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Occurrence of co-existing bla VIM-2 and bla NDM-1 in clinical isolates of Pseudomonas aeruginosa from India
Annals of Clinical Microbiology and Antimicrobials volume 15, Article number: 31 (2016)
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.
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.
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.
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.
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, bla VIM-2 has now been spread as prevalent MBL among P. aeruginosa in all European countries  whereas bla NDM-1 and bla VIM-18 has known to be originated from India [4, 5] where irrational use of antibiotic is a major contributor for their emergence . 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.
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 .
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 . 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  as well as other metallo-β-lactamase gene (bla NDM, bla IMP, bla GIM, bla SIM, bla SMB)  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 . 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  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 . 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 . 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 . 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′) .
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 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 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 in-gel 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).
PCR based replicon typing
The incompatibility type of the plasmids encoding bla VIM-2 and bla NDM-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 .
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 .
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 . 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 . Clonal relatedness within the isolates was determined by comparing the band patterns.
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.
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-aacA7-bla 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-1was 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).
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 . 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 , 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. . 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 bla VIM-2, thus making the phenotype resistance to amikacin and gentamicin as well. In the year 2012, Toleman et al.  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  and the same working group has reported that resistance determinant is chromosomally located in this particular organism . Similarly, a report from India also has established its chromosomal location . 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 . 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  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.
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.
Verona integron-encoded MBL
New Delhi metallo-β-lactamase
- Bla :
modified Hodge test
polymerase chain reaction
extended spectrum beta-lactamase
- 5CS & 3CS:
5′ & 3′-conserved segments
insertion sequence common region
minimum inhibitory concentration
pulse field gel electrophoresis
urinary tract infection
Clinical and Laboratory Standards Institute
Patel G, Bonomo RA. “Stormy waters ahead”: global emergence of carbapenemases. Front Microbiol. 2013;48:1–17.
Cornaglia G, Giamarellou H, Rossolini GM. Metallo-β-lactamase: a last frontier for β lactams? Lancet Infect Dis. 2011;11:381–93.
Tato M, Coque TM, Baquero F, Canton R. Dispersal of carbapenemase bla VIM-1 gene associated with different Tn402 variants, mercury transposons and conjugative plasmids in enterobacteriaceae and Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2010;54:320–7.
Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K. Characterization of a New Metallo-β-Lactamase Gene, bla NDM-1, and a Novel Erythromycin Esterase Gene Carried on a Unique Genetic Structure in Klebsiella pneumoniae Sequence Type 14 from India. Antimicrob Agents Chemother. 2009;53:5046–54.
Castanheira M, Bell JM, Turnidge JD, Mathai D, Jones RN. Carbapenem resistance among Pseudomonas aeruginosa strains from India: evidence for nationwide endemicity of multiple metallo-beta-lactamase clones (VIM-2,-5,-6, and -11 and the newly characterized VIM-18). Antimicrob Agents Chemother. 2009;53:1225–7.
Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, Chaudhary U, Doumith M, Giske CG, Irfan S, Krishnan P, Kumar AV, Maharjan S, Mushtaq S, Noorie T, Paterson D, Pearson A, Perry C, Pike R, Rao B, Ray U, Sarma JB, Sharma M, Sheridan E, Thirunarayan MA, Turton J, Upadhyay S, Warner M, Welfare W, Livermore DM, Woodford N. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis. 2010;10:597–602.
Colee JG, Diguid JP, Mackie Fraser AG, Microbiology McCartney Practical Medical. 14th edn. Edinburgh: Churchill, Livingstone; 1996.
Jh Y. Yi K, Lee H, Yong D, Lee K, Kim JM, Rossolini GM, Chong Y. Molecular characterization of metallo-β-lacatamase-producing Acinetobacter baumannii and Acinetbacter genomospecies 3 from Korea: identification of two new integrons carrying the bla VIM-2 gene cassettes. J Antimicrob Chemother. 2002;49:837–40.
Lee S, Park YJ, Kim M, Lee HK, Han K, Kang CS. Prevalence of Ambler class A and D β- lactamases among clinical isolates of Pseudomonas aeruginosa in Korea. J Antimicrob Chemother. 2005;56:122–7.
Koeleman JGM, Stoof J, Der Bijl MWV, Vandenbroucke-grauls CMJE, Savelkoul PHM. Identification of Epidemic Strains of Acinetobacter baumannii by Integrase Gene PCR. J Clin Microbiol. 2001;39:8–13.
Toleman MA, Spencer J, Jones L, Walsh TR. bla NDM-1 is a chimera likely constructed in Acinetobacter baumannii. Antimicrob Agents Chemother. 2012;56:2773.
Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005;63:219–28.
Clinical Laboratory Standard Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third Informational Supplement. 2013; M100-S23. CLSI, Wayne.
Locke JB, Rahawi S, LaMarre J, Mankin LS, Shawa KJ. Genetic Environment and Stability of cfr in Methicillin-resistant Staphylococcus aureus CM05. Antimicrob Agents and Chemother. 2012;56:332–40.
Tato M, Coque TM, Ruiz-Garbajosa P, Pintado V, Cobo J, Sader HS, Jones RN, Baquero F. Complex clonal and plasmid epidemiology in the first outbreak of enterobacteriaceae infection involving VIM-1 metallo-β-lactamase in Spain: towards endemicity? Clin Infect Dis. 2007;45:1171–8.
Toleman MA, Vinodh H, Sekar U, Kamat V, Walsh TR. bla VIM-2- harboring integrons isolated in India, Russia, and the United States arise from an ancestral class 1 integron predating the formation of the 3/conserved sequence. Antimicrob Agents and Chemother. 2007;51:2636–8.
Jovcic B, Lepsanovic Z, Suljagic V, Rackov G, Begovic J, Topisirovic L, Kojic M. Emergence of NDM-1 metallo-β-lactamase in Pseudomonas aeruginosa clinical isolates from Servia. Antimicrob Agents Chemother. 2011;55:3929–31.
Jovcic B, Lepsanovic Z, Begovic J, Rakonjac B, Perovanovic J, Topisirovic L, Kojic M. The clinical isolate Pseudomonas aeruginosa MMA83 carries two copies of the bla NDM-1 gene in a novel genetic context. Antimicrob Agents Chemother. 2013;57:3405–7.
Mishra S, Upadhyay S, Sen MR, Maurya AP, Choudhury D, Bhattacharjee A. Genetic Acquisition of NDM Gene Offers Sustainability among Clinical Isolates of Pseudomonas aeruginosa in Clinical Settings. PLoS ONE. 2015;10(1):e0116611. doi:10.1371/journal.pone.0116611.
Paul D, Dhar Chanda D, Maurya AP, Mishra S, Chakravarty A, Sharma GD, Bhattacharjee A. Co-Carriage of bla KPC-2 and bla NDM-1 in Clinical Isolates of Pseudomonas aeruginosa Associated with Hospital Infections from India. PLoS ONE. 2015;10(12):e0145823. doi:10.1371/journal.pone.0145823.
DP: Design and performed the experimental work, literature search, data collection, analysis and prepared the manuscript. DD: Participated in experiment designing and manuscript correction. APM: Participated in sample collection and part of experiments. SM: Carried out the Southern hybridization experiment and result analysis. AC: Participated in experiment designing and manuscript correction. GDS: Participated in drafting the manuscript. AB: Supervised the research work and participated in designing the study and drafting the manuscript. All authors read and approved the final manuscript.
The authors would like to acknowledge the support of HOD, Microbiology, Assam University for providing infrastructural facility. The authors sincerely acknowledge the financial support provided by Council of Scientific and Industrial Research and Department of Biotechnology (DBT-NER twinning Scheme) to carry out the work. Authors also acknowledge the help from Assam University Biotech Hub for providing laboratory facility to complete this work.
The authors declare that they have no competing interests.
Council of Scientific and Industrial Research (CSIR Grant Number: 37(1632)/14/EMR-II) and Department of Bio-technology (DBT-NER Twinning Scheme, BT/215/NE/TBP/2011 dtd.15/11/2011) Government of India.
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Paul, D., Dhar, D., Maurya, A.P. et al. Occurrence of co-existing bla VIM-2 and bla NDM-1 in clinical isolates of Pseudomonas aeruginosa from India. Ann Clin Microbiol Antimicrob 15, 31 (2016). https://doi.org/10.1186/s12941-016-0146-0
- bla VIM-2
- bla NDM-1
- Inc F type
- Pseudomonas aeruginosa