Molecular characterization and virulence potential of Staphylococcus aureus from raw milk artisanal cheeses

Canastra Minas Artisanal Cheese (CMAC), produced in Brazil's Serra da Canastra region, uses raw milk, rennet, and a natural starter culture called pingo. Raw milk can introduce pathogenic bacteria, including Staphylococcus aureus, leading to potential foodborne illnesses. This study analyzed 303 S. aureus isolates from CMAC, collected from 83 cheese farms through cross-sectional and longitudinal samplings, to assess their genetic diversity, virulence potential, and antimicrobial susceptibility. Pulsed Field Gel Electrophoresis (PFGE), agr quorum sensing, and spa typing were used for clonal and epidemiological profiling. Additional genes related to hemolysins, toxins, biofilm formation, and adhesive matrix molecules (MSCRAMMs) were PCR tested. Antimicrobial susceptibility was determined by disc diffusion, and biofilm formation was assessed using the crystal violet method. Agr typing revealed agr-I (27.4%), agr-II (31.4%), and agr-III (14.2%), with no agr-IV detected, and 27% of isolates lacking any agr type. PFGE identified 54 profiles, with dominant spa genotypes t127 (26%), t267 (19.57%), and t605 (10.87%). Enterotoxin genes were rare, but sea, seh, sei, sej, seg, seo, and sem were found. Nearly all isolates had the potential to be hemolytic (positive for hla and hlb). Most isolates possessed MSCRAMMs genes, including fnbA, fnbB, clfA, clfB, and eno. Biofilm genes icaA and icaD were present in 70.3% and 46.2% of isolates, respectively. Antibiotic resistance was observed in 46.3% (penicillin) and 24.0% (tetracycline) among the isolates. Three isolates carried the mecA gene. Biofilm formation varied from weak forming (59.3%) to moderate (24%). The study revealed substantial diversity and virulence potential of S. aureus in the Canastra region, highlighting the need for hygienic practice and training for prevention of contamination, as well as ongoing research into their genetic diversity and enterotoxin production profile to enhance safety strategies for these artisanal cheeses.