Molecular characterization and antibiotic susceptibility of Shiga toxin- producing Escherichia coli (STEC) isolated from raw milk of dairy bovines in Khyber Pakhtunkhwa, Pakistan

Abstract

This study investigated the virulence potential and antibiotic susceptibility analysis of non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups, which are significant cause of food borne diseases. A study collected 800 samples of dairy bovine raw milk through various sources, 500 from milk shops, 200 from dairy farms, 26 from milk collection centers, and 74 from street vendors. Using a standard method, E. coli was detected in 321 out of the 800 samples collected. Out of the 321 E. coli-positive samples isolated, 148 were identified as STEC using selective media, specifically Cefixime Tellurite Sorbitol MacConkey’s Agar (CT-SMA). Out of the 148 positive samples, 40 were confirmed as STEC non-O157 strains using multiplex PCR, indicating a prevalence of 5% (40 out of 800 samples). STEC isolates were subjected to antimicrobial susceptibility testing, and all isolates were resistant to at least one or more antimicrobials tested through the disk diffusion method, revealed high resistance to Amoxicillin 100%, Ceftriaxone 50%, and Penicillin 44.5%, and notably 44% of the strains exhibited Streptomycin resistance, while Enrofloxacin 55%, Florfenicol 50% and Norfloxacin 44%, demonstrated the highest susceptibility. Out of 40 STEC non-O157, twelve were subjected to Multi Locus Sequence Typing (MLST) sequencing through Illumina Inc. MiSeq platform’s next-generation sequencing technology, United States. The genome investigation evidenced the persistence of twelve serotypes H4:O82, H30:O9a, H4:O82, H16:O187, H9:O9, H16:O113, H30:O9, H32:O, H32:O, H32, H32, and H38:O187, linked to the potential infections in humans. Conclusion: STEC isolates showed resistance to multiple antimicrobials, raising concerns for both animal and public health due to widespread use of these drugs in treatment and prevention. The study contributes new insights into monitoring STEC in raw milk, emphasizing the critical role of whole genome sequencing (WGS) for genotyping and sequencing diverse isolates. Still a deficiency in understanding STEC pathogenesis mechanisms, ongoing surveillance is crucial for safeguarding human health and enhancing understanding of STEC genetic characteristics.