| Summary: | <i>Staphylococcus</i> species are amongst the bacteria that cause bovine mastitis worldwide, whereby they produce a wide range of protein toxins, virulence factors, and antimicrobial-resistant properties which are enhancing the pathogenicity of these organisms. This study aimed to detect <i>Staphylococcus</i> spp. from the milk of cattle with subclinical mastitis using MALDI-TOF MS and 16S rRNA PCR as well as screening for antimicrobial resistance (AMR) and virulence genes. Our results uncovered that from 166 sampled cows, only 33.13% had subclinical mastitis after initial screening, while the quarter-level prevalence was 54%. Of the 50 cultured bacterial isolates, MALDI-TOF MS and 16S rRNA PCR assay and sequencing identified <i>S. aureus</i> as the dominant bacteria by 76%. Furthermore, an AMR susceptibility test showed that 86% of the isolates were resistant to penicillin, followed by ciprofloxacin (80%) and cefoxitin (52%). Antimicrobial resistance and virulence genes showed that 16% of the isolates carried the <i>mecA</i> gene, while 52% of the isolates carried the <i>Lg G</i>-binding region gene, followed by <i>coa</i> (42%), <i>spa</i> (40%), <i>hla</i> (38%), and <i>hlb</i> (38%), whereas <i>sea</i> and <i>bap</i> genes were detected in 10% and 2% of the isolates, respectively. The occurrence of virulence factors and antimicrobial resistance profiles highlights the need for appropriate strategies to control the spread of these pathogens.
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