Staphylococcus aureus (S. aureus) is a major pathogen responsible for mastitis in dairy cows and can contaminate raw milk, thereby posing significant health risks to consumers. The emergence of methicillin-resistant S. aureus (MRSA) has further heightened public health concerns due to its antibiotic resistance and infectious potential. In this study, we examined the prevalence, virulence genes, antimicrobial resistance, spa types, and biofilm formation of S. aureus isolates from dairy farms in Guangxi Province, China. Among 242 randomly selected samples, 37 S. aureus strains were identified (15.3% infection rate), including 67.5% MRSA. Antibiotic resistance was observed in 78.4% of isolates, with 35.1% exhibiting multidrug resistance (MDR). Enterotoxin gene analysis showed sea as the most common (67.6%), followed by ser (54.1%) and seh (51.4%), whereas seb and selj were absent. All isolates formed biofilms in vitro, with 64.8% showing strong biofilm-forming ability. Staphylococcal protein A (spa) typing classified the 37 S. aureus strains into 11 spa types, with t030 being the most prevalent (43.2%). These findings indicate that S. aureus is moderately prevalent in raw milk, often carrying multiple virulence genes, forming robust biofilms, and showing antimicrobial resistance. The MRSA that is “latent” in raw milk reminds us of the need for monitoring at the farm level.

Staphylococcus aureus is an important pathogen that can cause widespread infections as well as severe outbreaks of food poisoning. Recent studies have drawn attention to foodborne pathogens such as S. aureus endowed with the ability to form biofilms and increase resistance to antimicrobial agents as well as environmental stress, posing challenges to food safety. The Clp (caseinolytic protease) protein complex plays a crucial role in energy-dependent protein hydrolysis processes. This mechanism is a common way to maintain intracellular homeostasis and regulation in both prokaryotic and eukaryotic cells, especially under stress conditions. In S. aureus, multiple genes encoding Clp ATPase homologues have been identified: clpC, clpB, clpY, clpX, and clpL. This study investigated the roles of clpC in stress tolerance and biofilm formation of foodborne S. aureus RMSA24 isolated from raw milk. Our results showed that the deletion of the clpC gene significantly reduced the bacterium’s tolerance to heat, desiccation, hydrogen peroxide, and high osmotic pressure compared to wild type (WT). Furthermore, the clpC knockout mutant also exhibited a marked decrease in biofilm formation using Crystal Violet Staining (CVS) and Scanning Electron Microscopy (SEM). Finally, compared to WT, there was a total of 102 DEGs (differentially expressed genes), with a significant downregulation of genes related to biofilm formation (isaA and spa) and heat-shock response (clpP and danJ). These findings suggest that clpC regulates environmental tolerance in S. aureus by modulating the expression of stress- and biofilm-related genes, positioning it as a potential biomarker and a novel target for controlling contamination in the food industry.