Investigation of NDM, VIM, KPC and OXA-48 genes, blue-carba and CIM in carbapenem resistant Enterobacterales isolates

Introduction: Carbapenem resistance is an emerging problem in Enterobactarales. We aimed to investigate the presence of carbapenemase genes blaNDM, blaKPC, blaVIM and blaOXA-48 and evaluate the phenotypic blue-carba method and carbapenem inactivation method (CIM) in Enterobacterales isolates. Methodology: Total of 153 Enterobacterales isolates were tested in the study. Presence of blaNDM, blaKPC, blaVIM and blaOXA-48 genes was investigated by polymerase chain reaction (PCR) method. Carbapenemase production of the isolates was also tested by blue-carba method and CIM. Results: The presence of blaOXA-48 gene was detected in 110 (71.4%) and blaNDM gene was detected in 2 (1.3%) of the Enterobacterales isolates by PCR method. None of the isolates were positive for blaKPC and blaVIM genes. The 121 (78.54%) of the isolates were found to be positive by blue-carba method and CIM. And 105 (68.18%) of the isolates were determined as positive by both PCR, blue-carba and CIM. Conclusions: In our study, 112 (72.7%) of the Enterobacterales isolates were found to be positive for carbapenemase genes (blaoxa-48 and blaNDM), and 121 (78.57%) of different isolates were found to be positive for blue-carba and CIM. However, 105 (68.18%) of the carbapenem resistance isolates found to be positive for all three methods.


Introduction
Enterobacterales are inhabitants of the intestinal flora and are among the most common human pathogens, causing infections such as cystitis, pyelonephritis, septicemia, pneumonia, peritonitis, meningitis, and device-associated infections [1]. The emergence and transmission of carbapenem-resistant Enterobacterales (CRE) over the past two decades has attracted worldwide attention, for its indication that most currently available broad-spectrum antibiotics may no longer be a therapeutic option for some patients [2]. It is therefore mandatory to maintain the clinical efficacy of carbapenems (imipenem, ertapenem, meropenem, doripenem), which have become antimicrobial drugs of last resort [1].
Multidrug-resistant organisms are a major public health concern worldwide; of particular concern has been the emergence of resistance to carbapenem antimicrobial drugs among Enterobacterales [3]. Therefore, preventing transmission of carbapenemaseproducing, carbapenem-resistant Enterobacterales is a public health priority [4].
Carbapenem resistance in Enterobacterales is often associated with presence of carbapenemases located on plasmids or other mobile genetic structures [5]. Carbapenem resistance due to the production of acquired carbapenemase genes is increasingly reported among members of Enterobacterales, these mechanisms are still less commonly observed in Enterobacter spp. than in Klebsiella spp. or Escherichia coli [6]. The other carbapenem resistance mechanisms for Enterobacterales species are combination of presence of extended-spectrum β-lactamases (ESBLs), increased efflux, porin alteration, and a strongly expressed (derepressed) endogenous AmpC enzyme [7].
Many phenotypic methods for detection of carbapenemase-producers have been described for the Enterobacterales [8]. Phenotypic methods defined by the Clinical and Laboratory Standards Institute (CLSI) that clinical microbiology laboratories can apply to detect carbapenemase producers include the Modified Hodge Test (to be removed from the M100 in 2018; CLSI, January 2017 meeting minutes), the Carba NP and most recently the modified Carbapenem Inactivation Method (mCIM) [9]. The blue-carba, has the same principle as the CarbaNP but uses bromothymol blue as a pH indicator and does not need the enzymatic extraction step [10]. The CIM utilizes readily available reagents not requiring reagent preparation and results are more objective in nature as a zone diameter reading is used for interpretation of results [9]. The CIM concept has the potential to also be applied to assess enzymatic hydrolysis of other antibiotics, e.g. allowing detection of ESBL activity. Preliminary experiments in laboratory have shown this maybe feasible [11].
In our study, we aimed to investigate the presence of blaNDM, bla VIM , bla KPC and bla OXA-48 genes, which are responsible for the development of resistance to carbapenem antibiotics in Enterobacterales isolates, and to evaluate the phenotypic carbapenemase detection methods blue-carba and CIM.

Methodology
Carbapenem resistant 153 Enterobacterales isolates that were isolated from various clinical samples sent to the Medical Microbiology Laboratory of Ondokuz Mayis University Faculty of Medicine were included in the study (Table 1). Identification of the isolates was done using conventional methods and Vitek-MS (Biomeirux, France) automated system. The antibiotic susceptibility was tested with the Vitek2 Compact (Biomeirux, France) automated system. Susceptibility of the isolates were evaluated according to the EUCAST criteria. For quality control, reference strains E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used. Isolates that were resistant to one of the carbapenems (imipenem, meropenem or ertapenem) were considered as carbapenem resistance and enrolled in the study. Enterobacterales isolates were stored at -20°C until the molecular study. The existence of bla NDM , bla VIM , bla KPC and bla OXA-48 genes were studied by polymerase chain reaction (PCR) using specific primers (Table 2) [12,13]. DNA extraction were made by the method of boiling. Positive strains for bla NDM , bla VIM , bla KPC and bla OXA-48 were as positive control and E. coli ATCC 25922 was used as negative control in the PCR assays. Positive strains for bla NDM , bla VIM , bla KPC and bla OXA-48 were obtained as a part of national quality control program. bla NDM and selected bla OXA-48 positive isolates were sequenced in Sentegen (Turkey), they confirmed positive for bla NDM and bla  .
Bromothymol blue was selected as the indicator in the blue-carba test. A commercially available imipenem (Tienam500; Merck Sharp & Dohme, France) was used as the substrate for carbapenemases. The test solution consisted of an aqueous solution of bromothymol blue at 0.04% (Merck Millipore, Germany) adjusted to pH 6.0, 0.1 mmol/liter ZnSO 4 , and 3 mg/mL of imipenem, with a final pH of 7.0. A negative-control solution (0.04% bromothymol blue solution, pH 7.0) was prepared. A loop (approximately 5 µl) of bacteria was directly suspended in 100 µl of both test and negativecontrol solutions in a plate and incubated at 37°C for 2 hours. Carbapenemase activity was revealed when the test and negative-control solutions, respectively, were (i) yellow versus blue, (ii) yellow versus green, or (iii)  [10]. In carbapenem inactivation method (CIM), a suspension was made by suspending a full 10 μL inoculation loop of culture, taken from blood agar plate in 400 μL water. Subsequently, a susceptibility-testing disk containing 10 μg meropenem was immersed in the suspension and incubated for a minimum of two hours at 35°C. After incubation, the disk was removed from the suspension, placed on a Mueller-Hinton agar plate inoculated with a susceptible E. coli ATCC 29522 and subsequently incubated at 35°C. The inhibition zone around the disc was evaluated after 6 or 24 hours. If the tested bacterial isolate produced carbapenemase, the meropenem in the susceptibility disk was inactivated allowing uninhibited growth of the susceptible E. coli ATCC 29522. Disks incubated in suspensions that do not contain carbapenemases yielded a clear inhibition zone. [11].
In this study, 110 (71.9%) isolates were determined positive for blaOXA-48 and 2 (1.3%) isolates were positive for bla NDM . Bla VIM and KPC genes were not detected ( Figure 1). Most of the isolates were sent from internal medicine clinics (77.92%). And majority of the bla OXA-48 positive isolates were isolated from the specimens that sent form internal medicine clinics (61.68%) bla NDM positive isolates were isolated the clinical specimens that sent from intensive care unit and internal medicine clinic ( Table 3). The sequenced bla NDM and selected bla OXA-48 positive isolates were confirmed positive for bla NDM and bla  .
For blue-carba test and CIM, 120 isolates were determined positive (78.43%). In our study 99 (64.7%) of the tested isolates were found positive by three methods (PCR, blue-carba and CIM). And 19 (12.4%) isolates were found to be negative by three methods. For the bla OXA-48 and bla NDM positive isolates 101 (90.2%) of them were found to be positive by bluecarba and 111(99.1%) of them were found to be positive by CIM.
But 12 of the PCR positive isolates were determined negative by blue-carba test, and two of the PCR positive isolates were found negative by CIM. Nineteen and nine of the PCR negative isolates were detected positive by blue-carba and CIM, respectively ( Table 4). The sensitivity and specitivity of the blue-carba was determined as 89.4% and 52.5%, respectively. The sensitivity and specitivity of CIM was determined as 98.2% and 77.5%.

Discussion
The Enterobacterales account for up to 25% of healthcare associated infections (HAIs) reported to the U.S. National Healthcare Safety Network [14]. Carbapenemases increasingly have been reported in Enterobacterales in the past 10 years [1]. The emergence and spread of carbapenemase-producing Enterobacterales (CPE) is a serious global threat that  considerably limits therapeutic options available for life threatening Gram-negative infections [15]. Detection of infected patients and carriers with carbapenemase producers is necessary for prevention of their spread, also may help prevent development of nosocomial outbreaks caused by carbapenemase producers, particularly K. pneumoniae [1].
Carbapenem-resistant Enterobacterales have been reported worldwide as a result of various kind of carbapenemase genes [16]. The first carbapenemase producer in Enterobacterales (NmcA) was identified in 1993 [17]. Bla KPC have been reported from worldwide with higher endemicity in USA and Greece. bla OXA-48 type have been reported mostly from European and Mediterranean countries and India [1].
NDM-1 firstly isolated in 2008 and now it is the focus of worldwide attention [18,19]. Deshpande et al. (2010) reported that of the 24 carbapenem resistant Enterobacterales, 22 were bla NDM producers while 2 were bla NDM non-producers. Amongst the 22 bla NDM producing organisms 10 were Klebsiella spp, 9 were Escherichia coli, 2 were Enterobacter spp and 1 was Morganella morganii [20]. Since mid-August 2010, bla NDM producers have been identified on all continents except in Central and South America with, in most of the cases, a direct link with the Indian subcontinent, also recent findings suggest that the Balkan states and the Middle East may act as secondary reservoirs of bla NDM producers [19]. Yanık  PCR, sequencing revealed that K. pneumoniae bla NDM carbapenemase. This study has also constituted to the very first report of an bla NDM positive isolate in Turkey [22].
The first acquired MBL, bla IMP , was reported in Serratia marcescens in Japan in 1991 [23]. IMP types and, more recently, of the New Delhi metallo-βlactamase-1 (bla NDM ) type. Endemicity of VIM-and IMP-type enzymes has been reported in Greece, Taiwan, and Japan [24,25]. Death rates, associated with MBL producers, range from 18% to 67% [26].
The first bla KPC producer (KPC-2 in K. pneumoniae) was identified in 1996 in the eastern United States [27]. KPC-mediated carbapenem resistance in members of the Enterobacterales has emerged recently in Israel, as observed in clinical strains of Escherichia coli [28], Enterobacter cloacae [29], and Klebsiella pneumoniae [30]. Bla KPC producers have been reported, mostly from nosocomial K. pneumoniae isolates and to a much lesser extent from E. coli (especially in Israel) and from other Enterobacterales species [2]. Institutional outbreaks of KPC-producing Enterobacterales due to the spread of a single strain have also been reported for other species, including Enterobacter spp. and Serratia marcescens [29,31].
The first identified bla OXA-48 producer was from a K. pneumoniae strain isolated in Turkey in 2001 [32]. More than 250 class D β-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for β-lactams [33]. Although bla OXA-48 βlactamase and its variants typically have low-level hydrolytic activity against many carbapenems, they can contribute to high-level carbapenem resistance in combination with other mechanisms [34]. Since then, bla OXA-48 producers have been extensively reported from Turkey as a source of nosocomial outbreaks [35,36]. Their worldwide distribution now includes countries in Europe, in the southern and eastern part of the Mediterranean Sea, and Africa [32,36]. Several bla OXA-48 producing clones have been identified, and dissemination of this resistance trait is associated with a 62.5-kb plasmid (previously identified as a plasmid of ≈ 70 kb) [35]. Although reported in various enterobacterial species, bla OXA-48 producers are mostly identified in K. pneumoniae and E. coli, and the level of resistance to carbapenems is usually higher when ESBL and permeability defects are associated [35,37]. Out of 296 carbapenem-nonsusceptible isolates, bla OXA-48 gene was detected in 12 K. pneumoniae isolates in Croatia [38]. Perçin et al. (2012) investigated the presence of bla IMP , bla OXA OXA, bla NDM , bla KPC genes in carbapenem-resistant K. pneumoniae from rectal swabs. Carbapenem susceptibility was evaluated by using E-test method, the presence of beta-lactamases was determined by using modified Hodge test (MHT). Of the 801 isolates 33 were determined as carbapenem resistant. No resistance gene were identified by PCR in 13 of 33 isolates, whereas bla OXA-48 was detected in 19 and bla IMP in 1 of 20 positive isolates [39]. Eser et al. (2014) investigated the presence of carbapenem resistance in 210 Enterobacterales isolates (E. coli (n = 153), K. pneumoniae (n = 47) and Klebsiella oxytoca (n = 10)) recovered from invasive infections, in Hacettepe University Hospital, Ankara, Turkey, between 2005-2009, by PCR (AmpC, CTX-M, KPC, NDM, OXA, IMP and VIM). One K. pneumoniae isolate was found to inhabit bla OXA-48 gene. Five isolates were positive for OXA-1 and one for OXA-10. Two isolates were positive for bla CTX-M , however bla IMP , bla VIM , bla KPC and bla NDM-1 genes were not detected among the isolates [40].
Studies have shown that locally class D OXA-48like enzymes and NDM are the most common carbapenemases in Enterobacterales with sporadic occurrence of KPC and VIM type enzymes [42,43]. Turkey is known to be an area of endemicity for OXA-48-producing Enterobacterales [22], and in this study most of the carbapenem resistant isolates were positive OXA-48 and NDM were determined in two isoates.
In Tamma et al. (2017) study, two collections of carbapenem-resistant Enterobacterales (CRE) isolates were evaluated including 191 retrospective isolates (122 CP-CRE and 69 non-CP isolates) as well as 45 prospective clinical isolates (15 CP-CRE and 30 non-CP-CRE) obtained over a 3-month period. Among the retrospective cohort, sensitivities ranged from 72% for the boronic acid synergy test for the detection of KPCproducers to ≥ 98% for the Modified Carba NP, the RAPIDEC® CARBA NP, the manual Blue-Carba, and the modified carbapenem inactivation method for the detection of any CPE. All assays had excellent specificity exceeding 95% with the exception of the boronic acid synergy test (88%) and modified Hodge test (91%). Prospectively, 45 CRE isolates were encountered over a three-month period including 15 CPE (33%) and 30 non-CP-CRE (67%) [44].
A new phenotypic test called the carbapenem inactivation method (CIM) was developed to detect carbapenemase activity in Gram-negative bacilli within six hours. This method has been found to be highly compatible with the results obtained with PCR to detect genes encoding bla KPC , bla NDM , bla OXA-48 , bla VIM , bla IMP and bla OXA-23 carbapenemases. Comparing the results of PCR and CIM determined by genes producing several carbapenemase in Enterobacterales, P. aeruginosa and A. baumannii isolates; CIM (92.1%) has been shown to be a phenotypic screening method that can reliably detect carbapenemase activity. It was observed that, 67 isolates (16.3%) whose carbapenemase gene determined by PCR method of 411 isolates tested in the study were positive and according to CIM result, 65 (97.0%) of 67 isolates were positive. It was seen that bla KPC gene was not detected in the Pseudomonas isolates used in the study [11].
A total of 125 isolates were included in Bayraktar et al. (2018) study. The strain collection of CPEs included 89 OXA-48 (80 K. pneumoniae, six E. coli, and three K. oxytoca), two KPC (two K. pneumoniae), five NDM (four K. pneumoniae and one E. cloacae), two VIM (one K. pneumoniae and one E. coli), one IMP (K. pneumoniae), 10 OXA-48+NDM (eight K. pneumoniae, one E. coli, and one E. cloacae), and one OXA-48+VIM (K. pneumoniae) producer. Of the 110 CPE isolates (including the positive control isolate), 100 were found to be positive by all three tests and 10 isolates showed discordant results. Carba NP-direct, CIM, and b-CARBA tests detected 109 (99.0%), 102 (92.7%) and 108 (93.6%) isolates as positive, respectively [46]. Tijet et al. (2015) reported that the sensitivity (98.8%) of the CIM test were higher than those of the Carba NP test (90.1% and 88.2%, respectively) [47]. In our study sensitivity of the CIM test was higher than blue-carba test. The sensitivity of the CIM and blue-carba tests found to be similar to the other studies. However specitivity of the blue-carba test was found to be 52.5%.

Conclusions
Carbapenem resistance is an emerging problem in world wide. Determination of carbapenem resistance determinants is important for prevention of health-care associated infection.