Journal of Infection and Chemotherapy 28 (2022) 192–198

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Journal of Infection and Chemotherapy

journal homepage: www.elsevier.com/locate/jic

Original Article

Molecular characterization of carbapenemase producing Klebsiella

pneumoniae strains by multiplex PCR and PFGE methods: The first K.
pneumoniae isolates co-producing OXA-48/KPC and KPC/NDM in Turkey
Serpil Genç a, b, *, Fetiye Kolaylı b, Eda Yazıcı Özçelik c
a
Microbiology Laboratory, Giresun University A.Ilhan Ozdemir Education and Research Hospital, Giresun, Turkey
b
Department of Medical Microbiology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey
c
Department of Medical Microbiology, Institute of Health Sciences, Kocaeli University, Kocaeli, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: Introduction: Carbapenems are frequently used in the treatment of multidrug-resistant infections caused by
K. pneumoniae Klebsiella pneumoniae. The aim of the study is to definition and incidence of transferable carbapenemase genes of
OXA-48 carbapenem resistant K. pneumoniae (CRKP) and to determine clonal relatedness of these strains in tertiary care
NDM
hospital in Turkey.
KPC
VIM
Methods: Identification of all 100 K. pneumoniae isolates and low sensitivity to any of the carbapenem group
IMP antibiotics were determined by Vitek-2 (BioMérieux, France). The frequency of carbapenemase genes (blaOXA-48,
blaNDM, blaKPC, blaVIM, blaIMP) and extended spectrum beta-lactamase (ESBL) genes (blaCTX-M, blaSHV, blaTEM)
which frequently detected in Turkey, have been investigated by multiplex polymerase chain reaction (PCR).
Clonal relatedness was determined using Pulsed-field gel electrophoresis(PFGE).
Results: Ninety five isolates carried at least one of the carbapenemase genes (81.05% blaOXA-48, 38.9% blaNDM,
9.47% blaKPC,1.05% blaVIM). One isolate was carried the blaOXA-48+KPC and the two isolates were carried the
blaKPC+NDM. PFGE demonstrated the presence of 24 pulse types and 63.09% of the isolates were in four main
pulse types.
Conclusions: This study demonstrated the incidence of blaNDM is beginning to reach endemic levels, in addition to
blaOXA-48 found endemic in Turkey. To our knowledge, this is the first report of the co-production of these two
genes (blaKPC + NDM and blaOXA-48 + KPC) in CRKP isolates.

1. Introduction According to the National Antimicrobial Resistance Surveillance

Carbapenem-resistant enteric bacteria, which have increased in fre­
quency in recent years, have become a public health threat all over the
world. Carbapenem resistance in Enterobacterales is usually due to beta
lactamases (carbapenemases) that hydrolyse carbapenem. Although the
most common carbapenemase in Turkey is OXA-48, different carbape­
nemases have been reported, such as VIM-5, IMP-1, NDM-1 and KPC-2
[1,2] .
The carbapenem resistant Klebsiella pneumoniae (CRKP) rate is re­
ported as 3.6–10.8% in the United States (USA) according to Centers for
Disease Control and Prevention (CDC) National Healthcare Safety
Network 2008, while the European Antimicrobial Resistance Surveil­
lance System (EARSS) report it as 0.6% in Europe [3].
System (UAMDSS) 2013 report, carbapenem resistance in invasive
K. pneumoniae strains has exceeded 10% in Turkey (imipenem 16%,
meropenem 15.4%) [4]. According to Central Asian and Eastern Euro­
pean Surveillance of Antimicrobial Resistance (CAESAR) data, this rate
was 28% in 2014 and 30% in 2015 [5].
The aim of this study was to perform the molecular characterization
(identification of the genetic platforms) of CRKP isolated from patients
intertiary care hospital in Turkey.

* Corresponding author. Microbiology Laboratory, Giresun University A.Ilhan Ozdemir Education and Research Hospital, Giresun, Turkey.
E-mail address: metin.serpil@yahoo.com (S. Genç).

https://doi.org/10.1016/j.jiac.2021.10.009
Received 16 March 2021; Received in revised form 24 August 2021; Accepted 12 October 2021
Available online 26 October 2021
1341-321X/© 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
S. Genç et al.
2. Methods
2.1. Study design and bacterial isolates
One hundred K.pneumoniaestrains isolated from various clinical
specimens between January 2015 - February 2017, as single strains from
each patient, were included in this study.
İdentificationandantibioticsusceptibilitytests of the isolates were stud­
ied by Vitek-2 (BioMérieux, France) and determined moderately sensi­
tive or resistant to at least one of the carbapenem (ertapenem,
meropenem, imipenem) antibiotics [6].
The patient’s information was analyzed from the hospital informa­
tion system and the hospital-acquired (HA) infection/community-
acquired (CA) infection was differentiated according to the CDC diag­
nostic criteria [7].
2.2. Antimicrobial susceptibility
Sensitivities of amoxicillin/clavulanic acid (AMC-30μg), piper­
acillin/tazobactam (TZP-36μg) and trimethoprim/sulfomethaxazole
(SXT-25μg) were determined by Kirby-Bauer disc diffusion method.
Minimum Inhibitory Concentration (MIC) values were determined by
agar dilution method for other several antimicrobials including cefta­
zidime (CAZ), cefotaxime (CTX), ciprofloxacin (CIP), gentamicin (CN),
amikacin (AK), tigecycline (TG), ertapenem (ETP), imipenem (IPM),
meropenem (MEM). The results were interpreted according to the rec­
ommendations of European Committee on Antimicrobial Susceptibility
Testing (EUCAST) [8].
2.3. Molecular analysis
Total DNAs were extracted by boiling method [9]. Multiplex PCR
using previously described primers was used to test the presence of
carbapenemase and ESBL genes (blaOXA-48, blaNDM-1, blaIMP, blaVIM, and
blaKPC, blaCTX-M, blaSHV, blaTEM)by considering the most common genes
responsible for carbapenem resistance in Turkey [10–12].
Multiplex PCR for carbapenemase genes were optimized from the
work of Poirel et al. and were performed in two different multiplex re­
actions, with no.1 including detection of blaIMP and blaVIM, no.2
including detection of blaOXA-48, blaNDM and blaKPC [10].
Final concentrations for each reaction (multiplex PCR no.1 and
no.2); 23 μl PCR mixture was prepared to mix 1X Hot Start Buffer, 4 mM
MgCl2, 200 μM dNTP mix, 2.5 μl Hot Start Taq DNA Polymerase, 0.2 μM
Primer (2 pairs). 2 μl of DNA was added. For the multiplex PCR no.2, 3
mM MgCl2 was used instead of multiplex PCR no.1 for the lower MgCl2
concentration.
Amplification was carried out with the following thermal cycling
conditions: 5 min at 94 ◦ C and 30 cycles of amplification consisting of
30 s at 94 ◦ C, 40 s at 52 ◦ C (51 ◦ C for no.2) and 50 s at 72 ◦ C, with 5 min
at 72 ◦ C for the final extension.
For blaTEM, blaSHV and blaCTX-M, due to the similarity of amplicon size
(bp) and different annealing temperatüre multiplex PCR could not be
performed, separate reaction was established for each.PCR screenings
were accomplished in a final volume of 25 μL with a 2 μL DNA. The
master mixture was composed of 1X buffer, 3 mM MgCl2, 200 μM dNTPs,
0.2 μM primers each and 2.5 U DNA polymerase. For the blaSHV, 2 mM
MgCl2 and 2 U DNA polymerase were used. Amplification was accom­
plished after a 5 min denaturation at 94 ◦ C by 30 cycles of 30 s at 94 ◦ C,
40 s at 52 ◦ C and 50 s at 72 ◦ C, with 5 min at 72 ◦ C for the final extension.
For the blaSHV, thermal cycling conditions: 5 min at 94 ◦ C and 35 cycles
of amplification consisting of 35 s at 94 ◦ C, 1 min at 56 ◦ C and 2 min at
72 ◦ C, with 5 min at 72 ◦ C for the final extension. PCR products were run
on a 2% agarose gel at 110 V for 50 min and visualized on an ultraviolet
lamp.
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Journal of Infection and Chemotherapy 28 (2022) 192–198
2.4. Molecular epidemiology
Pulsed-field gel electrophoresis (PFGE) was performed using XbaI
DNA restriction enzyme according to PulseNet protocol developed by
Centers for Diseases Control and Prevention for 84CRKP isolates which
are considered to be HA infection agent [13].
The PFGE patterns were analyzed using Phoretix 1D Pro (Total Lab
Ltd) software. Similarity analysis was performed by Dice coefficients.
Clustering was created using unweighted pair group with arithmetic
averages (UPGMA).
2.5. Statistical analysis
Statistical evaluation was performed with IBM SPSS 20.0 (SPSS Inc.,
Chicago, IL, USA). Numerical variables were given as median (25th -
75th percentile) and frequency (percentages). Differences between the
groups were assessed by Mann Whitney U test for numerical variables
with no normal distribution, Fisher’s Exact Kikare analysis for categor­
ical variables, Yates’ Kikare analysis and Monte Carlo Kikare analysis. p
< 0.05 was considered adequate for statistical significance.
3. Results
3.1. Description of isolates
Of the 100 K.pneumoniae isolates studied, 19 were internal care unit
(ICU), 63 were serviced, 18 were isolated from clinic patient samples.
The median age of patients was 61.5 years old. Most of the isolates were
collected from urine/urinary catheters (n = 45, 45%), skin soft tissue (n
= 33, 33%), respiratory system (n = 17, 17%), blood (n = 3, 3%),
peritoneal fluid (n = 1, 1%) and genital discharge (n = 1, 1%).
When the patient data were evaluated, infection of 16 patients were
accepted as CA and infection of 84 patients were accepted as HA
infection.
3.2. Antimicrobial susceptibility
Susceptibility profiles against 12 antimicrobial agents are listed in
Table 1. As expected, the majority of the isolates were resistant to ß-
lactams, CIP, CN and SXT. The least resistant antibiotics were TG (37%
R) and AK (38% R).
3.3. B-lactamase content
At least one carbapenemase-encoding gene responsible for carba­
penem resistance has been identified at 95 of 100 CRKP isolates. 81.05%
of the detected genes were blaOXA-48, 38.9% were blaNDM and 9.47%
were blaKPC. The frequency of carbapenemase encoding genes and
sample gel images after PCR-electrophoresis are shown in Table 2 and
Fig. 1 respectively.
The most commonly detected ESBL gene while being blaSHV, the most
commonly detected profile was determined in 54 isolates were found to
have coexisting blaSHV+CTX+TEM (n = 54). The isolates carried the blaSHV
(n = 90), blaCTX (n = 82), blaTEM (n = 67). ESBL genes which are found to
be accompanied by carbapenemase genes are also shown in Table 3.
blaOXA-48 (52.6%) was the most common gene in strains isolated from
ICU and all isolates were HA infection agents. The distribution of the
genes between the ICU and the non ICU departmens and the distribution
of HA/CA infection are shown in Table 2.
The only isolate co-producing the blaOXA-48 + KPC were isolated from
the wound culture of a 61 years old female patient. The isolate was
resistant to all tested antibiotics. Two isolates identified as co-producing
blaKPC + NDM were considered to be hospital infection agent from an
external center. An isolate was isolated from the wound site of a 55 years
old female patient. Isolate was resistant to other antibiotics with mod­
erate sensitivity to TG. The other was isolated from the urine specimen
S. Genç et al. Journal of Infection and Chemotherapy 28 (2022) 192–198

Table 1
Antimicrobial susceptibility profiles of isolates.
Antibiotics Susceptible (S) Intermediate (I) Resistant (R) MIC (mg/L)

MIC50 MIC90 MIC range

AMC 0% 0% 100% - - -
TZP 0% 0% 100% - - -
CAZ 1% 1% 98% >32 >32 0.5->32
CTX 1% 1% 98% >16 >16 1->16
CIP 2% 4% 94% 8 >8 0.25->8
CN 18% 1% 81% 16 >32 ≤0.25->32
ETP 0% 2% 98% 32 64 1–64
MEM 11% 19% 70% 16 32 0.5–64
IPM 16% 28% 56% 16 32 0.5–64
AK 39% 23% 38% 16 128 ≤1->128
TG 21% 42% 37% 2 8 0.25->16
SXT 34% 3% 63% - - -

AMC: amoxicillin/clavulanic acid, TZP: piperacillin/tazobactam, SXT: trimethoprim/sulfomethaxazole, CAZ: ceftazidime, CTX: cefotaxime, CIP: ciprofloxacin, CN:
gentamicin, AK: amikacin, TG: tigecycline, ETP: ertapenem, IPM: imipenem, MEM: meropenem.

Table 2
The frequency of carbapenemase-encoding genes and the distribution of the carbapenemase-encoding genes between the ICU and the non ICU departmens.
Carbapenemase-encoding genes blaOXA48 blaNDM blaKPC blaOXA-48+NDM blaOXA-48+KPC blaVIM+NDM blaKPC+NDM Total

ICU HA 10 (52.6%) - - 8 (42.1%) - 1 (5.3%) - 19 (100%)

CA - - - - - - - -
Non ICU departmens HA 30 (37%) 9 (11.1%) 6 (7.4%) 12 (14.8%) 1 (1.2%) - 2 (2.5%) 60 (79%)
CA 11 (13,6%) - - 5 (6.2%) - - - 16 (21%)
Total 51 (53.7%) 9 (9.5%) 6 (6.3%) 25 (26.3%) 1 (1.05%) 1 (1.05%) 2 (2.1%) 95

HA: hospital-acquired CA: community-acquired ICU: Intensive care unıt.

Fig. 1. Sample gel images after PCR-electrophoresis. (A) Gel images of KPC + NDM positive samples. 1, Marker (50 bp); 2, multiplex PCR no.2 positive control; 3, 4,
patient samples. (B) Gel images of KPC + OXA-48 positive sample. 1, Marker (50 bp); 2, multiplex PCR no.2 positive control; 3, patient sample.

Table 3
ESBL genes accompanying carbapenemase genes.
ESBL-encoding genes

Carbapenemase-encoding genes blaSHV blaCTX blaSHVþCTX blaSHVþTEM blaCTXþTEM blaSHVþCTXþTEM Total

blaOXA-48 2 1 10 4 2 32 51
blaNDM 0 0 3 3 0 3 9
blaKPC 0 0 0 0 1 5 6
blaOXA-48+NDM 2 0 9 1 1 12 25
blaKPC+NDM 0 0 1 0 0 1 2
blaOXA-48+KPC 0 0 0 0 0 1 1
blaVIM+NDM 0 0 0 1 0 0 1
Negative 0 0 1 0 1 3 5
Total 4 1 23 9 4 54 100

ESBL: extended spectrum beta lactamase.

of a 69 years old female patient. The isolate was susceptible to TG and were detected in 5 isolates which considered to be CA infection agent.
SXT, resistant to other antibiotics.
blaOXA-48 was detected in 11 of 16 isolates, and blaOXA-48 and blaNDM

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3.4. Examination of carbapenem susceptibilities in the presence of genes

responsible for carbapenemase production

Fifty one isolates that were OXA-48 positive were resistant to ETP. Of
these strains only 13.72% (n = 7) and 21.56% (n = 11) were sensitive to
MEM and IPM respectively. When the detected genes were compared in
terms of sensitivity to meropenem and imipenem, only a significant
difference was found in blaOXA-48 positive isolates (p = 0.003, p <
0.001).Only blaOXA-48 positive isolates were significantly more suscep­
tible to meropenem and imipenem than isolates containing other genes.
There was no significant difference in sensitivity of genomes and erta­
penem (p = 0.495).
While of the 81 strains which were isolated from non ICU were
sensitive to MEM and IPM 13,58%(n = 11) and 19,75%(n = 16)
respectively, non of 19 ICU isolates were sensitive to MEM and IPM.
Comparing carbapenem susceptibility with clinical trials, it was deter­
mined that the susceptibility of meropenem and imipenem decreased
significantly in the strains isolated from the ICU samples (p < 0.05).
There was no significant difference for ertapenem (p > 0.05).
Fig. 3. PFGE band profiles. M, S. aureus NCTC 8325; 1- 3- 5- 7, Pulsotype A
3.4.1. PFGE strains; 2, Pulsotype B strain; 4- 10, Pulsotype C strains; 6, Pulsotype G strain; 8,
Four major patterns (21/84), B (11/84), C (11/84), and D (10/84) Pulsotype R strain; 9, Pulsotype E strain; 11, Pulsotype U strain.
were detected in 84 HA infection agents by PFGE.
The dendrogram of the PFGE patterns and clonal relationships of the the sensitive limits in 60% of the CPE. Therefore, CDC recommends the
isolates is shown in the Fig. 2. The PFGE band profiles of some strains are use of ETP for phenotypic screening of carbapenemase activity, espe­
shown in the Fig. 3. cially not to skip the carbapenem susceptible carbapenemase-producing
Enterobacterales [20,21].In this study, although all isolates with carba­
4. Discussion penemase genes were resistant or intermediate to ETP (sensitivity 100%,
specificity 95%), seven(7.4%) of the isolates were found to be MEM and
K. pneumoniae is the most common species of carbapenem resistant 11(11.6%) were sensitive to IPM. Sensitivity - specificity of MEM and
Enterobacterales(CRE) [14]. Percentage of carbapenem resistance in IPM were found as 92.6%-88.4% and 60%- 100%, respectively. ETP
invasive K. pneumoniae isolates was reported as 11%, 28%, 30% in 2013, seems to be a sensitive agent in screening carbapenemases, which car­
2014 and 2015, respectively, according to UAMDSS- "CAESAR" data [5, bapenemases are investigated, and it can be useful in centers where
15]. The carbapenem resistance rate in Europe was 8.1% in 2015 [16]. molecular tests are not available.
Even blaKPC is seen as endemic in the USA, Greece and Israel, blaVIM in Aminoglycoside, quinolone resistance and ESBL are also common in
Greece, blaNDM in India and Balkan countries, blaOXA-48 in Turkey and CRKP and the therapeutic agents that can be used for treatment are very
North Africa [17–19]. limited [22].Most of the carbapenem resistant isolates in this study were
EUCAST recommends the use of MEM, which is the best balance of also resistant to this group of antibiotics. In addition, resistance rates
sensitivity and specificity in carbapenem resistance screening in Enter­ against the last choice of antibiotics such as TG and polymyxins are
obacterales [17]. However, due to the low level of carbapenemase pro­ increasing [23].According to European survey of
duction, and especially the activity of OXA-48 against carbapenems is carbapenemase-producing Enterobacteriaceae (EuSCAPE) report, TG
not as high as KPC and MBLs, MIC values for MEM and IPM are among resistance rate was determined 25.9% in isolates sent from the Turkey

Fig. 2. Dendrogram obtained by PFGE analysis of isolates.

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S. Genç et al.
[24]. TG resistance was found to be 37% in this study. High resistance
rates for TG show how quickly antibiotic resistance spreads and is
alarming.
Although OXA-48 positive isolates were reported to be susceptible to
IPM and MEM, in this study, 51 isolates with only OXA-48 positivity
were found to have high MIC values for IPM and MEM, and resistance
rates were 80.4% and 84.4%, respectively. Infact, Azap et al. [25] found
high MIC values for imipenem and meropenem, which were thought to
be due to ESBL enzymes of TEM and SHV type determined in strains and
loss of outer membrane porin. In these 51 isolates, at least one ESBL gene
was accompanied by blaOXA-48 positivity (Table 3).
In general, it is known that MBL producing isolates cause higher
resistance than OXA-48 and KPC type enzyme producing strains. Plas­
mids carrying the blaNDM can cause multiple drug resistance by
harboring other carbapenemase and ESBL genes [22,26]. In this study, it
was found that 37 isolates with blaNDM positive were resistant with high
MIC values against all three carbapenems. Nine of the 11 pan-drug
resistant (PDR) isolates that were found to be resistant to all of the an­
tibiotics, were blaOXA-48 + NDM, two isolates were only blaNDM.
Although carbapenem resistance level can be significantly differ in
KPC producing isolates [22], in this study, high MIC values were
determined for all three carbapenems in 9 isolates with blaKPC. KPC
producers are generally multipl drug resistantant only gentamicin has
been reported to have a good efficacy from aminoglycosides [26].
Similarly, blaKPC positive isolates were found to be resistant to almost all
antibiotics and the most sensitive antibiotic was determined as
gentamicin.
Carbapenemases can be expressed at various levels. In addition, they
differ from each other in terms of both their biochemical properties and
the beta-lactam spectrum they affect. The activity of OXA-48 against
carbapenems is not as high as KPC and MBLs. Therefore, Enterobacterales
producing OXA-48 may have a more susceptible phenotype [20]. A 2016
study suggested that carbapenem susceptible carbapenemase producers
in carbapenemase isolates 14% (26/188) including 25% (14/63) of KPC
producers and 40% (12/30) of OXA-48 producers [27]. In this study,
carbapenem-susceptible CPE were only detected in OXA-48 producers.
The susceptibility rates in OXA-48 producers were 13.7% (7/51) and
21.6% (11/51) for MEM and IPM respectively.
When the detected genes were compared in terms of sensitivity to
MEM and IPM, only a significant difference was found in blaOXA-48
positive isolates (p = 0.003, p < 0.001). As aspected only blaOXA-48
positive isolates were significantly more susceptible to MEM and IPM
than isolates containing other genes.
Reduction in carbapenem susceptibility, compared to the clinics
where the samples were sent; it was determined that the susceptibility of
MEM and IPM decreased significantly in the strains isolated from the
ICU samples (p < 0.05). Higher rates of antimicrobial resistance are
expected in the ICU. Broad-spectrum and multiple antimicrobial agents
are more commonly used for empiric therapy in the ICU due to the
higher probability of antimicrobial resistance and the increased severity
of the infection. Similarly, Sader et al. [28] reported in their study that
they found isolates in ICU more resistant than those in non-ICU. Addi­
tionally, in this study, three of the investigated ESBLs (SHV+CTX+TEM)
were also present in 18 of 19 ICU isolates. Of the 81 non ICU isolates,
only 36 were accompanied by three ESBLs. The other isolates were
accompanied by ESBLs in 2-combinations (Tables 2 and 3). This situa­
tion can also be considered as a factor in carbapenem susceptibility
difference.
Infections with carbapenemase producing bacteria are mostly HA,
although CA infections are seen [29].The weak immunity of patients in
the ICU and critical conditions such as multiple comorbidities, invasive
procedures, antibiotic exposure make it easier for patients to become
infected. Also stay in ICU is determined as an independent risk factor for
CRKP infections [30,31].While all isolates in ICU were HA pathogen,
79% of non ICU isolates were HA (Table 2) in this study. In our hospital,
the rates of nosocomial infection in non ICU were unfortunately high in
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Journal of Infection and Chemotherapy 28 (2022) 192–198
terms of CRKP. Active surveillance was carried out but rectal swaps were
only screened for vancomycin resistant enterococ, a routine CRKP
screening was not performed. Unfortunately, it is thought that the
infection control measures are not adequately implemented in our
hospital. Compliance with routine infection control measures should be
reviewed, effective infection control measures should be taken by
investigating the causes of transmission.
While blaKPC is mostly detected in nosocomial isolates, blaNDM and
blaOXA-48 can be found in both nosocomial and CA pathogens [32].In this
study, all of the strains found to have blaKPC positivity were HA infection
agents. 11 of 16 isolates evaluated as CA infection only blaOXA-48 posi­
tivity and blaOXA-48 + NDM positivity were detected in five strains
(Table 2).In Turkey, where OXA-48 enzyme is endemic, it should be kept
in mind that NDM positive Enterobacterales members, which may cause
multiple drug resistant and PDR, may also be seen in CA infections.
In this study, at least one carbapenemase gene except blaIMP was
determined in 95 of 100 isolates, none of the carbapenemase genes were
detected in five isolates. Three of them were positive for blaSHV+CTX+TEM,
one of them was positive for blaSHV+CTX and one was blaCTX+TEM. When
the VITEK-2(Bio-Mérieux, France) results were examined, it was
observed that high level Amp C production was detected in three out of
five isolates. We thought that the carbapenem resistance in these isolates
was due to the presence of high level Amp C production with ESBL.
In Turkey, the most commonly detected carbapenemase gene in
enteric bacteria is reported as blaOXA-48 [25,33–35]. The most common
gene in this study was blaOXA-48 (81.05%), consistent with our country
data.
The first blaNDM declaration in our country was made in 2012 [36],
and then, notifications from our country continued [34,37,38]. The
second most common carbapenemase gene in this study was blaNDM
(38.9%). According to the data of our country, we found a much higher
percentage of blaNDM. It can be predicted that the rapidly spreading
blaNDM will begin to be endemic in our country in the coming years.
blaKPC, which was reported by Labarka [39] in 2014 for the first time
in Turkey, was reported to be isolated rarely but not detected in most
studies [19,34,37,38]. In this study, the third most common gene was
blaKPC (9.5%). It is seen that this gene, which we found at a very high
rate compared to the studies in our country, has become frequently
determined in our country over the years.
In Turkey, the first blaVIM-1 report in K.pneumoniae isolate was per­
formed by Yıldırım et al. [40] in 2007. Çizmeci et al. [34]reported that
they had detected blaVIM in two of 76 isolated CRE isolates (K.pneumo­
niae, Enterobacter cloacae) between 2014 and 2016. It was determined
that the only isolate they identified together with blaVIM and blaNDM was
sent from Kocaeli University Hospital within the scope of EuSCAPE
Project. One year after this study, we found only one isolate that pro­
duced blaVIM + NDM in 100 CRKP, and although this gene pattern existed
in our hospital in previous years, it is pleasing that the incidence has not
increased over the years.
Co-production of different carbapenemases in a single isolate in
enteric bacteria has been remarkably reported in many publications. K.
pneumoniae isolates were reported to have blaKPC-2 + VIM-1 [41]in Italy,
blaNDM-1 + IMP-4 and blaKPC-2 + NDM-1 [42,43]in China, blaOXA-48 + NDM-1
[44] has been reported in Switzerland. blaOXA-181 + VIM-5 has also been
reported in India [45]. A K.pneumoniae isolate was also identified, which
produced the blaNDM-1+VIM-1+OXA-48 from Morocco [46].
The first OXA-48 + NDM-1 producing K.pneumoniae strain in Turkey
was reported by Kılıç et al. [47].Afterwards, many studies reported
OXA-48 + NDM-1 [34,35,37].
Approximately two years after the above-mentioned studies, of the
95 carbapenemase enzyme producer K.pneumoniae isolates, 25 (26.3%)
had blaOXA-48 + NDM, two (2.1%) had blaKPC + NDM, and one blaOXA-48 +
KPC (1.05%) and blaNDM + VIM (1.05%) were detected. To our knowledge,
in the literature, there were no isolates reported to carry these two genes
(blaKPC + NDM and blaOXA-48 + KPC) together. We think this is the first
report from Turkey. For blaOXA-48 + KPC first report in the world.
S. Genç et al.
Compared to other studies, the ratio of blaOXA-48 + NDM producing strains
were very high. The probable cause of this situation may be the fact that
Kocaeli, an industrial city, has received more immigration from different
geographic regions.
PFGE is a distinctive method for clonal typing of isolates in short-
term outbreaks, and has a distinctive power in identifying the source,
but in studies where isolates that last more than six months are collected,
the distinguishing feature is reduced [48]. In this study, 84 HA agent
K. pneumoniae strains isolated from 2015 to 2017 were identified by
PFGE and 24 types were determined. Although a significant portion of
the K.pneumoniae strains in our hospital were clonally related, the
presence of different clones makes it possible for outbreaks to consist of
many different origins. In the twenty four patterns, Pulsotype A
constituted the largest group and it was determined that 21 isolates
originated from the same origin. Four of the 6 isolates in Pulsotype E
carry the blaKPC and one of the strains was found to carry the blaOXA-48 +
KPC together. Both of the isolates in pulsotype I, which are considered as
a different pattern, carry the blaKPC + NDM. It was isolated from the pa­
tients who were referred from our center to our hospital in 2017.
In an epidemic assessment, PFGE is a valuable method. However,
multi-locus sequence typing (MLST) has a higher discriminative power
to detect major epidemic clones and to determine its origin. The most
important limitation of this study was that the obtained PFGE types were
not determined by MLST.
5. Conclusion
Besides blaOXA-48 positive isolates that are endemic in Turkey, it is an
undeniable fact that blaNDM positive isolates are beginning to reach
endemic levels and that antibiotics that can be used in the treatment of
infections they cause are increasingly limited. In order to prevent this
situation, in addition to current strict infection control measures, it
would be appropriate to carry out the control fecal screening tests for the
management of CPE in the risky sections such as ICU as well as the strict
implementation of the necessary infection control measures for the CPE.
There is no protocol to detect carbapenemase production in most
countries, the actual prevelance of carbapenemase production and at
what rate it is still unknown. In the future, a possible epidemic may
occur with HA K.pneumoniae strains producing carbapenemase. For this
reason, early identification of these resistant microorganisms has an
important role in preventing epidemics.
Authorship statement
All authors meet the ICMJE authorship criteria. Serpil Genç and
Fetiye Kolaylı developed the trial designs and were responsible for the
organization and coordination of the trial. Serpil Genç and Eda Yazıcı
Özçelik collected isolates and performed the experiments. Serpil Genç
and Fetiye Kolaylı were responsible for the data analysis and wrote the
manuscript. All authors contributed to the writing of the final
manuscript.
Ethical approval
This study was approved by the ethics review committee of Kocaeli
University(14.07.2015–2015/214).
Funding source
This study was supported by grant from Kocaeli University Scientific
Research Projects(KOU BAP project number:2016/069).
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
197
Journal of Infection and Chemotherapy 28 (2022) 192–198
the work reported in this paper.
Acknowledgements
We thanks Canan Baydemir for statistical analaysis and Doğanhan
Kadir Er, Hüseyin Uzuner and Agim Osmani for their help in the mo­
lecular methods experiments.
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