Bacterial diversity, antimicrobial resistance profiles, and farm-level risk factors in bovine mastitis: A retrospective cohort study in dairy herds.

Bovine mastitis is the costliest infectious disease in dairy farming worldwide and a major driver of antimicrobial use. The increasing prevalence of antimicrobial resistance (AMR) in mastitis pathogens has elevated this condition to a One Health perspective. A retrospective analytical observational study was conducted from 540 quarter-milk samples collected from 419 cows across 123 farms collected between June 2023 and January 2026, from dairy cows with clinical or subclinical mastitis. Bacterial identification and antimicrobial susceptibility testing were performed using the Mastatest® automated system. Risk factors were assessed using binomial generalized linear mixed models (GLMMs), including dairy farm as a random intercept. Environmental pathogens such as Escherichia coli (25.7%) and Streptococcus uberis (23.7%) were the predominant pathogens identified. Conventional milking, udder alterations, and impaired general condition were associated with higher odds of E. coli isolation, whereas recurrent mastitis was associated with lower odds (all p < 0.05). Recurrent mastitis was the only factor significantly associated with S. uberis isolation (p = 0.037). Among Gram-positive monobacterial isolates, co-resistance to ≥2 antibiotic classes was detected in 10.5% overall; Streptococcus spp. showed the highest resistance rates, particularly to cloxacillin (42.9%), benzylpenicillin (25.0%) and macrolides (21.4%). No significant predictors of co-resistance were identified. Antibiotic resistance patterns observed in mastitis pathogens support the implementation of culture-guided antimicrobial therapy and provide baseline data for regional AMR surveillance within a One Health approach.