Single-isolate genomic characterization and cgMLST allelic comparison of a bovine mastitis-associated Staphylococcus...

Bovine mastitis caused by Staphylococcus aureus remains a major challenge for dairy production, but whole-genome data on mastitis-associated S. aureus from Kazakhstan are limited.

This single-isolate study characterized a milk-derived S. aureus isolate obtained from a six-year-old cow with clinically severe, treatment-refractory mastitis in Kostanay Region, Kazakhstan. The isolate was confirmed by bacteriological examination, Gram staining, and species-specific nuc gene PCR. Experimental virulence assessment in mice, histopathological examination of mammary tissue from the same clinical case, Oxford Nanopore MinION whole-genome sequencing, MLST, cgMLST, virulence gene profiling, antimicrobial resistance gene analysis, and supportive disk diffusion screening were performed.

The isolate showed dose-dependent mortality in mice, with an LD50 of approximately 6.3 × 10⁶ CFU. Histopathological examination revealed lesions consistent with acute suppurative mastitis. Whole-genome sequencing and Flye assembly via EPI2ME produced a de novo genome assembly of 2,771,674 bp, with 77× mean coverage and a GC content of 32.83%. MLST assigned the isolate to ST151, and cgMLST identified cgST-32654 as the closest profile, with 1,654 of 1,716 loci matched and 62 allelic differences. Virulence profiling detected genes encoding exoenzymes, hemolysins, leukocidins, and egc-associated enterotoxin-like proteins. CARD RGI detected mainly efflux-associated and regulatory antimicrobial resistance-associated loci, whereas ResFinder/PointFinder did not detect acquired antimicrobial resistance genes or known resistance-associated point mutations.

Comparative cgMLST minimum-spanning tree analysis indicated allelic relatedness to publicly available bovine-associated ST151 profiles. This genome provides a reference point for future genomic surveillance of mastitis-associated S. aureus in Kazakhstan and Central Asia, but population-level conclusions require larger isolate collections.