Antimicrobial Resistance and Virulence Determinants of <i>Escherichia coli</i> Isolates from Raw Milk of Dairy Cows with Subclinical Mastitis

Subclinical mastitis (SCM) is a stealthy but devastating challenge in the dairy industry, leading to economic losses and hindering efforts to achieve milk self-sufficiency. This study investigated the prevalence of SCM, antimicrobial resistance, and virulence profiles of <i>Escherichia coli</i>. A total of 174 milk samples were analyzed using the California mastitis test (CMT), somatic cell counts (SCCs), bacteriological culture, MALDI-TOF MS, and polymerase chain reaction (PCR). The findings revealed that the SCM prevalence was 68/174 (39.08%) based on CMT and SCC. Among SCM-positive samples, 60/68 (88.23%) were identified as <i>E. coli,</i> confirmed by MALDI-TOF MS and PCR assay. The most frequently detected serogroups were 0113 (11.6%) and 0113 (3.3%). Additionally, the genes for <i>Stx1</i> and <i>Stx2</i> were also detected in nine (15%) and one (1.7%), respectively. Antimicrobial susceptibility tests showed widespread resistance, with <i>E. coli</i> isolates demonstrating resistance to penicillin in 43 (71.6%), followed by ciprofloxacin in 42 (70%) and gentamicin in 18 (30%). A larger proportion of the <i>E. coli</i> strains (100%) harbored the <i>bla</i>_VIM gene, while 23 (38.3%), 20%, 20%, and 1.47% contained <i>bla</i>_KPC, <i>bla</i>_NMD, <i>suli1</i>, and <i>msrA</i>. Thirty (50%) isolates were considered multidrug-resistant (MDR). These findings underscore the urgent need for enhanced surveillance and antibiotic stewardship in dairy farming. The presence of MDR <i>E. coli</i> in SCM poses a dual threat of potential transmission to humans and treatment failures in mastitis management. This study highlights the importance of proactive control strategies to mitigate the spread of antimicrobial resistance in livestock and beyond.