Antimicrobial Susceptibility and Targeted Molecular Detection of Methicillin Resistance Determinants in Staphylococcus spp. Isolated from Broiler BCO Lesions

Background/Objectives: Antimicrobial resistance (AMR) in Staphylococcus spp. associated with poultry production is an emerging concern with implications for animal and public health. This study aimed to characterize antimicrobial susceptibility patterns and detect targeted methicillin resistance determinants in Staphylococcus isolates recovered from broiler chickens affected by bacterial chondronecrosis with osteomyelitis (BCO). Methods: A total of 200 bacterial isolates were evaluated, of which 167 were confirmed as Staphylococcus spp. Species identification was performed using presumptive phenotypic characterization followed by 16S rRNA gene sequencing. Antimicrobial susceptibility was assessed using disk diffusion, while presumptive methicillin-resistant phenotypes were evaluated using oxacillin screening and CHROMagar MRSA. Targeted molecular detection of mecA and mecC was performed by PCR. Results: The isolates demonstrated substantial species diversity, with S. aureus as the predominant species. Antimicrobial resistance was mainly observed against β-lactam antibiotics, particularly penicillin (33.5%), whereas high susceptibility was retained for non-β-lactam agents, including ciprofloxacin, tetracycline, trimethoprim–sulfamethoxazole, and azithromycin. A targeted PCR detected mecA in 7.2% of isolates, while mecC was not detected. The detection of mecA in oxacillin-susceptible isolates suggested genotype–phenotype discordance. Conclusions: BCO-associated Staphylococcus spp. from broiler chickens showed diverse species distribution, penicillin-dominant resistance, and targeted mecA detection across multiple species, supporting the use of combined phenotypic and molecular approaches for methicillin resistance surveillance.