Environmental modulation of cytotoxicity and virulence factor stability in psychrotolerant Bacillus cereus group isolates.

The Bacillus cereus group comprises strains relevant to food spoilage, agriculture, and human disease, with psychrotolerant members associated with refrigerated food spoilage. However, the food safety risk posed by these psychrotolerant strains remains poorly understood. This study characterized psychrotolerant B. cereus group strains from panC groups II and VI by quantifying (i) cytotoxicity at different temperatures and media (10, 32, and 37 °C in brain heart infusion broth (BHIB), and 10 °C and 32 °C in skim milk broth (SMB)), (ii) transcription of virulence genes at these same conditions, and (iii) reduction in supernatant cytotoxicity due to protein stressors. Isolates showed higher cytotoxicity (p = 0.00086) when grown in BHIB at 32 °C (0.40 ± 0.23) compared to 37 °C (0.28 ± 0.20). A subset of tested isolates also showed higher cytotoxicity (p = 0.0054) and transcription (p < 0.001) of tested virulence genes at 32 °C than at 10 °C in SMB and BHIB. Thermal (65, 75, 100 °C for 1-2 min), low-pH (2), and trypsin (10 μg/μL) treatments significantly reduced cytotoxicity of supernatants collected from isolates grown at 32 °C in BHIB, though residual cytotoxicity remained above the level of sterile BHIB. This study highlights that psychrotolerant B. cereus isolates express cytotoxic virulence factors, although at a low level, in a dairy food model under conditions mimicking temperature abuse during refrigerated storage. Furthermore, the persistence of residual cytotoxicity following thermal, pH, and proteolytic treatments highlights the need to better understand the biological relevance of these residual activities during human exposure.