| Summary: | The core objective of this study was to genetically and phenotypically characterize subclinical mastitis-causing multidrug-resistant <i>Staphylococcus aureus</i> (MDRSA). In addition, risk factors associated with subclinical mastitis caused by MDRSA were investigated. Bacterial cultures were performed on 2120 mammary quarters, 40 swabs of milk utensils, 5 bulk tank milk samples, and 11 nostril and 11 hand swabs from milkers from five dairy farms. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was conducted for <i>S. aureus</i> identification. Antimicrobial resistance was screened phenotypically using the disk diffusion test in all <i>S. aureus</i> isolates. A biofilm formation assay; detection of genes associated with beta-lactam resistance, efflux pump, and biofilm formation; and pulsed-field gel electrophoresis (PFGE) were performed in all MDRSA isolates. Multi-locus sequence typing (MLST) was carried out in cefoxitin-resistant MDRSA isolates. A total of 188 <i>S. aureus</i> isolates from milk as well as two from milking utensils and one from bulk tank milk were identified. Most of the isolates (92.7%; 177 of 191) showed beta-lactam resistance, and 7% (14 of 191) were MDRSA. Interestingly, 36% (5 of 14) of MDRSA isolates were cefoxitin-resistant, but none carried <i>mecA</i> or <i>mecC</i> genes. Based on PFGE results, it was observed that <i>S. aureus</i> strains were more likely to be unique to a specific herd. Two clonal complexes were identified, CC97 (ST126; commonly livestock-associated) and CC1 (ST7440; usually community-associated). To the best of our knowledge, this is the first report of ST7440 isolated from bovine mastitis in Brazil. The risk factor results underscored the importance of considering parity, stage of lactation, SCC, milk production, and herd size when studying the risk of subclinical mastitis and antimicrobial resistance in <i>S. aureus</i>. Thus, to implement effective strategies to prevent subclinical mastitis in dairy herds and to minimize MDRSA spread, it is important to understand MDRSA strains’ distribution and their antimicrobial resistance profile.
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