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Note: Tables, appendix and figures
of the article can be accessed and seen in the PDF file.
Introduction
Klebsiella pneumoniae is an important nosocomial
pathogen that has the potential to cause severe
infections, particularly in intensive care units and
amongst pediatric patients1. The
increased use of extended spectrum cephalosporins has
led to the emergence of resistant strains and outbreaks
due to these organisms have been associated with higher
morbidity and mortality2. Spreading of
extended spectrum beta lactamase (ESBL) producing K.
pneumoniae in a hospital may be a complex event
involving several modes of spread, such as dissemination
of several unrelated strains or the propagation of a
single clone from patient to patient3.
Mostly,
ESBL-encoding genes are located within transposons or
integrons, which strongly facilitates
antibiotic-resistant gene transfer between bacterial
species resulting in cross-transmission, thereby
spreading resistance among related and unrelated
gram-negative bacteria4. Investigation of a
presumed outbreak by Klebsiella species
often requires strain typing data to identify
outbreak-related strains. Traditional techniques for
typing K. pneumoniae are based on phenotypic
characteristics and include biotyping, antibiogram
typing, O-serotyping, bacteriocin and phage typing; but
they all have poorly discriminatory power.
Unfortunately, biochemical reaction patterns are usually
invariable among clinical isolates5.
Although, the molecular methods such as plasmid-profile
analysis, ribotyping, small-fragment restriction
endonuclease analysis, gene sequencing and pulsed-field
gel electrophoresis are available, they cannot be used
routinely in many laboratories in developing countries
like India, due to high cost and poor resource settings.
Random
Amplification of Polymorphic DNA (RAPD) is a molecular
typing technique, which is based on PCR amplification of
random DNA fragments with short primers 6-12 bp of
arbitrary sequence. The resulting amplified fragments
function as polymorphisms for DNA fingerprinting. In
contrast to traditional target-specific PCR, no prior
sequence information is required and the technique is
potentially applicable to all bacteria. This technique
has been applied successfully to epidemiological
investigations of many bacterial and fungal species6.
In this
background, the aim of this study was to investigate the
epidemiology and molecular characterization of
multiresistant ESBL-producing K pneumoniae
strains associated with an outbreak of bloodstream
infection (BSI) in a busy neonatal intensive care unit (NICU)
in a tertiary care hospital, South India.
Material
and Methods
Source
of the isolates
During the
month of June 2008, an increased frequency of K
pneumoniae was isolated in the blood culture from
NICU. Blood for culture was collected from these
patients on clinical suspicion of neonatal sepsis. Blood
cultures were done using biphasic medium consisting of
Brain Heart Infusion (BHI) agar and BHI broth with
sodium polyanethol sulphonate as an anticoagulant.The
clinical detail of the patients were recorded and environmental
sampling, personal surveillance was done in NICU on mid
June 2008.
A total of
31 isolates of K.pneumoniae were included. Twenty
seven strains were isolated from neonates during the
outbreak and four strains from environment sampling done
in NICU. All the isolates were identified as per
the standard bacteriological procedures7 and
they were stocked in 0.2% semi-solid agar tubes and
stored under 4◦C until further
characterization.
Antimicrobial susceptibility testing
Antimicrobial susceptibility of the isolates was done by
the disk diffusion method on Mueller Hinton agar
(Hi-Media, Mumbai) following the zone size criteria
recommended by the Clinical Laboratory Standards
Institute (CLSI) 8. The antibiotic used were;
ampicillin(10μg), amikacin(30μg), gentamicin(30μg),
piperacillin(100μg), piperacillin/ tazobactum 100/10μg),
cefoperazone/sulbactum (75/10µg), cefoxitin (30μg),
cefotaxime (30μg), ceftazidime (30μg),
ceftriaxone(30μg), ciprofloxacin(5μg) , cefepime (30μg),
Trimethoprm-Sulfmethoxazole (7.5/2.5µg),
meropenem(10μg), imipenem (10μg). The Minimum inhibitory
concentration (MIC) of third generation cephalosporins
(Hi-Media, Mumbai) and meropenem (Astra-Zeneka, UK) was
done by agar dilution method as per CLSI guidelines9.
Control strain Escherichia coli ATCC 25922 was
included in each series.
Phenotypic detection of ESBL
The
isolates showing resistance to one or more third
generation cephalosporins (3GCs) were tested for ESBL
production by the combination
disc method using cefotaxime(30μg), cefotaxime/clavulanic
acid (30/10μg) and cefatzidime(30μg), ceftazidime/clavulanic
acid (30/10 μg). A ≥5mm
increase in diameter of the inhibition zone of the
cephalosporin-plus-clavulanate disc, when compared to
the cephalosporin disc alone, was interpreted as
phenotypic evidence of ESBL production8.
K.
pneumoniae ATCC 700603 was used as positive control
and Escherichia coli ATCC 25922 was used as
negative control.
Molecular detection of ESBL-gene types
For the
molecular analysis, the template DNA was prepared from
an overnight culture (18–24 h) on a Mueller–Hinton
plate. Two colonies were suspended in 100 µl of
distilled water and the cells were lysed by heating at
95°C for 10 min. Cellular debris was removed by
centrifugation at 15,000 rpm for 2 min, and the
supernatant was used as a source for DNA for PCR.
Isolates with the ESBL phenotype were examined for the
presence of β -lactamases genes responsible for the
resistance such as blaTEM, blaSHV, and
blaCTX-M by PCR using the primers and conditions
used elsewhere 10,11,12. .
Typing
by RAPD analysis
K.pneumoniae isolates from the infected neonates and
isolated from the environment were typed by RAPD
technique to investigate the role of environment in
transmission of infection. Two individual primers, AP4
(5'-TCA CGA TGC A-3'), HLWL74 (5'-ACG TAT CTG C- 3')
13 were used. The DNA was prepared by
boiling method as above mentioned. Amplification was
performed on a Corbett Research thermal cycler (HP,
USA). The reaction mixtures comprised of 12.5 µl of red
eye PCR master mix (Amplicon III), 2µl of lM AP4 and
HLWL74 primers each individually, 7.5µl of sterile nano
pure water and 3µl of template DNA. The final volume was
25µl. The amplification condition were initial
denaturation for 7 min at 94°C; followed by 30cycles of
1 min at 94°C, 1 min at 36°C, and 2 min at 72°C; and
with a final extension for 10min at 72°C. The amplified
products were separated by electrophoresis in a 2%
agarose gel and stained with ethidium bromide. The band
patterns were visually interpretated and a difference of
more than 2 bands were considered a given major type,
one to two bands difference was considered a minor
variant.
Sequencing
Representative amplified products of TEM, SHV, CTX-M
group- 1 PCRs from each cluster were outsourced to
Macrogen, Korea for sequencing by the Sanger method
using an ABI 373A DNA sequencer, using the primers
described previously10,11,12.
Results
Antimicrobial susceptibility testing
As
determined by disc-diffusion antibiotic susceptibility
testing, all the 27 clinical isolates and 2
environmental isolates exhibited the same pattern of
resistance to β-lactam agents,
demonstrating resistance to penicillins (ampicillin and
piperacillin), extended-spectrum cephalosporins (ceftriaxone,
ceftazidime and cefotaxime) and cefepime. In addition,
they exhibited resistance to gentamicin, chloramphenicol,
piperacillin/tazobactam and trimethoprim/sulfmethoxazole.
Based on the susceptibility to other aminoglycoside (amikacin),
carbapenem (meropenem) and cefperazone/sulbacatam
combinations, 2 antibiotypes were defined for the 29
isolates (Table 1). Nine isolates belonged to resistance
pattern I, which was characterized by resistance to
amikacin, meropenem, and cefperazone/sulbactam. The 20
isolates belonged to antibiotype pattern II, which
differed from I by susceptibility to amikacin, meropenem,
and cefperazone/sulbactam .All isolates tested remained
susceptible to imipenem. The MIC of 3GCs for both
antibiotype I and II were >256 μg/ml, and the MIC of
meropenem for antibiotype I and II were 16 μg/ml and <2
μg/ml respectively. The two environmental isolates E3
and E4 were susceptible to all drugs except ampicillin
(Table 1).
ESBL types
All the 27 isolates from
neonates and 2 environmental isolates were ESBL positive
phenotypically and they carried TEM, SHV and CTX-M group
I genes by PCR. Sequencing reveals that TEM-1, SHV-12
and CTX-M 15 type of ESBLs were present in both clinical
and environmental strains.
RAPD typing
In RAPD
analysis, two distinct band
patters were generated individually by primers AP4 and
HLWL74. An average of 2-4 bands per pattern was
generated with AP4 primer and with HLWL74 an average of
9-11 bands was generated, thus two RAPD clusters was
recorded among the outbreak strains. All the strains
from each RAPD clusters shows 100% similarity to the
environmental isolates and the two clusters showed a
major pattern difference (Figure 1a, 1b). The
environmental source of cluster I was found to be the
oxygen circuit (at patient end) and for cluster II, the
suction tube. The susceptible environmental E3, E4
strains showed varied RADP patterns (Figure 2).
The mean
age of the infected patients is 4.8 days and standard
error is (.508), the male distribution is 51.9% and
female distribution is 48.1%. The distribution of
antibiotype II is higher 66.7% and the RAPD type B is
18%. The observed mortality rate among the infected
patients is (.67).
Discussion
ESBL
producing K. pneumoniae are usually resistant to
the first line of antibiotics. Additionally, their
ability to spread rapidly amongst patients often leads
to nosocomial outbreaks constitutes a persistent problem
in many parts of the world, especially in intensive care
units. Moreover, these bacteria are increasing sources
of resistance to other group of antibiotics also13,14.
In the hospital environment, spreading of these
organisms may be a complex event involving several modes
of epidemic spread, such as dissemination of several
epidemic strains or the propagation of a single clone
from patient to patient15.
K.pneumoniae that produces ESBL has been associated
with infection acquired in NICU. A major risk factor for
colonization or infection with ESBL producing bacteria
is long term antibiotic exposure and frequent use of
antimicrobial agents in NICUs. It is a common practice
to use aminogycosides and 3GC in a neonatal septicemic
cases16,17. Usually prescribed
antimicrobials fail to inhibit these pathogens, and the
most effective antimicrobials against ESBL producing
K.pneumoniae are carbapenems. Meropenem and imipenem
are the two carbapenem available in India; their
prescription is based on individual physician criterion
that could lead to inappropriate use of antibiotics
favoring selection of resistant strains.
This study
describes an outbreak due to multiresistant ESBL-producing
K pneumoniae in a neonatal ICU. Apparently, the
outbreak started at the end of may reached peak on June
and subsided on July (Figure 3). We suggest that
successive small outbreaks, as reported here, may be
partly due to changes in the multiplication rate of
strains. The micro-organisms generally multiply at a low
rate but this may increase for some reason, resulting in
the spread of a micro-organism to several patients and
possible infections. After transmission, the organisms
may regress, leaving patients susceptible to
colonization and infection by other strains of K.
pneumoniae.
In the
present study, all the 27 clinical isolates and the 2
environmental isolates were multidrug resistance and
ESBL producers nine meropenem-resistant isolates were
detected from the patients during the same outbreak
period, which suggests that exposure to meropenem
induced expression of resistant strains. This resistance
may be associated with a modification of a major outer
membrane protein or changes in lipopolysaccharide
18. The widespread use of meropenem in ICUs is of
concern as this selects meropenem-resistant isolates.
Since both meropenem sensitive and resistant strains
caused the outbreak at the same time, this raises the
question whether multiple colonization involved
sequential exposure to different strains or whether
genotypic variations within strains were induced in
vivo in response to changes in therapy. It is
difficult to determine the role, if any, of antibiotic
therapy in the evolution of different isolates of
K.pneumoniae.
Antibiotic-resistant Gram-negative organisms are a
significant risk to severely ill children in ICUs and in
many instances these are imported into the unit or
rapidly acquired from environmental reservoirs19.
In our study, the isolates from two environmental
sites such as oxygen circuit (at patient end) and the
suction tube were found to be the cause for the outbreak
by the horizontal transfer of these strains. The other 2
RAPD types III and IV from the environment were the
susceptible wild strains.
Prior to
testing of the multiresistant K. pneumoniae
strains, RAPD was performed with a random of 6
epidemiologically unrelated strains of K. pneumoniae
that had the usual susceptibility pattern, showing
resistance to ampicillin alone. The RAPD patterns
produced were all dissimilar to those seen with the
strains from NICU. Typing of the strains using AP4 and
HLWL74 gave good discriminatory bands and we suggest
that both can be used individually or simultaneously for
better interpretation in typing a large number of
strains.
The high
level of mortality (Table 1) arising from intra hospital
infections with multi-drug resistant strains of
K.pneumoniae, as seen, in this study, is alarming.
We did not find any statistically significant
associations between patient mortality and any of the
factors like sex, age, or antibiotype. The P value is
(>0.05). However, this negative result still may be
worth mentioning. It appears that the lack of control of
contamination sources and hygiene has caused the
dissemination if the pathogen among the patients. Simple
hygienic measures, such as hand-washing practices, the
use of sterile equipment (particularly for intravenous
access and when possible), and patient cohorting (i.e.,
grouping patients with similar infections in the same
location) can help prevent the further spread of these
resistance traits. Also, the empirical
use of carbapenems for nosocomial sepsis where ESBL
Klebsiella is prevalent should be evaluated and
parameters to prevent over usage should be placed.
K.
pneumoniae infections have been caused by a variety
of strain genotypes that could be transmitted from one
patient to another in different ways, and it is
important to monitor such strains closely to prevent
their spread. In this situation, the design of rational
infection control measures that require the adoption of
new antibiotic policies in addition to improving
hospital hygiene becomes even more challenging20.
Nosocomial
infections in neonatal ICUs are the most difficult and
tedious to manage and control. The outcome of neonatal
infections can be improved if illness is recognized
early and appropriate agents are promptly administered.
Knowledge of epidemiological and anti-microbial
susceptibility pattern of common pathogens in a given
area helps to inform the choice of antibiotics.
Epidemiological surveillance studies such as the current
one should provide useful information base to guide
practice and policies on rational use of anti-infective
agents and to eradicate the source of environmental
reservoir.
In
conclusion, this is the one of the first reports from
our hospital in which the causative organism of an
outbreak of infection with multi-drug resistant CTX-M-15
producing K.pneumoniae associated with a high
mortality has been characterized at the molecular level.
The RAPD typing is good and can be used as a screening,
rapid and inexpensive test for ESBL producing K.
pneumoniae during investigation of outbreaks.
Acknowledgement
The authors
wish to extend their gratitude to the medical and
nursing staff of the Neonatal Intensive Care Unit,
JIPMER Hospital, Pondicherry for their cooperation in
this study.
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