The emergence of carbapenem resistant Klebsiella pneumoniae in Malaysia: correlation between microbiological trends with host characteristics and clinical factors
Low et al. Antimicrobial Resistance and Infection Control
The emergence of carbapenem resistant Klebsiella pneumoniae in Malaysia: correlation between microbiological trends with host characteristics and clinical factors
Ye-Mun Low 0
Polly Soo-Xi Yap 0
Kartini Abdul Jabar 0
Sasheela Ponnampalavanar 2
Rina Karunakaran 0
Rukumani Velayuthan 0
Chun-Wie Chong 1
Sazaly Abu Bakar 0 3
Mohd Yasim Md Yusof 0
Cindy Shuan-Ju Teh 0
0 Department of Medical Microbiology, University of Malaya , 50603 Kuala Lumpur , Malaysia
1 Department of Life Sciences, International Medical University , Bukit Jalil, 57000 Kuala Lumpur , Malaysia
2 Department of Medicine, University of Malaya , 50603 Kuala Lumpur , Malaysia
3 The Tropical Infectious Diseases Research & Education Centre (TIDREC), University of Malaya , 50603 Kuala Lumpur , Malaysia
Background: Carbapenem resistant Enterobacteriaceae is a growing concern worldwide including Malaysia. The emergence of this pathogen is worrying because carbapenem is one of the 'last-line' antibiotics. The main objective of this study was to determine the prevalence of genetic mechanisms and clinical risk factors of carbapenem resistant Klebsiella pneumoniae (K. pneumoniae) in Malaysia. Methods: In this study, seventeen carbapenem resistant K. pneumoniae strains isolated from a tertiary teaching hospital in 2013 were studied. Minimal inhibitory concentration (MIC) of the bacterial strains was determined and genes associated with carbapenemases and extended-spectrum-beta-lactamases (ESBLs) were sequenced and compared with the closest representatives published in public domains. All strains were also sub-typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Statistical analyses were performed to determine the correlation between risk factors for acquiring carbapenem resistant K. pneumoniae and in-hospital mortality. Results: The predominant carbapenemase was blaOXA-48, detected in 12 strains (70.59%). Other carbapenemases detected in this study were blaKPC-2, blaIMP-8, blaNMC-A and blaNDM-1. Nine different pulsotypes were identified and nine strains which were affiliated with ST101, the predominant sequence type had similar PFGE patterns (similarity index of 85%). Based on univariate statistical analysis, resistance to imipenem and usage of mechanical ventilation showed a statistically significant effect separately to in-hospital mortality. Conclusion: The diverse genetic mechanisms harbored by these carbapenem resistant K. pneumoniae facilitates its spread and complicates its detection. Thus, correlation between microbiological trends with host characteristics and clinical factors will provide a better insight of rational treatment strategies and pathogen control.
Carbapenem resistant K; pneumoniae; OXA-48; KPC-2; ST101; Enterobacteriaceae
Klebsiella pneumoniae (K. pneumoniae) is an important
pathogen responsible for many healthcare associated
infections. This Gram-negative bacterium affiliated with
the Enterobacteriaceae family is the fourth and fifth
most common cause of pneumonia and bacteremia
respectively, among intensive care units (ICU), newborn
units and in immunocompromised patients [1, 2]. In
hospital environments, Klebsiella species survive and
multiply in wet environmental sites and colonize the
human bowel, bladder, upper respiratory tract and skin .
During the 1990s, extended-spectrum beta-lactamases
(ESBLs) producing Klebsiella species that are able to
hydrolyze broad and extended-spectrum cephalosporins,
monobactams and penicillins were reported . Thus,
carbapenems, one of the 'last resort antibiotics' were
often used to treat serious infections caused by ESBL
K. pneumoniae was first reported to harbor Klebsiella
pneumoniae carbapenemase (KPC), one of the
epidemiologically important carbapenemases first detected in
North Carolina, USA in 1996 . It was later identified
in outbreaks in USA and was soon detected in many
European countries and South America . Initially, it
was thought that carbapenemases were attributed to
chromosomally encoded beta-lactamases and can only
be transferred through clonal spread. However, plasmid
encoded blaIMP-1 and blaKPC-1 reported in the 1990s 
confirmed the presence of these resistance genes in
mobile genetic elements which implicates potential for
resistance transmission through horizontal gene transfer.
The first report of carbapenem resistant K. pneumoniae
in Malaysia was an imipenem resistant strain isolated from
blood culture of a 42-year old woman in 2004 . To date,
carbapenemases harbored by K. pneumoniae isolated in
Malaysia identified using Pubmed search were NDM-1 [9,
10], OXA-232  and IMP-4 .
In this study, we investigated the genotypic
characteristics of carbapenem resistant K. pneumoniae isolated
from patients in a tertiary teaching hospital in Malaysia.
Their association with Tn4401 and the loss of porin as
well as their subtypes were identified. Further risk
factors associated with in-hospital mortality rate were also
A total of seventeen Klebsiella pneumoniae strains
isolated within an eight-month period in 2013 since the
first report of carbapenem resistant Enterobacteriaceae
in this tertiary teaching hospital were revived from stock
cultures. Antimicrobial susceptibility testing (AST) was
conducted and carbapenemase production of the strains
was detected by the Modified Hodge Test (MHT) (K.
pneumoniae ATCC® BAA-1705 and K. pneumoniae
ATCC® BAA-1706 as MHT positive and negative
reference strain respectively). Clinical records of the patients
from whom strains were isolated were retrieved from
the hospital database and ethics approval (MEC:1059.15)
was obtained from University of Malaya Medical Centre
(UMMC) Ethics Committee prior to the start of this
Determination of minimal inhibitory concentration (MIC)
These seventeen strains which had been previously
detected as resistant to imipenem and/or meropenem
based on Clinical and Laboratory Standards Institute
(CLSI) guidelines  by the hospital's diagnostic
microbiology lab were subjected to E-test (bioMérieux, USA)
using E. coli ATCC® 25922 as the quality control strain
for susceptibility testing. The MIC values (μg/ml) for
tigecycline, tetracycline, cefoxitin, cefotaxime,
ceftazidime, ceftriaxone, cefepime, imipenem, meropenem,
ertapenem, gentamicin, tobramycin, amikacin,
aztreonam, ciprofloxacin, levofloxacin and colistin were
determined. The results were interpreted using CLSI
guidelines  while susceptibility to colistin and
tigecycline were interpreted according to European
Committee on Antimicrobial Susceptibility Testing
(EUCAST) breakpoints . In brief, imipenem and
meropenem resistance were defined as MIC ≥4 μg/ml
while ertapenem resistance was defined as MIC ≥2 μg/
Pulsed-field gel electrophoresis (PFGE)
The clonal relatedness of these seventeen strains was
determined using PFGE. Briefly, plugs containing whole
genomic DNA of K. pneumoniae strains were digested
with XbaI. The DNA fragments were separated in a
PFGE CHEF-DR III system (Bio-Rad, UK). PFGE
conditions of XbaI macrorestriction analysis were 6Vcm-1 for
20 hours, with pulse times ranging from 5 s to 30s at a
temperature of 14 °C and at an angle of 120°. The
banding patterns were analyzed using BioNumerics software
and similarity >85% upon dendrogram analysis was
considered to represent the same PFGE pattern groups.
Multilocus sequence typing (MLST)
Seven housekeeping genes of K. pneumoniae namely,
gapA, infB, mdh, pgi, phoE, rpoB and tonB were
sequenced and analyzed using Institut Pasteur MLST and
whole genome MLST database for K. pneumoniae,
Each locus was assigned an allele number and the
sequence type (ST) of each strain was determined based
on the allelic profile generated using the allele numbers
of the seven loci.
Detection of carbapenemases, AmpC beta-lactamases,
extended-spectrum beta-lactamases (ESBL) and outer
membrane porin genes
Carbapenemase genes such as KPC , OXA-48
, IMP, VIM , NDM , NMC, IMI, SIM, SPM
, GIM , AmpC beta-lactamases (CMY, DHA,
ACC, FOX)  as well as other ESBL associated
genes such as TEM, SHV , OXA-1 , OXA-9
, CTXM-1, CTXM-2  were amplified via PCR.
The amplified products were sequenced and gene
types were compared to the nearest gene homology
using a BLAST search. The presence of porin
associated genes namely ompK35, ompK36 and ompK37
 were also determined by PCR.
Determination of genetic elements of Tn4401
Genetic elements of Tn4401, a 10 kb Tn3 based
transposon commonly found to harbor blaKPC was
determined as previously described [14, 22]. The unconserved
region located between ISKpn7 and blaKPC gene was also
amplified to determine the isoforms of Tn4401.
Risk factors associated with in-hospital mortality were
compared statistically depending on the level of
measurement. Specifically nominal parameters (eg.
mechanical ventilation, resistance to antibiotics, etc) were
compared using chi-square or Fishers' exact test as
appropriate while continuous variables (age and length of
hospitalization) were analyzed using either student t test
or Mann Whitney U test depending on data normality. P
values less than 0.05 were deemed as statistically
significant. Multiple logistic regression was not included in
this study due to the small sample size. Continuous
variables were summarized as mean ± standard deviation or
median (inter quartile range) for both normal and
nonnormal distribution respectively.
Overview of carbapenem resistant Klebsiella pneumoniae
The first case of carbapenem resistant K. pneumoniae in
this study was detected from blood culture of a
57-yearold male patient in April 2013 in this 1,000 bed tertiary
teaching hospital in Kuala Lumpur, Malaysia. Since then,
no new cases were reported until another carbapenem
resistant K. pneumoniae isolation from peritoneal
drainage of a 67-year-old male patient in August 2013. In the
following weeks, an average of one new carbapenem
resistant case was detected every 2 weeks and as of 19
December 2013, a total of seventeen strains which were
resistant to imipenem and/or meropenem were
identified from blood (n = 4), urine (n = 4), swabs (stoma,
perirectal, foot) (n = 3), drainage fluids (percutaneous
transphetic biliary drainage (PTBD), peritoneal) (n = 2),
tracheal secretion (n = 1), tracheal aspirate (n = 1),
sputum (n = 1) and tissue (n = 1)). The time-line of events
during in-patient admission period including the wards
stayed and carbapenem resistant K. pneumoniae strains
isolation from the sixteen patients from March 2013 to
April 2014 was illustrated in Fig. 1. Four of the patients
stayed in surgical wards, four in medical wards, two in
orthopedic wards and six others moved from different
wards. A total of four patients stayed in ICU during their
These strains were the first carbapenem resistant K.
pneumoniae isolate from each patient with the exception
of two strains which were isolated from a 60-year-old
male patient. The first strain isolated from this patient
was in October 2013 from tracheal aspirate.
Subsequently, we did not identify carbapenem resistant K.
pneumoniae from respiratory samples with the Vitek2
automated susceptibility testing system in 2013 while
urine samples had no growth from October until
November 2013. However, carbapenem resistant K.
pneumoniae was detected in this patient's urine samples in
This study was conducted in a multiracial community
in Malaysia which comprises of three major races
namely Malays, Chinese and Indians. There were nine
male patients and seven female patients. The mean age
of the patients was 66.13 years where the youngest
patient was 48-years-old while the eldest was 85-years-old
(Table 1). The average length of hospitalization prior to
strain isolation was 65.18 days. Two carbapenem
resistant K. pneumoniae strains (K/1309-38 and K/1311-26)
were isolated from urine and blood respectively from
two different patients within 48 hours of admission and
one of the patients (K/1309-38) had came into contact
with healthcare facilities in the past 30 days for
endoscopic procedures. All patients in this study had
comorbidities such as diabetes mellitus, hypertension,
chronic kidney disease and solid organ tumors. Diabetes
mellitus and hypertension were the predominant
noncommunicable diseases identified in these patients.
All 16 patients had been administered antibiotics over
a period of 90 days prior to carbapenem resistant K.
pneumoniae strain isolation. Cephalosporins and
betalactam/beta-lactamase inhibitor combinations were the
two most common classes of antibiotics being
administered to them. These patients had been treated
empirically with antibiotics such as beta-lactam/beta-lactamase
inhibitors, carbapenems, cephalosporins, ciprofloxacin,
vancomycin and colistin prior to carbapenem resistant
K. pneumoniae identification (Table 1).
Eight patients (47.06%) in this study were infected with
carbapenem resistant K. pneumoniae while others were
considered to be colonized. Of the eight infected
Fig. 1 Time-line of events during patient admission period.
patients, one of them had changed their antimicrobial
regimen to colistin while five others were given
carbapenem and colistin combination. One patient (12.5%)
died before changing therapy and another continued
with colistin therapy.
Risk factors such as surgical procedures undergone,
usage of mechanical ventilation and the usage of
catheter were also taken into consideration. However, based
on univariate analyses, only the usage of mechanical
ventilation (P = 0.043) shows significant effect to
inhospital mortality rate (Table 2).
Determination of minimal inhibitory concentration (MIC)
The antibiotic profiles were tabulated in Table 3. Briefly,
all strains were resistant to tetracycline (two strains were
intermediate) but only one strain was resistant to
tigecycline. Meanwhile, all strains were sensitive to colistin.
Eight strains (47.06%) were resistant to all
cephalosporins and all strains showed resistance to at least one
carbapenem (MIC = 4 - >32 μg/ml) in this study. Resistance
to imipenem was significantly associated with in-hospital
mortality (P = 0.043) (Table 2). It was also noted that
two strains (K/1311-35 and K/1304-16) were resistant to
imipenem (MIC = 4 - 6 μg/ml) but were sensitive to
meropenem and ertapenem while one strain (K/1312-3)
was sensitive to imipenem but resistant to meropenem
and ertapenem (MIC = 6 - 16 μg/ml). Furthermore, three
strains (17.65%) were resistant to all aminoglycosides
(MIC = 24 - >256 μg/ml for gentamicin and tobramycin;
MIC = 64 - >256 μg/ml for amikacin) while thirteen
strains (76.47%) were resistant to all fluoroquinolones
(MIC = 8 - >32 μg/ml).
Molecular typing via PFGE & MLST
Based on the PFGE patterns obtained, the seventeen
strains formed nine different pulsotypes while MLST
analysis showed eight STs (Fig. 2). Nine strains affiliated
with the predominant sequence type, ST101
demonstrated similar PFGE patterns (Dice index >85%
homology) suggesting close intra ST-type genomic similarity.
Five of the ST101 strains were isolated within a week
apart from one another in August until September 2013
from patients who had stayed in surgical wards. Two
weeks later, this sequence type was isolated from one
patient in the medical ward and in the following months
from two patients in medical wards and one patient
from the orthopedic ward. It was also noted that three
other sequence types, ST789, ST11 and ST495 were
isolated from patients who had stayed in surgical wards
two months after the first ST101 isolation and one of
the patients had stayed in the surgical ward in August
2013. All ST101 strains in this study were highly
resistant to meropenem (MIC >32 μg/ml) and ertapenem
(MIC = 24 - >32 μg/ml). High MIC was also observed
for imipenem (MIC >32 μg/ml) with the exception of
three strains which had lower MICs ranging from 2 μg/
ml (intermediate) to 4 μg/ml (resistant).
Similarly, two strains (K/1304-16 and K/1309-39)
(Dice index = 84.2% homology) from the same
subcluster were categorized into the same sequence type,
ST147. However, two other strains, K/1310-35 and K/
1312-3 which were isolated two months apart from a
60-year-old male patient exhibited distinct PFGE pattern
and sequence type (ST397 and ST13 respectively);
indicating low relatedness.
Detection of carbapenemases, AmpC beta lactamases,
extended spectrum beta lactamases (ESBL) and porin
AmpC (blaFOX-7) and ESBLs (blaSHV, blaTEM, blaCTXM-2
blaCTXM-15, blaOXA-1, blaOXA-9) were present in all 17
strains while carbapenemases (blaKPC-2, blaOXA-48,
s o n
o th c ro
-h ae se se o se o o o o o o o se o o se o se lse te
In d Y Y N Y N N N N N N N Y N N Y N Y if M
o T P
rtaeg iitnb T ,PC ,TC d T il il d il ,TZA il eil ,TC ,TC ,TC il il nU cy r3
T a C M IP IP C N N IP N V N N IP IP IP N N , m fo
ia nd ru ro o se se se kn se se o se se se se se se se kn se sae ,IP O ca
n U s p N Y Y Y U Y Y N Y Y Y Y Y Y Y U Y is en TCb,r
Table 2 Characteristics of carbapenem resistant Klebsiella pneumoniae associated with in-hospital mortality. (Continued)
Carbapenem (Meropenem / Imipenem)
Vancomycin & Piperacillin-tazobactam
Colonization / Infection
Resistance to antibiotics (MIC)
Values are expressed as n (%) except where otherwise noted.
atargeted therapy was not administered as strain was colonizer or patient passed away before targeted therapy was initiated
P-values were obtained using Fishers' exact test unless noted otherwise. bP-values obtained using student t test, cP-values obtained using chi-square, dP-values
obtained using Mann Whitney U test. The significant P values were highlighted in bold.
blaIMP-8, blaNDM-1, blaNMC-A,) were detected in only 16
strains where blaOXA-48 was the dominant
carbapenemase (Table 4). All strains also exhibited the presence of
three outer membrane porins (ompK35, ompK36,
ompK37) with the exception of K/1310-24 where
ompK36 was absent. This strain, K/1310-24 also showed
the highest resistance towards imipenem (MIC = 6 μg/
ml), meropenem (MIC = >32 μg/ml) and ertapenem
(MIC = 6 μg/ml) among the group of six strains made
up of sequence type other than ST101 and ST147.
Characterization results for Tn4401
Primer pairs specific for different genetic elements
on Tn4401 were used to deduce the structure of it.
A schematic representation of a typical Tn4401
structure is shown in Fig. 3a. All strains harboring
blaKPC-2 showed the presence of Tn4401 elements
which indicated that blaKPC-2 gene was carried
within Tn4401 transposon (Table 4).
Isoforms of Tn4401 were determined based on the
fragment size of primer pair hybridizing ISKpn7 and
blaKPC; upstream of blaKPC . All six strains which
showed positive amplification were of the same DNA
fragment size (703 bp) and they belonged to isoform
b where no deletion in the variable region is
observed. Among these six strains, one was sensitive,
two were intermediate while three exhibited resistance
to imipenem (MIC = 4 - >32 μg/ml) but all strains
were resistant to meropenem and ertapenem (MIC = 6
- >32 μg/ml). Both flanking sequences of Tn4401,
inverted repeat left (IRL) and inverted repeat right
(IRR) were present in three out of 17 strains (17.65%)
while three others (17.65%) showed only one flanking
sequence. Complete Tn4401 was not detected in this
study as some elements were absent in all strains.
T :2 :R 9 8 25 6 9 25 25 9 25 5 5 25 25 8 8 9 ,
2 .5 .1 .3 .1 .0 .1 .1 .1 .1 .1 .2 .2 .1 .1 .3 .3 .1 am
aC ≤( > 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 n
6 6 6 5 C ib w
5 5 5 2 M tp se
PM≤(2 -48 ≥1 2> 2> 2> 42 2> .30 16 1 > 1 > 2 6 2 > 3 0 ,P ce l
2 .5 2 4 4 2 .1 u
i o S C
n , x I
oum :S1 ,I= :)4R 652 652 652 652 652 652 .5 652 652 652 652 ifrtae ,tSDDa.eM
e TX ≤( 2 ≥ > > > > > 8 4 2 > 1 > 3 > > > 1 1 C n is
n X ta w
n :S ,I )R 6 6 6 6
ta 1 = :4 52 65 52 52 52 2 .7 2 2 4 2
is TC ≤( 2 ≥ > 2 > > > 4 6 4 > 0 > 6 > 6 > 1 .07 ,e ten su
s imII e
re txa ,e lien
o iv d
f t i
e is u
,S ,I )R
em /m : : 6 6 6 6 6 6 6
n g 8 = 2 5 5 5 82 .5 82 8 52 4 52 52 52 C n g
e μ FX ≤( 16 ≥3 2> 2> 2> 2 1 1 1 4 > 2 6 > > 3 > 6 3 TC Se 15
: 2 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 m
1 = 4 3 .0 3 3 3 .1 3 3 3 .5 3 3 3 3 3 3 .0 A
IC ≤( 2 ≥ > 0 > > > 0 > > > 0 > > > > > > 0 K
:S ,I :R 6 6 6 6 5 6 7 6 6 6 5 7 y
T 4 = 61 52 52 52 8 52 4 .21 .91 52 .40 52 .52 52 29 52 .21 .40 am
A ≤( 8 ≥ > > > 4 > 2 0 0 > 0 > 0 > 1 > 0 0 rb
Fig. 2 Dendogram of PFGE banding patterns of carbapenem resistant Klebsiella pneumoniae and their sequence type (ST) determined via MLST
together with their antibiotic and genotypic profile. IP: imipenem, MP: meropenem, ETP:ertapenem
Table 4 PCR results of gene specific primers depicting Carbapenemase, AmpC, ESBL, porin protein and Tn4401-like elements in
carbapenem resistant Klebsiella pneumoniae strains
Genetic elements surrounding Tn4401
FOX-7 SHV-11, CTXM-15, CTXM-2
OXA-48, KPC-2, FOX-7 TEM-135, SHV-148, CTXM-15,
FOX-7 TEM-1, SHV-28, OXA-1, OXA-9,
FOX-7 TEM-1, SHV-28, OXA-1, OXA-9,
FOX-7 SHV-12, CTXM-2
FOX-7 SHV-28, OXA-1, CTXM-2
FOX-7 SHV-28, OXA-1, CTXM-2
FOX-7 TEM-1, SHV-28, OXA-1, OXA-9,
FOX-7 SHV-121, CTXM-2
FOX-7 TEM-1, SHV-28, OXA-1, OXA-9,
FOX-7 SHV-28, OXA-1, CTXM-2
FOX-7 TEM-1, SHV-28, OXA-1, OXA-9,
FOX-7 SHV-28, OXA-1, CTXM-2
FOX-7 SHV-1, CTXM-2
FOX-7 TEM-1, SHV-11, OXA-9, CTXM-15, OmpK35, OmpK36,
IRL inverted repeat left, tnpA transposase, ISKpn7 insertion sequence 7, ISKpn6 insertion sequence 6, IRR inverted repeat right
Fig. 3 Shaded triangles represent inverted repeats (IR) sequences. Empty triangles represent IRs of insertion sequences, ISKpn6 and ISKpn7. The
location of the variable region is as labeled. (Adapted from  and ). a: Schematic representation of intact classical Tn4401. b: Examples of
truncated Tn4401 detected in this study. ISKpn7, encodes two consecutive open reading frames (ORF); istA which encodes a 341-amino-acid
putative transposase while istB encodes a 259-amino-acid transposition helper protein. TnpA encodes a transposase of 1,009 amino acids while
tnpR is a 1,713 bp resolvase gene
Examples of truncated Tn4401 detected in this study
were shown in Fig. 3b.
Among the seventeen carbapenem resistant K.
pneumoniae strains investigated, two of them (K/1310-35 and K/
1312-3) showed distinct pulsotypes, sequence types and
resistotypes, indicating non-clonal relationship despite
being isolated from the same patient. During an
outbreak investigation of ESBL producing K. pnemoniae in
a neonatal intensive care unit in Germany, Haller and
co-workers  found two isolates from the same
patient taken 138 days apart to be affiliated with separate
clusters on phylogenetic tree. This could indicate the
presence of diverse pathogen populations within
individuals which complicates treatment. These two strains
isolated in this study were colonizers (strains isolated from
tracheal aspirate and urine) and this patient died after
229 days of hospitalization. Dautzenberg and co-workers
 have reported that patients colonized with
carbapenem resistant Enterobacteriaceae have higher
mortality rates as compared to non-colonized patients due to
long hospital stay.
The administration of multiple antibiotics has been
reported as a risk factor for carbapenem resistance
acquisition  and all sixteen patients in this study were given
at least one antibiotic in the past 90 days prior to
carbapenem resistant K. pneumoniae isolation. Five patients
had been administered with carbapenems for more than
seven days and another for three days while the ten
other patients were exposed to other beta-lactams
(penicillins, beta-lactam/beta-lactamase inhibitors and
cephalosporins), ciprofloxacin, vancomycin, metronidazole,
oxytetracycline and colistin. This is agreeable with
findings as shown by Patel and co-workers  that
carbapenem resistance is not attributed only to previous
exposure to carbapenem but also exposure to other
One strain, K/1310-23 harbored only AmpC (blaFOX-7)
and ESBLs (blaSHV-11, blaCTXM-15 and blaCTXM-2)
without the presence of carbapenemases or loss of porin but
it was resistant to meropenem (MIC = 6 μg/ml) and
ertapenem (MIC = 8 μg/ml). This may be due to reduced
porin expression  in the strain or this strain may
have harbored rare carbapenemases or variants that
were not tested for in this study. Only strain, K/1310-24
was associated with loss of porin; ompK36 and it also
harbored carbapenemases (blaOXA-48 and blaKPC-2).. This
strain also exhibited the highest resistance towards
carbapenems in the group of strains other than ST101 and
ST147 which supports the findings that loss of porin
together with the presence of blaKPC-2 is associated with
elevated carbapenem MIC .
It was also observed that three strains exhibited
varying susceptibility patterns to carbapenems tested in this
study. K/1311-35 and K/1304-16 were resistant to
imipenem but sensitive to meropenem and ertapenem while
K/1312-3 was sensitive to imipenem but resistant to
meropenem and ertapenem. This discrepany in
resistance pattern often poses a challenge in carbapenem
resistant detection especially if either imipenem or
meropenem is used as a representative carbapenem in the
screening system [29, 30]. Imipenem resistant,
meropenem sensitive Pseudomonas aeruginosa and imipenem
sensitive, meropenem resistant K. pneumoniae strains
have been previously reported [30, 31]. This difference
in susceptibility patterns can be attributed to the
variation in outer membrane porin structure and the efflux
pumps expression. The eight amino acid sequence
deletion on loop L7 of OprD, a carbapenem-specific porin in
P. aeruginosa sufficiently opens the porin channel,
allowing optimal penetration of meropenem and
increases its activity without affecting the susceptibility of
smaller carbapenem molecules such as imipenem .
The overexpression of resistance-nodulation-cell division
(RND) efflux pump in P. aeruginosa has also been
reported to cause varying carbapenem MIC levels .
Thus, we postulate that there may be similar porin
structure and efflux pump variations in K. pneumoniae
that contribute to this discrepancy in carbapenem
Overall, PFGE and MLST produced comparable
clustering results. The predominant sequence type,
ST101 (n = 9) shared the same pulsotype. This
characteristic had been reported by Kitchel and co-workers
 where K. pneumoniae strains with ≥ 80%
similarity in PFGE patterns strongly agreed with their MLST
results. It was observed that ST101 spread occurred
in the surgical ward in August 2013 as five of ST101
strains were isolated from five different patients who
had stayed in surgical wards during this one month
period. However, ST101 was also isolated from three
other patients from medical wards and one patient
from orthopedic ward in the following months. We
postulate that more complicated transmission routes
such as asymptomatic patients who were never
detected, via health care personnel or medical devices
 may have contributed to the spread of ST101 to
patients from other wards. ST789, ST11 and ST495
were also isolated from three separate patients in
surgical wards in October and November 2013 and one
of the patients had came into contact with the ST101
harboring patients as they shared the same ward in
August and was colonized. Despite being
phylogenetically unrelated to ST101, these three strains harbor
similar ESBL and carbapenemase genes indicating
horizontal gene transfer may occur since these genes
are often found on transposons and plasmids [36, 37].
Our finding of ST101 as a predominant strain is
similar to the report of ST101 as a predominant K.
pneumoniae clone in an acute general hospital in Italy .
ST101 was reported to be responsible for blaOXA-48
outbreaks in Spain and Tunisia  and harboring blaKPC-2
in Italy  which was comparable to our results where
ST101 isolated were blaOXA-48 with four co-producing
BlaOXA-48, which was first reported in Turkey  was
also known to exhibit susceptibility towards
extendedspectrum cephalosporins and carbapenems  which
complicates the detection via routine clinical
microbiology laboratory tests. Similarly, K/1310-35 (sensitive to all
cephalosporins), K/1310-24 (intermediate to ceftriaxone
while being sensitive to all other cephalosporins) and K/
1311-26 (susceptible dose dependent (SDD) to cefepime
while being sensitive to all other cephalosporins)
harbored blaOXA-48. Interestingly, these three strains also
harbored blaSHV and blaCTXM which was known to
hydrolyze cefotaxime and ceftazidime  but all three
strains were sensitive to both antibiotics in this study.
BlaKPC is endemic in northeastern regions in USA,
Greece and Israel but cross regional spreading into
United Kingdom, Brazil, Sweden, India and China had
been reported recently . This gene is often detected
on mobile genetic elements such as plasmids and
transposons which facilitates its rapid dissemination
worldwide . BlaKPC-2 is generally associated with Tn4401
isoforms  and in this study, Tn4401b (no deletion in
the variable region) was associated with six blaKPC-2
harboring strains. This isoform had been reported to exhibit
lower resistance to carbapenems as compared to other
isoforms . However, such conclusions cannot be
made in this study as only one isoform type was
detected in this study. To the best of our knowledge, this
report presented the first identification of K.
pneumoniae harboring blaOXA-48 and blaKPC-2 in Malaysia.
Two strains, K/1304-16 and K/1309-39 demonstrated
slight differences in PFGE banding patterns (Dice homology
= 84.2%) despite having the same sequence type, ST147.
PFGE is able to detect chromosomal rearrangements 
which may contribute to differences in their antibiotic and
genotypic profiles. K/1309-39 was resistant to imipenem,
meropenem and ertapenem (MIC = 12 - >32 μg/ml) while
K/1304-16 demonstrated low resistance to imipenem, (MIC
= 4 μg/ml) and was sensitive to both meropenem and
ertapenem. Furthermore, blaTEM carried by both strains was of
different gene types. TEM-1 was present in K/1309-39 while
TEM-135 was detected in K/1304-16. These two strains can
also be distinguished further based on the presence of
blaNDM-1 in K/1309-39 and the presence of inverted repeats
(IR) of Tn4401 in K/1304-16.
A majority of NDM cases reported worldwide were
related to travel or hospitalization in the Indian
subcontinent such as India and Pakistan  and ST147 has been
shown to be associated with blaNDM-1 in India .
However, in our study, the blaNDM-1 positive patient had
no record of prior travel outside Malaysia. The presence
of blaNDM without any association with international
travel has also been reported by Rimrang and
coworkers  which indicated that the NDM gene was
This study is limited by its small sample size as this
study only includes the carbapenem resistant K.
pneumoniae strains which were isolated during an eight months'
period since the first carbapenem resistant K. pneumoniae
isolation in this hospital. Thus, the findings cannot be
generalized to a broader population based on this study
alone as it may include potential biases. The significance
of mortality rates with imipenem resistance as compared
to meropenem and ertapenem resistance cannot be firmly
drawn since all the studied strains were imipenem and /
or meropenem resistant strains. The increased use of
imipenem may have contributed to increased imipenem
resistance  since five patients were administered with
imipenem while only two patients were given meropenem.
However, our findings that mechanical ventilation is
associated with mortality can be justified as it was also
reported in other studies [25, 49].
The emergence of carbapenem resistant K pneumoniae
harboring various carbapenemases coupled with the
presence of transposon and the loss of porin may contribute
to the increasing cases being detected. Thus,
microbiological, molecular and clinical data of these strains are
important to provide information for a better
understanding and to facilitate carbapenem resistance control.
AST: Antimicrobial susceptibility testing; CLSI: Clinical and Laborartory
Standards Institute; ESBL: Extended spectrum beta-lactamase;
EUCAST: European Committee on Antimicrobial Susceptibility Testing;
ICU: Intensive care unit; MHT: Modified Hodge test; MIC: Minimal inhibitory
concentration; MLST: Multilocus sequence typing; PFGE: Pulsed-field gel
electrophoresis; PTBD: Percutaneous transphetic biliary drainage;
RND: Resistance-nodulation-cell division; SDD: Susceptible dose dependent;
ST: Sequence type
This work was supported by Fundamental Research Grant Scheme (FRGS)
from the Ministry of Education, Malaysia (grant number: FP023-2014A),
University of Malaya Research Grant (UMRG) from University of Malaya (grant
number: RP026B-14HTM) and International Society for Infectious Diseases
(ISID) research grant (grant number: IF016-2015).
CSJT and MYMY designed the study. YML and PSXY carried out the
experiments and collected the data. CWC performed the statistical analysis.
KAJ, SP, RK and RV interpreted the clinical data. YML, KAJ, RK, CWC, SAB and
CSJT wrote and revised the manuscript. All authors' read and approved the
final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Ethics approval (MEC:1059.15) was obtained prior to the start of this study.
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