Epidemiology and pathogenesis of Bacillus cereus infections

Bacillus cereus is a causative agent in both gastrointestinal and in nongastrointestinal infections. Enterotoxins, emetic toxin (cereulide), hemolysins, and phoshpolipase C as well as many enzymes such as beta-lactamases, proteases and collagenases are known as potential virulence factors of B. cereus. A special surface structure of B. cereus cells, the S-layer, has a significant role in the adhesion to host cells, in phagocytosis and in increased radiation resistance. Interest in B. cereus has been growing lately because it seems that B. cereus-related diseases, in particular food poisonings, are growing in number.     

普洱茶中微生物的筛选及其安全性初步评价

目的在对普洱茶中微生物筛选和鉴定基础上对蜡样芽胞杆菌毒素基因的分布、普洱茶下调毒素基因的表达和改善肠上皮细胞的损伤进行研究。方法分别采用无菌水和沸水泡制普洱茶,获得分离株,通过16SrDNA测序以及生理生化试验确定其归属;对所筛选的菌株进行耐模拟胃肠液能力评价和毒力基因的检测;采用细胞实验和荧光定量PCR技术,研究普洱茶对蜡样芽胞杆菌毒素的抑制作用。结果无菌水浸泡普洱茶获得的45株菌中44株为芽胞杆菌属,沸水浸泡获得的7株菌均为蜡样芽胞杆菌(FBCE01、FBCE06、FBCE10、FBCE14、FBCE20、FBCE26和FBCE29),多重PCR技术结果表明其分别含有毒力基因cytK、nheA和hblD的2种或3种。耐模拟胃肠液实验表明,7株菌均具有很强的耐模拟胃肠液消化能力;细胞实验结果发现,普洱茶汤能显著降低蜡样芽胞杆菌对Caco-2细胞的粘附(P0.05);MTT实验结果显示,普洱茶能有效降低蜡样芽胞杆菌对细胞的损伤;荧光定量PCR技术结果进一步说明,普洱茶使蜡样芽胞杆菌肠毒素的mRNA表达水平下调。结论普洱茶具有抑制蜡样芽胞杆菌毒素的作用。     

Cereulide, the emetic toxin of Bacillus cereus, is putatively a product of nonribosomal peptide synthesis

AIMS: To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a nonribosomal peptide synthetase (NRPS). METHODS AND RESULTS: NC Y, an emetic strain of Bacillus cereus, was examined for a NRPS gene using PCR with primers recognizing a fragment of a NRPS gene from the cyanobacterium Microcystis. The amplicon was sequenced and compared with other gene sequences using BLAST analysis, which showed that the amplicon from strain NC Y was similar in sequence to peptide synthetase genes in other micro-organisms, including Bacillus subtilis and B. brevis, while no such sequence was found in the complete genome sequence of a nonemetic strain of B. cereus. Specific PCR primers were then designed and used to screen 40 B. cereus isolates previously implicated in outbreaks of foodborne illness. The isolates were also screened for toxin production using the MTT cell cytotoxicity assay. PCR and MTT assay screening of the B. cereus isolates revealed a high correlation between the presence of the NRPS gene and cereulide production. CONCLUSIONS: The results indicate that cereulide is produced by a NRPS complex. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide evidence identifying the mechanism of production of cereulide, the emetic toxin of B. cereus. The PCR primers developed in the study allow determination of the potential for cereulide production among isolates of B. cereus.     

An improved method for detecting cytostatic toxin (emetic toxin) of Bacillus cereus and its application to food samples

Abstract We developed an improved HEp-2 cell assay method for the detection of Bacillus cereus toxin, which affects the proliferation of HEp-2 cells. The cytostatic toxin was stable upon exposure to heat, pH 2, pH 11 and trypsin, which suggests it is an emetic. Using the HEp-2 cell assay, we examined the distribution and contamination of B. cereus strains that produced an emetic toxin in various foods. Although there were 228 enterotoxin producers among 310 B. cereus strains obtained from foods, 16 of them produced the cytostatic type (emetic toxin). All of the strains that produced the cytostatic toxin were of the H.1 serotype.     

Growth conditions of and emetic toxin production by Bacillus cereus in a defined medium with amino acids

The growth and emetic toxin (cereulide) production of Bacillus cereus strains in defined culture media were studied. We found that a fully synthetic medium (CADM) allowed the production of emetic toxin and the addition of glucose enhanced it. By subtracting each amino acid from CADM, we found that only three amino acids, valine, leucine and threonine, were essential for growth and toxin production by B. cereus. The addition of high levels (50 mM) of leucine, isoleucine and glutamic acid decreased the toxin production. Other amino acids had no effect at this concentration.     

Heat-stable toxin production by strains of Bacillus cereus, Bacillus firmus, Bacillus megaterium, Bacillus simplex and Bacillus licheniformis

Strains of Bacillus cereus can produce a heat-stable toxin (cereulide). In this study, 101 Bacillus strains representing 7 Bacillus species were tested for production of heat-stable toxins. Strains of B. megaterium, B. firmus and B. simplex were found to produce novel heat-stable toxins, which showed varying levels of toxicity. B. cereus strains (18 out of 54) were positive for toxin production. Thirteen were of serovar H1, and it was of interest that some were of clinical origin. Two were of serovars 17B and 20, which are not usually implicated in the emetic syndrome. Partial purification of the novel B. megaterium, B. simplex and B. firmus toxins showed they had similar physical characteristics to the B. cereus emetic toxin, cereulide.     

Improved cytotoxicity assay for Bacillus cereus diarrhoeal enterotoxin

An improved McCoy cell cytotoxicity assay for Bacillus cereus diarrhoeal toxin, which includes a staining procedure in addition to visual examination, was developed and the method was compared with two commercially available kits (Oxoid BCET-RPLA and Tecra BDE-VIA). A total of 71 strains of 15 different Bacillus, Brevibacillus and Paenibacillus species, including 16 strains of B. cereus from outbreaks of food-borne illness, were evaluated for toxin production. Eleven of the outbreak strains exhibited cytotoxicity, including all six B. cereus strains associated with diarrhoeal illness. Several other isolates of B. cereus, and its relatives B. anthracis, B. mycoides and B. thuringiensis, exhibited similar cytotoxicity. The other species showed no cytotoxicity, with the exception of certain B. subtilis strains. The cytotoxicity assay was more sensitive than the Oxoid kit and unlike the Tecra kit, did not give false positive results with supernatant fluids heat-treated to destroy the toxin.     

Production of diarrheal enterotoxins and other potential virulence factors by veterinary isolates of bacillus species associated with nongastrointestinal infections

With the exceptions of Bacillus cereus and Bacillus anthracis, Bacillus species are generally perceived to be inconsequential. However, the relevance of other Bacillus species as food poisoning organisms and etiological agents in nongastrointestinal infections is being increasingly recognized. Eleven Bacillus species isolated from veterinary samples associated with severe nongastrointestinal infections were assessed for the presence and expression of diarrheagenic enterotoxins and other potential virulence factors. PCR studies revealed the presence of DNA sequences encoding hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T (BceT) in five B. cereus strains and in Bacillus coagulans NB11. Enterotoxin HBL was also harbored by Bacillus polymyxa NB6. After 18 h of growth in brain heart infusion broth, all seven Bacillus isolates carrying genes encoding enterotoxin HBL produced this toxin. Cell-free supernatant fluids from all 11 Bacillus isolates demonstrated cytotoxicity toward human HEp-2 cells; only one Bacillus licheniformis strain adhered to this test cell line, and none of the Bacillus isolates were invasive. This study constitutes the first demonstration that Bacillus spp. associated with serious nongastrointestinal infections in animals may harbor and express diarrheagenic enterotoxins traditionally linked to toxigenic B. cereus.     

Semiautomated metabolic staining assay for Bacillus cereus emetic toxin

This paper describes a specific, sensitive, semiautomated, and quantitative Hep-2 cell culture-based 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay for Bacillus cereus emetic toxin. Of nine Bacillus, Brevibacillus, and Paenibacillus species assessed for emetic toxin production, only B. cereus was cytotoxic.     

A complete physical map of a Bacillus thuringiensis chromosome.

Bacillus thuringiensis is the source of the most widely used biological pesticide, through its production of insecticidal toxins. The toxin genes are often localized on plasmids. We have constructed a physical map of a Bacillus thuringiensis chromosome by aligning 16 fragments obtained by digestion with the restriction enzyme NotI. The fragments ranged from 15 to 1,350 kb. The size of the chromosome was 5.4 Mb. The NotI DNA fingerprint patterns of 12 different B. thuringiensis strains showed marked variation. The cryIA-type toxin gene was present on the chromosome in four strains, was extrachromosomal in four strains, and was both chromosomal and extrachromosomal in two strains. A Tn4430 transposon probe hybridized to 5 of the 10 cryIA-positive chromosomal fragments, while cryIA and the transposon often hybridized to different extrachromosomal bands. Ten of the strains were hemolytic when grown on agar plates containing human erythrocytes. Nine of the strains were positive when assayed for the presence of Bacillus cereus enterotoxin. We conclude that B. thuringiensis is very closely related to B. cereus and that the distinction between B. cereus and B. thuringiensis should be reconsidered.     

Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay

Bacillus cereus causes two types of gastrointestinal diseases: emesis and diarrhea. The emetic type of the disease is attributed to the heat-stable depsipeptide cereulide and symptoms resemble Staphylococcus aureus intoxication, but there is no rapid method available to detect B. cereus strains causing this type of disease. In this study, a polymerase chain reaction (PCR) fragment of unknown function was identified, which was shown to be specific for emetic toxin producing strains of B. cereus. The sequence of this amplicon was determined and a PCR assay was developed on this basis. One hundred B. cereus isolates obtained from different food poisoning outbreaks and diverse food sources from various geographical locations and 29 strains from other species belonging to the B. cereus group were tested by this assay. In addition, 49 non-B. cereus group strains, with special emphasis on food pathogens, were used to show that the assay is specific for emetic toxin producing B. cereus strains. The presented PCR assay is the first molecular tool for the rapid detection of emetic toxin producing B. cereus strains.     

Distribution of genes encoding putative virulence factors and fragment length polymorphisms in the vrrA gene among Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

One hundred twenty-one strains of the Bacillus cereus complex, of which 80 were isolated from a variety of sources in Brazil, were screened by PCR for the presence of sequences (bceT, hblA, nheBC, plc, sph, and vip3A) encoding putative virulence factors and for polymorphisms in variable-number tandem repeats (VNTR), using a variable region of the vrrA open reading frame as the target. Amplicons were generated from isolates of B. cereus and Bacillus thuringiensis for each of the sequences encoding factors suggested to play a role in infections of mammals. Intriguingly, the majority of these sequences were detected more frequently in Bacillus thuringiensis than in B. cereus. The vip3A sequence, which encodes an insecticidal toxin, was detected exclusively in B. thuringiensis. VNTR analysis demonstrated the presence of five different fragment length categories in both species, with two of these being widely distributed throughout both taxa. In common with data generated from previous studies examining European, Asian, or North American populations, our investigation of Brazilian isolates supports the notion that B. cereus and B. thuringiensis should be considered to represent a single species.     

Cholera- and anthrax-like toxins are among several new ADP-ribosyltransferases

Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins we identified and characterized using in silico and cell-based techniques. We also uncovered medically relevant toxins from Mycobacterium avium and Enterococcus faecalis. We found agriculturally relevant toxins in Photorhabdus luminescens and Vibrio splendidus. These toxins belong to the ADP-ribosyltransferase family that has conserved structure despite low sequence identity. Therefore, our search for new toxins combined fold recognition with rules for filtering sequences--including a primary sequence pattern--to reduce reliance on sequence identity and identify toxins using structure. We used computers to build models and analyzed each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. We confirmed activity using a yeast growth test. In this era where an expanding protein structure library complements abundant protein sequence data--and we need high-throughput validation--our approach provides insight into the newest toxin ADP-ribosyltransferases.     

From soil to gut: Bacillus cereus and its food poisoning toxins

Bacillus cereus is widespread in nature and frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insects and mammals. From these habitats it is easily spread to foods, where it may cause an emetic or a diarrhoeal type of food-associated illness that is becoming increasingly important in the industrialized world. The emetic disease is a food intoxication caused by cereulide, a small ring-formed dodecadepsipeptide. Similar to the virulence determinants that distinguish Bacillus thuringiensis and Bacillus anthracis from B. cereus, the genetic determinants of cereulide are plasmid-borne. The diarrhoeal syndrome of B. cereus is an infection caused by vegetative cells, ingested as viable cells or spores, thought to produce protein enterotoxins in the small intestine. Three pore-forming cytotoxins have been associated with diarrhoeal disease: haemolysin BL (Hbl), nonhaemolytic enterotoxin (Nhe) and cytotoxin K. Hbl and Nhe are homologous three-component toxins, which appear to be related to the monooligomeric toxin cytolysin A found in Escherichia coli. This review will focus on the toxins associated with foodborne diseases frequently caused by B. cereus. The disease characteristics are described, and recent findings regarding the associated toxins are discussed, as well as the present knowledge on virulence regulation.     

Bacillus cereus is common in the environment but emetic toxin producing isolates are rare

AIMS: To determine the incidence of emetic toxin producing Bacillus cereus in soil, animal faeces and selected vegetable produce to compare the results with the previously reported high incidence in rice paddy fields. To examine whether the emetic toxin has antibiotic activity. METHODS AND RESULTS: The incidence of emetic toxin producing B. cereus was evaluated by plating on selective agar 271 samples of soils, animal faeces, raw and processed vegetables. Overall, 45.8% of samples were positive for B. cereus. One hundred and seventy-seven B. cereus isolates were recovered at 30 degrees C with the grand mean spore count being 2.6 +/- 1.7 log(10) CFU g(-1) and 148 B. cereus isolates were recovered at 7 degrees C with the grand mean spore count being 2.2 +/- 1.2 log(10) CFU g(-1) of the 177 B. cereus isolated at 30 degrees C, only 3 were positive for emetic toxin production at a titre of 1/64, 1/32, 1/16, respectively. Also, 1 of 148 B. cereus isolated at 7 degrees C was positive for emetic toxin production to a titre of 1/128. All positive isolates came from washed or unwashed potato skins, one was psychrotrophic as determined by PCR and growth at 7 degrees C on subculture. The emetic toxin was not shown to have any antibiotic effects in growth inhibition studies. CONCLUSIONS: While B. cereus was a common isolate, the incidence of the emetic strain was rare. This is in contrast to previous findings of the high incidence in rice paddy fields and the processing environment, which may suggest rice is a selective area for growth of the emetic strain of B. cereus. SIGNIFICANCE AND IMPACT OF STUDY: The finding that a psychrotrophic isolate of B. cereus can produce emetic toxin is the first ever such observation and suggests the possibility that psychrotrophic isolates could grow in refrigerated fresh foods and cause emesis. The incidence of emetic B. cereus strains in rice paddy fields now requires further study for comparison with the low incidence found in other soils. The emetic toxin failed to inhibit the growth of other bacterial, fungal and yeast species. Whether the toxin (which is similar in structure to the antibiotic valinomycin) plays a competitive role in the environment therefore remains unclear.