Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates

We examined 154 Norwegian B. cereus and B. thuringiensis soil isolates (collected from five different locations), 8 B. cereus and 2 B. thuringiensis reference strains, and 2 Bacillus anthracis strains by using fluorescent amplified fragment length polymorphism (AFLP). We employed a novel fragment identification approach based on a hierarchical agglomerative clustering routine that identifies fragments in an automated fashion. No method is free of error, and we identified the major sources so that experiments can be designed to minimize its effect. Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli. The ATCC reference strains were restricted to two of the genetic groups, clearly not representative of the diversity in these bacteria. Both B. anthracis strains analyzed were closely related and affiliated with a B. cereus milk isolate (ATCC 4342) and a B. cereus human pathogenic strain (periodontitis). Across the entire study, pathogenic strains, including B. anthracis, were more closely related to one another than to the environmental isolates. Eight strains representing the five distinct phylogenetic clusters were further analyzed by comparison of their 16S rRNA gene sequences to confirm the phylogenetic status of these groups. This analysis was consistent with the AFLP analysis, although of much lower resolution. The innovation of automated genotype analysis by using a replicated and statistical approach to fragment identification will allow very large sample analyses in the future.     

Diversity of culturable root-associated/endophytic bacteria and their chitinolytic and aflatoxin inhibition activity of peanut plant in China

A total of 72 isolates of root-associated/endophytic (RAE) bacteria were isolated from peanut plants grown in the main producing areas of 6 provinces in China. The 16S rRNA gene sequences of these isolates were determined and phylogenetic analyses revealed that 72 isolates belonged to the classes Bacilli (49 isolates) and Gammaproteobacteria (23 isolates). The majority of RAE bacteria in Bacilli belonged to 2 genera, Bacillus and Lysinibacillus (48 and 1) while those in Gammaproteobacteria belonged to the genera Enterobacter, Serratia, Stenotrophomonas, and Pseudomonas (7, 11, 3 and 2 isolates, respectively). This is the first report of Lysinibacillus xylanilyticus isolate as biocontrol agent against AFs. All of the selected RAE bacteria showed inhibitory activities against Aspergillus parasiticus (A. parasiticus) growth and/or aflatoxins (AFs) production by visual agar plate assay and tip culture method. Most of the RAE bacteria strains (96?% strains) were determined to have decreased mycelia growth or AFs production levels by >50?% (p?<?0.05). Bacterial isolates were further characterized for chitinolytic activity and 22 strains (30?% strains) of identified RAE bacteria degraded colloidal chitin on the chitin medium plate. Ten selected chitinolytic RAE bacteria were tested for antifungal activity on peanuts and most of them significantly decreased mycelial growth and AFs production levels by >90?%. These results showed a wide distribution of biological control bacteria against AFs in Chinese peanut main producing areas and the selected RAE bacteria could potentially be utilized for the biocontrol of toxicogenic fungi.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Isolates

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.     

Production of extracellular enzymes and degradation of biopolymers by saprotrophic microfungi from the upper layers of forest soil

Production of extracellular enzymes participating in the degradation of biopolymers was studied in 29 strains of nonbasidiomycetous microfungi isolated from Quercus petraea forest soil based on the frequency of occurrence. Most of the isolates were ascomycetes and belonged to the genera Acremonium, Alternaria, Cladosporium, Geomyces, Hypocrea, Myrothecium, Ochrocladosporium, and Penicillium (18 isolates), and two isolates were zygomycetes. Only six isolates showed phenol oxidation activity which was low and none of the strains were able to degrade humic acids. Approximately half of the strains were able to degrade cellulose and all but six degraded chitin. Most strains produced significant amounts of the cellulolytic enzymes cellobiohydrolase and ??-glucosidase and the chitinolytic enzymes chitinase, chitobiosidase, and N-acetylglucosaminidase. The highest cellulase activities were found in Penicillium strains, and the highest activity of chitinolytic enzymes was found in Acremonium sp. The production of the hemicellulose-degrading enzymes ??-galactosidase, ??-galactosidase, and ??-mannosidase was mostly low. The microfungal strains were able to produce significant growth on a range of 41?C87, out of 95 simple C-containing substrates tested in a Biolog? assay, monosaccharides being for all strains the most rapidly metabolized C-sources. Comparison with saprotrophic basidiomycetes from the same environment showed that microfungi have similar cellulolytic capabilities and higher chitinase activities which testifies for their active role in the decomposition of both lignocellulose and dead fungal biomass, important pools of soil carbon.     

Bacterial chitin utilisation at extremely haloalkaline conditions

Chitin is produced in large amounts in hypersaline habitats with neutral pH due to the high biomass production of brine shrimp Artemia. Recently, a high abundance of Artemia was also noticed in hypersaline soda lakes in the Kulunda Steppe (Altai, Russia), which prompted us to survey the possibility of microbial chitin utilization at extremely haloalkaline conditions in soda brines. Most active chitin utilisation-supporting microbial growth was found at anaerobic conditions at pH 10 and up to 3.5?M total Na⁺. At aerobic conditions, the degradation of chitin was slower, mostly incomplete and active at <2?M total Na⁺, although very slow partial degradation was possible up to 4?M Na⁺. Anaerobic enrichments at pH 10 yielded two different groups of obligately haloalkaliphilic fermentative anaerobes, exclusively specialized to utilise insoluble chitin as the only growth substrate. One group was represented by a single strain growing at moderate salinity, and another comprised multiple isolates growing up to 3.5?M Na⁺. These groups represent two novel bacterial phyla not closely related to any other cultured bacteria. Aerobic enrichments from the lake sediments were dominated by several obligately haloalkaliphilic members of the genus Marinimicrobium in the Gammaproteobacteria. They were less specialised than the anaerobes and grew with chitin and its monomer and oligomers at a pH of 10 up to 2.5?M Na⁺. Furthermore, several strains of haloalkaliphilic Gram-positive chitinolytics belonging to bacilli and actinobacteria were isolated from soda lake sediments and surrounding soda soils. In general, the results indicate the presence of an active and diverse haloalkaliphilic chitinolytic microbial community in hypersaline soda habitats.     

Members of <Emphasis Type="Italic">Gammaproteobacteria</Emphasis> and <Emphasis Type="Italic">Bacilli</Emphasis> represent the culturable diversity of chitinolytic bacteria in chitin-enriched soils

Culturable chitinolytic bacterial diversity was studied in chitin-rich soils collected from two industries involved in chitin production. A total of 27 chitinolytic isolates were isolated among which only 10 showed zone of clearance ≥4 mm on colloidal chitin agar plate. Using morphological, biochemical and 16S rDNA analysis, isolates were identified as Bacillus, Paenibacillus, Stenotrophomonas and Pseudomonas. Molecular phylogenetic analysis revealed that Gammaproteobacteria and Bacilli were found to be the predominant classes in these chitin-enriched soils. Chitinolytic bacterial population densities were significantly high and showed a rather simple community composition dominated by genus Bacillus and Stenotrophomonas (74%). This is the first report on assessing the chitinolytic bacterial diversity of soils from industries involved in chitin production.     

Esterase electrophoretic polymorphism ofBacillus thuringiensis andBacillus cereus reference strains

The genomic diversity and relationship among 61Bacillus thuringiensis andBacillus cereus reference strains were investigated by electrophoretic analysis of esterase enzymes on native polyacrylamide gel. Polymorphism of the esterolytic bands revealed seven esterases, designed as Est A to Est G in order of decreasing anodal migration. Each esterase showed two to three mobility variants that assigned the analysed strains into 35 electrophoretic types (ETs). This high diversity allowed the identification of several serovar or strain-specific ETs. Cluster analysis of ETs showed three major groups in which the strains belonging to the serovartolworthi were the most distant. The ETs distribution showed thatB. thuringiensis andB. cereus are intermingled in the dendrogram with the resolution of some common serovars ofB. thuringiensis in tight phylogenetic lineages. These results indicate that the esterase enzyme electrophoresis, applied as a sole typing method for the closely related speciesB. thuringiensis andB. cereus is suitable to highlight the intraspecific genetic diversity.     

Prevalence,Genetic Diversity,and Host Range of Tectiviruses among Members of the Bacillus cereus Group

GIL01, Bam35, GIL16, AP50, and Wip1 are tectiviruses preying on the Bacillus cereus group. Despite the significant contributions of phages in different biological processes, little is known about the dealings taking place between tectiviruses and their Gram-positive bacterial hosts. Therefore, this work focuses on characterizing the interactions between tectiviruses and the B. cereus group by assessing their occurrence and genetic diversity and evaluating their host range. To study the occurrence of tectiviruses in the B. cereus group, 2,000 isolates were evaluated using primers designed to be specific to two variable regions detected in previously described elements. PCR and propagation tests revealed that tectivirus-like elements occurred in less than 3% of the isolates. Regardless of this limited distribution, several novel tectiviruses were found, and partial DNA sequencing indicated that a greater diversity exists within the family Tectiviridae. Analyses of the selected variable regions, along with their host range, showed that tectiviruses in the B. cereus group can be clustered mainly into two different groups: the ones infecting B. anthracis and those isolated from other B. cereus group members. In order to address the host range of some novel tectiviruses, 120 strains were tested for sensitivity. The results showed that all the tested tectiviruses produced lysis in at least one B. cereus sensu lato strain. Moreover, no simple relationship between the infection patterns of the tectiviruses and their diversity was found.     

Production and purification of a chitinase from Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus

The chitinolytic properties of Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus, are reported. E. americana was shown to produce chitinolytic activity in the absence of chitin and in the presence of glucose and N-acetylglucosamine, indicating constitutive synthesis by these strains. A single 33-kDa protein with chitinolytic activity was purified to homogeneity from culture filtrates, by hydrophobic interaction chromatography using a phenyl-group substituted matrix. This enzyme, by virtue of differential activity against chromogenic chitooligosaccharides and against dye-labelled soluble carboxymethylated chitin (CM-chitin-RBV), was demonstrated to be an endochitinase. Our data suggest this 33-kDa chitinase appeared to be the only chitinolytic enzyme produced by E. americana, strains of which do not grow using chitin as a carbon source. The significance of these findings in the context of mushroom disease is discussed.     

Peptidoglycan from Bacillus cereus Mediates Commensalism with Rhizosphere Bacteria from the Cytophaga-Flavobacterium Group

Previous research in our laboratory revealed that the introduction of Bacillus cereus UW85 can increase the populations of bacteria from the Cytophaga-Flavobacterium (CF) group of the Bacteroidetes phylum in the soybean rhizosphere, suggesting that these rhizosphere microorganisms have a beneficial relationship (G. S. Gilbert, J. L. Parke, M. K. Clayton, and J. Handelsman, Ecology 74:840-854, 1993). In the present study, we determined the frequency at which CF bacteria coisolated with B. cereus strains from the soybean rhizosphere and the mechanism by which B. cereus stimulates the growth of CF rhizosphere strains in root exudate media. In three consecutive years of sampling, CF strains predominated among coisolates obtained with B. cereus isolates from field-grown soybean roots. In root exudate media, the presence of B. cereus was required for CF coisolate strains to reach high population density. However, rhizosphere isolates from the phylum Proteobacteria grew equally well in the presence and absence of B. cereus, and the presence of CF coisolates did not affect the growth of B. cereus. Peptidoglycan isolated from B. cereus cultures stimulated growth of the CF rhizosphere bacterium Flavobacterium johnsoniae, although culture supernatant from B. cereus grown in root exudate media did not. These results suggest B. cereus and CF rhizosphere bacteria have a commensal relationship in which peptidoglycan produced by B. cereus stimulates the growth of CF bacteria.     

Inhibition of Bacillus cereus Strains by Antimicrobial Metabolites from Lactobacillus johnsonii CRL1647 and Enterococcus faecium SM21

Bacillus cereus is an endospore-forming, Gram-positive bacterium able to cause foodborne diseases. Lactic acid bacteria (LAB) are known for their ability to synthesize organic acids and bacteriocins, but the potential of these compounds against B. cereus has been scarcely documented in food models. The present study has examined the effect of the metabolites produced by Lactobacillus johnsonii CRL1647 and Enterococcus faecium SM21 on the viability of select B. cereus strains. Furthermore, the effect of E. faecium SM21 metabolites against B. cereus strains has also been investigated on a rice food model. L. johnsonii CRL1647 produced 128 mmol/L of lactic acid, 38 mmol/L of acetic acid and 0.3 mmol/L of phenyl-lactic acid. These organic acids reduced the number of vegetative cells and spores of the B. cereus strains tested. However, the antagonistic effect disappeared at pH 6.5. On the other hand, E. faecium SM21 produced only lactic and acetic acid (24.5 and 12.2 mmol/L, respectively) and was able to inhibit both vegetative cells and spores of the B. cereus strains, at a final fermentation pH of 5.0 and at pH 6.5. This would indicate the action of other metabolites, different from organic acids, present in the cell-free supernatant. On cooked rice grains, the E. faecium SM21 bacteriocin(s) were tested against two B. cereus strains. Both of them were significantly affected within the first 4 h of contact; whereas B. cereus BAC1 cells recovered after 24 h, the effect on B. cereus 1 remained up to the end of the assay. The LAB studied may thus be considered to define future strategies for biological control of B. cereus.     

Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp. konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.     

Direct ethanol production from N-acetylglucosamine and chitin substrates by Mucor species

Chitin, which is a polymer of β-(1–4) linked N-acetyl-d-glucosamine (GlcNAc) residues, is one of the most abundant renewable resources in nature, after cellulose. In this study, we found some native Mucor strains, which can use GlcNAc and chitin substrates as carbon sources for growth and ethanol production. One of these strains, M. circinelloides NBRC 6746 produced 18.6 ± 0.6 g/l of ethanol from 50 g/l of GlcNAc after 72 h and the maximum ethanol production rate was 0.75 ± 0.1 g/l/h. Furthermore, M. circinelloides NBRC 4572 produced 6.00 ± 0.22 and 0.46 ± 0.04 g/l of ethanol from 50 g/l of colloidal chitin and chitin powder after 16 and 12 days, respectively. We also found an extracellular chitinolytic enzyme producing strain M. ambiguus NBRC 8092, and successfully improved ethanol productivity of NBRC 4572 from colloidal chitin using crude chitinolytic enzyme derived from NBRC 8092. The ethanol titer reached 9.44 ± 0.10 g/l after 16 days. These results were the first bioethanol production from GlcNAc and chitin substrates by native organisms, and also suggest that these Mucor strains have great potential for the simultaneous saccharification and fermentation (SSF) of chitin biomass.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates

We examined 154 Norwegian B. cereus and B. thuringiensis soil isolates (collected from five different locations), 8 B. cereus and 2 B. thuringiensis reference strains, and 2 Bacillus anthracis strains by using fluorescent amplified fragment length polymorphism (AFLP). We employed a novel fragment identification approach based on a hierarchical agglomerative clustering routine that identifies fragments in an automated fashion. No method is free of error, and we identified the major sources so that experiments can be designed to minimize its effect. Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli. The ATCC reference strains were restricted to two of the genetic groups, clearly not representative of the diversity in these bacteria. Both B. anthracis strains analyzed were closely related and affiliated with a B. cereus milk isolate (ATCC 4342) and a B. cereus human pathogenic strain (periodontitis). Across the entire study, pathogenic strains, including B. anthracis, were more closely related to one another than to the environmental isolates. Eight strains representing the five distinct phylogenetic clusters were further analyzed by comparison of their 16S rRNA gene sequences to confirm the phylogenetic status of these groups. This analysis was consistent with the AFLP analysis, although of much lower resolution. The innovation of automated genotype analysis by using a replicated and statistical approach to fragment identification will allow very large sample analyses in the future.     

Molecular Characterization of Korean Bacillus anthracis Isolates by Amplified Fragment Length Polymorphism Analysis and Multilocus Variable-Number Tandem Repeat Analysis

We analyzed the genetic relationships and molecular characteristics of 34 Bacillus anthracis isolates from soil and clinical samples in various regions of Korea and 17 related Bacillus species, using the amplified fragment length polymorphism (AFLP) and multilocus variable-number tandem repeat (MLVA) approaches. Triplicate AFLP profiles of these strains showed high reproducibility and identified 376 polymorphisms. AFLP phylogenetic analysis of B. anthracis isolates showed a high level of similarity, 0.93, and this monomorphic fragment profile proved to be useful to differentiate B. anthracis strains from other Bacillus species. The B. cereus group was separated from other Bacillus species at a level of similarity of 0.68. Among them, some B. cereus strains showed genetic interspersion with B. thuringiensis strains. The evolutionary pattern of nucleotide differences among B. anthracis strains with the eight MLVA markers showed nine MLVA types. Three MLVA types, M1 to M3, were pathogenic B. anthracis isolates and were assigned as new genotypes belonging to the A4 and B3 clusters, compared with 89 genotypes deduced from previous data. This indicates that differences in cluster prevalence and distribution may be influenced more by MLVA markers on two plasmids loci and human activity. Consequently, we suggest that the novel MLVA type may represent significant evidence for historic adaptation to environmental conditions of the Asian continent, particularly Korea. Therefore, MLVA techniques may be available for molecular monitoring on anthrax-release-related bioterrorism and further study is required for the continuous epidemiological study of variable anthrax collections.     

Identification of Bacillus anthracis Spore Component Antigens Conserved across Diverse Bacillus cereus sensu lato Strains

We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection.The anthrax causing bacterium Bacillus anthracis is a member of the Bacillus cereus sensu lato (s.l.)¹ group, a term given to the polyphyletic species consisting of Bacillus thuringiensis, Bacillus cereus, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus pseudomycoides (1). Genomics studies of B. cereus s.l. strains have shown a similar chromosomal gene composition within this group (2–7). Many phenotypes that distinguish B. cereus s.l. members, such as crystalline toxin production (8), emesis in humans (9), and anthrax virulence (10), are encoded by genes on large plasmids. Experimental conjugative transfer of plasmids between B. cereus s.l. strains has been demonstrated in vitro, in complex media, and in vector species (11–13). Therefore there is a concern about transfer of virulence genes between genetic backgrounds creating new pathogen lineages. In this regard, there is an emerging evidence of natural dissemination of the pXO1 and pXO2 plasmids that encode the anthrax lethal toxin and capsule, respectively. For example, B. cereus G9241 carries a pXO1 plasmid and lethal toxin genes almost identical to those in B. anthracis (6), and a B. cereus strain, which causes anthrax-like illness in African great apes, apparently contains both pXO1 and pXO2 plasmids (14).The infectious agent of most if not all human B. cereus s.l. diseases is the spore. The spore is a dormant, environmentally resistant structure that persists in nutrient- or water-limiting conditions. Anthrax infection occurs after introduction of the B. anthracis spore into a skin abrasion or via inhalation or ingestion (10). The spore germinates inside host cells, and the resulting vegetative bacteria express toxins and capsules that elicit an immune response (10, 15, 16). Formation of the B. cereus spore involves asymmetric cell division during which a copy of the genome is partitioned into each of the sister cells. The smaller cell (prespore) develops into mature endospore, and the larger cell (mother cell) contributes to the differentiation process but undergoes autolysis following its completion to release the endospore into the surrounding medium. Synthesis of cortex, coat, and exosporium are a function mainly of the mother cell. The cortex and coat layers are in close proximity to one another, whereas the exosporium tends to appear as an irregularly shaped, loosely attached, balloon-like layer (17–20). The coat and the exosporium contribute to the remarkable resistance of spores to extreme physical and chemical stresses including the exposure to extraterrestrial conditions (21, 22). Recent work on the structure, composition, assembly, and function of the spore coat and exosporium of pathogenic organisms like B. anthracis and B. cereus have highlighted the crucial link that exists between the origin of these layers (19, 23). There are differences in the appearance and thickness of the coat layers among the spores of various strains and species. In some B. thuringiensis strains, the inner coat is laminated but consists of a patchwork of striated packets, appearing either stacked or comblike, and the outer coat is granular (24), whereas in B. anthracis and other B. cereus s.l. isolates the coat appears compact (25–27). The coat layers comprise about 30% of the total proteins present in the spore (19, 28). Intraspecies variation in the structure and composition of the spore surface layers may reflect the environmental conditions under which these spores are formed (29–31).Because the spore is crucial to infection and persistence of B. anthracis and its close relatives, we undertook an investigation of its protein profile variability across the B. cereus s.l. group. Our goal in this study was to identify conserved antigenic spore proteins that may be transitioned in the future as candidates for immunodiagnostics, therapeutics, or vaccines. We used two high throughput approaches: genome-based bioinformatics analysis and comparative proteomics analysis of spores of B. cereus s.l. to select conserved targets. Our analysis revealed a list of conserved spore proteins within B. cereus but relatively few cross-reacting antigens. Two of these spore conserved antigens (Crd and MetN) have not been described previously for B. anthracis.     

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.     

Multilocus Sequence Typing Scheme for Bacteria of the Bacillus cereus Group

In this study we developed a multilocus sequence typing (MLST) scheme for bacteria of the Bacillus cereus group. This group, which includes the species B. cereus, B. thuringiensis, B. weihenstephanensis, and B. anthracis, is known to be genetically very diverse. It is also very important because it comprises pathogenic organisms as well as bacteria with industrial applications. The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil. A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria. Primers were designed for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes, adk, ccpA, ftsA, glpT, pyrE, recF, and sucC, were sequenced for all strains. The number of alleles at individual loci ranged from 25 to 40, and a total of 53 allelic profiles or sequence types (STs) were distinguished. Analysis of the sequence data showed that the population structure of the B. cereus group is weakly clonal. In particular, all five B. anthracis isolates analyzed had the same ST. The MLST scheme which we developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the B. cereus group.     

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.