title: Toxigenic potential and heat survival of spore-forming bacteria isolated from bread and ingredients.
abstract: Fifty-four spore-forming bacterial strains isolated from bread ingredients and bread, mainly belonging to the genus Bacillus (including Bacillus cereus), together with 11 reference strains were investigated to evaluate their cytotoxic potential and heat survival in order to ascertain if they could represent a risk for consumer health. Therefore, we performed a screening test of cytotoxic activity on HT-29 cells using bacterial culture filtrates after growing bacterial cells in Brain Heart Infusion medium and in the bread-based medium Bread Extract Broth (BEB). Moreover, immunoassays and PCR analyses, specifically targeting already known toxins and related genes of B. cereus, as well as a heat spore inactivation assay were carried out. Despite of strain variability, the results clearly demonstrated a high cytotoxic activity of B. cereus strains, even if for most of them it was significantly lower in BEB medium. Cytotoxic activity was also detected in 30% of strains belonging to species different from B. cereus, although, with a few exceptions (e.g. Bacillus simplex N58.2), it was low or very low. PCR analyses detected the presence of genes involved in the production of NHE, HBL or CytK toxins in B. cereus strains, while genes responsible for cereulide production were not detected. Production of NHE and HBL toxins was also confirmed by specific immunoassays only for B. cereus strains even if PCR analyses revealed the presence of related toxin genes also in some strains of other species. Viable spore count was ascertained after a heat treatment simulating the bread cooking process. Results indicated that B. amyloliquefaciens strains almost completely survived the heat treatment showing less than 2 log-cycle reductions similarly to two strains of B. cereus group III and single strains belonging to Bacillus subtilis, Bacillus mojavensis and Paenibacillus spp. Importantly, spores from strains of the B. cereus group IV exhibited a thermal resistance markedly lower than B. cereus group III with high values of log-cycle reductions. In conclusion, our results indicate that spore-forming bacteria contaminating bread ingredients and bread could represent a source of concern for consumer health related to the presence of strains, such as strains of B. cereus group III and single strains of other species, showing the ability to produce toxic substances associated to a thermal resistance enough to survive the bread cooking conditions.
authors.affiliation: Institute of Sciences of Food Production, National Research Council, Via Amendola 122/O, 70126 Bari, Italy.
authors.forename: Palmira
authors.initials: P
authors.lastname: De Bellis
authors.affiliation: Institute of Sciences of Food Production, National Research Council, Via Amendola 122/O, 70126 Bari, Italy.
authors.forename: Fiorenza
authors.initials: F
authors.lastname: Minervini
authors.affiliation: Institute of Sciences of Food Production, National Research Council, Via Amendola 122/O, 70126 Bari, Italy.
authors.forename: Mariaelena
authors.initials: M
authors.lastname: Di Biase
authors.affiliation: Institute of Sciences of Food Production, National Research Council, Via Amendola 122/O, 70126 Bari, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Valerio
authors.affiliation: Institute of Sciences of Food Production, National Research Council, Via Amendola 122/O, 70126 Bari, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Lavermicocca
authors.affiliation: Institute of Sciences of Food Production, National Research Council, Via Amendola 122/O, 70126 Bari, Italy. Electronic address: angelo.sisto@ispa.cnr.it.
authors.forename: Angelo
authors.initials: A
authors.lastname: Sisto
mesh_terms: D001407:Bacillus / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D001409:Bacillus cereus / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001427:Bacterial Toxins / Q000032:analysis / Q000235:genetics / Q000633:toxicity*; D001939:Bread / Q000382:microbiology*; D015169:Colony Count, Microbial; D019073:HT29 Cells; D006358:Hot Temperature; D006801:Humans; D050296:Microbial Viability; D013170:Spores / Q000502:physiology
chemical_list: D001427:Bacterial Toxins
keywords: Bread contamination; Cytotoxicity; Food-related risk; Thermal resistance; Toxin genes; Toxin production