RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
TOPIC
CHARACTERIZATION AND ANTIBIOGRAM OF
KLEBSIELLA SPP. ISOLATED FROM CLINICAL SPECIMEN
AND HOSPITAL PERSONNEL IN A RURAL TEACHING
HOSPITAL
Dr. NAMRATHA K.G.
POST GRADUATE STUDENT
DEPARTMENT OF MICROBIOLOGY
K.V.G MEDICAL COLLEGE AND HOSPITAL
KURUNJIBHAG SULLIA – 574327.
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
NAME OF THE CANDIDATE AND Dr. NAMRATHA K.G.
ADDRESS
POST GRADUATE STUDENT
DEPARTMENT OF MICROBIOLOGY
K.V.G MEDICAL COLLEGE AND
HOSPITAL
KURUNJIBHAG SULLIA – 574327.
2.
NAME OF THE INSTITUTION
K.V.G. MEDICAL COLLEGE, SULLIA.
3.
COURSE OF STUDY AND SUBJECT
M.D. MICROBIOLOGY
4.
DATE OF ADMISSION TO COURSE
30/05/2009
5.
TITLE OF TOPIC
CHARACTERIZATION AND
ANTIBIOGRAM OF KLEBSIELLA SPP.
ISOLATED FROM CLINICAL SPECIMEN
AND HOSPITAL PERSONNEL IN A
RURAL TEACHING HOSPITAL.
6.
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the study
The genus Klebsiella belongs to the Tribe Klebsiellae of the family
Enterobacteriaceae. The members of genus Klebsiella are Gram negative, lactose
fermenting, non-motile, usually capsulated, rod shaped, urease producing, lysine
decarboxylase producing and non- ornithine decarboxylase producing, generally VogesProskauer test positive bacteria.1 Klebsiella spp. is ubiquitous in nature. They inhabit the
environment and intestinal tract of humans and animals. 1, 5 Seven species of Klebsiella
– K.pneumoniae, K.oxytoca, and K.granulomatis are associated with human illness;
K.ozaenae and K. rhinosleromatis are associated with specific diseases6; K.planticola
(SYN.K.trevisanii), K.terrigena and K. ornithinolytica are now transferred to a new
genus Raoultella .9
K.pneumoniae is a primary pathogen and can cause a classic form of primary
pneumonia.5 K.pneumoniae can also cause urinary tract infection, nosocomial infections,
wound and biliary tract infection, peritonitis, meningitis, bacteremia, enteritis,
septicemia.16, 17 Plasmid mediated multiple drug resistance by extended spectrum beta
lactamase (ESBL) production; development of resistance to  lactum / lactamase
inhibitors, quinolones and amino glycosides, colistin and tigecyclins is a serious threat to
therapeutic armamentarium.8
Over the years K.pneumoniae infections have been showing demographic and
geographic variations. Total community acquired pneumonia due to K.pneumoniae has
severally declined. But community acquired K.pneumoniae has been responsible for
increased number of liver abscess especially in Central Asia.12 Nosocomial Klebsiella
infections continue to be heavy burden on the economy and life expectancy of patients.
Therefore, study of Klebsiella associated infections and the antibiogram pattern of
Klebsiella isolates are the requirement in the implementation of preventive and control
measures of infections caused by Klebsiella spp.
6.2 Review of the literature
The genus Klebsiella was named after Edwin Klebs, a late 19th century German
microbiologist. In 1884 the Danish scientist Hans Christian Gram developed the gram
staining technique to discriminate K.pneumoniae and S.pneumoniae .Friedlander in 1882
discovered Klebsiella to be a pathogen that caused pneumonia.5
In humans, K. pneumonia infrequently inhabits the nasopharynx and G.I tract as a
saprophyte. The carrier rate of gastro intestinal tract ranges from 5%-38%, while the rate
for nasopharynx range from 1%-6%. The carrier rate drastically increases on
hospitalization, specially in patient who had previous antibiotic therapy with broad
spectrum antibiotics. K.pneumoniae is commonly found in the G.I tract and hands of
hospital personnel12, medical equipment’s, blood products, G.I tracts of patients and
hands of hospital personnel are considered to be the reservoirs for transmission of
Klebsiella in the hospital.1
K. pneumoniae as primary pathogen causes pneumonia and UTI in healthy
individuals6. Oropharyngeal colonization may be a source of lung infection in patients
with debilitating conditions like alcoholism, diabetes mellitus and chronic obstructive
pulmonary disease.20 Extra-pulmonary infection includes meningitis, UTI, liver abscess,
septicemia, arthritis. K.oxytoca like K.pneumoniae can cause a variety of nosocomial
infections. 6
All strains of K.pneumoniae are resistant to ampicillin as result of the presence of
a chromosomal gene encoding a penicillin specific beta-lactamase.18 The occurance of
extended spectrum beta-lactamase (ESBL) producing isolates among K.pneumoniae was
first observed in 1983.10 Extended spectrum beta lactamases are enzyme that confers
resistance to oxyimino-beta-lactams such as ceftaxime, ceftazidime, ceftriaxone and the
monobactum aztreonam but not to cephamycins or carbapenemases.23 ESBL production
is plasmid mediated multiple drug resistant strains of K.pneumoniae causing nosocomial
infections are frequently ESBL producers.1 Major outbreaks involving ESBL strains
have been reported from all over the world; thus making them emerging pathogens.24
ESBL producing K.pneumoniae were frequently found to be resistant to the other
class of antibiotics such as beta-lactam/beta lactamase inhibitors combinations,
quinolones and aminoglycosides, limiting the treatment option to 4th generation
cephalosporins or carbapenemeses. Emergence of carbapenem resistant K.pneumoniae
(KPC-Kp) has been reported at an alarming rate from western hemisphere. ESBL
producers KPC-kp are also resistant to amino-glycosides and quinolones. The
therapeutic options against KPC-kp are limited to colistin and tigecycline. Tigecycline
may not reach the desired serum levels to treat blood stream infections leaving colistin
the last choice. Colistin resistant KPC-kp is also reported from USA.8
Aslwin et al from Pondicherry in 2000 reported that 48.75% of K.pneumoniae
isolated from wound and burns infections were ESBL producers. All of these were also
resistant to gentamycin, ciprofloxacin, and chloramphenicol.
In another study from Chennai 6.6% of Klebsiella isolated from various clinical
specimens collected from patients aged 0-5 years of age were ESBL producers. These
were also reported to be resistant to aminoglycosides, sulfonamides and tetracyclins.
Both were sensitive to imipenem.2
Jemina and Verghese4 described an ESBL designated SHV-28 produced by a
strain of K.pneumoniae isolated from urine of a 65-year-old male from Chennai.
In a study from Davanagere, Karnataka 28 isolates of K.pneumoniae from various
clinical specimens were screened for ESBL production by double disc synergy test and
phenotypic confirmation test recommended by CLSI and found that 62.2% of
K.pneumoniae were ESBL producers.7
6.3 Objectives of the Study
A. Isolation and identification of Klebsiella spp. from clinical specimens and hospital
Personnel.
B. Study of the antibiotic pattern of the isolates.
C. Detection of ESBL producing and other drug resistant strains among the Klebsiella
spp.
D. Data analysis by relavent statistical methods.
7.
MATERIALS AND METHODS
7.1 Source of Data
Clinical isolates of Klebsiella spp. isolated in the clinical laboratory at KVG
Medical College and Hospital Sullia during one and half year from December 2011 to
June 2013 will be processed for detection of ESBL and carbapenemases resistance
pattern as per the standard procedures.
7.2 Method of Collection of Data
Study design, Sample size and procedure of sampling.
A total of 100 strains of Klebsiella spp. isolated from clinical specimens and
hospital personnel will be further screened for antibiotic susceptibility and the
production of ESBL. The procedure in brief is as follows:
Clinical samples like sputum, urine, throat swab, wound swabs, stool, ulcer
material, blood and any other material received at the departmental diagnostic laboratory
and the swab from oropharyngeal region collected from hospital personnel willing to
participate in the study will be inoculated on blood agar and MacConkey agar, incubated
at 370C for 24 to 48h and the growth occurred will be identified by standard procedure.22
All strains Klebsiella spp. so isolated will be screened further for antibiotic
susceptibility and ESBL production.
Antimicrobial susceptibility testing:
The screening of Klebsiella isolates for antibiotic susceptibility will be done by
Kirby-Bauer disk diffusion method.14 The
antimicrobial disks used will be
chloramphenicol (30g), gentamicin (15g), ampicillin(10units), ciprofloxacin (5g)
and cephotaxime (30g) and also third generation cephalosporins(3GC) viz.,
ceftazidime,
ceftaxime,
ceftriaxone,
each
30
µg
/disc2,
piperacillin(100µg),
cefoxitin(30g,) tetracycline (30 g), cefpime (30 g) and imipenem (10g).7 The plates
will be incubated at 370C overnight. The diameter of the zone of inhibition for each
antimicrobial will be measured and recorded as resistant, intermediate or susceptible
according to the standard NCCLS interpretative criteria.13
Extended spectrum beta lactam detection by Double Disc Diffusion Synergy
Test(DDST):
In the DDST, synergy will be determined between a disc of augmentin (20 g
amoxicillin and 10 g clavulanic acid) and a 30 g disc of each 3GC test antibiotic
placed at a distance of 30mm apart on a lawn culture of the resistant isolates under test
on Muller-Hinton Agar (MHA, Hi-Media).21 The test organism was considered to
produce ESBL, if the zone size around the test antibiotic disc increased towards the
augmentin disc. Escherichia coli ATCC 25922 were used as the negative control and
ESBL producer was used as the positive control.2
Inclusion criteria
1. All patients in the hospital during the study period except those excluded.
2. Health care personnel including doctors and nursing staff.
Exclusion criteria
1. Hospital environment.
2. Health care personnel other than doctors and nursing staff.
Follow up
No follow up.
Follow up period
Not applicable.
Analysis of data
The data will be analyzed on IBM SPSS version 19 software. Chi-square test will
be applied to test the difference between proportions, at 5% level of significance.
7.3 Does the study require any investigation\intervention to be conducted on
patients\ humans\ animals? If so, please describe briefly:
a. Investigations: Yes, Oropharyngeal swab will be collected from health care
personnel.
Informed consent will be taken.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Yes.
Copy of Ethical Committee Clearance attached.
Informed written consent will be obtained from each interviewee before enrolment into
the study.
8
REFERENCES
1.
Podschun R and Ullmann U. Klebsiella spp. As Nosocomial pathogens:
Epidemiology, Taxonomy, Typing Methods, and Pathogenicity Factors. Clinical
Microbiology Reviews, Oct 1998; (11):589-603.
2.
Shubha A, Ananthan S. Extended Spectrum Beta Lactamase(ESBL) mediated
resistance to third generation cephalosporins among Klebsiella pneumonia in
Chennai. Indian J Med Microbiology 2002; 20(2):92-5.
3.
Ananthakrishnan AN, Kanungo R, Kumar A and Badrinath S. Detection of
Extended Spectrum Beta-Lactamase producers among surgical wound infections
and burns patients in JIPMER. Indian J Med Microbiology 2000; 18(4): 160-5.
4.
Jemima SA, Verghese S. SHV-28 An Extended Spectrum Beta Lactamase
produced by a clinical isolate of Klebsiella pneumonia in South India. Indian J
Med Microbiology, 2009; 27(1): 51-4.
5. Washington Winn, Jr., Stephen Allen et al. Chapter 6. The Enterobacteriaceae:
Taxonomy of the Enterobacteriaceae. In: Koneman’s Color Atlas and Textbook
of Diagnostic Microbiology. Sixth edition. Lippincott Williams & Wilkins 2006:
259-64.
6. Michael S. Donnenberg .Chapter 215. Enterobacteriaceae. In: Gerald L. Mandell,
John E. Bennett, Raphael Dolin. Editors. Mandell, Douglas, and Bennett’s
Principles and Practice of Infectious Diseases. Sixth edition. Churchill
Livingstone 2005: 2578-9.
7.
Rao PNS, Basavarajappa KG, Leela Krishna G. Detection of Extended Spectrum
Beta- lactamase from clinical isolates in Davangere. Indian Journal of Pathology
and Microbiology-51(4), October-December 2008; 51(4):497-9.
8. Endimiani A, Hugen K M, Huger A M, Armstrong E S, Chaudhary Y, Aggen J
B, and Bonomo R A. ACHN- 490, a Neoglycoside with potent in vitro activity
against multi drug resistant K. pneumonia isolates. Antimicrobial agent and
chemotherapy2009; 53(10):4504-7.
9. Drancourt M, Bollet C, Carta A, et al. Phylogenetic analysis of Klebsiella species
delineate Klebsiella and Raoultella gen. nov. with description of Raoultell
ornithinolytica comb. nov. Raoultella terrigena comb.nov. Int J syst Evol
Microbial 2001; 51:925-32.
10. Pitout JDD, Lavpland KB. Extended Spectrum Beta- Lactamase producing
Enterobacteriaceae: An emerging public- health concern. Lancet infet Dis 2008;
8:159-66.
11. Alberti, S, Marques G, Camprubi S, Merino S, Tomas JM, Vivanco F, and
Benedi VJ.1993. Clq binding and activation of the complement classical pathway
by K.pneumoniae outer membrane proteins. Infect. Immun; 61:852-60.
12. Wen-Chien Ko, Paterson DL, Sagnimani AJ & Hansen DS. Community acquired
k. pneumonia Bacteraemia; Global differences in clinical patterns. Emreging
Infect Dis 2002; 8(2): 160-6.
13. National committee for Clinical Laboratory standards: Performance standards for
antimicrobial disk susceptibility test, 5th ed., (Villanova, PA: NCCLS) 1993:
document M2-A5.
14. Bauer AW, Truck H. and Saerries JC. Antibiotic Susceptibility Testing by
standardized single disc method. Am J Clin Pathol 1966; 45: 493-6.
15. Jarvis WR, Munn VP, Highsmith AK, et al. The nosocomial infections caused by
K.pneumoniae. Infect Control. 1985; 6: 68-74.
16. Geerdes HF, Ziegler D, Lode H, et al. Septicemia in 980 patient at a university
hospital in Berlin: Prospective studies during 4 selected years between 1979 and
1989. Clin Infect Dis. 1992; 15: 991-1002.
17. Garcia de la Torre M, Romero-vivas J, Martinez-Beltran J, et al. Klebsiella
Bacteremia: An analysis of 100 episodes. Rev Infect Dis 1985; 7: 143-50.
18. Haeggman S, Lofdahl S, Burman LC. An allelic variant of the chromosomal gene
for class A beta-lactamase K2, specific for Klebsiella pneumonia, is the ancestor
of SHV-1. Antimicrobial Agents Chemotherapy 1997; 41:2705-9.
19. Izard D, Ferragat C, Yarina F, et al. Klebsiella terrigina, a new species from soil
and water. Int J Syst Bacteriol 1981; 31:116-27.
20. Reynolds; HY, Pneumonia due to Klebsiella (Fridlander’s pneumonia) In:
Wyngaarden JB, Smith LH, eds. Cecil text book of Medicine. 16th ed.
Philadelphia: Saunders 1982: pp-1430-2.
21. Vercauteren E, Descheemaeker P, Ieven M, Sanders, Goossens H. Comparison of
screening methods for detection Extended- Spectrum- beta-Lactamases and their
Prevalence among Blood isolates of Escherichia coli and Klebsiella spp. in a
Belgian Teaching Hospital. J Clini Microbiology 1997; 35:2191-7.
22. Pamela B Chrichton. Enterobacteriaceae: Escherichia, Klebsiella, Proteus and
other genera. In: Collee JG, Fraser AG, Marmion BP, Simmon A, editors.
Mackie and Mc Cartney Practical Medical Microbiology. 14th edition Churchill
Livingston: New York 2006. pp-361-84.
23. Rasheed JK, Anderson GJ, Yigit H, Queenan AM, Domenech-Sanchez A,
Swenson JM, et al. Characterization of the extended-spectrum β-Lactamase
reference strain, Klebsiella pneumonia K6(ATCC 700603), which produces the
novel enzyme SHV-18. Antimicrob Agents Chemother 2000; 44:2382-8.
24. Chou T. Emerging infectious diseases and pathogens. Nurs Clin North Am 1999;
34(2):427-42.
9.
SIGNATURE OF CANDIDATE
:
10.
REMARKS OF THE GUIDE
11
11.NAME AND DESIGNATION OF :
:
(in block letters)
11.1 Guide
: Dr. SUBBANNAYYA KOTIGADDE
Professor,
Department of Microbiology,
K.V.G Medical College and Hospital
Sullia, D.K.
11.2 Signature
11.3 Head of the Department
:
: Dr. MEERA D MEUNDI
Professor and HOD,
Department of Microbiology,
K.V.G. Medical College,
Sullia, D.K.
11.4 Signature
12.
:
12.1 Remarks of the Principal
:
12.2 Signature
:
ETHICAL COMMITTEE CLEARANCE
1.
TITLE OF DISSERTATION
CHARACTERIZATION AND ANTIBIOGRAM
OF KLEBSIELLA SPP. ISOLATED FROM
CLINICAL
PERSONNEL
SPECIMEN
IN
A
AND
HOSPITAL
RURAL
TEACHING
HOSPITAL
2.
NAME OF THE CANDIDATE
Dr. NAMRATHA K.G
3.
NAME OF THE GUIDE
Dr. SUBBANNAYYA KOTIGADDE
4.
APPROVED/NOT APPROVED
Sri. KRISHNAMURTHY, Chairperson.
Dr. SUBBANNAYYA KOTIGADDE, Secretary.
Dr. S. GOPALRAO, Member
Dr. C.S. MOHANRAJ , Member
Dr. H.R.SHIVAKUMAR , Basic scientist
LAW EXPERT : Sri KRISHNAMURTHY, Advocate
PRINCIPAL
K.V.G MEDICAL COLLEGE AND HOSPITAL, SULLIA.

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