Co-Carriage of bla KPC-2 and bla NDM-1 in Clinical Isolates of Pseudomonas aeruginosa Associated with Hospital Infections from India

Global spread of KPC poses to be a serious threat complicating treatment options in hospital settings. The present study investigates the genetic environment of bla KPC-2 among clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital of India. The study isolates were collected from different wards and clinics of Silchar Medical College and Hospital, India, from 2012–2013. The presence of bla KPC was confirmed by genotypic characterization followed by sequencing. Cloning of the bla KPC-2 gene was performed and the genetic environment of this gene was characterized as well. Transferability of the resistance gene was determined by transformation assay and Southern hybridization. Additionally, restriction mapping was also carried out. Two isolates of P. aeruginosa were found to harbor bla KPC-2, were resistant towards aminoglycosides, quinolone and β-lactam-β-lactamase inhibitor combination. In both the isolates, the resistance determinant was associated with class 1 integron and horizontally transferable. Both the isolates were co-harboring bla NDM-1. The first detection of this integron mediated bla KPC-2 coexisting with bla NDM-1 in P. aeruginosa from India is worrisome, and further investigation is required to track the gene cassette mediated bla KPC-2 in terms of infection control and to prevent the spread of this gene in hospitals as well as in the community.


Introduction
Klebsiella pneumoniae carbapenemase (KPC) is a major contributor for carbapenem resistance across the globe within the members of Enterobacteriaceae [1] and in recent past they are also been reported in Pseudomonas spp. from Brazil and China [2,3]. In Indian subcontinent, this gene has so far been reported only in Enterobacteriaceae family [4]. Their coexistence has been witnessed with other carbapenemase genes as well [4]. However, no study from this country has attempted to investigate genetic background of this gene. In this study, we have reported coexistence of bla KPC-2 and bla NDM-1 with different genetic context in P. aeruginosa in a tertiary referral hospital of India.

Clinical isolates
A total number of 88 non duplicate carbapenem nonsusceptible P. aeruginosa were collected from the patients admitted to different wards or attended clinics of Silchar Medical College and Hospital, Silchar, India for a period of one year from July 2012 to June 2013 (S1 Table). All the isolates were subjected to PCR assay for detection of class A and class B carbapenemases as described below.

Molecular characterization & cloning
Multiplex PCR assay was performed for characterizing the bla genes using the primers as mentioned (Table 1) and the reaction conditions as described previously [5][6][7]. The amplicons were sequenced to confirm the variant of amplified gene. For cloning of bla KPC-2 , primers were designed (Table 1) from the flanking region of bla KPC-2 to amplify the whole gene including the promoter region. The amplified products were purified using MinElute PCR Purification Kit (Qiagen, Hilden, Germany) and was ligated into pGEM-T Vector (Promega, Madison, USA), transformed into E. coli JM107. Coexistence of class B β-lactamase was determined by multiplex PCR targeting IMP, VIM and NDM gene (Table 1) as described earlier [8][9]. IMI/NMC-R 5'-CTACCGCATAATCATTTGC-3' SME SME-F 5'-AACGGCTTCATTTTTGTTTAG-3' 831 7 Determination of genetic environment Integron carriage was assessed by performing integrase gene PCR [10] targeting integrase gene of class 1 and class 2 integron. To determine the cassette array within class 1 integron, four sets of PCR reactions were performed, in first set, primers of 5 / conserved region (5CS) and reverse primer of bla KPC (KPC-R) were used. In second PCR, a set of primers targeting 5 / conserved region (5CS) and forward primer of bla KPC (KPC-F) were used. In the third reaction, 3 / conserved region (3CS) of class 1 integron and forward primer of bla KPC (KPC-F) were taken and the fourth PCR assay was performed using 3CS primer and reverse primer of bla KPC (KPC-R).
All the primers are mentioned in the Table 1. The PCR conditions were as described previously [5,10]. The visible amplification was observed in 2 nd and 4 th PCR assay for both the samples. The amplified products were sequenced and were analyzed using the software available on National Centre of Biotechnology Information website (http://www.ncbi.nlm.nih.gov) and the sequencing results could confirm the location and cassette array of bla KPC within class 1 integron. The whole class 1 integron gene cassette was also amplified using primers 5CS and 3CS with the following reaction condition, initial denaturation at 95°C for 3mins, 34 cycles at 95°C for 30s, 49°C for 1min, 72°for 3mins and final extension at 72°C for 7 mins [11].

Plasmid analysis and Southern blot hybridization
Plasmids were purified by Gene Jet plasmid Miniprep kit (Thermo Scientific, Lithuania) and were subjected to transformation by heat shock method using E.coli JM107 as recipient. Transformants were selected on LB agar with 100μg/ml of ampicillin and 0.25μg/ml of imipenem. Southern blotting was performed on agarose gel by in-gel hybridization with the bla KPC and bla NDM probe labeled with digoxigenin DIG-high prime labeling mix (Roche, Germany) detection Kit. Plasmid DNA from transformants was separated by pulsed field gel electrophoresis (PFGE) (CHEF DR-III System, Bio-Rad; USA) and was transferred to nylon membrane (Hybond N, Amersham, UK), hybridized with prepared probes. Detection was performed using NBT color detection Kit (Roche, Germany).

Restriction digestion of plasmid encoding bla KPC-2
Plasmids of the transformants were digested with XbaI, EcoR1 and HindIII separately. The digested products were separated by PFGE for 14 hour at 4 V/cm at an included angle of 120°a nd band patterns were analyzed. Further, the fragments were cloned in pGEM-T Vector (Promega, Madison, USA) as per manufacturer's instruction and were sequenced.

Strain typing
The heterogeneity in the isolates was determined by Pulse field gel electrophoresis (PFGE) as well as by repetitive extragenic palindromic (REP) PCR and enterobacterial repetitive intergenic consensus (ERIC) PCR [15]. Pulse field gel electrophoresis was performed using Xba1 restriction enzyme and the DNA fragments were separated with a CHEF-DR III apparatus for 22 hours at 4 V/cm.

Results
Out of 88 P. aeruginosa tested, two were harboring bla KPC gene, confirmed as encoding KPC-2.
The first isolate (PA-529) was obtained from a 13 years-old male patient, who attended the clinic in September 2012 and was diagnosed with urinary tract infection and the second isolate (PA-551) was collected from pus sample of 18-years-old female patient admitted to surgery ward in November 2012. Restriction analysis and sequencing revealed KPC-2 was present in two different locations within the host. In first, it was not associated with any mobile genetic element, whereas the second one was class 1 integron mediated attaining reverse orientation (Fig 1). Furthermore, 59 base element PCR confirmed bla KPC-2 was flanked by attC site. bla KPC-2 was also flanked by dfr16 and aadA2 in the upstream region while qac was located in the downstream area within the cassette. In both the isolates, bla KPC-2 was horizontally transferable as when transformants were selected with ampicillin and subsequently confirmed by Southern hybridization assay. An approximate 35 kb plasmid of Inc F type was isolated and restriction map of plasmids isolated from both the strains showed similar band pattern. Additionally, both the isolates were co-harboring bla NDM-1 . This gene could not be selected with ampicillin but selection with imipenem could successfully transfer NDM-1 and the gene was found to be located in Inc N type plasmid of approximately 26 kb in size. Interestingly, selection with imipenem could only select bla NDM-1 and not bla KPC-2 . The 35 kb plasmid was hybridized only with bla KPC-2 probe. MIC results ( Table 2) were indicative that bla KPC-2 was responsible for ertapenem resistance. Besides carbapenem resistance, PA-529 also showed resistance to co-trimoxazole, amikacin, gentamicin, netilmicin, ciprofloxacin and polymixin B whereas PA-551 was found to be susceptible towards gentamicin and polymixin B. PFGE pattern showed both the isolates were of different pulse types and which was also supported by ERIC and REP PCR results where two different clones were observed. In remaining test samples, carriage of bla NDM-1 was also observed in nine isolates and 16 were phenotypically positive for carbapenemase by modified Hodge test where molecular basis of carbapenem resistance could not be established by our target primers.

Discussion
KPC producing organisms have been described quite often from different parts of the world and more frequently from South America, US and Europe [16]. Carriage of KPC within an unnatural host i.e. P. aeruginosa is less frequently reported [2] as from India it has been on the record only from E. coli, K. pneumoniae and Proteus mirabilis [1]. Integron borne KPC is not very common around the world and there are few studies on the record that claim their presence within the gene capture mechanism [17]. Thus, our study suggests integron as a tool for horizontal dissemination of this resistance determinant in our hospital setting. However, the role of bla KPC-2 exhibiting beta-lactamase activity in reverse orientation is not clear and possibly the other copy of the same gene as observed in the study could play a role in carbapenem resistance. We have also recorded coexistence of bla NDM-1 in both the isolates as well as resistance towards most of the tested antibiotics, particularly indicates the selection pressure of different groups of beta-lactam antibiotics in the hospital environment of this part of the world. The antibiogram pattern was supported by the existing flanking resistance determinants of bla KPC-2 within integron conferring a phenotype with trimethoprim, quinolone and aminoglycosides resistance. In this study, it was observed that both the resistance determinants (bla KPC-2 and bla NDM-1 ) were located in two different genetic vehicles. Previous studies have already pointed out the need of investigation on extent of spread of KPC and its role in carbapenem resistance in India [18]. However, recent reports have been from western and southern part of India [3,18], we identified their expansion in northeastern part of this country as well. This study also established that co-acquisition or subsequent acquisition of bla KPC-2 and bla NDM-1 contributed a complete carbapenem resistance in this hospital setting.

Conclusion
To the best of our knowledge this is the first report of integron borne KPC-2 producing P. aeruginosa in India. This study advocates implementation of proper antimicrobial policy and infection control management in hospital settings so as to prevent horizontal spread of this resistant determinant within different host range.

Ethical Approval
The work was approved by Institutional Ethical committee of Assam University, Silchar vide Reference Number: IEC/AUS/C/2014-001. The authors confirm that participants provided their written informed consent to participate in this study.
Supporting Information S1 Table. Clinical history of patients & details of the samples collected for this study. No ethical, third party or legal restriction limit exists in sharing the data. (DOCX)