Phenotypic & genotypic characterization of vancomycin resistant Enterococcus isolates from clinical specimens

Abstract

Background & objectives: Enterococci have emerged as important nosocomial pathogens and emergence of resistance to many of the antimicrobials used for Gram-positive organisms has made the management of infections due to Enterococcus species difficult. Resistance to glycopeptide antibiotics, especially vancomycin is of special concern. This study was undertaken to perform a phenotypic and genotypic characterization of vancomycin resistant Enterococcus (VRE) isolates obtained from clinical samples in a tertiary care hospital in southern India.

Methods: Susceptibility testing was performed for Enterococcus isolates collected over a period of one year (November 2008-October 2009). Minimum inhibitory concentrations (MIC) of vancomycin and teicoplanin were determined for the isolates by the agar dilution method. Genotypic characterization of VRE isolates was done by performing multiplex polymerase chain reaction (PCR) for detecting the various vancomycin resistance genes.

Results: Of the 367 isolates of Enterococcus species isolated, 32 were found to be resistant to vancomycin after MIC testing. VanA was the commonest phenotype of vancomycin resistance and the commonest genotype was vanA. Among the other important findings of the study was the presence of heterogeneity in isolates of VRE with the vanA gene cluster with regards to resistance to teicoplanin and the coexistence of vanA and vanC1 gene clusters in an isolate of E. gallinarum which conferred high level glycopeptide resistance to the isolate.

Interpretation & conclusions: Enterococcus species have emerged as important nosocomial pathogens in our patients with a capacity to cause a variety of infections. The vancomycin resistance among Enterococcus isolates was 8.7 per cent in our study which was high compared to other Indian studies. VanA was the commonest phenotype of glycopeptide resistance and vanA was the commonest vancomycin resistance gene. The study also demonstrates phenotypic as well as genotypic heterogeneity among isolates of VRE from clinical specimens.

Fig. 1

Gel electrophoresis of amplified products by PCR for vancomycin resistance genes. Agarose gel electrophoresis of the amplified products by PCR for Vancomycin resistance genes. M-100 base pair DNA ladder (Bangalore Genei, India). PC, positive control for vanA gene; NC, negative control; Lanes 1-5 positive for vanA gene (732 bp).

Fig. 2

Gel electrophoresis of amplified products by multiplex PCR for detection of vancomycin resistance genes. Agarose gel electrophoresis of amplified products by PCR for vancomycin resistance genes. M-100 bp DNA ladder; Lane 9-Positive for vanA (732bp), vanC1 (822bp) and 16SrRNA internal control (320 bp)-multiplex PCR; Lane 8-Positive only for 16SrRNA internal control (no van resistance genes) by multiplex PCR; Lane 6-Positive for vanC1 (822 bp) by PCR targeting only vanC1 gene; Lanes 2 & 4-Positive for E.gallinarum (173 bp) by using species-specific primers.