Summary

Introduction: One of the most common mechanisms in the development of carbapenem resistance is acquiring carbapenemases. A new and different carbapenemase, Klebsiella pneumoniae carbapenemase (KPC), has recently emerged in Turkey. Klebsiella pneumoniae carbapenemases are encoded by the bla_KPC gene and can be transferred between different species. Reports showing the presence of bla_KPC gene in isolates from Turkey are limited. The current study aimed to investigate the presence of bla_KPC gene in carbapenem resistant K. pneumoniae and Escherichia coli isolates in a tertiary care hospital in Turkey.
Materials and Methods: Patient samples from various clinical units in Adana Numune Training and Research Hospital between November 2013 and October 2014 were sent to our laboratory at the same hospital. K. pneumoniae and E. coli strains isolated from different clinical samples were identified using both conventional methods and the VITEK^® 2.0 automated identification system (bioMérieux, France). Imipenem-cilastatin, meropenem, and ertapenem susceptibility was analyzed using a VITEK^® 2.0 antibiotic susceptibility testing system (bioMérieux, France). The presence of bla_KPC gene was investigated by polymerase chain reaction.
Results: A total of 49 resistant K. pneumoniae and 33 E. coli isolates were included in the study. Most were isolated from urine specimens. Among the carbapenems we tested, highest resistance rates were to ertapenem (98.0% in K. pneumoniae and 84.8% in E. coli isolates). One K. pneumoniae isolate was found positive for the bla_KPC gene.
Conclusion: According to our literature survey, this study is among the first reports from Turkey presenting the isolation of KPC-producing K. pneumoniae. This finding indicates the need to monitor carbapenem resistance arising from bla_KPC, which may spread horizontally. Moreover, surveillance of antibiotic sensitivity rates and observation of regional differences will be useful guidelines in determining infection control and antimicrobial use management policies.

Introduction

Carbapenems are highly effective antibiotics in the treatment of Enterobacteriaceae infections. However, carbapenem-resistant Enterobacteriaceae (CRE) strains have been reported all over the world in recent years^[1]. Carbapenem-resistant Enterobacteriaceae infections comprise public health threat due to the limited treatment options^[2].

Different mechanisms are known in the development of carbapenem resistance.

These include (i) decreased permeability of the outer membrane, (ii) beta-lactamase (carbapenemase) production, (iii) decreased affinity of penicillin-binding proteins, (iv) high-level production of cephalosporinase, and (v) loss or alteration of porins^[3]. One of the most common mechanisms of carbapenem resistance is carbapenemases such as VIM, IMP, NDM, and OXA. Another carbapenemase, Klebsiella pneumoniae carbapenemase (KPC), has recently been detected in Turkey^{[2, 4, 5]}. Klebsiella pneumoniae carbapenemase is encoded by the bla_KPC gene and can be transferred between different species; its universal spread is mainly attributable to the Tn3-type transposon (Tn4401)^[5]. The presence of carbapenemase in Enterobacteriaceae was first reported in 1993^[6]. Klebsiella pneumoniae carbapenemase was first reported in the Eastern United States in 1996 and caused epidemics, mainly in New York. Subsequently, the gene was also reported in Europe and South Asia^[6-8].

The current study aimed to investigate the presence of bla_KPC gene in carbapenem-resistant K. pneumoniae and E. coli strains isolated from different clinical units between November 2013 and November 2014 in the microbiology laboratory of Adana Numune Training and Research Hospital.

Methods

This study was performed in accordance with the principles of the Declaration of Helsinki and was approved by a Local Ethics Committee (Adana Numune Training and Research Hospital protocol number: 2014/7). The study was conducted using patient samples sent to our laboratory from different clinical units of the hospital between November 2013 and November 2014. Each sample was used to inoculate three types of agar plates: 5% sheep blood agar (GBL, Turkey); Eosin methylene blue agar (GBL, Turkey), and chocolate agar (GBL, Turkey). The plates were incubated in aerobic conditions for 24-48 hours at 37 °C and growth was evaluated. Catalase-positive, oxidase-negative, non-fermentative bacteria with Gram-negative bacillary appearance on stained microscopic examination were identified by classical methods. In addition, isolates were identified at the species level in accordance with the manufacturer’s recommendations with VITEK^® 2.0 automated bacterial identification and antibiotic susceptibility system (bioMérieux, France). Isolates identified as K. pneumoniae or E. coli were aliquoted and stocked at -20 °C.

Antibiotic susceptibility patterns of isolates identified as K. pneumoniae and E. coli to imipenem-cilastatin (IMP), ertapenem (ETP), and meropenem (MEM) were assessed with the VITEK^® 2.0 automated system (bioMérieux, France). The results were categorized as sensitive or resistant according to Clinical and Laboratory Standards Institute (CLSI) criteria^[9]. Intermediate strains were regarded as resistant because they could produce carbapenemases^[4]. K. pneumoniae ATCC BAA-1705 was employed as a KPC-positive control strain and K. pneumoniae ATCC BAA-1706 was used as a KPC-negative control strain. Any detected carbapenem resistance was reported to the clinical units of origin for implementation of necessary contact isolation procedures.

Investigation of extended-spectrum beta-lactamase positivity was confirmed by either double-disc synergy test method or combined disc method^[9]. AmpC beta-lactamase detection in the isolates was performed according to CLSI recommendations. ATCC 25922 E. coli and ATCC 700603 K. pneumoniae isolates were used as controls in all tests^[10].

Modified Hodge test was used to detect carbapenemases phenotypically. E. coli ATCC 25922, K. pneumoniae ATCC BAA-1705 (positive control), K. pneumoniae ATCC BAA-1706 (negative control), and ETP disc (10 μg) were used. After 24 hours of incubation at 35 °C on a Mueller-Hinton agar (MHA) medium, a clover leaf appearance was considered positive^[10].

In the boronic acid inhibition test, an inhibition zone at least 5 mm larger in diameter around the ETP/boronic acid (10 μg/400 μg) disc compared to the ETP-only disc (10 μg) after 24 h incubation on MHA medium was accepted as positive^[11].

Isolates were stored at -20 °C in tryptic soy broth medium (Fluka, 22092, USA) containing 15% glycerol (Merck, Germany) until analysis^[12].

The isolates were passaged onto 5% sheep blood agar from stock medium. DNA extraction was performed using GF-1 nucleic acid extraction kit (Vivantis, Malaysia) according to manufacturer’s recommendations. Thus, the template DNA samples to be used in the PCR process were obtained. Polymerase chain reaction was performed employing bla_KPC forward (5’-ATGTCACTGTATCGCCGTCT-3’) and reverse (5’-TTTTCAGAGCCTTACTGCCC-3’) primers (IDT, USA) in order to detect all known bla_KPC types (bla_KPC-1-7)^[13]. For electrophoresis, 1% agarose gel was prepared and ethidium bromide was added to obtain a final concentration of 0.5 μg/ml. In order to evaluate product size, 5 μl of a 100 base pair (bp) DNA ladder (New England BioLabs, UK) was added to the first and last wells. K. pneumoniae ATCC BAA-1705 and ATCC BAA-1706 were used as KPC positive and negative controls, respectively.

Bands formed during electrophoresis were observed on a UV transilluminator and images were obtained with the VersaDoc™ Imaging System (Bio-Rad, USA).

Results

During the study period, the microbiology laboratory isolated 3802 K. pneumoniae and 2576 E. coli strains from patient samples in various wards. In total, 152 of these isolates were determined to be resistant to carbapenems. Forty-nine carbapenem-resistant K. pneumoniae and 33 E. coli isolates were included in the study after excluding subsequent isolates of the same strain and antibiotic susceptibility pattern from a patient. The most common type of specimen included in the study was urine samples, followed by blood and catheter samples. Distribution of isolates in terms of sample type is shown in Table 1, and distribution of clinical units from which the samples were sent is shown in Table 2.

Table 2: Distribution of carbapenem-resistant isolates according to clinical units

As can be seen in Table 3, the highest susceptibility rate of the tested carbapenems was detected against IMP in E. coli isolates, with 78.8%. The lowest sensitivity was found against ETP in K. pneumoniae isolates, with 2%.

The MHT, which is a phenotypic screening test for carbapenemase, was positive in 61.5% of K. pneumoniae isolates and 45.5% of E. coli isolates. Boronic acid inhibition test, which is a phenotypic screening test for class A carbapenemases, was positive in 14.3% of K. pneumoniae isolates. Data regarding extended-spectrum beta-lactamase, boronic acid inhibition test, and MHT positivity rates are shown in Table 3.

Amplification with the generic bla_KPC primers yielded a band of 882 bp in one of the isolates (Figure 1). The positive band belonged to a K. pneumoniae isolate from the urine specimen of a 77-year-old Turkish man who presented to the urology department for nephrolithiasis and benign prostatic hyperplasia. He had no history of travel abroad in the recent past. This isolate was determined to be ESBL-positive, resistant to ceftriaxone, cefotaxime, ceftazidime, and all three carbapenems tested; susceptible to gentamicin, amikacin, cefepime, and tigecycline.

Discussion

Carbapenem resistance in Enterobacteriaceae was first reported in 1997 in the United States and in 2001 in Turkey^[14]. Subsequently, CRE epidemics were reported at different times in various regions^[14]. Carbapenem-resistant Enterobacteriaceae can be encountered in almost every specimen type^[15]. In our study, carbapenem-resistant E. coli and K. pneumoniae isolates were detected mostly in urine specimens. Similar to our study, Tseng et al.^[8] studied 163 carbapenem-resistant K. pneumoniae strains in Taiwan and found that they were most commonly in urine samples 36.2%. Balkan et al.^[15] examined bloodstream infections caused by CREs and showed that 5.5% of these infections originated from the urinary tract. Us et al.^[7] examined 26 carbapenem-resistant K. pneumoniae strains, 50% of which originated from blood and 23% from urine.

Gram-negative bacteria, which cause serious hospital infections, are responsible for a significant proportion of infections in intensive care units (ICUs)^[14]. Members of the Enterobacteriaceae family are among the most common pathogens in ICUs^[16]. K. pneumoniae is reported to be the fourth most common cause of pneumonia and the fifth most common cause of bacteremia in patients receiving treatment in ICUs, while, E. coli is the most common cause of urinary tract infections^{[17, 18]}. Severe infections in the ICU are usually treated with carbapenem group antibiotics^[7]. When the distribution of CRE isolates in our study were analyzed, K. pneumoniae was found to be mostly isolated from patients treated in ICU (44.9%) and E. coli was mostly isolated from the other inpatients (48.5%). Similarly, in a study conducted in Malatya, Kuzucu et al.^[19] reported that 70% of E. coli strains and 71.7% of Klebsiella strains were isolated from inpatient wards. In a study conducted by Us et al.^[7] in Ankara, 54% of carbapenem-resistant K. pneumoniae strains were detected in the ICU.

Isolates belonging to the Enterobacteriaceae family may be resistant to antibiotics such as carbapenems that are used as last-resort treatment for serious infections. Carbapenem-resistance may further limit treatment options and lead to treatment failure^[20]. Differential susceptibility to carbapenems may occur due to relative penetration rates through minor porins, differential susceptibility to efflux, or relative susceptibility to slow hydrolysis by AmpC enzymes or ESBLs^{[21, 22]}. Higher than data from other countries but similar to those of other Turkish studies, the highest rate of resistance to the carbapenems that we tested in our study was to ETP (98.0% in K. pneumoniae, 84.8% in E. coli). In 2008, Güzel Tunçcan et al.^[23] conducted a study in Ankara and found reported no carbapenem resistance in 58 E. coli and 37 Klebsiella spp. strains isolated from various clinical specimens. However, in 2011, Kuzucu et al.^[19] reported 0.7% IMP and MEM resistance and 1.4% ETP resistance in ESBL-positive E. coli and Klebsiella spp. strains in Malatya. In 2015 in the USA, Pecora et al.^[24] detected carbapenem resistance in 1.5% of 41 K. pneumoniae isolates from various clinical specimens. In Brazil, Biberg et al.^[25] detected carbapenem resistance in 12.2% of 360 Klebsiella spp. isolates.

There are many mechanisms of carbapenem resistance among Enterobacteriaceae. One of these, carbapenemase production, has become a major public health problem worldwide^[1]. The KPC type carbapenemase is one of these critical mechanisms^[2]. The presence of the bla_KPC gene was first detected in the USA in 1996^[8]. Studies have shown that the bla_KPC gene is gradually spreading among K. pneumoniae strains^[11]. In France, China, Colombia, and Scotland, sporadic cases of KPC-producing strains were observed, while prolonged outbreaks were reported in Israel and Greece in 2004 and 2007, respectively^[26].

Reports of K. pneumoniae isolates carrying the bla_KPC gene have been very limited in Turkey. A strain showing KPC gene positivity was first reported by Labarca et al.^[27] in 2014 in a case report of an 80-year-old Romanian patient treated in an ICU in Kocaeli. In the same year, Poirel et al.^[28] identified two K. pneumoniae isolates producing KPC-2 carbapenemase in İstanbul. In 2016, as part of the European Survey of Carbapenemase Producing Enterobacteriaceae (EuSCAPE) project, 155 carbapenemase suspected K. pneumoniae (n=134, 86.5%) and E. coli (n=21, 13.5%) isolates submitted from 18 centers in various regions of Turkey were investigated, but none was positive for KPC^[20]. Recently, Sağıroğlu et al.^[29] reported the presence of KPC in K. pneumoniae strains isolated from two patients. In 2016, Kuskucu et al.^[30] reported two KPC-2-producing E. coli isolates in Turkey. In this study, we investigated the presence of bla_KPC gene in carbapenem-resistant E. coli and K. pneumoniae isolates and detected bla_KPC gene in one of the K. pneumoniae isolates.

According to our literature survey, this report is one of the few reports reporting the presence of KPC-producing K. pneumoniae isolates in Turkey.

This study was carried out in a state hospital and has some limitations due to financial and technical issues resulting in lack of sequencing the positive gene, and screening other carbapenemase genes in the laboratory. The bla_KPC-positive strain and the other isolates used in the study could not survive because of technical problems in the hospital.

Conclusion

In conclusion, continuous monitoring of sensitivity rates and observation of regional differences will provide guidance in determining infection control and antimicrobial usage policies. In addition, surveillance of resistance genes with the potential for horizontal spreading, like in this study, will likely contribute to the prevention of carbapenemase-induced resistance.

Ethics

Ethics Committee Approval: The study was approved by the Adana Numune Training and Research Hospital of Local Ethics Committee (protocol number: 2014/7).

Informed Consent: It was not received.

Peer-review: Externally and internally peer-reviewed.

Authorship Contributions

Concept: G.Ö., E.Ö., H.K., B.M.S., F.B., Design: G.Ö., E.Ö., H.K., B.M.S., C.K.B., Data Collection or Processing: G.Ö., E.Ö., S.K.S., H.A., C.K.B., Analysis or Interpretation: G.Ö., E.Ö., Literature Search: E.Ö., F.B., C.K.B., Writing: G.Ö., E.Ö., C.K.B.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.