This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Castanheira, M.
Right arrow Articles by Jones, R. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Castanheira, M.
Right arrow Articles by Jones, R. N.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, March 2009, p. 1225-1227, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.01011-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Carbapenem Resistance among Pseudomonas aeruginosa Strains from India: Evidence for Nationwide Endemicity of Multiple Metallo-β-Lactamase Clones (VIM-2, -5, -6, and -11 and the Newly Characterized VIM-18){triangledown}

Mariana Castanheira,1* Jan M. Bell,2 John D. Turnidge,2 Dillip Mathai,3 and Ronald N. Jones1,4

JMI Laboratories, North Liberty, Iowa 52317,1 Women's and Children's Hospital, Adelaide, Australia,2 Christian Medical College, Vellore, India,3 Tufts University School of Medicine, Boston, Massachusetts 021104

Received 28 July 2008/ Returned for modification 31 August 2008/ Accepted 21 December 2008


arrow
ABSTRACT
 
Among 57 metallo-β-lactamase (MβL)-producing Pseudomonas spp. detected in India during 2006, five blaVIM genes were found, including a newly characterized blaVIM gene. Pseudomonas aeruginosa strains were clustered in 33 ribotypes with clones found in multiple hospitals. Several types of blaVIM-2-carrying integrons were detected. The newly characterized variant VIM-18 showed a 4-amino-acid deletion compared to other VIM variants. In this study, we show that VIM-producing Pseudomonas spp. were highly prevalent in India with a great diversity of blaVIM types and MβL-carrying integrons.


arrow
INTRODUCTION
 
Among the types of metallo-β-lactamases (MβLs) reported to date, IMP and VIM variants are the most prevalent and have been described from numerous geographic locations (9). VIM-2 was first reported from a Pseudomonas aeruginosa strain recovered during 1996 in a hospital located in Marseilles, France (5). Among VIM enzymes, VIM-2 appears to be the most dominant allelic variant and isolates carrying this enzyme have been described in more than 23 countries, including India (7).

In this study, initiated to characterize MβL-producing Pseudomonas isolates, we observed that such strains are endemic in India, with a great diversity of MβL types (five VIM variants) and different MβL-carrying integrons. In addition we characterized a VIM variant, herein named VIM-18.

(This study was partially presented as abstracts at the 18th European Congress of Microbiology and Infectious Diseases, Barcelona, Spain, 2008.)

A total of 301 Pseudomonas isolates were consecutively collected as previously described (3) in 10 Indian hospitals. The isolates were susceptibility tested against 28 antimicrobial agents according the Clinical and Laboratory Standards Institute (1) broth microdilution procedure and interpretation criteria. Among those isolates, 107 (35.5%) showed elevated MICs for imipenem or meropenem (MIC, ≥8 µg/ml). These isolates belonged to five different species: P. aeruginosa (97/107), Pseudomonas putida (7/107), and Pseudomonas stutzeri, Pseudomonas fluorescens, and Pseudomonas mendocina (1/107 each). Isolates nonsusceptible to imipenem or meropenem (MIC, ≥8 µg/ml) were tested with a multiplex PCR strategy, as recently described (4). Amplicons obtained were sequenced on both strands and analyzed.

MβL genes were detected in 57 (53.2% [18.9% of overall Pseudomonas spp.]) of the carbapenem-nonsusceptible strains. Five blaVIM variants were detected, and blaVIM-2 was detected in 38 (66.7% of the MβL producers) isolates, including 35 P. aeruginosa isolates, 2 P. putida isolates, and 1 P. stutzeri isolate. The second most common VIM-encoding gene was blaVIM-6, found in 12 (21.0%) P. aeruginosa isolates. blaVIM-11 was detected in one P. aeruginosa isolate and one P. stutzeri isolate, while blaVIM-5 was observed in one P. aeruginosa isolate. Additionally, one P. aeruginosa isolate was found to carry a new VIM variant, named VIM-18.

VIM-producing isolates were detected in 9 of 10 participating medical institutions, and VIM-2-producing strains were observed in 8 of the 10 hospitals (Table 1). In 5 of 10 hospitals, isolates harbored multiple MβL types (Table 1). VIM-encoding genes were detected in non-P. aeruginosa Pseudomonas isolates in three medical centers (Indore, Mumbai, and Rajkot). The VIM-2-producing P. putida and P. stutzeri isolates (two isolates and one isolate, respectively) were detected in hospitals where P. aeruginosa isolates carrying the same blaVIM gene were present; however, in one hospital (Rajkot) only a P. stutzeri isolate carrying blaVIM-11 was detected.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Distribution of carbapenem-resistant Pseudomonas spp. in medical centers in various Indian cities participating in the SENTRY Program in 2006

MβL-producing isolates were ribotyped using the Riboprinter microbial characterization system (Qualicon, Wilmington, DE). Isolates were clustered in 33 ribogroups, and 12 clones were detected. Seven of these clones were detected in multiple institutions. Six clones had isolates carrying distinct blaVIM genes and were further evaluated by pulsed-field gel electrophoresis, as previously described (6). The results obtained demonstrated that these isolates were not genetically related. Pulsed-field gel electrophoresis is usually considered to have a better discriminatory power for Pseudomonas spp.(8); however, ribotyping has been shown to be a valuable tool to identify long-term epidemiological events for other bacterial species, such as isolates from geographically distinct sources, like in this study, or over a broad time frame (2). Thus, clonal strains showing different VIM alleles could have had the same ancestor, but these isolates could have more recently evolved and acquired distinct blaVIM-carrying genetic elements.

MβL-carrying integrons were evaluated, and PCR experiments using primers annealing into blaVIM-2 and the qacE{Delta}1 gene showed amplicons in 12 isolates that ranged from 2.0 to 2.2 kb in size. In several isolates, reactions failed to amplify blaVIM-2-carrying class 1 integrons when primers annealing to the 3' conserved sequence (3'CS) were used. However, reactions using blaVIM primers in combination with primers for tniC, which was found to be associated with blaVIM-2-carrying integrons in India (7), amplified 21 of the 38 VIM-2-producing isolates. Amplicons of six molecular sizes were detected (1.2, 1.8, 2, 2.2, 3.5 and 4 kb). Sau96I restriction digestions of these amplicons generated identical patterns for all of the 2-kb integrons (11 isolates from five hospitals), indicating that they possess an identical gene arrangement. Additionally, integrons of 1.8 kb detected in two isolates from the same hospital also showed identical Sau96I patterns. Only one isolate showed a Sau96I restriction profile, as expected for the blaVIM-2-carrying integron previously reported in India (GenBank accession no. AM296017) (7). blaVIM-2 was carried in the first or second position of the integrons harboring the integrase gene (intI1) in the 5'CS. All blaVIM-6 strains possessed the 5'CS and 3'CS typical of class 1 integrons. Integrons harboring blaVIM-6 genes of 3.5 and 5 kb (five and seven isolates, respectively) were detected. Both integron types were identified in Indore and Hyderabad.

The gene encoding VIM-18 was identified in a P. aeruginosa (243-31C) strain isolated from a patient sputum specimen (Kolkatta). The susceptibility profile for isolate 243-31C is presented in Table 2. P. aeruginosa isolate 243-31C was genetically distinct from other MβL-producing strains from India (Table 1). blaVIM-18 was carried as a single gene cassette in a class 1 integron that contained the integrase gene in the 5'CS and the qacE{Delta}1/sul1 gene in the 3'CS. This integron was flanked upstream by a resolvase gene (tnpR) from a Tn5051-like transposon. Isolate 243-31C appeared to be without plasmid DNA in preparations carried out with the Plasmid DNA Midi kit; Qiagen, Hilden, Germany).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Antimicrobial susceptibility profile of the VIM-18- producing P. aeruginosa clinical isolate and β-lactam susceptibility profiles of the E. coli strain carrying the recombinant plasmid vector pPCRScript with or without blaVIM-18 and blaVIM-2 inserts

VIM-18 showed the highest homology to VIM-6 (99.6%), followed by VIM-2, -3, and -11 (99.2%). Amino acid sequence analysis demonstrated that VIM-18 possessed 262 amino acids and contained a deletion of 4 amino acids in position 140 compared with other VIM enzymes. blaVIM-18 cloned into pPCRScriptCam SK+ (Stratagene Cloning Systems, La Jolla, CA) and expressed in XL10-Gold Kan ultracompetent Escherichia coli cells showed lower MICs for ampicillin, cefoxitin, cefazolin, and ertapenem (>3-, >6-, 4-, and 9-fold, respectively) compared with VIM-2 expressed in the same background (Table 2). MICs for imipenem, meropenem, piperacillin, cefotaxime, ceftazidime, and cefepime were similar for both MβL enzymes (Table 2).

This initial nationwide MβL survey for India suggests a complex dissemination pattern and evolution of VIM enzymes. The high prevalence, continuous dissemination, and expansion of these resistance determinants in this region pose a serious therapeutic challenge.


arrow
Nucleotide sequence accession number.
 
The nucleotide sequence of blaVIM-18 has been submitted to GenBank nucleotide database and assigned accession no. AM778091.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: 345 Beaver Kreek Centre, Suite A, North Liberty, IA 52317. Phone: (319) 665-3370. Fax: (319) 665-3371. E-mail: mariana-castanheira{at}jmilabs.com Back

{triangledown} Published ahead of print on 29 December 2008. Back


arrow
REFERENCES
 
    1
  1. CLSI. 2008. M100-S18. Performance standards for antimicrobial susceptibility testing: 18th informational supplement. Clinical and Laboratory Standards Institute, Wayne, PA.
    2. 2
  2. Hermans, P. W. M., M. Sluijter, T. Hoogenboezem, H. Heersma, A. van Belkum, and R. de Groot. 1995. Comparative study of five different DNA fingerprint techniques for molecular typing of Streptococcus pneumoniae strains. J. Clin. Microbiol. 33:1606-1612.[Abstract]
    3. 3
  3. Jones, R. N. 2003. Global epidemiology of antimicrobial resistance among community-acquired and nosocomial pathogens: a five-year summary from the SENTRY Antimicrobial Surveillance Program (1997-2001). Semin. Respir. Crit. Care Med. 24:121-134.[CrossRef][Medline]
    4. 4
  4. Mendes, R. E., K. A. Kiyota, J. Monteiro, M. Castanheira, S. S. Andrade, A. C. Gales, A. C. C. Pignatari, and S. Tufik. 2007. Rapid detection and identification of metallo-β-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J. Clin. Microbiol. 45:544-547.[Abstract/Free Full Text]
    5. 5
  5. Poirel, L., T. Naas, D. Nicolas, L. Collet, S. Bellais, J.-D. Cavallo, and P. Nordmann. 2000. Characterization of VIM-2, a carbapenem-hydrolyzing metallo-β-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob. Agents Chemother. 44:891-897.[Abstract/Free Full Text]
    6. 6
  6. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.[Medline]
    7. 7
  7. Toleman, M. A., H. Vinodh, U. Sekar, V. Kamat, and T. R. Walsh. 2007. blaVIM-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 Chemother. 51:2636-2638.[Abstract/Free Full Text]
    8. 8
  8. van Belkum, A., P. T. Tassios, L. Dijkshoorn, S. Haeggman, B. Cookson, N. K. Fry, V. Fussing, J. Green, E. Feil, P. Gerner-Smidt, S. Brisse, and M. Struelens. 2007. Guidelines for the validation and application of typing methods for use in bacterial epidemiology. Clin. Microbiol. Infect. 13(Suppl. 3):1-46.[Medline]
    9. 9
  9. Walsh, T. R., M. A. Toleman, L. Poirel, and P. Nordmann. 2005. Metallo-β-lactamases: the quiet before the storm? Clin. Microbiol. Rev. 18:306-325.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, March 2009, p. 1225-1227, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.01011-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Castanheira, M.
Right arrow Articles by Jones, R. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Castanheira, M.
Right arrow Articles by Jones, R. N.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»