Identification, antimicrobial resistance, and biofilm-forming capability of bacterial isolates from bovine mastitis in the high-altitude region of Shannan, Tibet.

Bovine mastitis imposes substantial economic losses on the dairy industry worldwide, yet systematic etiological data from high-altitude regions remain scarce, limiting evidence-based treatment decisions. This study aimed to characterize the bacterial isolates, antimicrobial resistance phenotypes, and biofilm-forming capabilities associated with bovine mastitis in the high-altitude region of Shannan, Tibet, China. A total of 177 raw milk samples were collected from cows diagnosed with mastitis based on clinical signs (udder swelling, redness, heat, and milk abnormalities including clots or watery appearance) and/or positive California Mastitis Test (CMT) results. Samples were inoculated onto trypticase soy agar for bacterial isolation, with preliminary phenotypic identification performed based on colony morphology and coloration. Purified single colonies were subjected to 16 S rRNA gene sequencing to enable species-level identification. Antimicrobial susceptibility testing was conducted using the Kirby-Bauer disk diffusion method as a screening tool, with the broth microdilution method used for confirmation, covering β-lactams, macrolides, fluoroquinolones, aminoglycosides, and glycopeptides. Biofilm-forming capability was assessed using the crystal violet staining method, with classification according to Stepanović criteria. A total of 188 bacterial isolates were obtained, among which Staphylococcus accounted for the highest proportion (20.2%, 38/188), followed by Bacillus (10.1%, 19/188) and Enterococcus (7.0%, 13/188). Within the staphylococci, Staphylococcus aureus was the most frequently identified species (8 isolates). Antimicrobial susceptibility testing revealed that all Streptococcus and Enterococcus isolates exhibited multidrug resistance (MDR), whereas a high proportion of Staphylococcus (76.3%) and Bacillus (63.2%) isolates also exhibited MDR. Notably, 62% of Enterococcus isolates exhibited a vancomycin-resistant phenotype, though this was not confirmed by molecular methods. In contrast, the majority of isolates (96%) remained highly susceptible in vitro to fluoroquinolones. Biofilm formation assays demonstrated that most isolates possessed biofilm-forming capability (93%, 174/188), and isolates with higher levels of multidrug resistance (≥ 4 MDR, defined as resistance to four or more classes of antimicrobial agents) tended to exhibit stronger biofilm-forming ability (Spearman's [Formula: see text]). Collectively, these findings indicate that bovine mastitis in the high-altitude region of Shannan, Tibet, is characterized by a predominance of staphylococci, accompanied by pronounced multidrug resistance and a considerable potential for biofilm formation. This study provides a foundational basis for etiological surveillance and targeted antimicrobial therapy for bovine mastitis in this region, however, the lack of molecular characterization of resistance determinants, limited sample sizes for certain genera, and the absence of high-resolution clonal typing represent important limitations that should be addressed in future investigations.