Starvation-induced metabolic adaptation of Bacillus cereus during biofilm formation: Phenotypic characterization and...

Bacillus cereus forms robust biofilms that enhance its persistence in food processing environments, leading to substantial safety risks and deterioration of dairy products. Nutrient availability is a vital factor to regulate biofilm formation; however, adaptive strategies of B. cereus under nutrient stress remain poorly understood. This study employs integrated phenotypic characterization and label-free quantitative proteomics to systematically decode the starvation-induced (in 1/10 TSB and 1/100 TSB) biofilm formation mechanism in B. cereus 12-1. The results showed that nutrient stress inhibited the growth of B. cereus 12-1 in its planktonic state, but increased its spore formation by 1.04 (1/10 TSB) and 1.79 (1/100 TSB) Log CFU/mL, protease production by 76.7 (1/10 TSB) and 140.37 U (1/100 TSB), cell surface hydrophobicity by 35.2 % (1/100 TSB), and self-aggregation capacity by 7.39 % (1/10 TSB) and 1.98 % (1/100 TSB). In addition, starvation induced its biofilm formation on stainless steel, resulting in elevated bacterial cell density within biofilms by 1.23 Log CFU/cm² (1/10 and 1/100 TSB), enhanced metabolic activity by 36.7 % (1/10 TSB) and 60.1 % (1/100 TSB). The confocal laser scanning microscopy results revealed more complex biofilm architectures with increased thickness by 3.54 % (1/10 TSB) and 35.2 % (1/100 TSB) and greater surface coverage of 52.46 (1/10 TSB) and 52.44 μm (1/100 TSB). Proteomics analysis identified 589 (in 1/10 TSB) and 442 (in 1/100 TSB) differentially expressed proteins (DEPs). These DEPs were mainly involved in 11 upregulated pathways, including quorum sensing, ATP-binding cassette (ABC) transporters, fatty acid degradation, several amino acid metabolic pathways, and oxidative phosphorylation, but were also significantly enriched in 12 downregulated pathways like ribosome, HIF-1 signaling pathway, nucleotide metabolism, and several carbohydrate metabolic pathways. In this regard, this investigation provides initial insights into the survival adaptation of B. cereus under nutrient stress conditions, serving as a foundational step toward understanding its stress responses and offering a preliminary scientific basis to inform future efforts aimed at mitigating biofilm-related risks in dairy processing environments.