The Impact of Mutations in the Pore-Forming Cytotoxins NheA and NheB of Bacillus cereus Isolated from four Traditional Fermented Beverages

Bacillus cereus is commonly associated with foodborne illnesses but also plays a significant role in food and beverage fermentations. Mutations in the non-hemolytic enterotoxin (Nhe) genes, which encode the NheA, NheB, and NheC components, can have significant functional consequences. This study aims to determine whether hemolytic enterotoxin Hbl, non-hemolytic enterotoxin (NheA, NheB, and NheC), and CytK components contain mutations that could alter the 3D structure of these proteins. Using microbiological and molecular techniques, including 16S rRNA analysis, 300 isolates were identified as B. cereus, with 11% (34/300) confirmed through NCBI accession, demonstrating its ubiquity. Genotypic and phenotypic analyses revealed strains with enzymatic activities, suggesting potential benefits in fermentation processes. Strains were also tested for the presence of the Hbl, Nhe complex, and cytK genes using PCR and sequencing. A total of 170 PCR amplifications targeting the hblA, nheA, nheB, nheC, and cytK genes were performed on 34 randomly selected strains. Of these, 69% (116/170) of the genes were successfully amplified, while 31% (54/170) were not detected. In-silico analysis of the wild-type hblA, nheA, nheB, nheC, and cytK genes confirmed their relation to B. cereus. Seven mutations were detected in the NheA60-220 region, including Q62N, Y64S, L82K, W96E, Y122D, D135N, and I213H. Four mutations were found in the NheA270-380 peptide sequence: Q280N, I316V, D317E, and W330Y. In NheB, truncated tripeptides NheB∆VKQ at position 34 and NheB∆SQD at position 211 were identified. Additional NheB mutations included A213S, D154E, Y231L, G273V, and K283Y. No mutations were detected in the NheC or HblA proteins. Some key mutations significantly altered the predicted 3D structure of the NheA and NheB components. These conformational changes may explain the non-pathogenicity of B. cereus in traditional fermented foods and beverages, which are often consumed without sterilization treatment.