Detection of enterotoxin genes in mosquito-larvicidal Bacillus species

The presence of the B component of hemolysin BL (hblA), enterotoxin BceT (bceT), and enterotoxin S (entS) genes in mosquito-larvicidal Bacillus sp., including 25 B. sphaericus and 4 B. thuringiensis subsp. israelensis strains, has been analyzed by multiplex PCR in this study. The results showed that all four B. thuringiensis strains contain the hblA gene and the sequences of bceT and entS genes. However, none of the enterotoxin gene sequences were detected in the B. sphaericus strains. The enterotoxin production in all strains has also been analyzed by using two commercial immunoassay kits (TECRA and RPLA), and it has been proved that all the B. thuringiensis strains and one B. cereus strain can produce enterotoxins during growth. No enterotoxin activity could be detected in B. sphaericus strains. Received: 20 November 2001 / Accepted: 31 December 2001     

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.     

Detection of Enterotoxic Bacillus cereus and Bacillus thuringiensis Strains by PCR Analysis

Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereus and B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceT gene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of the bceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes in B. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.     

苏云金芽胞杆菌肠毒素基因的PCR检测

采用多重引物PCR进行了 45株苏云金芽胞杆菌、2株蜡状芽胞杆菌和 2株球形芽胞杆菌溶血素BL ,肠毒素T和entS基因的检测 ,结果表明 95 6%苏云金芽胞杆菌含溶血素hblA基因 ,91 1 %含bceT基因 ,93 3%含entS基因。用两种商业化肠毒素检测试剂盒TECRA和RPLA进行所有菌株肠毒素的体外免疫测定 ,大部分苏云金芽胞杆菌和阳性蜡状芽胞杆菌都能产生不同水平的肠毒素活性 ,同hblA基因PCR检测结果基本相符。尽管DBT0 0 7和T2 4 0 0 1含有hblA基因 ,但用TECRA却检测不到肠毒素 ;Dmu39菌株不含肠毒素基因 ,但用TECRA却检测出高的肠毒素活性。苏云金芽胞杆菌BDT2 4 8和球性芽孢杆菌不含肠毒素基因和肠毒素。结果表明昆虫病原菌苏云金芽胞杆菌的安全性有待进一步研究     

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.     

The Hemolytic Enterotoxin HBL Is Broadly Distributed among Species of the Bacillus cereus Group

The prevalence of the hemolytic enterotoxin complex HBL was determined in all species of the Bacillus cereus group with the exception of Bacillus anthracis. hblA, encoding the binding subunit B, was detected by PCR and Southern analysis and was confirmed by partial sequencing of 18 strains. The sequences formed two clusters, one including B. cereus and Bacillus thuringiensis strains and the other one consisting of Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis strains. From eight B. thuringiensis strains, the enterotoxin gene hblA could be amplified. Seven of them also expressed the complete HBL complex as determined with specific antibodies against the L₁, L₂, and B components. Eleven of 16 B. mycoides strains, all 3 B. pseudomyoides strains, 9 of 15 B. weihenstephanensis strains, and 10 of 23 B. cereus strains carried hblA. While HBL was not expressed in the B. pseudomycoides strains, the molecular assays were in accordance with the immunological assays for the majority of the remaining strains. In summary, the hemolytic enterotoxin HBL seems to be broadly distributed among strains of the B. cereus group and relates neither to a certain species nor to a specific environment. The consequences of this finding for food safety considerations need to be evaluated.     

Determination of Toxigenic Potentials of <Emphasis Type="Italic">Bacillus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Okpehe</Emphasis>, a Nigerian Fermented Condiment

The toxigenic potential of Bacillus species isolated from the traditional fermented condiment okpehe was determined; this is aimed at selection of non-toxigenic bacilli as starter cultures to bring about production of safe product. B. subtilis and B. cereus strains isolated from okpehe were evaluated for their possible possession of virulence characteristics. Fifty isolates were screened for their ability to produce diarrhoea enterotoxin by reversed passive latex agglutination (BCET-RPLA) test kit; the result showed that 40% of the B. cereus strains were toxigenic. The ability of the selected isolates to compete in situ and in vitro toxin production during the fermentation was also determined. The enterotoxin was not detected using BCET-RPLA kit in the spontaneously fermented samples of okpehe, but the toxin was detected in the okpehe samples fermented using B. cereus enterotoxin producer in mixed starter culture fermentation. The PCR amplification of virulence genes revealed that Bacillus cereus and B. licheniformis, a strain from the B. subtilis group, contained DNA sequences encoding the haemolysin BL (hblD) enterotoxin complex. The growth ability of B. cereus strains to high population during the fermentation and the presence of detectable diarroheagenic genes in B. cereus and B. licheniformis showed that strains carrying virulence characteristics cannot be totally ruled out in traditionally fermented okpehe.     

Detoxification of enterotoxigenic Bacillus cereus (JX455159) isolated from meat by a local strain of Lactobacillus plantarum (JX282192)

Raw minced meat samples (25) were randomly collected from different slaughterhouses in Dakhlia and Sharkyia Governorates, Egypt. One hundred and fifty Bacillus species related to the cereus group were isolated from the collected meat samples using Mannitol Yolk Polymyxin (MYP) agar plates. Purified bacterial cultures were then tested for their virulence factors with respect to hemolysin, protease and lecithinase. Of the tested Bacillus strains (150), 81, 95.3 and 76 % of total tested Bacillus strains were positive for hemolysin, protease and lecithinase tests, respectively. The identity of one of the most potent strains suspected and encoded as Bacillus cereus F23 was confirmed by amplifying its 16S rRNA gene. The partial nucleotide sequence of the amplified 16S rRNA gene of the tested strain was submitted to GenBank with accession number JX455159. Multiplex PCR amplification of enterotoxin genes in the tested strain, using specific primers, yielded amplicons of molecular sizes 695 and 565 bp for enterotoxins hblC and cytK, respectively. Thermal resistance of B. cereus F23 (JX455159) spores was determined by calculating D values at 65, 75, 85 and 95 °C for 36, 25, 19 and 16 min, respectively, and the calculated Z value was recorded as 0.119 °C. A lactic acid bacteria (LAB) strain isolated from pickles was preliminary identified as Lactobacillus plantarum F14 (LBF14) and later confirmed by detecting its 16S rRNA gene, and it was submitted to GenBank with accession number JX282192. The identified LAB strain was tested as a bioprotective agent against toxigenic B. cereus F23 spores both in minced meat samples and BHI broth medium. A reduction in B. cereus F23 population between 4 and 6 log cycles under different tested conditions was recorded. The activity of virulence factors (protease and lecithinase) decreased and hemolytic activity was completely inhibited in the presence of 10³ CFU/ml of Lactobacillus plantarum F14 (JX282192). Inthe presence of 10⁵ CFU/ml Lactobacillus plantarum F14 (JX282192), protease and lecithinase activities of B. cereus F23 were decreased by 85 and 71 %, respectively.     

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.     

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.     

Cloning and Nucleotide Sequence Analysis of gyrB of Bacillus cereus, B. thuringiensis, B. mycoides, and B. anthracis and Their Application to the Detection of B. cereus in Rice

As 16S rRNA sequence analysis has proven inadequate for the differentiation of Bacillus cereus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic marker. The gyrB genes of B. cereus JCM 2152^T, Bacillus thuringiensis IAM 12077^T, Bacillus mycoides ATCC 6462^T, and Bacillus anthracis Pasteur #2H were cloned and sequenced. Oligonucleotide PCR primer sets were designed from within gyrB sequences of the respective bacteria for the specific amplification and differentiation of B. cereus, B. thuringiensis, and B. anthracis. The results from the amplification of gyrB sequences correlated well with results obtained with the 16S rDNA-based hybridization study but not with the results of their phenotypic characterization. Some of the reference strains of both B. cereus (three serovars) and B. thuringiensis (two serovars) were not positive in PCR amplification assays with gyrB primers. However, complete sequencing of 1.2-kb gyrB fragments of these reference strains showed that these serovars had, in fact, lower homology than their originally designated species. We developed and tested a procedure for the specific detection of the target organism in boiled rice that entailed 15 h of preenrichment followed by PCR amplification of the B. cereus-specific fragment. This method enabled us to detect an initial inoculum of 0.24 CFU of B. cereus cells per g of boiled rice food homogenate without extracting DNA. However, a simple two-step filtration step is required to remove PCR inhibitory substances.     

Comparison of multiplex PCR, enzyme immunoassay and cell culture methods for the detection of enterotoxinogenic Bacillus cereus

Fast and reliable methods are needed for the detection of pathogenic Bacillus cereus which should provide consistent results. Therefore, we tested a panel of 176 strains, including B. cereus strains, B. cereus group strains and other Bacillus spp. with polymerase chain reaction, immunoassays and cytotoxicity tests and assessed the consistency of the results. A screening multiplex PCR for the detection of hbl, nhe, ces and cytK1 as well as two multiplex PCRs for the differentiation of Hbl genes (hblC, hblD, hblA) and Nhe genes (nheA, nheB, nheC) was applied. All PCRs included an internal amplification control. Component specific antibody based immunoassays were used for the detection of the three components of Hbl and Nhe and the overall cytotoxicity to Vero cells and HEp-2 cells was checked. An overall excellent correlation was obtained for the results of the three, methodically independent assays and no false-negative PCR results were seen for any of the strains tested positive in immunoassays and cytotoxicity tests. The three multiplex PCRs proved to be a facile method for the identification of enterotoxinogenic B. cereus isolates.     

Development of a Fluorogenic Probe-Based PCR Assay for Detection of Bacillus cereus in Nonfat Dry Milk

A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5′-to-3′ exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72 B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereus strains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negative B. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with B. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.     

Hemolytic and Nonhemolytic Enterotoxin Genes are Broadly Distributed among Bacillus thuringiensis Isolated from Wild Mammals

The presence of cytotoxin K (cytK), nonhemolytic (NHE), and hemolytic (HBL) enterotoxin genes was investigated in 74 Bacillus thuringiensis strains recovered from the intestines of wild mammals from northeast Poland, using polymerase chain reaction amplification and Southern hybridization. All the isolates harbored genes coding for toxin(s) that could cause diarrhea. The B. thuringiensis strains containing the nhe genes were found more frequently (nheA 100%, nheB 77%, nheC 96%) than those with the hblACD (74%) and cytK (73%) genes. The presence/absence of the nheA, hblA, and cytK genes was confirmed in all of the B. thuringiensis strains by Southern hybridization. Interestingly, these experiments also indicated that the nheA locus is located on a more variable chromosome region compared with hblA and, to a lesser degree, cytK. Detection of the 41-kDa component of NHE enterotoxin by the TECRA assay revealed various protein levels by B. thuringiensis strains. These results indicate the existence of environmental B. thuringiensis strains bearing the potential virulence arsenal for the production of diarrheal toxins, and emphasize the importance of small animals in the spread of B. cereus–like enterotoxin genes in nature. However, further investigation is needed to clarify any possible involvement of environmental B. thuringiensis strains in human health issues.     

Genotyping and Toxigenic Potential of Bacillus subtilis and Bacillus pumilus Strains Occurring in Industrial and Artisanal Cured Sausages

Artisanal and industrial sausages were analyzed for their aerobic, heat-resistant microflora to assess whether new emerging pathogens could be present among Bacillus strains naturally contaminating cured meat products. Sixty-four isolates were characterized by randomly amplified polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (fAFLP). The biotypes, identified by partial 16S rRNA gene sequence analysis, belonged to Bacillus subtilis, Bacillus pumilus, and Bacillus amyloliquefaciens species. Both RAPD-PCR and fAFLP analyses demonstrated that a high genetic heterogeneity is present in the B. subtilis group even in strains harvested from the same source, making it possible to isolate 56 different biotypes. Moreover, fAFLP analysis made it possible to distinguish B. subtilis from B. pumilus strains. The strains were characterized for their toxigenic potential by molecular, physiological, and immunological techniques. Specific PCR analyses revealed the absence of DNA sequences related to HBL, BcET, NHE, and entFM Bacillus cereus enterotoxins and the enzymes sphingomyelinase Sph and phospholipase PI-PLC in all strains; also, the immunological analyses showed that Bacillus strains did not react with NHE- and HBL-specific antibodies. However, some isolates were found to be positive for hemolytic and lecithinase activity. The absence of toxigenic potential in Bacillus strains from the sausages analyzed indicates that these products can be considered safe under the processing conditions they were produced; however, great care should be taken when the ripening time is shortened, particularly in the case of traditional sausages, which could contain high amounts of Bacillus strains and possibly some B. cereus cells.     

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 L₂ component, and antibody 1C2 was specific for the L₁ 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.     

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.     

Genome Differences That Distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis

The three species of the group 1 bacilli, Bacillus anthracis, B. cereus, and B. thuringiensis, are genetically very closely related. All inhabit soil habitats but exhibit different phenotypes. B. anthracis is the causative agent of anthrax and is phylogenetically monomorphic, while B. cereus and B. thuringiensis are genetically more diverse. An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing Bacillus species, some of which show significant similarity to B. anthracis. Suppression subtractive hybridization was then used to characterize the genomic differences that distinguish three of the non-anthrax-causing bacilli from B. anthracis Ames. Ninety-three DNA sequences that were present in B. anthracis but absent from the non-anthrax-causing Bacillus genomes were isolated. Furthermore, 28 of these sequences were not found in a collection of 10 non-anthrax-causing Bacillus species but were present in all members of a representative collection of B. anthracis strains. These sequences map to distinct loci on the B. anthracis genome and can be assayed simultaneously in multiplex PCR assays for rapid and highly specific DNA-based detection of B. anthracis.     

Prevalence and expression of enterotoxins in Bacillus cereus and other Bacillus spp., a literature review

Members of the Bacillus genus are ubiquitous soil microorganisms and are generally considered harmless contaminants. However, a few species are known toxin producers, including the foodborne pathogen, B. cereus. This species produces two distinct types of foodborne illness, the emetic (vomit-inducing) syndrome, associated with consumption of toxin in cooked rice dishes, and the diarrheal illness seen occasionally following consumption of contaminated meats, sauces, and certain dairy products. In the latter case, illness results from the production of enterotoxins by vegetative cells in the small intestine of the host. In dairy products, the occurrence of Bacillus spp. is inevitable, and the spore-forming ability of this organism allows it to easily survive pasteurization. Many strains have been shown to grow and produce enterotoxin in dairy products at refrigeration temperatures. Evaluation of toxin gene presence and toxin expression in Bacillus spp. other than B. cereus has not been thoroughly investigated. However, the presence of natural isolates of Bacillus spp. harboring one or more enterotoxin gene(s) and subsequent demonstration of conditions which may support toxin expression holds crucial importance in the food safety arena.     

Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group

Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker.