Potential risk of biofilm-forming Bacillus cereus group in fresh-cut lettuce production chain

Bacillus cereus and Bacillus thuringiensis, which belong to the B. cereus group, are widely distributed in nature and can cause food poisoning symptoms. In this study, we collected 131 isolates belonging to the B. cereus group, comprising 124B. cereus and seven B. thuringiensis isolates, from fresh-cut lettuce production chain and investigated their potential risk by analyzing genotypic (enterotoxin and emetic toxin gene profiles) and phenotypic (antibiotic susceptibility, sporulation, and biofilm formation) characteristics. Enterotoxin genes were present only in B. cereus, whereas the emetic toxin gene was not detected in any of the B. cereus isolates. All isolates were susceptible to vancomycin, which is a last resort for treating B. cereus group infection symptoms, but generally resistant to β-lactam antimicrobials, and had the ability to form spores (at an average sporulation rate of 24.6 %) and biofilms at 30 °C. Isolates that formed strong biofilms at 30 °C had a superior possibility of forming a dense biofilm by proliferating at 10 °C compared to other isolates. Additionally, confocal laser scanning microscopy (CLSM) images revealed a notable presence of spores within the submerged biofilm formed at 10 °C, and the strengthened attachment of biofilm inner cells to the substrate was further revealed through biofilm structure parameters analysis. Collectively, our study revealed the prevalence and contamination levels of B. cereus and B. thuringiensis at fresh-cut lettuce production chain and investigated their genotypic and phenotypic characteristics, aiming to provide valuable insights for the development of potential risk management strategies to ensure food safety, especially along the cold chain.