Hemolysin BL Enterotoxin Research Articles

Short Communication: Enterotoxin Genes and Antibiotic Susceptibility of Bacillus cereus Isolated from Garlic Chives and Agricultural Environment.

This study aims to investigate the enterotoxin profiles and antibiotic susceptibility of Bacillus cereus isolated from garlic chives and environmental samples. A total of 103 B. cereus isolates were used to identify enterotoxin genes, including hblA, hblC, hblD, nheA, nheB, and nheC. The hemolysin BL enterotoxin complex (hblACD) was detected in 38 isolates (36.9%), and the non-hemolytic enterotoxin complex (nheABC) was detected in 8 (7.8%) isolates. Forty-five isolates (43.7%) had hblACD and nheABC genes. B. cereus was resistant to β-lactam antibiotics and susceptible to non-β-lactam antibiotics. However, some B. cereus strains showed intermediate resistance to β-lactam and non-β-lactam antibiotics. B. cereus isolated from garlic chives showed intermediate resistance to cefotaxime (7.7%), rifampin (15.4%), clindamycin (30.8%), erythromycin (7.7%), and tetracycline (7.7%). B. cereus isolates from the agricultural environment were moderately resistant to cefotaxime (18.9%), rifampin (15.6%), clindamycin (12.2%), erythromycin (4.4%), and tetracycline (5.6%). Moreover, B. cereus isolates from garlic chives and cultivation environments could change their antibiotic resistance profile from susceptible to intermediate-resistant to rifampin, clindamycin, erythromycin, and tetracycline and exhibit multidrug resistance. These results indicate that continuous monitoring of B. cereus contamination in the produce and agricultural environment might be needed to ensure the safety of consuming fresh vegetables.

시중 유통 중인 향신료가공품의 미생물학적 안전성 평가

The study investigated the level of contamination by total aerobic bacteria, coliform bacteria and food poisoning bacteria in commercial spices for the evaluation of microbiological safety. A total of 119 commercial spices was used for this study. The total aerobic bacterial count was 6.2 log CFU/g in HACCP certificated spices and 5.4 log CFU/g in HACCP non-certificated spices. Coliform bacteria were detected in 13 (35.1%) out of 37 HACCP certificated spices and 27 (32.9%) out of 82 HACCP non-certificated spices. Bacillus cereus was detected in 7 (18.9%) out of 37 HACCP certificated spices and 10 (11.8%) out of 82 non-certificated spices. All the B. cereus detected in this study possessed at least one toxin gene, and hemolysin BL enterotoxin was detected in 9 (52.9%) out of 17 B. cereus isolates. Overall, these results indicated that contamination with total aerobic bacteria was higher in HACCP certified spices, with more coliform bacteria and B. cereus detected, than in non-certificated spices. Hence, it is necessary to refine the HACCP system and introduce good agricultural practices for the improvement of microbiological safety of spices.

Prevalence and Toxin Characteristics of Bacillus thuringiensis Isolated from Organic Vegetables.

The prevalence and toxin characteristics of Bacillus thuringiensis isolated from 39 organic vegetables were investigated. B. thuringiensis was detected in 30 out of the 39 organic vegetables (76.9%) with a mean value of 2.60 log CFU/g. Twenty-five out of the 30 B. thuringiensis isolates (83.3%) showed insecticidal toxicity against Spodoptera exigua. The hblCDA, nheABC, and entFM genes were found to be the major toxin genes, but the ces gene was not detected in any of the tested B. thuringiensis isolates. The hemolysin BL enterotoxin was detected in all 30 B. thuringiensis isolates (100%). The non-hemolytic enterotoxin complex was found in 27 out of 30 B. thuringiensis isolates (90.0%). The B. thuringiensis tested in this study had similar toxin gene characteristics to B. cereus, which possessed more than one toxin gene. B. thuringiensis could have the potential risk of foodborne illness based on the toxin genes and toxin-producing ability.

Isolation and Characterization of Spore-Forming Bacilli (SFB) from Shepherd's Purse (Capsella bursa-pastoris).

Shepherd's purse (Capsella bursa-pastoris), native to Europe, is commonly consumed fresh and sometimes inadequately washed before consumption in Korea. The objective of this study was to characterize isolates of spore-forming bacilli (SFB) in samples of fresh Shepherd's purse. Three genera were identified: Bacillus (9 species), Paenibacillus (3 species), and Brevibacillus (1 species). None of the genes of the hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE) complexes, or of the emetic toxin, was detected in the 25 SFB isolates, except for 2 Bacillus pseudomycoides isolates, where all 3 genes of the HBL enterotoxin complex were detected. There were significant sequence variations between the 2 species (Bacillus cereus and B. pseudomycoides) in the 3 genes of the HBL enterotoxin complex. These findings may provide insights into the diverse characteristics of the B. pseudomycoides HBL enterotoxin complex. Antibiotic resistance was assessed using 8 antibiotics. Among the 25 SFB isolates, 11 showed resistance to antibiotics, of which 5 were multiresistant. Assessment of the spoilage potential showed that all 25 SFB isolates could produce enzymes that can cause spoilage of foods. In conclusion, our findings may serve as integrative information for food research and industrial sectors.

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.

Growth of enterotoxigenic Bacillus cereus on salmon (Oncorhynchus nerka).

We previously demonstrated the widespread presence of enterotoxigenic Bacillus cereus in marine foods. In view of the widespread consumption of raw fish, we sought to determine the ability of this organism to grow on the surface of wild Alaskan salmon at abusive temperatures (12, 16, and 20°C), using an isolate able to produce elevated levels of hemolysin BL enterotoxin and nonhemolytic enterotoxin. An incubation temperature of 37°C for colony formation was found to be selective for B. cereus grown on salmon held for up to 24 h at each temperature. A fivefold increase in log CFU per gram was observed after 26 and 22 h at 16 and 20°C, respectively, while a >4-log CFU/g increase occurred on salmon held at 12°C for 48 h. Generation times of 169.7, 53.5, and 45.6 min were observed at 12, 16, and 20°C. Nonhemolytic enterotoxin was detected when levels of B. cereus were in excess of 10(8) CFU/g. Nisin, at concentrations of 1 and 15 m g/g of salmon, reduced levels of B. cereus 2.5- and 25-fold, respectively. Our results indicate that fresh salmon can serve as an excellent substrate for enterotoxigenic B. cereus and that this organism can reach levels associated with foodborne illness following moderate temperature abuse.

Genetic diversity, antimicrobial resistance, and toxigenic profiles of Bacillus cereus strains isolated from Sunsik.

Bacillus cereus can cause emetic and diarrheal types of food poisoning, but little study has been done on the toxins and toxin-encoding genes of B. cereus strains isolated from Sunsik, a Korean ready-to-eat food prepared from grains, fruits, and vegetables. In this study, 39 unique B. cereus strains were isolated and identified from Sunsik samples, with an average contamination level of 10 to 200 CFU/g. The detection rates of the hblACD, cytK, and bceT genes among all the strains were 48.7, 66.7, and 87.1%, respectively. All 39 B. cereus strains carried nheABC and entFM genes, and 36 strains also had the ces gene, which encodes an emetic toxin. Nonhemolytic enterotoxin and hemolysin BL enterotoxin were produced by 39 and 26 strains, respectively. The strains were separated into 13 profiles based on the presence or absence of toxins and their genes, as determined by antibody tests and PCR analysis. Profile 1 was the largest group, comprising 30.7% (12 of 39) of the B. cereus strains tested; these strains harbored all toxins and their genes. The B. cereus strains were susceptible to most of the antibiotics tested but were highly resistant to b -lactam antibiotics. The repetitive element sequence polymorphism PCR fingerprints of the B. cereus strains were not influenced by the presence of toxin genes or antibiotic resistance profiles. Our results suggest that B. cereus strains from Sunsik could cause either the diarrheal or emetic types of food poisoning because all strains isolated contained at least one toxin and its gene, although the level of B. cereus contamination in Sunsik was low.

Toxin Genes Profiles and Toxin Production Ability of Bacillus cereus Isolated from Clinical and Food Samples

Bacillus cereus can cause diarrheal and emetic type of food poisoning but little study has been done on the main toxins of food poisoning caused by B. cereus in Korea. The objective of this study is to characterize the toxin gene profiles and toxin-producing ability of 120 B. cereus isolates from clinical and food samples in Korea. The detection rate of nheABC, hblCDA, entFM, and cytK enterotoxin gene among all B. cereus strains was 94.2, 90.0, 65.8, and 52.5%, respectively. The ces gene encoding emetic toxin was not detected in all strains. Bacillus cereus strains carried at least 1 of the 8 enterotoxin genes were classified into 12 groups according to the presence or absence of 8 virulence genes. The 3 major patterns, I (nheABC, hblCDA, entFM, and cytK gene), II (nheABC, hblCDA and entFM gene), and VI (nheABC and hblCDA gene), accounted for 79.2% of all strains (95 out of 120 B. cereus isolates). Non-hemolytic enterotoxin (NHE) and hemolysin BL (HBL) enterotoxins were produced by 107 and 100 strains, respectively. Our finding revealed that NHE and HBL enterotoxins encoded by nhe and hbl genes were the major toxins among B. cereus tested in this study and enterotoxic type of B. cereus was predominant in Korea.

Detection of toxigenic Bacillus cereus and Bacillus thuringiensis spores in U.S. rice

Bacillus cereus is a gram-positive, endospore forming pathogenic bacterium that is ubiquitous in the environment and is frequently associated with emetic and diarrheal types of foodborne illness. In this study, 178 samples of raw rice from retail food stores were analyzed for the presence of B. cereus spores. Spores of Bacillus species were found in 94 (52.8%) of the rice samples with an average concentration of 32.6 CFU/g (3.6–460 CFU/g for B. cereus and 3.6–23 CFU/g for Bacillus thuringiensis). Eighty three of the 94 isolates were identified as B. cereus and 11 were identified as B. thuringiensis. Bacillus mycoides (240 CFU/g) was the predominant isolate in one rice sample. Using PCR the isolates were checked for the presence of the cereulide synthetase gene ( ces), the hblA and hblD genes of the hemolysin BL (HBL) complex and the nheA and nheB genes of the nonhemolytic (NHE) enterotoxin complex. The ces gene was not identified in any of the isolates. By contrast 47 (56.6%) B. cereus isolates possessed the hblA and hblD genes and 74 (89.1%) isolates possessed the nheA and nheB genes. As determined by commercial assay kits, forty four (53.0%) of the 83 B. cereus isolates produced both NHE and HBL enterotoxins whereas 78 (93.9%) were positive for either one or the other. Protein toxin crystals were detected visually in the 11 B. thuringiensis isolates. PCR analysis revealed 10 (90.9%) of those 11 isolates carried the cry gene. All the B. thuringiensis isolates were positive for NHE and HBL enterotoxins. Our results suggest that foodborne illness in the U.S. due to B. cereus with rice as the vehicle would be most likely associated with the diarrheal-type syndrome.

X‐ray crystal structure of the B component of Hemolysin BL from Bacillus cereus

Bacillus cereus Hemolysin BL enterotoxin, a ternary complex of three proteins, is the causative agent of food poisoning and requires all three components for virulence. The X-ray structure of the binding domain of HBL suggests that it may form a pore similar to other soluble channel forming proteins. A putative pathway of pore formation is discussed.

Effects of porcine bile on survival of Bacillus cereus vegetative cells and Haemolysin BL enterotoxin production in reconstituted human small intestine media

To determine the effects of porcine bile (PB) on Bacillus cereus vegetative cells and Haemolysin BL (HBL) enterotoxin production in reconstituted small intestine media (IM). The effects of PB on the growth of B. cereus vegetative cells in reconstituted IM at PB concentrations ranging between 0 and 3.0 g l(-1) were examined. Four gastric media (GM) named GM-J broth (JB), GM-chicken, GM-milk and GM-pea were prepared by mixing equal volumes of a gastric electrolyte solution containing pepsin with JB, chicken, semi-skimmed milk and pea soup, respectively. Bacillus cereus was inoculated at approx. 2 x 10(4) CFU ml(-1) into each GM at pH 5.0 for 30 min at 37 degrees C, then mixed to the same volume of double-strength JB (IM) and PB to give concentrations of between 0 and 3.0 g of PB per litre at pH 6.5 and incubated at 37 degrees C. The diarrhoeal B. cereus strain F4430/73 grew in IM-JB, IM-chicken and IM-milk at PB concentrations of up to 0.6, 1.5 and 1.2 g l(-1), respectively. Growth was observed in IM-pea at all concentrations tested. The highest PB concentrations allowing a 3 log B. cereus increase in IM-JB, IM-chicken, IM-milk and IM-pea after a 7-10 h incubation period were 0.3, 0.9, 0.9 and 3.0 g l(-1), respectively. The effect of PB on B. cereus cells was strongest in IM-JB, followed by IM-chicken, IM-milk and IM-pea. Haemolysin BL enterotoxin was detectable in IM-chicken, IM-whole milk, IM-semi-skimmed milk and IM-pea up to PB concentrations of only 0.6, 0.6, 0.3 and 0.9 g l(-1), respectively. The diarrhoeal B. cereus strain F4433/73 behaved similarly to B. cereus strain F4430/73, whereas the food strain TZ415 was markedly more susceptible to bile. The tolerance of B. cereus cells to PB strongly depends on the type of food contained in the IM. Bile tolerance is also subject to strain variation. The probability that B. cereus cells will grow in the small intestine, produce toxins and cause diarrhoea is likely to depend on the food they are ingested with, on the bile tolerance of the B. cereus strain, and on bile concentration.

Colony immunoblot assay for the detection of hemolysin BL enterotoxin producingBacillus cereus

Bacillus cereus strains involved in food poisoning cases of the diarrheal type may produce two different enterotoxin complexes. To facilitate the identification of hemolysin BL-enterotoxin complex (HBL) and/or the nonhemolytic enterotoxin (NHE) producing colonies a colony immunoblot procedure was developed, which allows a fast and easy identification of the respective colonies from blood agar plates. The enterotoxins were transferred from the blood agar medium to a nitrocellulose membrane and the immobilized toxins were probed with monoclonal antibodies. The antibodies 2A3 and 1A8 allowed the specific detection of the B component of HBL and the nheA component of NHE. The assay enabled the reliable identification of HBL expressing colonies and differentiation from NHE producing but HBL negative colonies.

Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing Bacillus cereus

Bacillus cereus strains involved in food poisoning cases of the diarrheal type may produce two different enterotoxin complexes. To facilitate the identification of hemolysin BL-enterotoxin complex (HBL) and/or the nonhemolytic enterotoxin (NHE) producing colonies a colony immunoblot procedure was developed, which allows a fast and easy identification of the respective colonies from blood agar plates. The enterotoxins were transferred from the blood agar medium to a nitrocellulose membrane and the immobilized toxins were probed with monoclonal antibodies. The antibodies 2A3 and 1A8 allowed the specific detection of the B component of HBL and the nheA component of NHE. The assay enabled the reliable identification of HBL expressing colonies and differentiation from NHE producing but HBL negative colonies.

Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae.

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.

Bacillus cereus Group Strains, Their Hemolysin BL Activity, and Their Detection in Foods Using a 16S RNA and Hemolysin BL Gene-Targeted Multiplex Polymerase Chain Reaction System

Hemolysin BL (HBL) is a major virulence factor for Bacillus cereus group strains. It is also a target enterotoxin for the most commonly used B. cereus detection kit, i.e., the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (BCET-RPLA) test kit. A survey of the HBL activities and the cytotoxicities to the Chinese hamster ovary (CHO) cells for the B. cereus group strains, however, showed that although only part of the B. cereus group strains are HBL active, all strains show cytotoxicity to the CHO cells. Thus, methods that allow the detection of not only the HBL but also of the B. cereus group strains are important. In this study, by comparison of the gene sequences of the 16S rRNA for B. cereus group and other bacteria strains, we designed primers B16S1 and B16S2 specific to all the B. cereus group strains. In addition, because HBL is a major enterotoxin, we also designed HBL gene-specific polymerase chain reaction (PCR) primers, i.e., Hm1 and Hm2, that generated the same results as those of the hemolysis and BCET-RPLA assays. Primers B16S1/B16S2 and Hm1/Hm2 could be combined into a multiplex PCR system for the simultaneous detection of B. cereus group cells and the possible presence of their HBL enterotoxins. Also, all these PCR systems allowed the detection of n × 100 CFU B. cereus cells per g of food sample if an 8-h enrichment step was performed prior to the PCR.

Production and characterization of monoclonal antibodies against the hemolysin BL enterotoxin complex produced by Bacillus cereus.

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereus were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L(2) component, and antibody 1C2 was specific for the L(1) protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains of B. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity with B. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.

Evidence for a further enterotoxin complex produced by Bacillus cereus

Out of 321 strains of Bacillus cereus from several sources and isolated in four different countries, 239 (74%) produced cytotoxins. Only 127 (53%) of the cytotoxic strains were positive for the B-component gene of the haemolysin BL (enterotoxin) by polymerase chain reaction (PCR). Western blots using antiserum produced against enterotoxin(s) gave positive results for 199 (83%) of the cytotoxic B. cereus strains. On closer examination of seven of the strains, involved in food poisoning, we found that two strains completely lacked the L 2- and B-components (of the haemolysin BL), and two strains were negative for the B-component gene by PCR, but were positive for the L 2-component. From our experiments we concluded that there is at least one enterotoxin complex in addition to the haemolysin BL enterotoxin and enterotoxin T.

Evidence for a further enterotoxin complex produced by Bacillus cereus.

Out of 321 strains of Bacillus cereus from several sources and isolated in four different countries, 239 (74%) produced cytotoxins. Only 127 (53%) of the cytotoxic strains were positive for the B-component gene of the haemolysin BL (enterotoxin) by polymerase chain reaction (PCR). Western blots using antiserum produced against enterotoxin(s) gave positive results for 199 (83%) of the cytotoxic B. cereus strains. On closer examination of seven of the strains, involved in food poisoning, we found that two strains completely lacked the L2- and B-components (of the haemolysin BL), and two strains were negative for the B-component gene by PCR, but were positive for the L2-component. From our experiments we concluded that there is at least one enterotoxin complex in addition to the haemolysin BL enterotoxin and enterotoxin T.