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 the 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, which is likely the principal reason for the menadione-dependent SCV phenotype. The SCV strain presented not only typical characteristics, such as slow growth and increased resistance to aminoglycoside antibiotics, but also atypical ones, inculding 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 suggesting that the SCV strain reduced biofilm-forming ability may stem from compromised metabolic activity. Moreover, the SCV strain potentially enabling the acquisition of exogenous heme via unidentified pathways, thereby restoring biofilm formation capacity. The regulatory mechanisms involved require further investigation. These findings provide new insights into the biofilm regulation and survival/pathogenicity mechanisms of Staphylococcus aureus SCVs.