Bacillus cereus group is widespread in nature and foods. Several members of this group are recognized as causing food spoilage and/or health issues. This study was designed to determine the prevalence and genetic diversity of the B. cereus group strains isolated in Tunisia from different foods (cereals, spices, cooked food, fresh-cut vegetables, raw and cooked poultry meats, seafood, canned, pastry, and dairy products). In total, 687 different samples were collected and searched for the presence of the B. cereus group after selective plating on MYP agar and enumeration of each sample. The typical pink-orange uniform colonies surrounded by a zone of precipitate were assumed to belong to the B. cereus group. One typical colony from each sample was subcultured and preserved as cryoculture. Overall, 191 (27.8%) food samples were found positive, giving rise to a collection of 191 B. cereus-like isolates. The concentration of B. cereus-like bacteria were below 103 cfu/g or ml in 77.5% of the tested samples. Higher counts (>104 cfu/g or ml) were found in 6.8% of samples including fresh-cut vegetables, cooked foods, cereals, and pastry products. To verify whether B. cereus-like isolates belonged to the B. cereus group, a PCR test targeting the sspE gene sequence specific of the group was carried out. Therefore, 174 isolates were found to be positive. Food samples were contaminated as follows: cereals (67.6%), pastry products (46.2%), cooked food (40.8%), cooked poultry meat (32.7%), seafood products (32.3%), spices (28.8%), canned products (16.7%), raw poultry meat (9.4%), fresh-cut vegetables (5.0%), and dairy products (4.8%). The 174 B. cereus isolates were characterized by partial sequencing of the panC gene, using a Sym'Previous software tool to assign them to different phylogenetic groups. Strains were distributed as follows: 61.3, 29.5, 7.5, and 1.7% in the group III, IV, II, and V, respectively. The genetic diversity was further assessed by ERIC-PCR and PFGE typing methods. PFGE and ERIC-PCR patterns analysis allowed discriminating 143 and 99 different profiles, respectivey. These findings, associated to a relatively higher prevalence of B. cereus group in different foods, could be a significant etiological agent of food in Tunisia.

Background Despite the importance of the B. cereus group as major foodborne pathogens that may cause diarrheal and/or emetic syndrome(s), no study in Tunisia has been conducted in order to characterize the pathogenic potential of the B. cereus group. The aim of this study was to assess the sanitary potential risks of 174 B. cereus group strains isolated from different foodstuffs by detecting and profiling virulence genes ( hblA , hblB, hblC , hblD , nheA , nheB , nheC , cytK , bceT and ces ), testing the isolates cytotoxic activity on Caco-2 cells and antimicrobial susceptibility towards 11 antibiotics. Results The entertoxin genes detected among B. cereus isolates were, in decreasing order, nheA (98.9%), nheC (97.7%) and nheB (86.8%) versus hblC (54.6%), hblD (54.6%), hblA (29.9%) and hblB (14.9%), respectively encoding for Non-hemolytic enterotoxin ( NHE) and Hemolysin BL ( HBL). The isolates are multi-toxigenic, harbouring at least one gene of each NHE and HBL complexes associated or not to bceT , cytK-2 and ces genes. Based on the incidence of virulence genes, the strains were separated into 12 toxigenic groups. Isolates positive for cytK (37,9%) harbored the cytK-2 variant. The detection rates of bceT and ces genes were 50.6 and 4%, respectively. When bacteria were incubated in BHI-YE at 30 °C for 18 h and for 5 d, 70.7 and 35% of the strains were shown to be cytotoxic to Caco-2 cells, respectively. The cytotoxicity of B. cereus strains depended on the food source of isolation. The presence of virulence factors is not always consistent with cytotoxicity. However, different combinations of enterotoxin genetic determinants are significantly associated to the cytotoxic potential of the bacteria. All strains were fully sensitive to rifampicin, chloramphenicol, ciprofloxacin, and gentamycin. The majority of the isolates were susceptible to streptomycin, kanamycin, erythromycin, vancomycin and tetracycline but showed resistance to ampicillin and novobiocin. Conclusion Our results contribute data that are primary to facilitate risk assessments in order to prevent food poisoning due to B. cereus group.

The Bacillus cereus (B. cereus) group is a widespread foodborne pathogen with a persistent ability to form biofilm, and with inherent resistance to traditional treatment in the food industry. Bacteriophages are a promising biocontrol agent that could be applied to prevent or eliminate biofilms formation. We have described, in this study, the isolation from sewage samples and preliminary characterization of bacteriophages that are active against the B. cereus group. The effectiveness of phage treatment for reducing B. cereus attachment and biofilms on stainless steel surfaces has been also assessed using three incubation periods at different titrations of each phage. Out of 62 phages isolated, seven showed broad-spectrum lytic action against 174 B. cereus isolates. All selected phages appeared to be of the Siphoviridae family. SDS-PAGE proved that two phages have a similar profile, while the remainder are distinct. All isolated phages have the same restriction pattern, with an estimated genome size of around 37 kb. The isolated bacteriophages have been shown to be effective in preventing biofilm formation. Reductions of up to 1.5 log10 UFC/cm2 have been achieved, compared to the untreated biofilms. Curative treatment reduced the bacterial density by 0.5 log10 UFC/cm2. These results support the prospect of using these phages as a potential alternative strategy for controlling biofilms in food systems.