Biological characteristics of a menadione-dependent Staphylococcus aureus small colony variant (SCV) from bovine mastitis and heme promotion of biofilm formation.

While small colony variants (SCVs) of Staphylococcus aureus have been extensively documented, little is known about the properties of menadione-dependent SCVs from cow raw milk and how heme affects their ability to form biofilms. In this study, gentamicin-induced SCVs were generated from mastitis-derived Staphylococcus aureus, and the underlying mechanisms were investigated through nutrient dependency assays combined with genomic analysis. A comparative assessment of biological characteristics-including physiological and biochemical properties, antibiotic susceptibility, biofilm formation, and hemolytic activity-was conducted between the SCV and wild-type strains. Additionally, the effects of exogenous heme and menadione on biofilm formation were evaluated. The results revealed a frameshift deletion mutation due to a 95-nucleotide deletion in the menE gene. This mutation altered the corresponding amino acid sequence, correlating with the menadione-dependent SCV phenotype. Contrary to the typical SCV phenotype, the isolated SCV strain not only presented classic traits, such as slow growth and increased resistance to aminoglycoside antibiotics, but also exhibited atypical characteristics, including significantly decreased biofilm formation capacity and increased hemolytic activity. Exogenous supplementation with menadione or heme markedly promoted biofilm formation in the SCV strain. These results suggest that the reduced biofilm-forming ability of the SCV strain may stem from compromised metabolic activity. Furthermore, the SCV strain may be capable of acquiring exogenous heme via unknown pathways, a hypothesis that warrants further investigation, thereby restoring its biofilm formation capacity. In conclusion, this atypical SCV exhibits reduced biofilm formation and increased hemolytic activity, and heme partially restores biofilm via increased EPS and surface hydrophobicity. These findings are derived from a single SCV-parent pair; further isolates are needed to determine generalizability.