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J Med Microbiol 57 (2008), 72-79; DOI: 10.1099/jmm.0.47144-0
© 2008 Society for General Microbiology
ISSN 1473-5644

Identification and characterization of a vancomycin-resistant Staphylococcus aureus isolated from Kolkata (South Asia)

Biswajit Saha1, Anil K. Singh2, Abhrajyoti Ghosh3 and Manjusri Bal1

1 Microbiology Laboratory, Department of Physiology, University College of Science, Technology and Agriculture, University of Calcutta, Kolkata, India

2 Department of Chemistry, Bose Institute, Kolkata, India

3 Dr B. C. Guha Centre for Genetic Engineering and Biotechnology, University College of Science, Technology and Agriculture, University of Calcutta, Kolkata, India

Correspondence
Manjusri Bal
manjusrb{at}vsnl.net

Received 2 January 2007
Accepted 8 August 2007

A pathogenic vancomycin-resistant Staphylococcus aureus (VRSA) isolate (MIC ≥64 µg ml–1) was obtained from a Kolkata hospital in June 2005. Species identification was confirmed by Gram staining, standard biochemical tests and PCR amplification of the nuc gene, which encodes the thermostable nuclease that is highly specific for S. aureus. The VRSA isolate was also resistant to beta-lactams (amoxicillin, ampicillin, cefepime, cefotaxime, cefuroxime, cephalexin and meticillin), chloramphenicol, streptomycin, macrolides (erythromycin and roxithromycin), clindamycin, rifampicin and trimethoprim-sulfamethoxazole. However, the isolate was susceptible to gentamicin (an aminoglycoside) and ciprofloxacin (a fluoroquinolone). The resistance to vancomycin was inducible in vitro, because the MIC of vancomycin increased from 64 µg ml–1 initially to 1024 µg ml–1 during culture of this VRSA strain in the presence of vancomycin. The VRSA isolate contained a large plasmid (~53.4 kb) and four small plasmids of ~6, 5.5, 5.1 and 1.5 kb. The large plasmid of ~53.4 kb harboured the vancomycin-resistance genes vanHAX, which was confirmed by PCR amplification using the same plasmid as template and, separately, primers specific for the 2.61 kb vanHAX gene cluster, vanH (969 bp), vanA (1032 bp) and vanX (609 bp). The VRSA isolate was also positive for mecA. Vancomycin resistance was successfully transferred from this VRSA donor to a vancomycin-sensitive recipient S. aureus clinical isolate by a broth mating procedure. The MIC of vancomycin for the transconjugant was 32 µg ml–1, as against 2 µg ml–1 for the parent strain. Nucleotide sequencing of the PCR product showed partial homology with van genes of an enterococcal transposon Tn1546-like element. This is believed to be the first Indian S. aureus isolate that has been shown to be phenotypically vancomycin-resistant, presumably due to a vanHAX analogue.

Abbreviations: DAD, disc agar diffusion; PG, peptidoglycan; VRSA, vancomycin-resistant S. aureus; VSSA, vancomycin-sensitive S. aureus.

The GenBank/EMBL/DDBJ accession numbers for the S. aureus sequences determined in this paper are EU019995 and EU016096.






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