Emetic Toxin Genes Research Articles

Potential risk of biofilm-forming Bacillus cereus group in fresh-cut lettuce production chain

Bacillus cereus and Bacillus thuringiensis, which belong to the B. cereus group, are widely distributed in nature and can cause food poisoning symptoms. In this study, we collected 131 isolates belonging to the B. cereus group, comprising 124B. cereus and seven B. thuringiensis isolates, from fresh-cut lettuce production chain and investigated their potential risk by analyzing genotypic (enterotoxin and emetic toxin gene profiles) and phenotypic (antibiotic susceptibility, sporulation, and biofilm formation) characteristics. Enterotoxin genes were present only in B. cereus, whereas the emetic toxin gene was not detected in any of the B. cereus isolates. All isolates were susceptible to vancomycin, which is a last resort for treating B. cereus group infection symptoms, but generally resistant to β-lactam antimicrobials, and had the ability to form spores (at an average sporulation rate of 24.6 %) and biofilms at 30 °C. Isolates that formed strong biofilms at 30 °C had a superior possibility of forming a dense biofilm by proliferating at 10 °C compared to other isolates. Additionally, confocal laser scanning microscopy (CLSM) images revealed a notable presence of spores within the submerged biofilm formed at 10 °C, and the strengthened attachment of biofilm inner cells to the substrate was further revealed through biofilm structure parameters analysis. Collectively, our study revealed the prevalence and contamination levels of B. cereus and B. thuringiensis at fresh-cut lettuce production chain and investigated their genotypic and phenotypic characteristics, aiming to provide valuable insights for the development of potential risk management strategies to ensure food safety, especially along the cold chain.

An Outbreak of Bacillus cereus Emetic Toxin Mediated Food Poisoning After Consumption of Fried Rice in Assam.

Bacillus cereus is an emerging food-borne pathogen responsible for two types of food poisoning: emetic and diarrhoeal type. Here we report an emetic type of food-borne illness attributable to Bacillus cereus. On 2nd February, 2021, 202 people suffered from pain in abdomen and vomiting after consuming the rice provided during a public gathering in Diphu, Assam. Culture of leftover fried rice showed growth of Bacillus cereus group of organisms. Molecular detection of enterotoxin and emetic toxin genes was done in the isolated strains by polymerase chain reaction. Multi locus sequence typing (MLST) and phylogenetic analysis was done to characterise the isolated strains. A total of five strains of Bacillus cereus were isolated. Ces gene was found in isolates GMC22 & GMC24 and other enterotoxins producing genes were found in isolates GMC23 and GMC24. MLST identified four sequence types (STs) (ST1051, ST1616, ST998 and ST1000). Phylogenetic analysis clustered ST-1051 assigned to the GMC22 strain into the previously defined clade I and was in close relation with ST-144, representing a new cereulide-producing emetic cluster. As Bacillus cereus is a common contaminant of foods, it is essential to evaluate the pathogenic potential of the bacteria for a definite link between causative agents and the illness. MLST can be used to characterize the Bacillus cereus strains isolated from outbreak samples in order to link the probable pathogens with the illness. In this outbreak, we suggest that ST-1051 is the strain responsible for the food-borne illness, which was predominantly of emetic type.

Genomic Diversity and Virulence Genes Characterization of Bacillus cereus sensu lato Isolated from Processing Equipment of an Algerian Dairy Plant

Bacillus cereus is a ubiquitous spore-forming bacterium causing food spoilage. In this study, seventeen B. cereus were isolated from dairy processing equipment in Algeria and characterized. The pathogenic potential genes encoding hemolysin, nonhaemolytic enterotoxin, cytotoxin K and emetic toxin were investigated by PCR. Phylogeny and genetic markers for antimicrobial resistance and virulence were analyzed using whole-genome sequencing data using the Illumina MiSeq® technology. The identity of the isolated strains was confirmed as B. cereus sensu lato and the sequencing of the panC gene clustered 64.7% of them in phylogenetic group III and 35.3% in group IV. The results of PFGE showed that 8 (47.1%), 4 (23.5%), 3 (17.6%) and 2 (11.8%) were differentiated into four groups A, B, C and D, respectively. Each of the two panC phylogenetic groups contains two different types of isolates corresponding to PFGE group (A and C) and (B and D) for phylogenetic groups III and IV, respectively. None of the 14 isolates carried the emetic toxin (ces) gene, whereas other toxin genes were variably detected. Genotyping assigned all isolates to three different sequence types (ST 2226, ST1018 and ST1431) while one clonal complex (CC-142) was identified in three isolates. Hierarchical clustering with a threshold of 500 core genome single nucleotide polymorphism (cgSNPs) differences grouped the strains into three clusters while two strains were less than 20 SNPs different. Three B. cereus (25%) contained three enterotoxic HBL complex-encoding genes hblA/C/D. Enterotoxic NHE complex-encoding genes (nheA/B/C) and the ctyK1 gene were detected in all isolates. The overall results highlight the risk due to toxins produced by the B. cereus group in the food safety and dairy industries in Algeria. This study emphasized the potential of whole-genome sequencing for genotyping and predicting the virulence-associated genes and antimicrobial resistance. The present study will help to better assess the health and spoilage risk associated with B. cereus in dairy processing plants and to incorporate adequate preventive measures.

Profiling toxin genes and antibiotic resistance in Bacillus cereus isolated from pre-launch spacecraft.

Characterization of the microbiomes of pre-launch spacecraft in spacecraft assembly facilities is an important step in keeping crews healthy during journeys that can last several hundred days in small artificial environments in space. Bacillus cereus, a foodborne pathogenic bacterium, has the potential to be a significant source of food contamination in such environments. This bacterium is a spore-forming bacteria that resists different antimicrobial treatments in cleanrooms where spacecraft are assembled. This study evaluated 41 B. cereus isolates from four pre-launch spacecraft in spacecraft assembly facilities for their toxin gene profile and antibiotic resistance. Four enterotoxin genes (hlbC, cytK, nheA, and entFM) and two emetic toxin genes (ces and CER) were targeted for chromosomal DNA and plasmid DNA. Results showed 31.7, 7.3, 85, and 41.5% of isolates contained hblC, cytK, nheA, and entFM, respectively, in chromosomal or plasmid DNA. Overall, 37 isolates (90.2%) showed at least one enterotoxin gene. The emetic toxin gene, ces, was detected in the plasmid DNA of three isolates (7.3%). The antibiotic resistance of isolates was evaluated by the Kirby-Bauer disk diffusion procedure. All the isolates exhibited 100% susceptibility to gentamicin, 97% were susceptible to clindamycin, and 95% to chloramphenicol, imipenem, tetracycline, and vancomycin. The overall susceptibility average is 51%. However, 98% of the isolates were resistant to β-lactam antibiotics, 97.5% were resistant to sulfamethoxazole/trimethoprim, and 80% were resistant to rifampin. This study provides important information on B. cereus isolates from spacecraft assembly facilities for use in microbial monitoring programs of spacecraft.

Investigation of Virulence and Antibiotic-Resistance of Bacillus cereus Isolated from Various Spices.

Spices and herbs are potential vectors for virulent and pathogenic micro-organisms, which cause illness in consumers, contribute to spoilage, and reduce the durability of foodstuffs. The present study aims to provide relevant data about virulence and antibiotic resistance of Bacillus cereus isolated from various spices. A total of 200 samples of 8 types of spices (black pepper, chilli, white pepper, cumin, cinnamon, turmeric, curry powder, and sumac) were collected from various markets, retail shops, and sucuk production premises located in the Isfahan province of Iran. Presumptive B. cereus strains were obtained using Bacara Agar plates after enrichment in saline peptone water and final colonies were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Enterotoxin (HBL) and nonhaemolytic enterotoxin (NHE) production were assessed using the Duopath® Cereus Enterotoxins Test kit. The Kirby-Bauer disc diffusion method was applied as antibiotics susceptibility test. PCR was used to detect Emetic toxin gene (CES and CER) and enterotoxigenic toxin gene (cytK, nheA, hblC, and entFM). Results show a significant prevalence of B. cereus (42%) in spices. However, the spices meet food safety recommendations (<104 cfu/g). Antibiotics susceptibility test show alarming rate of resistance to beta-lactam antibiotics specially ampicillin (83.33%) and penicillin (82.14%). Concerning the toxin producing capacity more than half of the isolates (51.19%) produce NHE toxin and 27.38% produce HBL toxin. The most abundant gene were nheA, nheB, and nheC and a combination of 4 genes (entFM, nheA, hblC, and cytK) was detected in many isolates. In conclusion, the presence of multidrug resistant B. cereus strains carrying diarrhoeal toxin-encoding genes in spices intended for human consumption represents a serious health hazard. These results indicate the need for regular surveillance of the occurrence of B. cereus strains in spices and food products in Iran.

Prevalence and antimicrobial-resistant characterization of Bacillus cereus isolated from ready-to-eat rice products in Eastern China.

Bacillus cereus is a major food-borne bacterial pathogen in the world, which can cause diarrhea and emetic syndrome. This study aimed to reveal the quantitative prevalence of B. cereus in ready-to-eat (RTE) rice products in Eastern China and to gain essential information on the characteristics of B. cereus isolates. A total of 91 out of the 1071 samples were positive for B. cereus. The contamination level of B. cereus in 0.5 % of RTE rice product samples outnumbered 103 CFU/g. The number of B. cereus attained 105−106 CFU/g in one sample. The distribution patterns of virulence genes in B. cereus isolates were identified. 84.6% of the B. cereus isolates had at least one enterotoxin or emetic toxin gene. The predominant pattern was XXV. 9.9% of isolates belonged to it and possessed one enterotoxin gene entFM. The occurrence rate of hblACD and nheABC was 36.3% and 47.3%, respectively. Antimicrobial susceptibility tests revealed a high resistance rate toward penicillin, and 23.1% of the isolates were multi-drug resistant. B. cereus isolates were genotyped by using ERIC-PCR. 89 genotypes were determined. The Hunter Gaston Discriminatory Index (HGDI) attained 0.9995. Relationships analysis revealed that Group A B. cereus isolates tended to carry hblA, hblC, hblD, nheA, nheB, and show resistance to penicillin/trimethoprim/sulfamethoxazole. This study was useful for updating the knowledge of the contamination status of B. cereus in RTE rice products in China.

High Risk of Potential Diarrheagenic Bacillus cereus in Diverse Food Products in Egypt

Bacillus cereus is one of the important foodborne pathogens that can be found in various foodstuffs, causes diarrheal and/or emetic syndromes, and can cause severe systemic diseases that may lead to death. This study was conducted to evaluate the prevalence, antimicrobial susceptibility profile, pathogenic potential, and genotypic diversity of B. cereus isolates recovered from diverse food products collected from markets in Cairo, Egypt. Of 165 food samples investigated in this study, 39 (24%) were positive for B. cereus, with contamination levels of 2 to 6 log CFU/g or mL and a higher prevalence of levels >3 log CFU. Antimicrobial susceptibility testing revealed that the B. cereus isolates were fully sensitive to all tested antimicrobial agents except β-lactams. The pathogenic potential of the 39 B. cereus isolates was assessed by detecting and profiling genes encoding virulence factors or toxins: the chromosomal genes hblA, bceT, plc, sph, nheA, entFM, and cytK associated with the diarrheal syndrome and the plasmid ces gene associated with the emetic syndrome. The most frequently detected genes were hblA, nheA, and entFM. All isolates harbored more than one of the diarrheal enterotoxin genes, and the genetic profile hblA-bceT-nheA-entFM-cytK-plc-sph was the most prevalent (20 of 39 isolates). The emetic toxin gene ces was not detected in any isolate. Enterobacterial repetitive intergenic consensus analysis of the 20 B. cereus isolates harboring the most prevalent genetic profile revealed that these isolates were genetically distinct, with a Simpson index of diversity value of 0.989. These findings provide useful information for public health management and serve as a warning of the potential risk of diarrheagenic B. cereus in diverse food products. Therefore, extensive study of the epidemiology of this food pathogen in Egypt is warranted. Strict procedures should be developed to monitor, protect, and safely handle food products, particularly ready-to-eat foodstuffs that are usually consumed without heat treatment.

Detection of Bacillus cereus genes responsible for diarrheal and emetic toxins

Bacillus cereus isolated from different food sources. The diarrheal toxin genes such as cytK, hemolytic enterotoxin (hblA, hblC and hblD), non-hemolytic enterotoxin (nheA, nheB and nheC), bceT and entFM in addition to emetic toxin gene were detected by PCR. The cyt K gene was observed in 94.87 % of the isolates. entFM and emetic toxin gene were found very rare in all food samples at the percentage 2.56% and 7.69% respectively. Uncooked rice which has a highest number of bacterial isolation, also showed relatively high percentage of the cytK and bceT genes (90%). These two genes present in 100% of Bacillus cereus isolates in most food samples. Bacteria isolated from burger meat contain all investigated genes.

Prevalence, toxigenic profiles, multidrug resistance, and biofilm formation of Bacillus cereus isolated from ready-to eat cooked rice in Penang, Malaysia

Bacillus cereus is a foodborne pathogen which causes intoxication and toxico-infections in human. Spore- and biofilm-forming abilities of B. cereus enable its persistence in various ecological niches and food products, resulting in its presence even in processed foods such as cooked rice. Violation of time-temperature control creates favourable conditions for the growth of B. cereus which then produce emetic toxin and enterotoxins. Moreover, emerging antibiotic resistance in B. cereus promotes concern such as resistant strains of B. cereus facilitate in enrichment and emergence of more antibiotic resistant strains. Hence, this study was carried out to determine the prevalence of B. cereus in 100 ready-to-eat (RTE) cooked rice samples randomly collected from food outlets in Penang Island and to study the different B. cereus strains' characteristics which are relevant to foodborne illnesses, antibiotic resistance and potentiality to cause recurrent contamination. Aerobic plate count method was used for isolation and enumeration of B. cereus and the identities of the isolates were confirmed through 16S rRNA gene PCR and sequencing. PCR analysis was incorporated to detect 9 toxin-associated genes. The antibiogram of the B. cereus isolates against 13 antibiotics and biofilm-forming ability were determined by Kirby-Bauer disc diffusion method and submerged-biofilm assay respectively. A total of 34 B. cereus positive samples were identified with the bacterial loads ranging from 3.51 to 5.95 log CFU/g. High percentage (82.4%) of the isolates carried at least 1 toxin gene, with higher detection rates of most diarrheal genes (58.8–76.5%), than emetic toxin gene (14.7%). The percentage of bacterial isolates displaying resistance against ampicillin, penicillin and trimethoprim were in the range of 79.4–94.1%. High percentage was observed for sensitivity to antibiotics such as ciprofloxacin (94.1%), chloramphenicol (94.1%), gentamicin (88.2%), kanamycin (82.4%), vancomycin (82.4%) and tetracyline (73.5%). Twelve isolates were identified as multidrug-resistant (MDR) strains. The presence of antibiotic resistant B. cereus especially MDR strains in food commodities indicate the role of food systems as reservoir for resistance genes. High percentage (55.9%) of the isolates in this study were capable of biofilm formation, a phenotype that promotes recurrent contamination in food processing lines. In conclusion, RTE cooked rice samples may act as a vehicle for B. cereus dissemination and the characteristics displayed by the isolates reflect on their role in potential pathogenicity, due to the organism's resistance and multiple toxin genes.

Enterotoxin and Emetic Toxin Genes Profiles and Genetic Diversity of Bacillus cereus Isolated from Food, Environmental and Clinical Samples in Serbia

Abstract Bacillus cereus, usually ingested by food, can cause two types of disease due to the presence of toxins: vomiting and diarrhea syndrome. Systemic infections can also occur. The aim was to detect genes for enterotoxins (hblA, entFM) and emetic toxin (cer) and to investigate the genetic heterogeneity of B. cereus isolates from food, environment and human stool. Identification of B. cereus was performed by means of selective medium, classical biochemical test and polymerase chain reaction (PCR). Toxin genes were detected by PCR. Typing was performed by random amplified polymorphic DNA (RAPD). EntFM gene was present in all stool and food samples and in 28/30 environmental isolates. HblA gene was present in 29/30 stool, 23/30 food and 24/30 environmental isolates. Cer gene was present in 30/30 stool, 28/30 food and 25/30 environmental isolates. The RAPD results show high heterogeneity among the isolates from each group. In the cumulative dendrogram, representative isolates from all three groups formed two clusters with a difference of 53%. The detection of toxin genes in all B. cereus isolates indicated these bacteria as potentially pathogenic and a serious threat for human health. The presence of isolates from all three groups in the same cluster suggests the existence of similar strains in the environment, food and patients, which is in line with the circulation of strains in nature through the food chain.

Toxic potential of Bacillus cereus isolated from fermented alcoholic beverages

Pathogenicity of Bacillus cereus is associated with the production of various toxins. This study investigated the distribution of toxin genes encoding haemolysin BL (hblA, hblC, and hblD), nonhemolytic enterotoxin (nheA, nheB, and nheC), enterotoxin FM (entFM), cytotoxin K (cytK), and cereulide (ces) in 185 B. cereus strains isolated from draft beer, microbrewed beer, pasteurized beer, grape wine, other fruit wine, refined rice wine, traditional Korean pure liquor, and traditional Korean turbid rice wines. A total of 93.0% (172 isolates) of these isolates possessed at least one toxin gene. The nonhemolytic enterotoxin-encoding genes were highly prevalent in the isolates; the detection rate of enterotoxins was 91.4% for nheC, 81.6% for entFM, 62.7% for nheB, 57.3% for nheA, 53.0% for hblC, 48.6% for cytK, 36.8% for hblA, and 36.2% for hblD. Overall, 54.6% and 33.0% of strains carried the integrated Nhe-encoding gene cluster (nheA, nheB, nheC at the same time) and had the Hbl-encoding gene cluster, respectively. The cereulide synthetase gene was detected in only 2.2% of isolates. Toxin gene distribution patterns could be classified into 8 major profile clusters, and the most prevalent profile was the presence of enterotoxin genes only and no emetic toxin genes. Therefore, B. cereus in fermented alcoholic beverages was predominantly of the diarrhoeagenic type. Our results may provide important basic information when considering microbial standards and regulations for B. cereus in related products.

Investigation of the diversity and safety of the predominant Bacillus pumilus sensu lato and other Bacillus species involved in the alkaline fermentation of cassava leaves for the production of Ntoba Mbodi

The objective of the study was to investigate the identity, diversity, and safety of the Bacillus population involved in the fermentation of cassava (Manihot esculenta Crantz) leaves for the production of Ntoba Mbodi, a Congolese food. Ninety bacteria were identified by phenotyping and genotyping using ITS-PCR, rep-PCR, and sequencing of the 16S rRNA, gyrA, gyrB and rpoB genes. Moreover, the isolates were screened for the presence of genes coding for haemolytic (HblC, HblD) and non-haemolytic enterotoxins (NheA, NheB and NheC), cytotoxin K (CytK) and emetic toxin (EM1) as well as their ability to produce haemolysin.The investigations revealed the predominance (72.2%) of species of the Bacillus pumilus group i.e. B. safensis (48), B. pumilus (7), and B. pumilus sensu lato (10). Other species of Bacillus including B. cereus sensu lato (11), B. megaterium (4), B. subtilis (4), B. amyloliquefaciens (2), B. siamensis (2), B. licheniformis (1) and Lysinibacillus louembei (1) were also identified. Haemolytic, non-haemolytic and cytokin toxin genes were detected in the B. cereus strains which were also able to produce haemolysin. The emetic toxin gene was not detected in any isolates. The toxin genes screened were not detected in any of the non B. cereus species.

Genetic diversity, antimicrobial resistance, toxin gene profiles, and toxin production ability of Bacillus cereus isolates from doenjang, a Korean fermented soybean paste

AbstractThe objectives of this study were to determine the toxin gene profile, toxin production, and antibiotic resistance of Bacillus cereus isolates from doenjang, a Korean fermented soybean paste. Repetitive element sequence polymorphism‐PCR (rep‐PCR) was also used to assess the genetic diversity between these isolates. Thirty‐five B. cereus strains were isolated from 67 commercial doenjang samples, and all 35 isolates carried the nheABC and entFM genes and produced NHE enterotoxin. Of them, 12 strains harbored the emetic toxin gene (ces) and tested positive for the HEp‐2 cell emetic toxin assay. The B. cereus isolates were highly resistant to ß‐lactam antibiotics including ampicillin, cefepime, penicillin, and oxacillin. The rep‐PCR fingerprints of the B. cereus isolates were not influenced by the presence of toxin genes or antibiotic resistance profiles. However, rep‐PCR assay can be used to discriminate emetic B. cereus isolates from nonemetic isolates.Practical applicationsDoenjang has been the focus of recent concern in Korea because of its potential to cause foodborne illness via Bacillus cereus. The results of the present study suggest that B. cereus isolates from doenjang could cause either diarrheal or emetic types of foodborne illness because all isolates contained at least one toxin and its gene, although B. cereus levels in doenjang meet the Korean Food and Drug Administration guidelines (&lt;104 cfu/g). Thus, it is necessary to constantly monitor the levels of B. cereus contamination and its toxins in doenjang.

Surveillance of Bacillus cereus Isolates in Korea from 2012 to 2014.

ObjectivesTo investigate the prevalence and toxin production characteristics of non-emetic and emetic Bacillus cereus strains isolated via the laboratory surveillance system in Korea.MethodsA total of 667 B. cereus strains were collected by the Korea National Research Institute of Health laboratory surveillance system from 2012 to 2014. The collected strains were analyzed by geographical region, season, patient age, and patient sex. Additionally, the prevalence rates of enterotoxin and emetic toxin genes were evaluated.ResultsThe isolation rate of B. cereus strains increased during the summer, but the isolation rate was evenly distributed among patient age groups. Emetic toxin was produced by 20.2% of the isolated strains. The prevalence rates of five enterotoxin genes (entFM, nheA, cytK2, hblC, and bceT) were 85.0, 78.6, 44.5, 36.6, and 29.7%, respectively, among non-emetic strains and 77.8, 59.3, 17.8, 11.9 and 12.6%, respectively, among emetic strains. Thus, the prevalence rates of all five enterotoxin genes were lower in emetic B. cereus.ConclusionThe prevalence of enterotoxin genes differed between non-emetic and emetic B. cereus strains. Among emetic B. cereus strains, the prevalence rates of two enterotoxin genes (cytK2 and hblC) were lower than those among the non-emetic strains. In both the emetic and non-emetic strains isolated in Korea, nheA and entFM were the most prevalent enterotoxin genes.

In vitro evaluation of candidate Bacillus spp. for animal feed.

The aim of this study was to select aerobic spore-formers for animal feed based on their in vitro probiotic potential, including their enzyme-producing ability and safety assessment. Seven isolates out of 187 spore-forming bacteria were selected for their ability to produce cellulase (89.21-1668.32 U/ml), xylanase (1399.68-4351.10 U/ml), and phytase (2.72-28.70 U/ml). Among seven isolates, five had activities towards a broad range of p-nitrophenyl esters with acyl chain lengths from C2 to C12. The probiotic properties of all selected isolates varied with respect to their acid and bile salt tolerance under simulated gastrointestinal tract (GIT) conditions, and their adherence ability to human intestinal cell lines (Caco-2 and HT-29). The safety assessment revealed that the isolate CM40 was not cytotoxic to Caco-2 and HT-29, did not exhibit hemolytic activity, carried no enterotoxin or emetic toxin genes, and was susceptible to ten antibiotics, including six key antibiotics (chloramphenicol, erythromycin, gentamicin, tetracycline, streptomycin, and kanamycin) as recommended by the European Food Safety Authority (EFSA). Co-incubation of isolate CM40 with enteric bacteria (Salmonella Typhi, Salmonella Enteritidis 1781, and Escherichia coli) demonstrated that CM40 significantly decreased the number of pathogens (about 30-48%) adhering to Caco-2 and HT-29 (P < 0.05). Analysis of gene encoding 16S rRNA, gyrase A (gyrA) and the cheA histidine kinase revealed that CM40 belongs to Bacillus subtilis. On the basis of probiotic properties and basic safety aspects, the B. subtilis strain CM40 was found to possess desirable in vitro probiotic properties, and may be a potential candidate for supplementation of animal feed.

Detection of Emetic Toxin Genes in Bacillus cereus Isolated from Food and their Production of Cereulide in Liquid Culture

AbstractBacillus cereus is a Gram‐positive that is ubiquitous in foods and the wider environment. The B. cereus emetic toxin, cereulide, synthesized by nonribosomal peptide synthetase (NRPS), can cause either gastrointestinal disorders and/or emetic form of food poisoning. In this study, 108 B. cereus isolates from foods were tested for a correlation between the presence of emetic toxin genes and cereulide production. Based on 16S rRNA sequence analysis, 107 isolates were identical to B. cereus spp. Further experiments showed that fourteen isolates exhibited 1,271, 2,200 and 188‐bp bands, corresponding to amplification of the ces1, ces2 and NRPS gene, respectively. However, only 7 out of 13 strains possessing the NRPS, ces1, and ces2 genes produced cereulide in liquid culture. Strain number 93, isolated from fresh vegetables, produced the highest levels of cereulide (11.17 μg/mL); maximum cereulide levels were observed after 4 days of cultivation at 30C in LB broth. The amount of cereulide produced differed between each isolate.Practical ApplicationsThe incident of Bacillus cereus in food could cause food poisoning. The results of our study on the presence of B. cereus in spoiled foods demonstrated that not all the food poisoning B. cereus spp. have the ability to synthesize the emetic toxin, cereulide. Nonribosomal peptide synthetase and cereulide synthetase genes could be used as biological markers for differentiation of cereulide producing and nonproducing B. cereus. Estimation of cereulide level reveals that the amounts of cereulide produced by some strains were very high and could be a threat to human health if consumed in contaminated foods. This study presents proper methods to select cereulide producing food‐poisoning B. cereus spp. and gives more information on the condition of cereulide production to prevent future cases of emetic food poisoning.

Quantitative Prevalence and Toxin Gene Profile of Bacillus cereus from Ready-to-Eat Vegetables in South Korea.

Ready-to-eat (RTE) foods such as prepared vegetables are becoming an increasingly popular food choice. Since RTE vegetables are not commonly sterilized by heat treatment, contamination with foodborne pathogens such as Bacillus cereus (B. cereus) is a major concern. The objective of this study was to assess the quantitative prevalence and toxin gene profiles of B. cereus strains isolated from RTE vegetables. We found that 70 of the 145 (48%) tested retail vegetable salad and sprout samples were positive for B. cereus. The B. cereus isolates harbored at least one enterotoxin gene. The detection rates of nheABC, hblCDA, cytK, and entFM enterotoxin genes among all isolates were 97.1%, 100%, 81.4%, and 98.6%, respectively. No strain carried the emetic toxin genes. Only 4 strains (5.7%) from the 70 isolates were psychrotrophic and were able to grow at 7°C. All of the psychrotrophic isolates possessed at least 1 enterotoxin gene.

A novel pentaplex real time (RT)- PCR high resolution melt curve assay for simultaneous detection of emetic and enterotoxin producing Bacillus cereus in food

Bacillus cereus causing emetic and diarrheal type of food poisoning is widely distributed in nature and is therefore considered a major foodborne pathogen. There is a growing demand for fast, accurate, reliable and economic detection of potentially toxigenic B. cereus. To improve differential diagnosis of toxigenic B. cereus, a highly sensitive pentaplex RT- PCR high resolution melt curve assay was developed for simultaneous detection of 4 major enterotoxim genes (cytK, entFM, hblD, nheA) and emetic toxin gene (ces). The average melting temperatures (Tm) of PCR products were 72.2 °C (ces), 74.23 °C (cytK), 76.55 °C (nheA), 78.42 °C (entFM) and 81.90 (hblD). The multiplex assay was evaluated using 71 bacterial strains including 17 emetic B. cereus reference strains, 9 enterotoxic B. cereus reference strains, 4 B. cereus group members, 23 wild B. cereus strains, 18 non-target strains, and was further tested on artificially inoculated foods. The detection limit in food samples was approximately 103 CFU/g without enrichment and 101 CFU/g was observed following 7 h enrichment. The DNA intercalating dye SYTO9 used in this study generated high resolution melt curve peaks for the target strains and genes in which the peaks were sharp and easily distinguishable from each other. Thus, the developed multiplex real-time (RT) PCR approach can be a reliable tool for the identification of emetic and enterotoxic strains of B. cereus present in food and food-related samples.

Incidence, Antibiotic Susceptibility, and Toxin Profiles of Bacillus cereus sensu lato Isolated from Korean Fermented Soybean Products.

Korean fermented soybean products, such as doenjang, kochujang, ssamjang, and cho-kochujang, can harbor foodborne pathogens such as Bacillus cereus sensu lato (B. cereus sensu lato). The aim of this study was to characterize the toxin gene profiles, biochemical characteristics, and antibiotic resistance patterns of B. cereus sensu lato strains isolated from Korean fermented soybean products. Eighty-eight samples of Korean fermented soybean products purchased from retails in Seoul were tested. Thirteen of 26 doenjang samples, 13 of 23 kochujang samples, 16 of 30 ssamjang samples, and 5 of 9 cho-kochujang samples were positive for B. cereus sensu lato strains. The contamination level of all positive samples did not exceed 4 log CFU/g of food (maximum levels of Korea Food Code). Eighty-seven B. cereus sensu lato strains were isolated from 47 positive samples, and all isolates carried at least one enterotoxin gene. The detection rates of hblCDA, nheABC, cytK, and entFM enterotoxin genes among all isolates were 34.5%, 98.9%, 57.5%, and 100%, respectively. Fifteen strains (17.2%) harbored the emetic toxin gene. Most strains tested positive for salicin fermentation (62.1%), starch hydrolysis (66.7%), hemolysis (98.9%), motility test (100%), and lecithinase production (96.6%). The B. cereus sensu lato strains were highly resistant to β-lactam antibiotics such as ampicillin, penicillin, cefepime, imipenem, and oxacillin. Although B. cereus sensu lato levels in Korean fermented soybean products did not exceed the maximum levels permitted in South Korea (<10(4) CFU/g), these results indicate that the bacterial isolates have the potential to cause diarrheal or emetic gastrointestinal diseases.

Spore Prevalence and Toxigenicity of Bacillus cereus and Bacillus thuringiensis Isolates from U.S. Retail Spices

Recent incidents of foodborne illness associated with spices as the vehicle of transmission prompted this examination of U.S. retail spices with regard to Bacillus cereus. This study focused on the levels of aerobic-mesophilic spore-forming bacteria and B cereus spores associated with 247 retail spices purchased from five states in the United States. Samples contained a wide range of aerobic-mesophilic bacterial spore counts (< 200 to 8.3 × 10(7) CFU/g), with 19.1% of samples at levels above 10(5) CFU/g. For examples, paprika, allspice, peppercorns, and mixed spices had high levels of aerobic spores (> 10(7) CFU/g). Using a novel chromogenic agar, B. cereus and B. thuringiensis spores were isolated from 77 (31%) and 11 (4%) samples, respectively. Levels of B. cereus were <3 to 1,600 MPN/g. Eighty-eight percent of B. cereus isolates and 91% of B. thuringiensis isolates possessed at least one type of enterotoxin gene: HBL (hemolysin BL) or nonhemolytic enterotoxin (NHE). None of the 88 isolates obtained in this study possessed the emetic toxin gene (ces). Using commercially available immunological toxin detection kits, the toxigenicity of the isolates was confirmed. The NHE enterotoxin was expressed in 98% of B. cereus and 91% of B. thuringiensis isolates that possessed the responsible gene. HBL enterotoxin was detected in 87% of B. cereus and 100% of B. thuringiensis PCR-positive isolates. Fifty-two percent of B. cereus and 54% of B. thuringiensis isolates produced both enterotoxins. Ninety-seven percent of B. cereus isolates grew at 12°C, although only two isolates grew well at 9°C. The ability of these spice isolates to form spores, produce diarrheal toxins, and grow at moderately abusive temperatures makes retail spices an important potential vehicle for foodborne illness caused by B. cereus strains, in particular those that produce diarrheal toxins.