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  Table of Contents  
Year : 2014  |  Volume : 32  |  Issue : 2  |  Page : 191-193

Emergence of VIM-2 metallo-beta-lactamase producing Ralstonia pickettii clinical isolate in India

1 Department of Microbiology, Armed Forces Medical College, Pune, Maharashtra, India
2 All India Institute of Medical Sciences, Bhubaneshwar, Odisha, India

Date of Submission09-Jun-2013
Date of Acceptance06-Nov-2013
Date of Web Publication2-Apr-2014

Correspondence Address:
A Khajuria
Department of Microbiology, Armed Forces Medical College, Pune, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.129831

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 ~ Abstract 

A multidrug-resistant clinical isolate of Ralstonia pickettii from a woman was analysed. Modified Hodge test was positive for carbapenemase production. Conjugation experiment revealed the presence of conjugative plasmid of >140 Kb size typed as IncN type. This is the first report of emergence blaVIM-2 in R. pickettii in India.

Keywords: Bla VIM-2 , IncN type, R. pickettii, Ralstonia. pickettii

How to cite this article:
Khajuria A, Praharaj A K, Grover N, Kumar M. Emergence of VIM-2 metallo-beta-lactamase producing Ralstonia pickettii clinical isolate in India. Indian J Med Microbiol 2014;32:191-3

How to cite this URL:
Khajuria A, Praharaj A K, Grover N, Kumar M. Emergence of VIM-2 metallo-beta-lactamase producing Ralstonia pickettii clinical isolate in India. Indian J Med Microbiol [serial online] 2014 [cited 2020 Aug 11];32:191-3. Available from:

 ~ Introduction Top

Non-fermenting Gram-negative bacilli are an emerging cause of hospital-acquired infection. The main opportunistic pathogens in this group are Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. Ralstonia pickettii is an opportunistic pathogen from the non-fermenting group of Gram-negative organisms, commonly grouped with Burkholderia cepacia-like organisms. R. pickettii has also been recognized since the last decade as a nosocomial pathogen particularly associated with individuals who are debilitated or immune-suppressed and is involved in causing multiple infections such as endocarditis, meningitis, osteomyelitis, seminal infection and peritonitis. [1] Several outbreaks caused by this bacterium arising from contaminated intravenous products used in hospital care have been described. [2],[3],[4] This bacterium is inherently resistant to penicillins, narrow-spectrum cephalosporins, ceftazidime and aztreonam. In the literature, OXA-22 and OXA-60 are two types of class D β-lactamases that have been identified in this species. [5] However, so far, no R. pickettii clinical isolate carrying carbapenem-resistance genes have been described. In this report, we describe a carbapenem-resistant R. pickettii isolate producing a VIM-2 metallo- β -lactamase.

 ~ Case Report Top

In Nov 2012, a 55-year-old female patient was transferred from peripheral hospital to our tertiary care centre. The patient presented with complaints of multiple episodes of vomiting for 2 days, fever for 5 days associated with altered sensorium and had an episode of generalized tonic clonic seizures (GTCS). MRI showed a 7.1 × 4.5 × 6.9 cms intraxial mass lesion in right frontotemporal region without contrast enhancement. This lesion was causing mass effect in the form of subfalcine and transtentorial herniation. On investigation the patient was found to have persistent leucopenia (700-2100/cu mm 3 ). While being evaluated for leucopenia patient deteriorated neurologically and was taken up for an emergency surgery. Right fronto-temporal craniotomy and gross total resection of tumour was done. Histopathological examination of the tumour showed Anaplastic Astrocytoma (WHO grade III). Post operatively the patient was put on ventilator support and treated with antibiotics vancomycin, meropenem and amikacin. Extubation was done after 48 hrs and patient was shifted to Neurosurgical ward. Seventy-two hours post-surgery, the patient developed seropurulent discharge from the surgical site along with features suggestive of septicaemia. She was febrile with a temperature 39.5°C, pulse rate 110/min and blood pressure150/90 mmHg, laboratory parameters revealed Hb of 8.7 gm/dl, leucopenia with a total leucocyte count of 1500/cu mm 3 and peripheral smear showed microcytic hypochromic anaemia and toxic granules in neutrophils suggestive septicaemia. Pus from the surgical site and blood was sent for culture which grew non-lactose-fermenting Gram-negative bacilli identified by VITEK-2 as R. pickettii. Biochemical reaction also carried out manually showed catalase, urease and oxidase positive, 2-ketogluconate utilization and nitrate reduction were also positive. The isolate was negative for orthonitrophenyl-β-Dgalactopyranoside, aesculine hydrolysis, lysine and orinthine decarboxylaion. The biochemical reactions were in conformity with that of VITEK2. The antibiotic sensitivity test was performed by standard Kirby-Bauer disc diffusion technique as per the guidelines of the Clinical Laboratory Standards Institute (CLSI) with commercially available discs (Hi Media, Mumbai, India) on Mueller-Hinton agar plates. [6] Minimum inhibitory concentrations (MIC) of antibiotics were determined by VITEK-2 against imipenem, meropenem, ticarcillin, amikacin, gentamicin, tobramycin, moxifloxacin, ciprofloxacin, levofloxacin, tigecycline, trimethoprim/sulfamethoxazole, piperacillin/tazobactam, cefoperazone/sulbactam, ampicillin/sulbactam, cefepime, tetracycline, ceftazidime, ceftriaxone and colistin [Table 1]. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 strains were used for quality control. As the isolate showed a reduced susceptibility to meropenem and imipenem (diameter of zones of inhibition, ≤13 mm) by disc diffusion, screening for the production of Carbapenemase was done by the Modified Hodge Test using meropenem disc. The test was interpreted as positive with a clover leaf-type indentation at the intersection of the test organism and the E. coli ATCC 25922. Screening for metallo- β-lactamase (MBL) production was carried out by the MBL (IP/IPI) E-test method (AB Biodisk, Solna, Sweden) as per manufacturer's instructions and was found to be positive for MBL. Molecular detection for metallo-β-lactamases was carried out as follows: Briefly DNA was extracted using the spin column method (QIAGEN; GmbH, Hilden, Germany) as per manufacturer's instructions. PCR amplification for Ambler class B MBLs: bla IMP, blaVIM , blaSPM , blaGIM , blaSIM and blaNDM-1 and Ambler class D blaOXA-23 , blaOXA-24 and blaOXA-48 was carried out on the isolates by using Gene Amp 9700 PCR System (Applied Biosystems, Singapore). PCR conditions and primers used was described as earlier [7] and PCR was found to be positive for bla VIM gene [Figure 1]. The amplicon of bla VIM was purified using QIAquick PCR purification kit (QIAGEN; GmbH, Hilden, Germany) and sequenced with the ABI 3730XL capillary sequencer (Applied Biosystems, Foster City, CA, USA). Sequencing results were analyzed using the Blast software on the website of the National Center for Biotechnology Information ( and showed the presence of blaVIM-2 gene. For genotypic conformation the isolate was further subjected to 16S r RNA gene sequence analysis and the isolate showed a sequence identity value of 100% to R. pickettii. Plasmid DNA extraction was performed using a Plasmid Mini Prep Kit (QIAGEN; GmbH, Hilden, Germany) according to manufacturer's instructions. To determine transferability of resistance via plasmid, conjugation was assayed by mating experiment in Luria-Bertani broth using R. pickettii isolate (Parental strain) as donor and an azide-resistant E. coli J53 as the recipient strain using 1:10 ratio. The transconjugants were selected on Luria-Bertani agar with selection based on growth on agar in the presence of ceftazidime (30 mg/L) and sodium azide (100 mg/L). E. coli J53 recipient strain showed an MBL phenotype. Plasmids were separated by electrophoresis on horizontal 0.8% agarose gels at 50 V for 3 h. The size of the plasmid was compared by co-electrophoresis with plasmids of known sizes from E. coli strains V517 and 39R861. DNA bands were visualised with an UV transilluminator after staining with 0.05% ethidium bromide. In this study, R. pickettii harboured a plasmid of >140 kb in size. Presence of blaVIM-2 gene in the transconjugants was confirmed by PCR and sequencing. The plasmids purified from the clinical isolate and transconjugants were typed by PCR-based replicon typing (PBRT) [8] which demonstrated that plasmid belonged to the Inc N type.
Figure 1: Electrophoretic banding pattern on 1.5% agarose gel showing PCR amplifi ed products of blaVIM (399 bp). Lane1- E. coli ATCC 25922, Lane2- Negative Control, Lane 3-Positive control for VIM, Lane 4-Ralstonia pickettii and M represents; 100 bp DNA ladder used as the molecular size standard

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Table 1: Antibiogram of blavim-2-positive R. pickettii and its transconjugant E. coli J53 (mg/L)

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 ~ Discussion Top

We reported a case of a woman having Anaplastic Astrocytoma (WHO grade III). Seventy-two hours post-surgery the patient developed seropurulent discharge from the surgical site. From the blood and surgical site R. pickettii was isolated. Based upon antibiogram the patient was successfully treated with tigecycline with a loading dose of 100 mg given by infusion, followed by 50 mg intravenously twice a day. After 2 weeks of tigecycline therapy in addition to intensive daily wound care, patient recovered well. In our case, multidrug-resistant R. pickettii an emerging pathogen caused the infection, requiring therapy with tigecycline. The presence of blaVIM-2 in the organism poses a serious threat for the spread of carbapenemase-resistant hospital-acquired infection. The emergence of new opportunistic pathogen has been linked to a multi-resistant phenotype that makes them refractory to the common antibiotics used in clinical practice. Carbapenem resistance has not been reported earlier in R. pickettii. With the worldwide increase in the rate of dissemination of resistant determinant genes, early detection is important. The benefits of early detection include timely implementation of strict infection control practices as well as clinical guidance regarding the potential risks for therapeutic failure. As R. pickettii is present in hospital environment and in nature, it is important that the isolates from cases should be screened for MBL and genotypic studies for resistance should be carried out. The nucleotide sequence data of the 16S ribosomal RNA and blaVIM-2 gene from the isolate of R. pickettii reported in the present study have been assigned GenBank nucleotide numbers KC599270 and KC576908, respectively

 ~ References Top

1.Ryan MP, Pembroke JT, Adley CC. Ralstonia pickettii: A persistent gram-negative nosocomial infectious organism. J Hosp Infect 2006;62:278-84.  Back to cited text no. 1
2.Roberts LA, Collignon PJ, Cramp VB, Alexander S, McFarlane AE, Graham E, et al. An Australia-wide epidemic of Pseudomonas pickettii bacteraemia due to contaminated "sterile" water for injection. Med J Aust 1990;152:652-5.  Back to cited text no. 2
3.Fernandez C, Wilhelmi I, Andradas E, Gaspar C, Gomez J, Romero J, et al. Nosocomial outbreak of Burkholderia pickettii infection due to a manufactured intravenous product used in three hospitals. Clin Infect Dis 1996;22:1092-5.  Back to cited text no. 3
4.Labarca JA, Trick WE, Peterson CL, Carson LA, Holt SC, Arduino MJ, et al. A multistate nosocomial outbreak of Ralstonia pickettii colonization associated with an intrinsically contaminated respiratory care solution. Clin Infect Dis 1999;29:1281-6.  Back to cited text no. 4
5.Poirel L, Naas T Nordmann P. Diversity, epidemiology and genetics of class D beta-lactamases. Antimicrob Agents Chemother 2010;54:24-38.  Back to cited text no. 5
6.Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty second Informational Supplement M100-S22. Wayne: CLSI; 2012.  Back to cited text no. 6
7.Khajuria A, Praharaj AK, Grover N, Kumar M. First Report of blaNDM-1 in Raoultella ornithinolytica. Antimicrob Agents Chemother 2013;57:1092-3.  Back to cited text no. 7
8.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.  Back to cited text no. 8


  [Figure 1]

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