Comparative phenotypic and genotypic profiling of ESBL- and carbapenemase-associated gram-negative bovine mastitis pathogens in western Türkiye, 2025–2026

Bovine mastitis is an important veterinary disease and is relevant for antimicrobial resistance surveillance; however, gram-negative mastitis pathogens are often evaluated as isolated species or resistance phenotypes rather than within a comparative resistance-severity framework. This cross-sectional study examined gram-negative bovine mastitis isolates collected from western Türkiye between May 2025 and January 2026. A total of 504 cows from 15 dairy farms were screened, and 450 quarter-milk samples meeting clinical or California mastitis test-based mastitis criteria were collected. The culture yielded 160 nonduplicate gram-negative isolates, including 60 Escherichia coli , 68 Klebsiella pneumoniae , and 32 Acinetobacter baumannii strains. The isolates were classified into five resistance-defined groups: extended-spectrum beta-lactamase (ESBL)-producing E. coli , carbapenem-resistant E. coli , ESBL-producing K. pneumoniae , carbapenem-resistant K. pneumoniae , and carbapenem-resistant A. baumannii . The isolates were characterized by antimicrobial susceptibility testing, phenotypic ESBL confirmation, carbapenemase detection assays, and PCR detection of major beta-lactamase genes. Clinical mastitis accounted for 122/160 (76.3%) isolates, and carbapenemase-associated groups were concentrated almost entirely among clinical cases [84/85 (98.8%)]. The resistance burden increased across the groups, with the mean number of resistant antimicrobial classes increasing from 4.45 ± 1.38 in ESBL-producing E. coli to 8.00 ± 0.00 in carbapenem-resistant A. baumannii . The frequency of panel-defined extensively drug-resistant (XDR) strains increased from 25.0% in ESBL-producing E. coli to 100.0% in carbapenem-resistant A. baumannii . blaCTX-M predominated in the ESBL-associated groups, whereas the carbapenem-resistant groups presented broader carbapenemase-associated gene profiles, with the broadest profile observed in A. baumannii . Among the phenotypic carbapenemase assays, the optimized carbapenem inactivation method, UniCarb assay, and meropenem–EDTA test showed the most consistent positivity and PCR-supported agreement across the carbapenemase-associated groups. These findings support bovine mastitis as a relevant veterinary surveillance interface for ESBL- and carbapenemase-associated gram-negative resistance with potential One Health relevance.