Whole Genome Sequencing (WGS) Analysis of Antimicrobial Resistance (AMR) Milk and Dairy-Derived Pathogens from Anand, Gujarat, India

Abstract

This study aimed to evaluate the antimicrobial resistance (AMR) patterns and genomic characteristics of Enterococcus faecalis, Enterococcus faecium, and Staphylococcus aureus strains isolated from dairy products, including buttermilk, curd, ice cream, and sweets, in the Anand region of Gujarat, India. A total of 205 isolates were analyzed, with the highest contamination levels found in buttermilk and curd. The bacterial isolates were identified using biochemical tests and advanced Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility testing (AST) was performed using the Kirby-Bauer disk diffusion method, following CLSI guidelines, focusing on common antibiotics used for treating dairy-related bacterial infections. Resistance profiles were analyzed using WHONET software.s The findings revealed significant multidrug resistance (MDR), particularly among E. faecium and E. faecalis, with over 95% resistance to key antibiotics, including linezolid, ciprofloxacin, cefpodoxime, and carbapenems. Many strains were classified as MDR, XDR, and PDR. Staphylococcus aureus also exhibited substantial resistance to penicillin and enrofloxacin. whole-genome sequencing (WGS) and phylogenetic analysis to identify AMR determinants and conduct nucleotide sequence alignment. The study identified several antibiotic resistance genes, including LiaF, which regulates the expression of LiaR and LiaS genes. WGS revealed that genes such as GdpD, MprF, and PgsA encode intrinsic resistance determinants, contributing to antibiotic resistance. Additional AMR mechanisms were identified, including ABC transporter efflux pumps and the regulation of resistance genes by LiaR and LiaS. Phylogenetic analysis indicates a close relationship between Enterococcus faecium 640 1352.18624 and Enterococcus durans FB129-CNAB-4 883162.3.