Molecular characterization and replicon typing of plasmids encoding carbapenemases in Gram-negative bacteria


 Background Carbapenem resistance in Gram-negative bacteria is an ongoing public-health problem of global dimensions leaving very few treatment options for severely infected patients. This study focuses on the dissemination of plasmid-borne carbapenemase genes in Gram-negative bacteria in Tamil Nadu, India. A total of 151 non-repetitive isolates belonging to 11 genera were collected from a diagnostic center in Tamil Nadu. Minimal inhibitory concentration of imipenem and meropenem were determined using micro-broth dilution method. E. coli pathotyping, Klebsiella serotyping, screening for beta-lactamases and plasmid incompatibility grouping was performed.Results E. coli (n=57) isolates were classified as, Enteropathogenic (n=12), Enteroaggregative (n=9), Enterohemorrhagic (n=8), Enterotoxigenic (n=3), Enteroinvasive (n=1) and unclassified E. coli (n=24). Of the 45 Klebsiella species, 14 were K1 whereas 11 were K2 serotype and in 20 Klebsiella serotype could not be determined. Other isolates (n=49) consisted of P. aeruginosa , S. typhi , E. cloacae , A. baumannii , S. marcescens , A. xylosoxidans , P. mirabilis and E. meningoseptica . Of the total number of isolates, 71% (n=107) and 68% (n=103) were found to be resistant to meropenem and imipenem respectively. The most prevalent beta-lactamase gene was bla NDM-1 (21%, 12/57) followed by bla OXA-181 (16%, 9/57) both detected in E. coli . Other carbapenemase genes detected were bla GES-9 (n=8), bla OXA-23 (n=7) and bla IMP-1 (n=3). Interestingly bla GES-1 (n=11), bla OXA-51 (n=9) were also detected. The unusual presence of bla OXA-23 was seen in E. coli (n=4), and bla OXA-23 and bla OXA-51 (IncA/C) in K. pneumoniae (n=3). Plasmid incompatibility (inc/rep) typing results showed that the plasmids carrying resistance genes (n=11) belonged to IncX, IncA/C, IncFIA-FIB and IncFIIA groups. Six E. coli isolates and one K. pneumoniae were able to transfer plasmid-borne carbapenemase ( bla NDM-1 , bla OXA-181 , bla GES-1 , bla GES-9 ) via conjugation.Conclusions This study highlights the prevalence of carbapenem resistance and the acquisition of plasmid-borne carbapenemase genes in unusual Gram-negative bacteria highlighting the role of evolution by generating microbial diversity.

resistance and the acquisition of plasmid-borne carbapenemase genes in unusual Gramnegative bacteria highlighting the role of evolution by generating microbial diversity.

Introduction
Antibiotic resistance is an emerging global health problem due to the injudicious use of antibiotics [1]. It is considered a major clinical and public health problem because of increasing bacterial resistance to most of the available antibiotics including penicillin, cephalosporins, carbapenems, and colistin [1]. World Health Organization (WHO) recently listed carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Extended Spectrum Beta-Lactamases (ESBL) -producing Enterobacteriaceae as pathogens that are of critical importance [2]. Gram-negative bacteria (GNB) especially Enterobacteriaceae have developed resistance towards a broad spectrum of antibiotics responsible for significant mortality around the globe [3]. Carbapenems are considered as one of the last resort antibiotics against infections caused by multi-drug resistant GNB [4].
The emergence of carbapenem resistance especially in Enterobacteriaceae is a threat to the patients, particularly with complex infections, immunocompromised conditions and multiple diseases [5]. Because pathogens that are resistant to carbapenems often shows high resistance to other commonly used antibiotics that are often used for treatment, not only the mortality rates are high with increased hospital stay, but also huge medical expenditure placing emotional, economic and financial burden on families especially in resource limited countries [6].
The assessment of the rise in global antibiotic resistance has become very difficult due to the increasing rate of multi-drug resistance shown by pathogens with no proper harmonized surveillance systems in resource limiting countries [7]. Moreover, the coexistence of more than one carbapenem resistance gene with other genes like plasmid mediated AmpC, or plasmid mediated quinolone resistance has resulted in an increased acquisition of resistance among Enterobacteriaceae for community as well as hospital acquired infections [8,9]. The carbapenem-hydrolyzing oxacillinases (CHDL) are the major resistance mechanisms to carbapenems in A. baumannii. The first report of OXA-23 betalactamase in A. baumannii was from United Kingdom, in 1985 [10]. Later, OXA-23 was found to confer carbapenem-resistance in A. nosocomialis [11] and recently, it was reported in members of Enterobacteriaceae family [12][13][14]. In 1996, the first report of OXA-51 type beta-lactamase was from Argentina and at present, there are more than 150 variants of OXA-51 were reported globally [15]. These intrinsic enzymes in A. baumannii are naturally chromosomal-borne but rare cases of plasmid-borne genes are also reported [16]. Earlier, we reported the distribution of carbapenem and colistin resistance, and the role of integrons serving as the horizontal gene transfer agents in disseminating resistance among Gram-negative bacteria [17,18]. In the present study, molecular characterization of Gram-negative bacteria was performed and the role of interspecies plasmid transfer as evolutionary mechanism of carbapenem resistance was determined.

Distribution of carbapenemase resistance genes
The distribution of beta-lactamase resistance genes among 151 Gram-negative isolates is summarized in Table 2. Of the 57 E. coli, 32 isolates carried carbapenemase and five E.
coli isolates did not belong to any of the tested pathotypes.
Of the 45 Klebsiella species, 14 belonged to K1 serotype, 11 were K2, none of the isolates were of K5 serotypes and 20 were of unknown serotypes (Table 3). Of the 14 K.

Discussion
In India, the prevalence of carbapenem-resistant Gram-negative bacteria has been reported with an increasing frequency. In this study, the distribution of carbapenemresistant isolates among 11 genera of Gram-negative bacteria isolated from diagnostic centers in Tamil Nadu, India is reported. Previously, the increasing prevalence of ESBL and MBL producers among Gram-negative bacteria has been reported in India [27][28][29][30].
In this study, there was a strong dissimilarity observed between the results obtained by disc-diffusion and micro-broth dilution, in which, MIC results showed that 107/151 (71%) were resistant to meropenem in accordance with 128 isolates by the disk-diffusion method. All the 71 isolates harbouring carbapenem resistance genes were resistant with MIC and disc-diffusion method. The remaining 36 resistant isolates identified by MIC and 57 resistant isolates identified by disc-diffusion were negative for carbapenem resistant genes which indicate false-positive result or a novel mechanism of resistance. Though earlier studies have shown the similar outcome, it should be noted that in India majority of the clinical diagnostic laboratories use disk-diffusion as a standard method for assessing the antibiotic sensitivity pattern. So, the knowledge of the relationships between the phenotypic methods is very important in the clinical settings [31,32]. In the clinical practice, an accurate detection method, such as MIC as confirmed in this study, should be used to identify the resistance in order to treat patients with a precise antibiotic because an irrational and improper use of antibiotics is one of the reasons for increasing antibiotic resistance in developing countries like India [33,34].
The studied E. coli pathotypes (EPEC, EHEC, EIEC, EAEC and ETEC) are associated with intestinal diseases, they are collectively called as diarrheagenic E. coli (DEC) or intestinal pathogenic E. coli (IPEC) [35,36]. All these pathotypes are linked directly to their virulence properties and severity of infections. Though there are studies that showed the prevalence of DEC in India [37,38], still the studies on E. coli pathotypes (virulence) interrelation to carbapenem resistance is not well established in India. In this study, all the five E. coli pathotypes were found to harbour carbapenem resistance genes, namely EPEC (NDM-1, OXA-181, and OXA-23), EHEC (NDM-1, OXA-181, GES-1, and GES-9), EIEC (NDM-1, OXA-181), EAEC (GES-9, OXA-23, and GES-1), ETEC (NDM-1) and some are unknown pathotypes (Table 4). Adding to their virulence, the presence of resistance genes makes these bacterial infections (mostly diarrhoea) more complicated due to unavailability of treatment options. The Klebsiella isolates can be grouped into serotypes using surface antigens or surface exposed lipopolysaccharides [39]. The Klebsiella belonging to Kserotypes have K-antigen that relates to the capsule polysaccharide (CPS) [39]. Of the known capsular types (eight serotypes), the serotypes K1 and K2 are the most virulent among the hypervirulent K. pneumoniae (hvKP) [40]. In this study, of the 14 Klebsiella isolates belonging to K1 serotypes, six isolates carried carbapenem resistance genes, bla NDM-1 , bla OXA-181 and bla OXA-51 . Among the 11 Klebsiella isolates belonging to K2 serotype, two isolates were found to carry carbapenem resistance genes, bla OXA-181 and bla   (Table 4). In India, NDM-1 and OXA-48 genes were detected in Klebsiella belonging to K2 serotypes [40], but to the best of our knowledge, this is the first study to detect the presence of carbapenemase genes NDM-1, OXA-181 and OXA-51 among K1 serotypes.
But the unusual occurrence of bla OXA-23 in E. coli, and plasmid-borne (IncA/C) bla  and bla OXA-51 in K. pneumoniae is one of the important findings of this study. There are very few earlier studies that reported the presence of bla OXA-23 gene in E. coli [43,44]. To the best of our knowledge, this is the first study to report the plasmid-borne (IncA/C) OXA-23 and OXA-51 in K. pneumoniae. The OXA-23-like genes in Enterobacteriaceae may be carried within a transposon but was not characterized in this study. The resistance reports on E. meningoseptica are very rare in India [43,44] and in our study, it was found that one isolate of E. meningoseptica was resistant to imipenem and meropenem. Though earlier studies showed the presence of carbapenemase genes in E. meningoseptica, in this study no carbapenem resistance genes were amplified.
Carbapenem resistance among Gram-negative bacteria is becoming very common in India and the spread of carbapenem resistance genes are one of the troublesome problems.
These resistance genes that are located adjacent to the mobile genetic elements (integrons and transposons), which facilitates the easy transposition between replicons [45]. The most common plasmid replicon types for carbapenem resistance genes are IncF, IncA/C 2 , IncX3, IncL/M and IncH [46]. In this study, bla NDM-1 was found to be harboured in This clearly showed that the beta-lactamase or carbapenemase resistance genes were present in the plasmids with different replicon types in the study region. Earlier, the bla NDM IncFII plasmids were reported from India [46] and IncFIA-FIB plasmids carrying carbapenem resistance genes such as bla NDM was report from India in the samples collected from river and sewage treatment plants [46,47]. This study also showed that some plasmids were carrying more than one resistance genes which are an alarming threat to the public health. Conjugative plasmids are known to spread their resistance characteristics among the bacteria from the same or different genus. This study showed that all the six E. coli isolates carrying plasmid-borne resistance genes (bla NDM-1 , bla OXA-181 , bla GES-1 , bla GES-9 ) were conjugative and one K. pneumoniae isolate plasmid (IncFIA-FIB with bla NDM-1 ) was transferable which clearly shows the way by which resistance genes can rapidly spread in clinical bacteria.

Conclusion
The emerging antibiotic resistance in bacteria is a worrisome problem. This study highlighted the distribution of carbapenem resistant isolates in the study region with the extra emphasis on the existence of bla NDM-1 , bla OXA-48-like , bla IMP-1 , bla GES-1 , bla GES-9 , bla OXA-23-like , bla OXA-51-like among the clinical pathogens. Alternative therapeutic options such as phage therapy should be undertaken immediately to combat the problem of resistance especially to treat infections caused by carbapenem resistant bacteria. Our study shows that the 'conjugative plasmids' can strongly contribute to the resistance transfer in pathogens leading to dissemination of resistance genes. Alternative approaches are necessary to combat the problem of resistance and concepts such as 'onehealth approach' can be appreciated.

Isolate collection and classification
During January 2015 and December 2016, a total of 151 Gram-negative bacterial isolates were collected from Hi-Tech diagnostic center in Chennai, Tamil Nadu, India. Bacteria were isolated from urine, blood, pus, bronchial secretion, cerebrospinal fluid, pulmonary secretion and bile fluid. The collected isolates were received at the Antibiotic Resistance and Phage Therapy Laboratory, VIT, Vellore, for further analyses. Genomic DNA was extracted from all the isolates using boiling lysis method [18]. Bacterial identification was carried out using VITEK identification system (bioMerieux) and 16S rRNA analysis using universal primers 27F and 1492R [18]. The PCR products were sequenced and identified to the species level using the BLASTN tool.

Antibiotic susceptibility testing and Minimal Inhibitory Concentration
Antibiotic resistance profiling was performed using the disk-diffusion method according to CLSI guidelines [19]. The antibiotics used for this study were gentamicin (10 µg was determined by broth micro-dilution method for meropenem and imipenem as described previously [18] and the results were interpreted according to CLSI guidelines [19].
The PCR amplicons of the resistance genes were sequenced and genes were confirmed using NCBI BLASTN programme.

Plasmid isolation and plasmid incompatibility grouping
Plasmid isolation was performed for all the isolates harbouring resistance genes. The  [26].

Conjugation studies
Representative carbapenem-resistant isolates harbouring plasmid-borne resistances (n=11) were subjected for conjugation using broth-mating method [18]. Briefly, the donor strain (strains carrying resistance genes) and the recipient strain (E. coli AB1157, Str r ) were grown overnight, and mixed in 9:1 ratio each of donor and recipient. The cells were kept undisturbed for 6 hours at 37°C and plated on to antibiotic containing medium. The isolates which grew on both meropenem and streptomycin were considered as transconjugants. All the transconjugants were confirmed for the presence of respective carbapenem resistance genes using PCR.

Consent to publish:
Not applicable.

Availability of data and materials:
All the datasets are presented in the main manuscript. The raw datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Funding information:
This research work was not funded by any external agencies.

Competing interest:
The authors declare that they have no competing interest.

Authors' contribution:
Authors PM and NR, collected the isolates from the clinical samples. Authors PM, MK and NR undertook the laboratory work, NR and BSL interpreted the data, and PM and NR wrote the initial manuscript. Authors NR and BSL revised and edited the manuscript. All the authors' have read and approved the manuscript.
-GES-1 is an extended spectrum beta-lactamase which shows a significant degree of inhibition by imipenem indicating that it may be able to bind imipenem with storng affinity without being able to hydrolyze it. This enzyme was detected in 11 isolates.
-OXA-51 is a beta-lactamase which can only act as a carbapenemase if it is upregulated by insertion elements or has a Trp222 site mutation. This enzyme was detected in 9 isolates.   Figure 1 The distribution of Gram-negative bacteria and comparison of minimal inhibitory concentrations (MIC) of imipenem and meropenem resistance.