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

Abstract

A pathogenic vancomycin-resistant Staphylococcus aureus (VRSA) isolate (MIC > or =64 microg 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 microg ml(-1) initially to 1024 microg ml(-1) during culture of this VRSA strain in the presence of vancomycin. The VRSA isolate contained a large plasmid ( approximately 53.4 kb) and four small plasmids of approximately 6, 5.5, 5.1 and 1.5 kb. The large plasmid of approximately 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 microg ml(-1), as against 2 microg 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.