A multiplex PCR for rapid detection of vancomycin and linezolid resistance genes in clinical and environmental enterococci: A one health diagnostic approach.

BACKGROUND

Antimicrobial resistance (AMR) in Enterococcus, particularly vancomycin-resistance (VRE) and linezolid-resistance (LZRE) strains, presents a rising threat to public health globally.

METHODS

We designed an assay targeting vanA, vanB, vanC, vanD, optrA, poxtA, cfr and validated it across human and animal Enterococcus isolates. One hundred eighty-six (186) clinical isolates of Enterococcus were retrieved from Microbiology & Parasitology Laboratory, while 300 environmental isolates were obtained from poultry farms. Analytical sensitivity was assessed using successive dilutions of synthetic DNA constructs and bacterial lysates. The diagnostic performance was benchmarked against the automated system for testing of antimicrobial susceptibility, VITEK II.

RESULTS

The assay demonstrated high analytical sensitivity, detecting synthetic DNA as low as 1 pg and lysates down to 105 CFU/mL. Among 30 clinical VRE isolates, the assay identified VRE genes with a 71.8 % sensitivity, 100 % specificity and an overall 83.6 % accuracy. For LZRE detection, the assay showed 88.9 % sensitivity, 100 % specificity, and 97.1 % accuracy. In environmental isolates, LZRE genes were detected in 3.0 % of poultry samples.

CONCLUSIONS

This assay supports integrated AMR surveillance within a One Health framework.