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

Abstract

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.

Keywords

Bovine mastitis / Antimicrobial resistance / Extended-spectrum beta-lactamase / Carbapenemase / Gram-negative bacteria / One Health

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