A NOVEL REAL-TIME PCR BASED APPROACH FOR THE DETECTION OF VAN GENE DETERMINANTS IN VANCOMYCIN RESISTANT ENTEROCOCCI FROM A TERTIARY CARE HOSPITAL SETTING IN INDORE, CENTRAL INDIA.

Abstract

Introduction- Vancomycin-resistant enterococci (VRE) are opportunistic pathogens capable of causing a broad spectrum of clinical infections, particularly in immunocompromised and hospitalized patients. To date, eight distinct phenotypic variants of acquired glycopeptide resistance have been identified in Enterococci: vanA, vanB, vanD, vanE, vanG, vanL, vanM, and vanN. In contrast, VanC represents an intrinsic resistance mechanism naturally occurring in Enterococcus gallinarum and Enterococcus casseliflavus. Among these, the vanA genotype is the most clinically relevant and is predominantly associated with E. faecium, accounting for the majority of VRE infections reported worldwide.

Aim- The objective of this study was to detect van gene determinants in Vancomycin Resistant Enterococci (VRE) isolated from both outpatient attendees and inpatients admitted to various wards of the hospital using Real-Time PCR based approach.

Material and Methods- A prospective cross-sectional study was carried out over a period of two years in the Department of Microbiology at Index Medical College and Research Centre, Indore, Madhya Pradesh. All Enterococcus isolates recovered from clinical specimens—including blood, urine, pus, sputum, wound swabs, catheter tips, and other body fluids—were included in the study. A total of 112 Enterococcus isolates were obtained using standard conventional culture techniques and confirmed through biochemical identification. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disc diffusion method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentrations (MICs) for vancomycin were determined using the broth dilution method.

Results-Among the 21 vancomycin-resistant isolates, Enterococcus faecium was the predominant species, representing 15 (71.4%) of the total isolates. This was followed by E. faecalis (23.8%) and E. gallinarum (4.8%), as identified using the disc diffusion method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Confirmation of vancomycin resistance was further established through the broth microdilution method, with all isolates previously identified as resistant by disc diffusion showing elevated minimum inhibitory concentrations (MICs). Gender-wise distribution revealed that 9 (43%) of the VRE cases were from male patients, while 12 (57%) were from female patients. Detection of vancomycin resistance genes was performed using real-time quantitative PCR (qPCR). Among the isolates, 13 (62%) carried the vanA gene, 5 (24%) harbored the vanB gene, and 3 isolates (14%) did not exhibit either vanA or vanB, indicating the possible involvement of other resistance mechanisms or gene variants not targeted by the assay.