Occurrence of co-existing bla VIM-2 and bla NDM-1 in clinical isolates of Pseudomonas aeruginosa from India.

BACKGROUND: bla VIM-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 bla VIM-2 in clinical isolates of P. aeruginosa harbor bla NDM-1 as well which were collected from October 2012 to September 2013.
METHODS: Two P. aeruginosa strains harboring bla VIM-2 along with bla NDM-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: bla VIM-2 was found to be horizontally transferable through an Inc F type plasmid of approximately 30 kb in size. bla VIM-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 bla NDM-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 bla VIM-2 and bla NDM-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-β-lactamase [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, blaVIM-2 has now been spread as prevalent MBL among P. aeruginosa in all European countries [3] whereas blaNDM-1 and blaVIM-18 has known to be originated from India [4, 5] where irrational use of 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 blaVIM-2 and blaNDM-1 in clinical isolates of P. aeruginosa.

Methods

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 blaVIM and blaNDM gene

PCR assay was performed for detection of blaVIM as described earlier [8] as well as other metallo-β-lactamase gene (blaNDM, blaIMP, blaGIM, blaSIM, blaSMB) [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 blaTEM, blaPER, blaOXA-2, blaSHV, blaCTX-M, blaVEB and blaGES [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 blaVIM-2

Genetic environment of blaVIM-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 blaVIM were determined using two sets of primers 5′CS and the reverse primer of blaVIM, 3′CS and forward primer of blaVIM as described earlier [10]. Amplified products were sequenced to determine genetic map of blaVIM-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 blaNDM-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 blaNDM were determined using two sets of primers 5CS and the reverse primer of blaNDM, 3CS and forward primer of blaNDM [10]. The linkage of blaNDM-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 blaNDM-1 (5′-GTA GTG CTC AGT GTC GGC AT-3′) [11].

Plasmid analysis, transformation and conjugation assay

blaVIM 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 blaVIM-2 and blaNDM-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 A600. 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 blaNDM-1 and blaVIM-2 as donor and E. coli strain B as recipient.

Southern hybridization for detection of transferability

Southern blotting was performed on agarose gel by in-gel hybridization with the blaVIM-2 and blaNDM-1 probe labelled with DIG HIGH PRIME LABELING MIX (Roche, Germany) detection Kit. The digoxigenin-labeled blaVIM-2 and blaNDM-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 blaVIM and blaNDM specific probe. Detection was performed by using an NBT color detection Kit (Roche, Germany).

PCR based replicon typing

The incompatibility type of the plasmids encoding blaVIM-2 and blaNDM-1 were characterized by PCR based replicon typing targeting 18 different replicon types such as FIA, FIB, FIC, HI1, HI2, I1/Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F and FIIA [12].

Antibiotic susceptibility and minimum inhibitory concentration (MIC)

Antimicrobial susceptibility of parent strains as well as transformants was determined by Kirby-Bauer disc diffusion method against β-lactam and non-β-lactam antibiotics (Hi-Media, India). MIC was also determined by agar dilution method for the isolates and the transformants carrying PVIM−2 and PNDM−1 towards imipenem (Merck, France), meropenem (AstraZeneca, UK), cefepime, aztreonam (Aristo, India), amikacin (Zuche pharmaceuticals, India), gentamicin (Pharmakem, India), ciprofloxacin(Ranbaxy, India), piperacillin-tazobactam (Alkem, India) and polymixin-B (Samarth, India) and interpreted as per CLSI guidelines [13].

Stability of blaVIM-2 and blaNDM-1

Stability of blaVIM-2 and blaNDM-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 blaVIM and blaNDM by PCR assay.

Pulsed field gel electrophoresis

blaVIM and blaNDM 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 blaVIM were recovered from Silchar Medical College and Hospital, India. The gene was further sequenced and confirmed as blaVIM-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. blaVIM-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 blaVIM-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 blaVIM-2-aadB-dhfrA-orfC-qacE-sul1 (PA-37) and aadB-aacA7-blaVIM-2-dhfrA1-orfC-qacE-sul1 (PA-131). Both the isolates were co-harboring blaNDM-1 and further ESBL screening revealed the presence of blaVEB-1 gene in these isolates. However, blaNDM-1 gene could not be hybridized with 30 kb plasmid that was harboring blaVIM-2, but with a 24 kb plasmid which was successfully hybridized with blaNDM-1 specific probe (Fig. 2). Linkage of ISAba125 was observed in the upstream region of blaNDM-1 whereas no association with integron gene cassette could be established. Interestingly on analyzing the stability of MBL genes i.e. blaVIM-2 and blaNDM-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 blaVIM-2 while blaNDM-1was retained till ninety passages without any antibiotic pressure. These blaVIM-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 blaVIM-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).

Fig. 1

Genetic Context of bla VIM-2

Fig. 2

PFGE and hybridization analysis of bla NDM-1. Lane 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 PPA 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 PPA 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

Table 1

MIC (μg/ml) of the bla VIM-2 and bla NDM-1 harboring isolates and their transformants

Organisms	Imipenem	Meropenem	Cefepime	Aztreonam	Amikacin	Gentamicin	Ciprofloxacin	Piperacillin-tazobactam	Polymixin B
PA-37a	>256	>256	>256	>256	>256	>256	256	>256	>256
E. coli JM107(pVIM−2/37)b	64	32	128	64	128	64	64	64	32
E. coli JM107(pNDM−1/37)c	64	64	128	64	128	128	64	64	32
PA-131a	>256	128	>256	>256	>256	>256	256	>256	256
E. coli JM107(pVIM−2/131)b	16	16	64	64	64	64	64	64	32
E. coli JM107(pNDM−1/131)c	32	16	128	64	64	64	64	64	16
E. coli JM107	0.06	0.012	0.06	0.12	0.06	0.125	0.06	0.12	0.006

aDonor strain

bRecipient E. coli carrying plasmid of bla VIM-2

cRecipient E. coli carrying plasmid of bla NDM-1

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 blaNDM [4]. But in the present study, we described an additional carbapenem resistance determinant i.e. blaVIM-2 which played a significant role in carbapenem resistance. Although blaVIM-2 with integron gene cassette is reported in previous studies [8], while in the present study, the genetic context of blaVIM-2 underscores their diverse origin and persistence along with other resistant genes. In the present study blaVIM-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 aminoglycosides resistance genes aadB and aacA7 on the same gene cassette along with blaVIM-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 blaNDM-1 in Acinetobacter baumanii, we too observed the same insertion sequence present in the upstream region of blaNDM-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 blaNDM-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 blaVIM-2 and blaNDM-1 harboring wild strains and transformants of blaVIM-2 and blaNDM-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 blaVIM-2 may facilitate their mobilization to other susceptible organisms. On performing the transmission dynamics of the strains and it was evident that blaVIM-2 and blaNDM-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 blaVIM-2 and blaNDM-1 showed that blaNDM-1 gene is more stable compare to blaVIM-2, may be because E. coli is an unnatural host for blaVIM-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.