RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
CHARACTERIZATION AND ANTIBIOGRAM OF
KLEBSIELLA SPP. ISOLATED FROM CLINICAL SPECIMEN
AND HOSPITAL PERSONNEL IN A RURAL TEACHING
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
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
NAME OF THE CANDIDATE AND Dr. NAMRATHA K.G.
POST GRADUATE STUDENT
DEPARTMENT OF MICROBIOLOGY
K.V.G MEDICAL COLLEGE AND
KURUNJIBHAG SULLIA – 574327.
NAME OF THE INSTITUTION
K.V.G. MEDICAL COLLEGE, SULLIA.
COURSE OF STUDY AND SUBJECT
DATE OF ADMISSION TO COURSE
TITLE OF TOPIC
ANTIBIOGRAM OF KLEBSIELLA SPP.
ISOLATED FROM CLINICAL SPECIMEN
AND HOSPITAL PERSONNEL IN A
RURAL TEACHING HOSPITAL.
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
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
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
B. Study of the antibiotic pattern of the isolates.
C. Detection of ESBL producing and other drug resistant strains among the Klebsiella
D. Data analysis by relavent statistical methods.
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 (30g), gentamicin (15g), ampicillin(10units), ciprofloxacin (5g)
and cephotaxime (30g) and also third generation cephalosporins(3GC) viz.,
cefoxitin(30g,) tetracycline (30 g), cefpime (30 g) and imipenem (10g).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
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
1. All patients in the hospital during the study period except those excluded.
2. Health care personnel including doctors and nursing staff.
1. Hospital environment.
2. Health care personnel other than doctors and nursing staff.
No follow up.
Follow up period
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
Informed consent will be taken.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Copy of Ethical Committee Clearance attached.
Informed written consent will be obtained from each interviewee before enrolment into
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.
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.
Ananthakrishnan AN, Kanungo R, Kumar A and Badrinath S. Detection of
Extended Spectrum Beta-Lactamase producers among surgical wound infections
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Jemima SA, Verghese S. SHV-28 An Extended Spectrum Beta Lactamase
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B, and Bonomo R A. ACHN- 490, a Neoglycoside with potent in vitro activity
against multi drug resistant K. pneumonia isolates. Antimicrobial agent and
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SIGNATURE OF CANDIDATE
REMARKS OF THE GUIDE
11.NAME AND DESIGNATION OF :
(in block letters)
: Dr. SUBBANNAYYA KOTIGADDE
Department of Microbiology,
K.V.G Medical College and Hospital
11.3 Head of the Department
: Dr. MEERA D MEUNDI
Professor and HOD,
Department of Microbiology,
K.V.G. Medical College,
12.1 Remarks of the Principal
ETHICAL COMMITTEE CLEARANCE
TITLE OF DISSERTATION
CHARACTERIZATION AND ANTIBIOGRAM
OF KLEBSIELLA SPP. ISOLATED FROM
NAME OF THE CANDIDATE
Dr. NAMRATHA K.G
NAME OF THE GUIDE
Dr. SUBBANNAYYA KOTIGADDE
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
K.V.G MEDICAL COLLEGE AND HOSPITAL, SULLIA.