Occurrence of co-existing blaVIM-2 and blaNDM-1 in clinical isolates of Pseudomonas aeruginosa from India

Background blaVIM-2 harboring Pseudomonas aeruginosa has been reported worldwide and considered as the most prevalent metallo-β-lactamase after NDM which are found horizontally transferable and mostly associated with integron gene cassettes. The present study investigates the genetic background, transmission dynamics as well as stability of blaVIM-2 in clinical isolates of P. aeruginosa harbor blaNDM-1 as well which were collected from October 2012 to September 2013. Methods Two P. aeruginosa strains harboring blaVIM-2 along with blaNDM-1 were isolated from Silchar Medical College and Hospital, India. Genetic environment of these resistance determinants was determined and transferability was checked by transformation and conjugation assay which was further confirmed by Southern hybridization. Replicon typing was performed to determine the incompatibility group of the resistant plasmid and their stability was checked by serial passage method. Antimicrobial susceptibility pattern of the isolates was determined and their clonal relatedness was checked by pulsed field gel electrophoresis. Results blaVIM-2 was found to be horizontally transferable through an Inc F type plasmid of approximately 30 kb in size. blaVIM-2 was found to be associated with integron gene cassette and was flanked by two different types of cassette arrays. Both the isolates were co-harboring blaNDM-1 which was carried within Inc N type of plasmid with an approximate 24 kb in size and associated with ISAba125 in their upstream region. Reduced susceptibility rate as well as high MIC range was observed in case of wild strains and transformants carrying blaVIM-2 and blaNDM-1. Conclusions The detection of this co-existence of multiple carbapenem resistance genes in this part of world is worrisome and further investigation is required in order to trace the source and to initiate proper treatment option.


Background
Carbapenems are considered as the last drug of choice for most of the serious infections caused by Gram negative bacteria, but due to the prevalence of multidrug resistant organisms these lifesaving drugs were compromised in treating the patients with severe illness. Gram negative bacteria have been documented as the most serious threat in the management of acute infections, as of exhibiting resistance to antibiotics due to the production of carbapenemase enzyme especially metallo-βlactamase [1,2]. Pseudomonas aeruginosa is one of the most common nosocomial pathogen causing acute infections and frequently reported for carbapenem resistance. VIM metallo-β-lactamase, the second most predominant MBL type responsible for antimicrobial resistance, is pandemic and becoming a health hazard. Among the 37 variant of VIM enzymes, bla VIM-2 has now been spread as prevalent MBL among P. aeruginosa in all European countries [3] whereas bla NDM-1 and bla VIM-18 has known to be originated from India [4,5] where irrational use of

Annals of Clinical Microbiology and Antimicrobials
antibiotic is a major contributor for their emergence [6]. VIM and NDM type of MBLs are horizontally transferable and found to be associated with mobile genetic elements, however few molecular information and genetic context is available from this part of India. The potential for quick and extensive dissemination rate of these resistance genes make over a great concern. The present study describes genetic background, transmission dynamics as well as stability of bla VIM-2 and bla NDM-1 in clinical isolates of P. aeruginosa.

Bacterial collection
From October 2012 to September 2013, a total of seventeen P. aeruginosa with reduced susceptibility to at least a single carbapenem were collected from Silchar Medical College and Hospital, Silchar. The isolates were then identified by standard biochemical reactions, cytochrome oxidase activity, citrate utilization, pigment production and growth on Cetrimide agar [7].

Characterization of carbapenemases
For detection of carbapenemase production, isolates were subjected to modified Hodge test (MHT) and imipenem-EDTA disc test for metallo-β-lactamase production [4]. MHT uses Escherichia coli ATCC 25922 as an indicator organism. The presence of clover leaf like indentation in MHT was interpreted as a positive result for carbapenem hydrolysis.

Molecular characterization of bla VIM and bla NDM gene
PCR assay was performed for detection of bla VIM as described earlier [8] as well as other metallo-β-lactamase gene (bla NDM , bla IMP , bla GIM , bla SIM , bla SMB ) [4] and amplified products were further sequenced to confirm the presence of resistant gene.

Detection of co-existing of ESBL genes
Co-existence of ESBL genes were determined by multiplex PCR targeting bla TEM , bla PER , bla OXA-2 , bla SHV , bla CTX-M , bla VEB and bla GES [9]. Reactions were performed under the following conditions: initial denaturation at 94 °C for 5 min, 33 cycles of 94 °C for 35 s, 51 °C for 1 min, 72 °C for 1 min and final extension at 72 °C for 7 min. The amplified products were further sequenced to confirm the co-existence of ESBL genes.

Detection of genetic context of bla VIM-2
Genetic environment of bla VIM-2 was determined by performing integrase gene PCR [10] for characterizing the class 1 and class 2 integron. The conserved sequences 5′CS and 3′CS flanking the bla VIM were determined using two sets of primers 5′CS and the reverse primer of bla VIM , 3′CS and forward primer of bla VIM as described earlier [10]. Amplified products were sequenced to determine genetic map of bla VIM-2 . The reaction condition was: initial denaturation at 95 °C for 3 min, 34 cycles at 95 °C for 30 s, 46 °C for 1 min, 72 °C for 3 min and final extension at 72 °C for 7 min. The presence of mobile element like ISCR in each strain was determined by performing PCR using primers ISCR F (5′-RNSBATAGGAADWWNAAHMNV-3′) and ISCR R (5′-BNKDTTNWWHTTCCTATVSNY-3′).

Determination of genetic environment of bla NDM-1
Integron carriage was assessed by performing integrase gene PCR for characterizing the class 1 and class 2 integron. PCR reaction conditions followed was described as earlier [10]. The flanking region of bla NDM were determined using two sets of primers 5CS and the reverse primer of bla NDM , 3CS and forward primer of bla NDM [10]. The linkage of bla NDM-1 with insertion sequence ISAba125 was determined by using forward primer of ISAba125 (5′GAA ACT GTC GCA CCT CAT GTT TG-3′) and reverse of bla NDM-1 (5′-GTA GTG CTC AGT GTC GGC AT-3′) [11].

Plasmid analysis, transformation and conjugation assay
bla VIM positive bacterial isolates were cultured in Luria-Bertani broth (Hi-Media, India) containing 0.25 μg/ml of imipenem. After overnight incubation, plasmids were extracted by using QIAprep Spin Miniprep Kit (Qiagen, Germany). Isolated plasmids were transformed into recipient strain Escherichia coli JM107 by heat shock method and transformants were selected on LB agar with 0.25μg/ml of imipenem. Conjugation experiment was carried out using bla VIM-2 and bla NDM-1 harboring transformants as donors and a streptomycin resistant E. coli recipient strain B (Genei, India), both the donor and reciepient cells were cultured in Luria-Bertani Broth (Hi-Media, India) till it reach an O.D. of 0.8-0.9 at A 600 . Cells were mixed at 1:5 donor-to-recipient ratios and transconjugants were selected on imipenem (0.25 μg/ml) and streptomycin (400 μg/ml) agar plates. Additionally conjugation experiment was also tried using P. aeruginosa harboring bla NDM-1 and bla VIM-2 as donor and E. coli strain B as recipient.

Southern hybridization for detection of transferability
Southern blotting was performed on agarose gel by ingel hybridization with the bla VIM-2 and bla NDM-1 probe labelled with DIG HIGH PRIME LABELING MIX (Roche, Germany) detection Kit. The digoxigenin-labeled bla VIM-2 and bla NDM-1 specific probe was prepared using primers VIM-F, VIM-R, NDM-F and NDM-R. Plasmid DNA from transformants and transconjugants was separated by PFGE (CHEF DR-III System, Bio-Rad; USA) and transferred to nylon membrane (Hybond N, UK) and then hybridised with prepared bla VIM and bla NDM specific probe. Detection was performed by using an NBT color detection Kit (Roche, Germany).

Stability of bla VIM-2 and bla NDM-1
Stability of bla VIM-2 and bla NDM-1 gene was determined by serial passage of the isolates as well as of the transformants in 1:1000 ratios without antibiotic pressure [14].
After each passage the test isolates were subjected to phenotypic detection of MBL and further confirmed the presence of bla VIM and bla NDM by PCR assay.

Pulsed field gel electrophoresis
bla VIM and bla NDM positive isolates were typed by pulsed field gel electrophoresis where chromosomal DNA was prepared in agarose blocks and digested with restriction enzyme Xba1 (Promega, USA). DNA fragments were separated with CHEF-DR III apparatus (Bio-Rad, USA) and the electrophoresis conditions used were for 24 h at 6 V/cm with pulse rate of 10-40 s as described previously [15]. Clonal relatedness within the isolates was determined by comparing the band patterns.

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.

Results
Two clinical isolates of P. aeruginosa (PA-37 and PA-131) harboring bla VIM were recovered from Silchar Medical College and Hospital, India. The gene was further sequenced and confirmed as bla VIM-2 variant. The first isolate (PA-37) was recovered from pus samples of a 55 year old female patient suffering from wound infection admitted in surgery ward in December 2012 while the second one (PA-131) was isolated from urine of a 40 years old female patient with UTI, who attended gynecology outpatient department (OPD) in the month of February 2013. bla VIM-2 was found to be horizontally transferable as the gene could be successfully conjugatively transferred from transformed E. coli JM107 to recipient E. coli strain B through an Inc F type plasmid having approximate size of 30 kb. These findings were further confirmed by Southern hybridization results. However, conjugative transfer of plasmids from P. aeruginosa to E. coli was not successful with our experiment. In both the isolates bla VIM-2 was located within integron gene cassette and was flanked by other antimicrobial resistant determinant like gene for aminoglycoside resistance (Fig. 1). The two different types of cassette arrays observed were bla VIM-2 -aadB-dhfrA-orfC-qacE-sul1 (PA-37) and aadB-aacA7bla VIM-2 -dhfrA1-orfC-qacE-sul1 (PA-131). Both the isolates were co-harboring bla NDM-1 and further ESBL screening revealed the presence of bla VEB-1 gene in these isolates. However, bla NDM-1 gene could not be hybridized with 30 kb plasmid that was harboring bla VIM-2 , but with a 24 kb plasmid which was successfully hybridized with bla NDM-1 specific probe (Fig. 2). Linkage of ISAba125 was observed in the upstream region of bla NDM-1 whereas no association with integron gene cassette could be established. Interestingly on analyzing the stability of MBL genes i.e. bla VIM-2 and bla NDM-1 , in case of wild type isolates these resistant genes were stable even after hundred consecutive passages but among their transformants, complete plasmid was lost after fifty-six passages in case of bla VIM-2 while bla NDM-1 was retained till ninety passages without any antibiotic pressure. These bla VIM-2 positive isolates showed resistance towards most of the antibiotics including piperacillin/tazobactam, co-trimoxazole, amikacin, gentamicin, netilmicin and quinolone group of drugs. MIC values obtained for both the isolates were above breakpoint towards ciprofloxacin, aminoglycosides, β-lactam-β-lactamase inhibitor as well as to polymixin B ( Table 1). The two bla VIM-2 harboring isolates of P. aeruginosa were found to be clonally different from each other on the basis of their PFGE banding pattern (Fig. 2).

Discussion
It has been evidenced that the rapid emergence and dissemination of carbapenemase producing bacteria in this subcontinent is mainly due to the acquisition of bla NDM [4]. But in the present study, we described an additional carbapenem resistance determinant i.e. bla VIM-2 which played a significant role in carbapenem resistance. Although bla VIM-2 with integron gene cassette is reported in previous studies [8], while in the present study, the genetic context of bla VIM-2 underscores their diverse origin and persistence along with other resistant genes. In the present study bla VIM-2 was found to be associated with the gene cassette along with other resistance determinants which is in agreement to the reports of Toleman et al. [16]. The association of other resistance determinants along with VIM type MBL confers the phenotype to become resistant to most of the available antimicrobial agents. The study reports the presence of  1 and 4 The undigested total DNA of PA 37 and PA 131 respectively; Lane 2 and 3 The PA 37 total DNA is digested with S1 and Xba I respectively; Lane 5 and 6 PA 131 total DNA digested with S1 and Xba I respectively; Lane 7 The undigested plasmid of PA 37; Lane 8 and 9 E. coli transconjugant carrying plasmid of PA 37 digested with S1 and Xba I respectively; Lane 10 Hybridized P PA 37 with probe; Lane 11 The undigested plasmid of PA 131; Lane 12 and 13 E. coli transconjugant carrying plasmid of PA 131 digested with S1 and Xba I respectively; Lane 14 Hybridized P PA 131 with probe; Lane 15 The total DNA of recipient E. coli without plasmid; Lane 16 and 17 The total DNA of recipient E. coli without plasmid digested with S1 and Xba I respectively aminoglycosides resistance genes aadB and aacA7 on the same gene cassette along with bla VIM-2 , thus making the phenotype resistance to amikacin and gentamicin as well.
In the year 2012, Toleman et al. [11] reported the association of insertion sequence ISAba125 in the upstream region of bla NDM-1 in Acinetobacter baumanii, we too observed the same insertion sequence present in the upstream region of bla NDM-1 in P. aeruginosa. Presence of NDM-1 in P. aeruginosa was for first time recorded in 2011 from patients in Serbia [17] and the same working group has reported that resistance determinant is chromosomally located in this particular organism [18]. Similarly, a report from India also has established its chromosomal location [19]. However, in our recent study we found presence of bla NDM-1 on plasmid DNA indicating a possible shift from one to another genetic location [20]. Future experiments will show which direction (chromosome to plasmid or vice versa) of the transfer took place in the organism. High MIC range as well as reduced susceptibility rate against majority of the tested antibiotics was observed in case of bla VIM-2 and bla NDM-1 harboring wild strains and transformants of bla VIM-2 and bla NDM-1 . Earlier studies reported [16] that carbapenemase producing isolates remain susceptible to polymixin B whereas both of our study isolates were found to be resistant to this antibiotic, which could be a challenging situation with no or too limited treatment option. The association of mobile genetic element with bla VIM-2 may facilitate their mobilization to other susceptible organisms. On performing the transmission dynamics of the strains and it was evident that bla VIM-2 and bla NDM-1 was horizontally transferable. It may be noted that in our study, the plasmid of P. aeruginosa encoding the NDM-1 was conjugally transferred from the E. coli transformants to recipient E. coli strain although conjugation of the same plasmid was not successful from original host P. aeruginosa to E. coli. The reason for absence of plasmid conjugation could be due to presence of some physiological barriers within these two strains or we did not find the appropriate laboratory conditions for initiation of conjugation or present plasmid do not carry functional Tra operon in P. aeruginosa. Serial passage of transformants harboring both bla VIM-2 and bla NDM-1 showed that bla NDM-1 gene is more stable compare to bla VIM-2 , may be because E. coli is an unnatural host for bla VIM-2 . The distinguishable pulsotypes of the two strains along with their different genetic arrangements indicates horizontal acquisition from diverse source and antibiotic pressure in this hospital setting.

Conclusions
Co-existence of multiple carbapenem resistance determinants in hospital isolate is worrisome and a matter of concern for infection control management considering the treatment option and clonal expansion. Thus, the current finding is of epidemiological interest, which requires immediate steps to initiate proper treatment option.