Phylogenetic Footprints of Coagulase-Negative Staphylococci and Mammaliicoccus Isolated from Raw Caprine Milk

Coagulase-negative staphylococci (CNS) and Mammaliicoccus species recently reclassified from the Staphylococcus sciuri group are increasingly recognized as opportunistic pathogens in dairy animals and humans. This study investigated phylogenetic diversity and antimicrobial resistance in raw caprine milk from Sudan by integrating conventional bacteriological methods, molecular sequencing, and antimicrobial susceptibility testing. Raw goat milk samples were cultured, and presumptive CNS isolates were identified using phenotypic tests (novobiocin, oxidase, urease, and carbohydrate fermentation) following the standard Staphylococcus identification flow chart. PCR amplification of the elongation factor Tu ( tuf ) gene and the methicillin-resistance gene ( mecA ) enabled molecular confirmation and assessment of antimicrobial resistance. Sequenced tuf amplicons (~370 bp) were analysed by BLAST and aligned in MEGA 12 for maximum-likelihood phylogenetic reconstruction. Staphylococcus simulans and Mammaliicoccus lentus were isolated in this study; antimicrobial susceptibility testing revealed that S. simulans , but not M. lentus , was methicillin-resistant and carried the mecA gene. Partial tuf gene sequencing confirmed 99.6 - 99.7% identity with reference strains of respective species. Phylogenetic analysis revealed that the isolated S. simulans formed a distinct branch within the global clusters. Meanwhile, M. lentus was found to be closely related to the global strains, showing only minor divergence. This study reports the presence of S. simulans and M. lentus in caprine milk from Sudan using both phenotypic and genotypic identification methods. It underscores the importance of integrating traditional laboratory methods with molecular techniques for precise species identification. The identification of methicillin-resistant S. simulans highlights the necessity for ongoing monitoring of CNS in raw milk.