Risk Comparison of the Diarrheal and Emetic Type of Bacillus cereus in Tofu.
ABSTRACT: We investigated the ability of biofilm formation, survival, and behavior of diarrheal and emetic Bacillus cereus vegetative cells and spores in tofu. Both diarrheal and emetic B. cereus did not proliferate at a temperature below 9 °C in tofu. However, the emetic B. cereus grew faster than diarrheal B. cereus at 11 °C and had better survival ability at low temperatures. Both diarrheal and emetic B. cereus were able to form a biofilm on stainless steel. These biofilm cells were transferred to tofu in live state. The transferred biofilm cells could not grow at a temperature below 9 °C but grew over 11 °C, like planktonic cells. B. cereus contamination in tofu at a high concentration (>6 logs CFU/g) was not entirely killed by heating at 80, 85, or 90 °C for 2 h. Spores and emetic B. cereus had higher resistance to heat than vegetative cells and diarrheal B. cereus, respectively.
Project description:Cereulide-producing members of Bacillus cereus sensu lato group III (also known as emetic B. cereus) possess cereulide synthetase, a plasmid-encoded, nonribosomal peptide synthetase encoded by the ces gene cluster. Despite the documented risks that cereulide-producing strains pose to public health, the level of genomic diversity encompassed by emetic B. cereus has never been evaluated at a whole-genome scale. Here, we employ a phylogenomic approach to characterize group III B. cereus sensu lato genomes which possess ces (ces positive) alongside their closely related, ces-negative counterparts (i) to assess the genomic diversity encompassed by emetic B. cereus and (ii) to identify potential ces loss and/or gain events within the evolutionary history of the high-risk and medically relevant sequence type (ST) 26 lineage often associated with emetic foodborne illness. Using all publicly available ces-positive group III B. cereus sensu lato genomes and the ces-negative genomes interspersed among them (n?=?159), we show that emetic B. cereus is not clonal; rather, multiple lineages within group III harbor cereulide-producing strains, all of which share an ancestor incapable of producing cereulide (posterior probability = 0.86 to 0.89). Members of ST 26 share an ancestor that existed circa 1748 (95% highest posterior density [HPD] interval = 1246.89 to 1915.64) and first acquired the ability to produce cereulide before 1876 (95% HPD = 1641.43 to 1946.70). Within ST 26 alone, two subsequent ces gain events were observed, as well as three ces loss events, including among isolates responsible for B. cereus sensu lato toxicoinfection (i.e., "diarrheal" illness).IMPORTANCE B. cereus is responsible for thousands of cases of foodborne disease each year worldwide, causing two distinct forms of illness: (i) intoxication via cereulide (i.e., emetic syndrome) or (ii) toxicoinfection via multiple enterotoxins (i.e., diarrheal syndrome). Here, we show that emetic B. cereus is not a clonal, homogenous unit that resulted from a single cereulide synthetase gain event followed by subsequent proliferation; rather, cereulide synthetase acquisition and loss is a dynamic, ongoing process that occurs across lineages, allowing some group III B. cereus sensu lato populations to oscillate between diarrheal and emetic foodborne pathogens over the course of their evolutionary histories. We also highlight the care that must be taken when selecting a reference genome for whole-genome sequencing-based investigation of emetic B. cereus sensu lato outbreaks, since some reference genome selections can lead to a confounding loss of resolution and potentially hinder epidemiological investigations.
Project description:The Bacillus cereus group comprises multiple species capable of causing emetic or diarrheal foodborne illness. Despite being responsible for tens of thousands of illnesses each year in the U.S. alone, whole-genome sequencing (WGS) is not yet routinely employed to characterize B. cereus group isolates from foodborne outbreaks. Here, we describe the first WGS-based characterization of isolates linked to an outbreak caused by members of the B. cereus group. In conjunction with a 2016 outbreak traced to a supplier of refried beans served by a fast food restaurant chain in upstate New York, a total of 33 B. cereus group isolates were obtained from human cases (n = 7) and food samples (n = 26). Emetic (n = 30) and diarrheal (n = 3) isolates were most closely related to B. paranthracis (group III) and B. cereus sensu stricto (group IV), respectively. WGS indicated that the 30 emetic isolates (24 and 6 from food and humans, respectively) were closely related and formed a well-supported clade distinct from publicly available emetic group III genomes with an identical sequence type (ST 26). The 30 emetic group III isolates from this outbreak differed from each other by a mean of 8.3 to 11.9 core single nucleotide polymorphisms (SNPs), while differing from publicly available emetic group III ST 26 B. cereus group genomes by a mean of 301.7-528.0 core SNPs, depending on the SNP calling methodology used. Using a WST-1 cell proliferation assay, the strains isolated from this outbreak had only mild detrimental effects on HeLa cell metabolic activity compared to reference diarrheal strain B. cereus ATCC 14579. We hypothesize that the outbreak was a single source outbreak caused by emetic group III B. cereus belonging to the B. paranthracis species, although food samples were not tested for presence of the emetic toxin cereulide. In addition to showcasing how WGS can be used to characterize B. cereus group strains linked to a foodborne outbreak, we also discuss potential microbiological and epidemiological challenges presented by B. cereus group outbreaks, and we offer recommendations for analyzing WGS data from the isolates associated with them.
Project description:<h4>Background</h4>Bacillus cereus is ubiquitous in nature, found in environments such as soil, plants, air, and part of the insect and human gut microbiome. The ability to produce endospores and biofilms contribute to their pathogenicity, classified in two types of food poisoning: diarrheal and emetic syndromes. Here we report gap-free, whole-genome sequences of two B. cereus strains isolated from air samples and analyse their emetic and diarrheal potential.<h4>Results</h4>Genome assemblies of the B. cereus strains consist of one chromosome and seven plasmids each. The genome size of strain SGAir0260 is 6.30-Mb with 6590 predicted coding sequences (CDS) and strain SGAir0263 is 6.47-Mb with 6811 predicted CDS. Macrosynteny analysis showed 99% collinearity between the strains isolated from air and 90.2% with the reference genome. Comparative genomics with 57 complete B. cereus genomes suggests these strains from air are closely associated with strains isolated from foodborne illnesses outbreaks. Due to virulence potential of B. cereus and its reported involvement in nosocomial infections, antibiotic resistance analyses were performed and confirmed resistance to ampicillin and fosfomycin, with susceptibility to ciprofloxacin, tetracycline and vancomycin in both strains.<h4>Conclusion</h4>Phylogenetic analysis combined with detection of haemolytic (hblA, hblC, and hblD) and non-haemolytic (nheA, nheB, and nheC) enterotoxin genes in both air-isolated strains point to the diarrheic potential of the air isolates, though not emetic. Characterization of these airborne strains and investigation of their potential disease-causing genes could facilitate identification of environmental sources of contamination leading to foodborne illnesses and nosocomial infections transported by air.
Project description:<h4>Background</h4>Spores of psychrotrophic Bacillus cereus may survive the mild heat treatments given to minimally processed chilled foods. Subsequent germination and cell multiplication during refrigerated storage may lead to bacterial concentrations that are hazardous to health.<h4>Scope and approach</h4>This review is concerned with the characterisation of factors that prevent psychrotrophic B. cereus reaching hazardous concentrations in minimally processed chilled foods and associated foodborne illness. A risk assessment framework is used to quantify the risk associated with B. cereus and minimally processed chilled foods.<h4>Key findings and conclusions</h4>Bacillus cereus is responsible for two types of food poisoning, diarrhoeal (an infection) and emetic (an intoxication); however, no reported outbreaks of food poisoning have been associated with B. cereus and correctly stored commercially-produced minimally processed chilled foods. In the UK alone, more than 10<sup>10</sup> packs of these foods have been sold in recent years without reported illness, thus the risk presented is very low. Further quantification of the risk is merited, and this requires additional data. The lack of association between diarrhoeal food poisoning and correctly stored commercially-produced minimally processed chilled foods indicates that an infectious dose has not been reached. This may reflect low pathogenicity of psychrotrophic strains. The lack of reported association of psychrotrophic B. cereus with emetic illness and correctly stored commercially-produced minimally processed chilled foods indicates that a toxic dose of the emetic toxin has not been formed. Laboratory studies show that strains form very small quantities of emetic toxin at chilled temperatures.
Project description:Several Bacillus cereus strains possess the genetic fittings to produce two different types of toxins, the heat-stable cereulide or different heat-labile proteins with enterotoxigenic potential. Unlike the diarrheal toxins, cereulide is (pre-)formed in food and can cause foodborne intoxications shortly after ingestion of contaminated food. Based on the widely self-limiting character of cereulide intoxications and rarely performed differential diagnostic in routine laboratories, the real incidence is largely unknown. Therefore, during a 7-year period about 4.300 food samples linked to foodborne illness with a preliminary report of vomiting as well as food analysed in the context of monitoring programs were investigated to determine the prevalence of emetic B. cereus in food environments. In addition, a lux-based real-time monitoring system was employed to assess the significance of the detection of emetic strains in different food matrices and to determine the actual risk of cereulide toxin production in different types of food. This comprehensive study showed that emetic strains are much more volatile than previously thought. Our survey highlights the importance and need of novel strategies to move from the currently taxonomic-driven diagnostic to more risk orientated diagnostics to improve food and consumer safety.
Project description:The oxidation of unsaturated lipids by lipoxygenases in soybeans causes undesirable flavors in soy foods. Using a traditional and a nontraditional soy food user group, we examined the cultural difference in perceiving the sensory characteristics of soymilk and tofu produced from soybeans with or without lipoxygenases (Lx123). The two groups described the samples using similar terms. The traditional users preferred the control soy milk and lipoxygenase-free tofu while the nontraditional users preferred the lipoxygenase-free soymilk with no preference for tofu. In a separate study, a trained descriptive taste panel compared the odor of soymilk and tofu from control soybeans or those lacking lipoxygenase-1 and lipoxygenase-2 (Lx12) or all three isomers (Lx123). The rancid/grassy odor was rated the lowest in Lx123 products, followed by Lx12 products with the control products given the highest rating. The Lx12 and Lx123 products were also sweeter and less bitter than the controls. Taken together, our results demonstrated that soybeans lacking lipoxygenases can produce soy foods with less undesirable aromas and are therefore likely more acceptable to the consumers.
Project description:Bacillus cereus group isolates that produce diarrheal or emetic toxins are frequently isolated from raw milk and, in spore form, can survive pasteurization. Several species within the B. cereus group are closely related and cannot be reliably differentiated by established taxonomical criteria. While B. cereus is traditionally recognized as the principal causative agent of foodborne disease in this group, there is a need to better understand the distribution and expression of different toxin and virulence genes among B. cereus group food isolates to facilitate reliable characterization that allows for assessment of the likelihood of a given isolate to cause a foodborne disease.We performed whole genome sequencing of 22 B. cereus group dairy isolates, which represented considerable genetic diversity not covered by other isolates characterized to date. Maximum likelihood analysis of these genomes along with 47 reference genomes representing eight validly published species revealed nine phylogenetic clades. Three of these clades were represented by a single species (B. toyonensis -clade V, B. weihenstephanensis - clade VI, B. cytotoxicus - VII), one by two dairy-associated isolates (clade II; representing a putative new species), one by two species (B. mycoides, B. pseudomycoides - clade I) and four by three species (B. cereus, B. thuringiensis, B. anthracis - clades III-a, b, c and IV). Homologues of genes encoding a principal diarrheal enterotoxin (hemolysin BL) were distributed across all, except the B. cytotoxicus clade. Using a lateral flow immunoassay, hemolysin BL was detected in 13 out of 18 isolates that carried hblACD genes. Isolates from clade III-c (which included B. cereus and B. thuringiensis) consistently did not carry hblACD and did not produce hemolysin BL. Isolates from clade IV (B. cereus, B. thuringiensis) consistently carried hblACD and produced hemolysin BL. Compared to others, clade IV was significantly (p?=?0.0001) more likely to produce this toxin. Isolates from clade VI (B. weihenstephanensis) carried hblACD homologues, but did not produce hemolysin BL, possibly due to amino acid substitutions in different toxin-encoding genes.Our results demonstrate that production of diarrheal enterotoxin hemolysin BL is neither inclusive nor exclusive to B. cereus sensu stricto, and that phylogenetic classification of isolates may be better than taxonomic identification for assessment of B. cereus group isolates risk for causing a diarrheal foodborne disease.
Project description:The species of the Bacillus cereus group have the ability to adhere to and form biofilms on solid surfaces, including stainless steel, a material widely used in food industries. Biofilms allow for recontamination during food processing, and the "clean-in-place" (CIP) system is largely used by industries to control them. This study thus proposes to evaluate the efficacy of peracetic acid and sodium hypochlorite against biofilms induced on stainless-steel surfaces. The SAMN07414939 isolate (BioProject PRJNA390851), a recognized biofilm producer, was selected for biofilm induction on AISI 304 stainless steel. Biofilm induction was performed and classified into three categories: TCP (Tindalized, Contaminated, and Pasteurized milk), TCS (Tindalized milk Contaminated with Spores), and TCV (Tindalized milk Contaminated with Vegetative cells). Subsequently, the coupons were sanitized simulating a CIP procedure, on a pilot scale, using alkaline and acid solutions followed by disinfectants (peracetic acid and sodium hypochlorite). Microorganism adhesion on the surfaces reached 6.3 × 105 to 3.1 × 107 CFU/cm-2. Results did not show significant differences (p > 0.05) for surface adhesion between the three tested categories (TCP, TCS, and TCV) or (p > 0.05) between the two disinfectants (peracetic acid and sodium hypochlorite). Microbial populations adhered to the stainless-steel coupons are equally reduced after treatment with peracetic acid and sodium hypochlorite, with no differences in the control of B. cereus s.s. biofilms on AISI 304 stainless-steel surfaces.
Project description:Observational studies on the association between tofu intake and breast cancer incidence have reported inconsistent results. We reviewed the current evidence and quantitatively assessed this association by conducting a dose-response meta-analysis. The electronic databases PubMed and EMBASE were searched for relevant studies published up to August, 2018. We included epidemiological studies that reported relative risks (RRs) or odds ratios (ORs) with 95% confidence intervals (CIs) for the association between tofu intake and breast cancer risk. A total of 14 studies (2 cohort studies, 12 case-control studies) were included in the meta-analysis. The overall OR of breast cancer for highest vs lowest intake of tofu was 0.78 (95% CI 0.69-0.88), with moderate heterogeneity (P = 0.011, I2 = 49.7%). Dose-response analysis based on 5 case-control studies revealed that each 10 g/d increase in tofu intake was associated with 10% reduction in the risk of breast cancer (95% CI 7%-13%, P = 0.037, I2 = 40.8%). In summary, our findings suggest an inverse dose-response association between tofu intake and risk of breast cancer. However, owing to the limitations of case-control studies, more properly designed prospective studies are warranted to confirm this association.