Molecular epidemiology of ESBL-producing Escherichia coli causing clinical and subclinical bovine mastitis: associations between multidrug resistance, virulence genes, and phylogroups

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli are increasingly implicated in bovine mastitis and may serve as a reservoir of antimicrobial resistance with public health relevance. This study aimed to characterize the antimicrobial resistance, ESBL gene distribution, virulence-associated genes, integron carriage, and phylogenetic backgrounds of ESBL-producing E.coli isolated from bovine mastitis, and to compare these features between clinical and subclinical cases. A total of 400 bovine mastitis milk samples collected between June 2024 and May 2025 were screened for ESBL-producing bacteria, followed by phenotypic confirmation and antimicrobial susceptibility testing. Molecular characterization targeted the major ESBL genes, bla CTX−M, blaTEM, and bla SHV, class 1 and 2 integrons (intI1, intI2), 12 virulence-associated genes, and phylogenetic backgrounds determined by the Clermont method. Among 300 Gram-negative isolates, 135/300 (45.0%) E.coli isolates were confirmed as ESBL producers. Multidrug resistance was detected in 94/95 (98.9%) clinical isolates compared with 15/40 (37.5%) subclinical isolates, and clinical isolates showed significantly higher resistance to fluoroquinolones and aztreonam. Although bla CTX−M predominated overall [90/95 (94.7%) in clinical mastitis and 30/40 (75.0%) in subclinical mastitis], bla TEM and bla SHV were also more frequent among clinical isolates [65/95 (68.4%) and 55/95 (57.9%), respectively] than among subclinical isolates [15/40 (37.5%) for both]. Class 1 integrons were common in both groups and did not differ significantly by disease presentation, whereas dual intI1/intI2 carriage was more frequent among clinical isolates. Clinical isolates showed exclusive detection of colV [20/95 (21.1%) vs. 0/40], whereas subclinical isolates more frequently carried iroN [25/40 (62.5%) vs. 40/95 (42.1%)] and hlyF [30/40 (75.0%) vs. 50/95 (52.6%)]. Phylogroup B2 was more frequent among clinical isolates [45/95 (47.4%) vs. 10/40 (25.0%)], whereas phylogroup F was detected only among subclinical isolates [10/40 (25.0%) vs. 0/95]. In conclusion, ESBL-producing E.coli from clinical and subclinical bovine mastitis showed distinct molecular and phenotypic distributions, with clinical isolates more commonly associated with multidrug resistance, broader ESBL gene carriage, exclusive colV detection, and high-risk phylogenetic backgrounds. These findings support the value of integrated molecular surveillance to inform antimicrobial stewardship and One Health-oriented risk assessment in dairy production systems.