Molecular detection, serotyping, cytotoxicity, and antimicrobial resistance of STEC and EPEC isolated from milk and milk products in northern India

Shiga toxin-producing <i>Escherichia coli</i> (STEC) and Enteropathogenic <i>E. coli</i> (EPEC) are important foodborne pathogens posing significant public health threats. This cross-sectional study investigated the prevalence, virulence profiles, serotypes, cytotoxicity, and antimicrobial resistance profiles of STEC and EPEC from milk and milk products in Uttarakhand, Northern India. A total of 680 samples (260 raw milk and 420 milk product samples) were collected from dairy farms, milk shops, collection centers, and street vendors over 9 months and screened for <i>E. coli</i> using conventional and molecular methods. Multiplex PCR targeting <i>stx1, stx2, eaeA,</i> and <i>hlyA</i> genes was employed to identify virulent isolates, which were further serotyped, evaluated for cytotoxicity on Vero cells, and tested for antibiotic susceptibility against 19 antibiotics. Resistant isolates were screened for <i>tetA, tetB, sul1,</i> and <i>CITM</i> genes by PCR. <i>E. coli</i> was detected in 28.82% of samples, with higher prevalence in raw milk (31.15%) than milk products (27.38%). Among isolates, 39.8% harbored at least one virulence gene, with <i>stx1</i> being most prevalent. Serotyping revealed 22 O-serogroups, predominantly O18, O111, O120, O126, and O17. All <i>stx</i>-positive isolates showed cytopathic effects in Vero cells, enhanced after ciprofloxacin induction. High resistance was observed against ampicillin, tetracycline, oxytetracycline, cephalothin, and sulphonamides, while imipenem, gentamicin, and nalidixic acid were most effective. Among multidrug-resistant isolates, 95% carried <i>tetB, sul1,</i> or <i>CITM</i> genes, while <i>tetA</i> was absent. The study confirms the presence of virulent and multidrug-resistant STEC and EPEC in milk and dairy products, highlighting the need for improved hygiene, judicious antimicrobial use, and regular monitoring to mitigate food safety and zoonotic risks.