce microcins
64
(antimicrobial peptides secreted by bacteria as a means of disabling neighbouring
bacteria).
In Carlson et al (2001), it was found that Salmonella could develop resistance to an E.
coli-derived microcin, which gives an advantage to pathogenic E. coli while also
potentially selecting for multidrug resistant Salmonella (Carlson et al., 2001). Host
ecology and environment also factor into the patterns of antibiotic resistance in natural
populations. For example, strains of Salmonella are likely to have experienced different
selective pressures for resistance than Escherichia coli strains (Tara and Howard, 2000).
Indigenous chicken is a valuable asset to local populations as they contribute significantly
to food security and poverty alleviation especially in disadvantaged groups and less
favoured areas of rural Africa including Kenya (Njue, 2002; Gueye, 2000; Kiptarus,
2005). The indigenous chicken provides readily harvestable animal protein to rural
households and in some parts of Africa is raised to meet the obligation of hospitality to
honoured guests (Aichi, 1998). Egg dishes and chicken meat cook faster than red meat,
and therefore use less fuel wood (Aichi, 1998).
However, indigenous chicken (Family poultry) production in Kenya of free feed resources
available in the surrounding environment, agricultural grains and farm waste, expose
chicken to severe conditions (bioactive veterinary drug residues and resistant bacteria). The
extreme conditions in the environment can lead to depressed immuno-responsiveness that
stimulates the production of immunoglobulins, specifically IgA, which tends to influence
pathogen growth more than beneficial microbes (Edens, 2003).
65
Furthermore, during the quest for food, and other animal management parameters such as
rearing indigenous chicken with monogastric species (Guèye, 2002; Apajalahti et al.,
2004), may contribute to gut population of Salmonella typhimurium and E. coli directly
by providing a continuous source of bacteria and indirectly by influencing the physical
condition and defense of the birds (Apajalahti et al., 2004). These in turn play a vital role
in the development, proliferation, and persistence of antimicrobial resistance, which is
currently a major public health concern. In fact, this has negatively impacted on health
leading to more infectious diseases episodes nowadays.
Scavenging indigenous Gallus gallus sold in leading slaughterhouse cum market outlet in
Nairobi, intended for food shed resistant Salmonella typhimurium and E. coli pathogens
which may enter the food chain. The poultry litter also may find its way to surroundings
such as aquatic environment due to run-off leading to potential reservoirs of bacterial drug
resistance. This is in line with the high prevalence of seasonal intestinal infections as noted
by Kariuki et al. (2005) in tropical Africa during rainy seasons. Thus, animal litter is now
considered as a route of human exposure to antimicrobials used in food producing animals.
Escherichia coli O157:H7 and Salmonella typhimurium have been seen to survive in cow
manure, slurry (Sakchai et al., 1999), swine manure and environment (Tat and Richard,
2003).
66
CHAPTER FIVE
CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusions
The isolation of enteric pathogens (Salmonella typhimurium and E. coli) in asymptomatic
indigenous chicken in this study shows that they harbour foodborne pathogens which
may be a source of contamination of poultry farmers and farms; poultry vendors; chicken
carcass and organs during the process of evisceration at slaughter; and could play a role
in the spread of food borne illnesses and multidrug resistance posing a public health risk.
The study indicates that indeed there is significant appearance of drug resistant strains of
E. coli (P<0.0002) and Salmonella typhimurium (P<0.0001) in indigenous Gallus gallus.
The findings demonstrated that, of the antimicrobial agents used, Norfloxacin, Nalidixic
acid and Gentamicin are the most effective antibiotics against E. coli. While Norfloxacin,
Nalidixic acid, Cefuroxime and Kanamycin are the most effective against Salmonella
typhimurium in indigenous Gallus gallus.
5.2 Recommendations
This study recommends that effective prevention of enteric pathogens in indigenous
chicken such as Salmonella typhimurium and E. coli associated with food illnesses is
essential. This could be attained as follows:
♦ On-farm practices that reduce pathogen carriage such as pathogen free feeds, clean
water, regulated movement, increased hygiene at slaughter and poultry meat processing,
consumer-education efforts to protect public health and continued implementation of
67
HACCP systems. This will minimize indigenous chicken contamination with these
pathogens that can occur at multiple steps along the food chain, including production,
processing, distribution, retail marketing, and handling or preparation.
♦ Routine surveillance and timely reporting of antibiotic resistance patterns among
enteric pathogens should become a high priority to establish possible sources of bacterial
resistance and provide data that can be used to select appropriate treatment.
♦ Indigenous chicken producers in the country (Kenya) should be encouraged to change
from the traditional based scavenging systems to semi-intensive or backyard production
systems with improved husbandry.
♦ Establishing a national program focusing on the identification and molecular subtyping
of zoonotic food borne bacterial pathogens that could be present in retail food animals
(poultry).
68
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APPEDINCES
Appendix I: Reagents
Solution I
50mM glucose
25mM Tris Cl (pH 8.0)
10mM EDTA (pH 8.0)
Solution II
0.2 N NaOH (freshly diluted from a 10 N stock)
1% SDS
Solution III
5M Potassium acetate
Glacial acetic acid
Sterile H2O
60ml
11.5ml
28.5ml
McFarland standard 0.5
Barium chloride (1.175%)
Sulphuric acid (1%)
0.05ml
9.95ml
Appendix II: Table 12: Biochemical identification test kit (API20E)
Test
Reaction
Indole
Methyl red
Detect indole production
Detect acid production from
glucose
Detect acetoin
Detect citrate utilization
Glucose fermentation
Adonitol fermentation
Arabinose fermentation
Lactose fermentation
Sorbitol fermentation
Mannitol fermentation
Rhamnose fermentation
Sucrose fermentation
VP
Citrate
Glucose
Adonitol
Arabinose
Lactose
Sorbitol
Mannitol
Rhamnose
Sucrose
Colour change of
medium
Cream to pink
Cream to red
Cream to red
Green to blue
Red to yellow
Red to yellow
Red to yellow
Red to yellow
Red to yellow
Red to yellow
Red to yellow
Red to yellow
84
Appendix III: Antimicrobial regiment/ zone diameters in mm of
bacterial strains
Code. AUG CRX NAL NOR AMP GEN COT TET KAN CHL
S15Bo 25mm 20mm 22mm 26mm 22mm 0mm 0mm 0mm 18mm 23mm
S19Bo 28mm 22mm 22mm 29mm 28mm 20mm 28mm 21mm 18mm 22mm
S82Ke 24mm 21mm 23mm 30mm 0mm 16mm 0mm 0mm 19mm 0mm
E68Mu 20mm 19mm 20mm 25mm 20mm 19mm 29mm 21mm 19mm 25mm
E20Bo 20mm 19mm 21mm 24mm 19mm 16mm 22mm 21mm 19mm 23mm
E40Ki 21mm 20mm 21mm 24mm 21mm 17mm 28mm 0mm 20mm 25mm
E92Ke 21mm 20mm 20mm 25mm 20mm 16mm 25mm 20mm 17mm 25mm
E56Mu 16mm 20mm 20mm 21mm 0mm 17mm 0mm 7mm 18mm 25mm
E101Ke 19mm 16mm 22mm 25mm 20mm 16mm 20mm 20mm 19mm 24mm
E52Mu 23mm 24mm 24mm 26mm 25mm 20mm 28mm 21mm 18mm 23mm
E95Ke 22mm 21mm 23mm 27mm 23mm 18mm 29mm 22mm 16mm 25mm
E97Ke 22mm 21mm 21mm 25mm 21mm 18mm 29mm 21mm 19mm 24mm
E69Mu 20mm 19mm 22mm 21mm 21mm 20mm 24mm 20mm 18mm 24mm
E31Ki 19mm 17mm 21mm 24mm 20mm 18mm 23mm 19mm 18mm 23mm
E13Bo 21mm 20mm 21mm 24mm 22mm 16mm 23mm 20mm 16mm 25mm
S1Bo 25mm 19mm 22mm 31mm 23mm 19mm 25mm 19mm 19mm 23mm
S46Ki 18mm 22mm 24mm 29mm 0mm 16mm 0mm 0mm 18mm 23mm
S101Ke 29mm 24mm 23mm 30mm 23mm 22mm 30mm 19mm 17mm 22mm
S48Ki 16mm 24mm 25mm 30mm 20mm 20mm 33mm 19mm 15mm 24mm
S81Ke 16mm 20mm 23mm 32mm 0mm 19mm 0mm 0mm 17mm 21mm
S77Mu 21mm 24mm 24mm 30mm 21mm 19mm 27mm 20mm 15mm 22mm
E37Ki 21mm 19mm 21mm 23mm 21mm 16mm 25mm 21mm 20mm 29mm
E71Mu 22mm 19mm 18mm 21mm 0mm 16mm 0mm 0mm 16mm 28mm
E65Mu 21mm 19mm 23mm 25mm 21mm 16mm 26mm 23mm 19mm 30mm
E51Mu 20mm 20mm 22mm 24mm 21mm 18mm 0mm 0mm 15mm 26mm
E44Ki 22mm 21mm 22mm 27mm 23mm 21mm 0mm 21mm 19mm 25mm
E91Ke 23mm 20mm 21mm 23mm 22mm 19mm 25mm 22mm 19mm 26mm
E2Bo 20mm 20mm 23mm 24mm 0mm 16mm 0mm 0mm 19mm 25mm
E77Mu 20mm 18mm 19mm 23mm 19mm 16mm 26mm 21mm 19mm 25mm
E53Mu 22mm 21mm 23mm 28mm 23mm 20mm 21mm 20mm 19mm 25mm
E76Mu 20mm 21mm 23mm 29mm 22mm 18mm 25mm 0mm 18mm 25mm
E72Mu 20mm 19mm 21mm 31mm 0mm 20mm 0mm 0mm 16mm 25mm
E38Ki 21mm 19mm 21mm 25mm 21mm 17mm 25mm 20mm 18mm 24mm
E50Mu 20mm 20mm 21mm 24mm 19mm 16mm 0mm 0mm 15mm 25mm
E43Ki 18mm 19mm 21mm 25mm 19mm 16mm 0mm 22mm 19mm 26mm
E74Mu 21mm 20mm 21mm 27mm 20mm 19mm 26mm 8mm 19mm 26mm
85
E10Bo
E57Mu
E9Bo
E19Bo
E45Ki
E4Bo
E63Mu
E14Bo
E83Ke
E17Bo
E18Bo
E98Ke
E100Ke
E84Ke
E87Ke
E99Ke
E47Ki
E49Ki
E29Ki
E59Mu
E75Mu
E21Bo
E89Ke
E3Bo
E79Ke
S37Ki
E73Mu
E94Ke
E42Ki
E36Ki
E66Mu
E16Bo
E93Ke
E78Ke
E25Ki
E25922
E22Bo
E30Ki
E8Bo
11mm 16mm 24mm 31mm 0mm 22mm 0mm 0mm 16mm 30mm
20mm 19mm 22mm 29mm 20mm 19mm 0mm 20mm 20mm 28mm
21mm 22mm 25mm 26mm 22mm 17mm 22mm 22mm 19mm 26mm
0mm 20mm 21mm 27mm 0mm 16mm 0mm 0mm 0mm 7mm
19mm 18mm 20mm 25mm 19mm 18mm 23mm 22mm 20mm 26mm
12mm 16mm 20mm 21mm 0mm 16mm 0mm 20mm 15mm 26mm
20mm 20mm 22mm 24mm 19mm 17mm 19mm 19mm 18mm 26mm
17mm 0mm 20mm 27mm 0mm 18mm 0mm 0mm 14mm 0mm
21mm 18mm 21mm 20mm 19mm 17mm 14mm 0mm 17mm 27mm
22mm 19mm 23mm 25mm 0mm 15mm 0mm 0mm 19mm 24mm
21mm 20mm 22mm 27mm 0mm 17mm 0mm 0mm 18mm 26mm
22mm 21mm 22mm 25mm 21mm 17mm 19mm 0mm 19mm 24mm
21mm 19mm 22mm 24mm 20mm 18mm 23mm 19mm 18mm 25mm
20mm 21mm 23mm 29mm 0mm 22mm 0mm 0mm 19mm 25mm
20mm 20mm 21mm 24mm 21mm 18mm 26mm 23mm 19mm 28mm
23mm 21mm 24mm 25mm 21mm 18mm 24mm 22mm 19mm 29mm
18mm 19mm 20mm 23mm 20mm 18mm 25mm 22mm 19mm 28mm
19mm 19mm 21mm 30mm 21mm 17mm 27mm 21mm 17mm 26mm
21mm 19mm 23mm 27mm 21mm 18mm 23mm 20mm 17mm 27mm
16mm 20mm 20mm 30mm 0mm 20mm 0mm 0mm 17mm 26mm
19mm 20mm 22mm 25mm 20mm 19mm 24mm 21mm 17mm 26mm
22mm 19mm 21mm 20mm 20mm 17mm 20mm 21mm 17mm 25mm
21mm 22mm 22mm 30mm 22mm 19mm 27mm 20mm 19mm 26mm
19mm 19mm 21mm 25mm 18mm 19mm 25mm 22mm 19mm 26mm
20mm 22mm 21mm 30mm 0mm 20mm 0mm 0mm 19mm 26mm
21mm 20mm 22mm 25mm 21mm 17mm 27mm 20mm 18mm 24mm
20mm 21mm 21mm 24mm 21mm 19mm 29mm 21mm 19mm 25mm
19mm 19mm 20mm 27mm 19mm 19mm 27mm 19mm 19mm 26mm
21mm 24mm 22mm 27mm 22mm 20mm 27mm 20mm 18mm 25mm
20mm 19mm 21mm 28mm 18mm 18mm 26mm 19mm 18mm 26mm
18mm 18mm 21mm 25mm 20mm 18mm 25mm 22mm 19mm 27mm
14mm 0mm 20mm 24mm 0mm 18mm 0mm 0mm 16mm 24mm
25mm 20mm 22mm 26mm 23mm 18mm 27mm 19mm 18mm 26mm
22mm 20mm 23mm 29mm 0mm 21mm 0mm 0mm 18mm 0mm
20mm 19mm 21mm 27mm 0mm 19mm 0mm 21mm 16mm 25mm
22mm 20mm 25mm 29mm 22mm 18mm 27mm 23mm 18mm 29mm
18mm 18mm 20mm 23mm 0mm 16mm 0mm 0mm 16mm 0mm
20mm 19mm 20mm 21mm 17mm 17mm 20mm 21mm 17mm 24mm
19mm 18mm 18mm 23mm 18mm 17mm 11mm 0mm 21mm 24mm
86
E24Ki
E11Bo
E28Ki
E27Ki
E23Bo
E39Ki
S62Mu
S5Bo
S64Mu
10mm 21mm 20mm 22mm 0mm 20mm 0mm 0mm 0mm 0mm
10mm 18mm 20mm 23mm 0mm 18mm 0mm 0mm 18mm 22mm
10mm 16mm 19mm 21mm 0mm 14mm 0mm 0mm 0mm 0mm
11mm 19mm 19mm 24mm 0mm 13mm 0mm 0mm 19mm 0mm
19mm 17mm 19mm 20mm 0mm 16mm 0mm 0mm 18mm 25mm
8mm 17mm 20mm 25mm 0mm 16mm 0mm 0mm 16mm 25mm
20mm 16mm 24mm 22mm 18mm 16mm 25mm 20mm 20mm 24mm
22mm 20mm 21mm 24mm 23mm 18mm 18mm 20mm 20mm 26mm
20mm 15mm 23mm 22mm 0mm 18mm 0mm 0mm 14mm 0mm
Appendix IV: Table 13: Zone Diameter Interpretative Standards and equivalent
Minimum Inhibitory Concentration
Interpretation Amp
R
13
I
1416
S
17
Aug
13
1417
18
Tet
14
1518
19
Cot
10
1115
16
Chl
12
1317
18
Crx
14
1522
23
Kan
13
1417
18
Gen
12
1314
15
Nor
12
1316
17
Nal
13
1418
19