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Indian J Med Res 129, May 2009, pp 599-602
Faecal carriage of CTX-M-15-producing Klebsiella pneumoniae in
patients with acute gastroenteritis
Muzaheed, Yohei Doi*, J.M. Adams-Haduch*, C.T. Shivannavar*, D.L. Paterson*+ & S.M. Gaddad
Department of Microbiology, Gulbarga University, Gulbarga, Karnataka, India, *Division of Infectious Diseases
University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA & +University of Queensland, Royal
Brisbane & Women's Hospital, Brisbane, Australia
Received February 18, 2008
Background & objectives: Data on extended-spectrum β-lactamases (ESBLs) produced by Gram-negative
bacteria including Klebsiella pneumoniae especially molecular types of ESBL genes from India are limited.
The present study was conducted to investigate the carriage and ESBL contents of multidrug-resistant
K. pneumoniae recovered from patients with gastroenteritis in a rural village in southern India.
Methods: Nine K. pneumoniae isolates obtained from 45 stool samples from patients with gastroenteritis
from one rural and two urban sites, in southern India were included in the study. Antibiotic susceptibility
testing, PCR analysis and sequencing were conducted to characterize the ESBL genes. Clonal relatedness
was assessed by pulsed-field gel electrophoresis (PFGE).
Results: All the isolates were found to be resistant to at least one of the third generation cephalosporins
tested. All the study isolates were confirmed to produce ESBLs. PCR and sequencing revealed the
responsible gene to be blaCTX-M-15. blaTEM and blaSHV were absent. PFGE indicated that five of seven isolates
from villagers were genetically closely related, and in turn were related to isolates from patients in two
urban areas in this region.
Interpretation & conclusions: Our findings showed that genetically-related isolates of K. pneumoniae
producing CTX-M-15 were present in multiple areas in southern India. Larger studies need to be done
in various geographical regions of the country to better define the molecular epidemiology of ESBLproducing K. pneumoniae and its clinical implications.
Key words CTX-M-15 - extended-spectrum beta-lactamase - Klebsiella pneumoniae
β-lactamases (ESBLs) is the most common mechanism
of resistance to third-generation cephalosporins among
Enterobacteriaceae including K. pneumoniae2,3.
Infections caused by ESBL-producing organisms are
difficult to manage for several reasons. First, empiric
therapy consisting of β-lactam antimicrobials is
Klebsiella
pneumoniae
is
a
successful
opportunistic pathogen associated with various
ailments such as urinary tract infections, septicaemia,
respiratory tract infections and diarrhoea1. K.
pneumoniae is part of commensal gastrointestinal
flora in humans1. Production of extended-spectrum
599
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INDIAN J MED RES, MAY 2009
often ineffective. Second, these organisms tend to
be also resistant to other classes of antimicrobials
including fluoroquinolones and aminoglycosides2.
CTX-M-type β-lactamases are increasingly becoming
the predominant ESBLs globally in recent years, as
opposed to the conventional TEM and SHV-type
ESBLs2,3. Studies have reported high prevalence of
ESBL-producing Enterobacteriaceae in India, where
use of antimicrobials is relatively unrestricted. A study
from northern India showed that 26 to 48 per cent of
uropathogens belonging to Enterobacteriaceae were
ESBL producers4. A recent report, from a hospital in
rural southern India, described a high prevalence of
ESBL producers5. Some reports have described the
molecular epidemiology of ESBL producers, from the
northern parts of the country6-8.
Though presence of ESBLs in K. pneumoniae has
been reported from India, there is scarce information
on the molecular types from southern India5,8. From
the limited data available, CTX-M-15 appears to
be the predominant ESBL in northern India4,6-8. In
the present study, we investigated the occurrence of
ESBL-producing K. pneumoniae carried by patients
presenting with gastroenteritis in southern India.
Material & Methods
Bacterial isolates: A total of 45 stool samples were
collected randomly from patients with gastroenteritis
at three sites in Karnataka, southern India. Out of
45 samples collected, 43 were from Sedum, the
other one each from Gulbarga and Raichur cities.
K. pneumoniae was isolated from nine of these samples
and included in the study. Seven were isolated from
inpatients with diarrhoea in Sedum village in south
India in August, 2005. The other two were isolated
from patients with diarrhoea in the cities of Raichur
and Gulbarga in August, 2005 and February, 2006,
respectively. Basic microbiology work was done in
Department of Microbiology, Gulbarga University,
Gulbarga and molecular work was performed in Dr
David L. Paterson Laboratory, Pittsburgh, USA. The
susceptibility profiles of each study isolate were not
known when the study was initiated. The village of
Sedam is located in the northern part of Karnataka,
52 km away from the large regional center, Gulbarga.
Raichur is 147 km away from Gulbarga.
Antibiotic susceptibility testing: The antibiotic
susceptibilities were determined by disk diffusion
method on Mueller-Hinton agar plates (Beckton
Dickinson, Sparks, MD, USA) as recommended by the
Clinical Laboratory Standards Institute (CLSI)9. The
disks containing the following antibiotics (µg) were
used (Beckton Dickinson): cefoxitin (30), cefepime
(30), ceftazidime (30), cefotaxime (30), ertapenem
(10), imipenem (10), amikacin (30), gentamicin (10),
ciprofloxacin (5) and chloramphenicol (30).
ESBL screening and confirmation by phenotypic
methods: The isolates showing reduced susceptibility
to ceftazidime or cefotaxime or both were tested
for ESBL production by double disk-diffusion test
(DDDT) using four disks (µg): cefotaxime (CTX)
(30), cefotaxime + clavulanic acid (10), cefatzidime
(CAZ) (30), and ceftazidime +clavulanic acid (10).
The inoculum and incubation conditions were the same
as for standard disk diffusion recommendations. A ≥5
mm increase in zone diameter for either antimicrobial
agent tested in combination with clavulanic acid versus
its zone when tested alone was designated as ESBLpositive9. Escherichia coli ATCC 25922 was used as
the control strain.
PCR detection and sequencing of ESBLs: Genomic
DNA was prepared by boiling the isolates and used
as the templates for PCR reactions. Amplification
of TEM,
TEM SHV and CTXCTX M-type ESBL genes was
performed on GeneAmp PCR System 9700 (Applied
Biosystems, Foster City, CA, USA) with primers
and cycling conditions given in Table I10. The PCR
Table I. Primers used for the study
Gene
blaSHV
blaTEM
blaCTX-M
blaCTX-M-15
Source: Ref. 10
Primer
SHV-F-S1
SHV-R-S2
TEM-F
TEM-R
CTX-M/F’
CTX-M/R’
CTXM15F
CTXM15R
Sequence
5’-ATTTGTCGCTTCTTTACTCGC-3’
5’-TTTATGGCGTTACCTTTGACC-3’
5’-ATGAGTATTCAACATTTCCGTG-3’
5’-TTACCAATGCTTAATCAGTGAG-3’
5’-TTTGCGATGTGCAGTACCAGTAA-3’
5’-CGATATCGTTGGTGGTGCCATA-3’
5’-CACACGTGGAATTTAGGGACT-3’
5’-GCCGTCTAAGGCGATAAACA-3’
Annealing temperature (°C) Product size (bp)
60
1051
55
861
51
544
50
996
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Muzaheed et al: FAECAL CARRIAGE OF ESBL PRODUCING K. PNEUMONIAE
Table II. Characteristics of ESBL-producing isolates from stool samples of patients from southern India
Isolate
No.
Location
30
31
32
33
34
35
36
37
236
Raichur
Sedam
Sedam
Sedam
Sedam
Sedam
Sedam
Sedam
Gulbarga
CAZ
CTX
FEP
FOX
ETP
IPM
CIP
C
CN
AM
PCR
PFGE
TEM SHV CTX-M
R
R
I
I
R
R
R
R
R
R
R
R
R
R
R
R
R
R
I
R
S
S
I
R
R
I
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
R
R
R
R
R
R
R
R
R
R
S
S
R
R
R
R
R
R
R
S
S
R
R
R
R
R
R
R
S
S
R
R
I
R
R
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
pos
pos
pos
pos
pos
Pos
pos
pos
pos
A
A
B
B
A
A
A
A
A
CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; FOX, cefoxitin; ETP, ertapenem; IPM, imipenem; CIP, ciprofloxacin; C,
chloromphenicol; CN, gentamicin; AM, amikacin. Suceptibility results were interpreted according to the CLSI criteria: R, resistance; I,
intermediate; S, susceptible. neg, negative; pos, positive
products were separated on 1 per cent agarose gels and
visualized under UV light.
The PCR products were purified with a QIAquick
PCR purification kit (Qiagen,Valencia, CA, USA) and
were sequenced on an ABI Prism 3100 automated
sequencer (Applied Biosystems).
Genomic fingerprinting by pulsed field gel
electrophoresis (PFGE): Agarose plugs containing
chromosomal DNA were prepared as described by
Yuan et al11. The chromosomal DNA was digested
overnight with 30 U of Xbal (New England Biolabs,
Ipswich, MA, USA). PFGE was run in a CHEF-DRIII
apparatus (Bio-Rad, Hercules, CA, USA) at 6V/cm for
22 h. The pulse times were 5-40s. The banding patterns
were interpreted according to the criteria of Tenover
et al12.
Results
The results of antibiotic susceptibility tests
indicated that all nine isolates in the study were resistant
to cefotaxime and ciprofloxacin and intermediate or
resistant to ceftazidime (Table II). Susceptibility to
cefepime, chloramphenicol, gentamicin and amikacin
was variable. All were susceptible to cefoxitin,
ertapenem and imipenem. The inhibitory zones of
cefotaxime and ceftazidime were increased in the
presence of clavulanic acid by more than 5 mm in
all isolates, which confirmed ESBL production. PCR
analysis was positive for CTX-M-type ESBL gene in
all nine isolates but negative for TEM and SHV-type
β-lactamase genes. Upon sequencing, the deduced
amino acid sequences of representative amplicons for
CTX-M-type ESBL gene matched that of CTX-M-15.
Fig. PFGE profile of ESBL-producing K. pneumoniae isolates
recovered from stool samples (Lanes 31 to 37, K. pneumoniae from
Sedam village; Lanes 30 and 236, K. pneumoniae from Raichur and
Gulbarga, respectively; Lanes M, molecular weight marker).
Macrorestriction polymorphism of genomic
DNA determined by PFGE revealed the presence of
two pulsotypes among the nine study isolates. Seven
belonged to one and two the other (Fig.). The two
isolates from Raichur and Gulbarga showed the same
pulsotype with five of the seven isolates from Sedam
(Table II, Fig.).
Discussion
Asymptomatic carriage of ESBL-producing
K. pneumoniae in the gastrointestinal tract may serve
as the source of subsequent infections such as urinary
tract infection13. Although the sample size was small
in the study, isolates originating from stool were
confirmed as ESBL producers. The results of our
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INDIAN J MED RES, MAY 2009
study revealed that K. pneumoniae producing CTXM-15 is already present in various geographic areas
in southern India, as it has been observed in northern
India6. CTX-M-15 is the predominant ESBL among
E. coli causing community-associated infections
worldwide3. It differs from CTX-M-3 by one amino
acid substitution (Asp240 to Gly), which is known
to extend the spectrum of activity to ceftazidime in
addition to cefotaxime14. The isolates in the present
study were all intermediate or resistant to ceftazidime,
which is consistent with this enzymatic property of
CTX-M-15. In addition to the cephalosporins all
isolates were resistant to ciprofloxacin as well. Orally
administered antimicrobials including ciprofloxacin
are available over-the-counter and frequently used for
the purpose of self-medication in India, which may
account for the accumulation of multi-drug resistance
among ESBL producing organisms15. Investigation of
the mechanisms of ciprofloxacin resistance among the
study isolates is underway.
Of note, study isolates recovered from patients
at three geographically distant sites shared identical
macrodigestion patterns by PFGE. This finding
suggests that a particular clone of K. pneumoniae
may be associated with the spread of CTX-M-15 in
southern India. Further studies are needed to determine
the potential of this particular clone of CTX-M-15producing K. pnemoniae in causing communityassociated infections, as has happened with CTX-M15-producing E. coli.
In
conclusion,
CTX-M-15-producing
K.
pneumoniae isolates from stool samples of patients
with gastroenteritis in rural and urban areas in southern
India were found, many of which belonged to a single
clone. Hence routine diagnosis of ESBL-producing K.
pneumoniae should be done in hospitals, coupled with
prudent use of antimicrobials to reduce propagation of
multidrug-resistant and ESBL-producing organisms.
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Reprint requests: Dr David L. Paterson, University of Queensland, Royal Brisbane & Women’s Hospital, Brisbane, Australia
e-mail: david.antibiotics@gmail.com