Genotyping of Bacillus cereus strains by microarray-based resequencing

The ability to distinguish microbial pathogens from closely related but nonpathogenic strains is key to understanding the population biology of these organisms. In this regard, Bacillus anthracis, the bacterium that causes inhalational anthrax, is of interest because it is closely related and often difficult to distinguish from other members of the B. cereus group that can cause diverse diseases. We employed custom-designed resequencing arrays (RAs) based on the genome sequence of Bacillus anthracis to generate 422 kb of genomic sequence from a panel of 41 Bacillus cereus sensu lato strains. Here we show that RAs represent a "one reaction" genotyping technology with the ability to discriminate between highly similar B. anthracis isolates and more divergent strains of the B. cereus s.l. Clade 1. Our data show that RAs can be an efficient genotyping technology for pre-screening the genetic diversity of large strain collections to selected the best candidates for whole genome sequencing.     

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.     

Complete sequence analysis of novel plasmids from emetic and periodontal Bacillus cereus isolates reveals a common evolutionary history among the B. cereus-group plasmids, including Bacillus anthracis pXO1

The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical approximately 272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one approximately 270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.     

Biology and taxonomy of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis

Three species of the Bacillus cereus group (Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis) have a marked impact on human activity. Bacillus cereus and B. anthracis are important pathogens of mammals, including humans, and B. thuringiensis is extensively used in the biological control of insects. The microbiological, biochemical, and genetic characteristics of these three species are reviewed, together with a discussion of several genomic studies conducted on strains of B. cereus group. Using bacterial systematic concepts, we speculate that to understand the taxonomic relationship within this group of bacteria, special attention should be devoted also to the ecology and the population genetics of these species.     

Diversity of commensal Bacillus cereus sensu lato isolated from the common sow bug (Porcellio scaber, Isopoda)

Although Bacillus cereus sensu lato are important both from an ecological and an economical point of view, little is known about their population structure, ecology, and relationships with other organisms. In the present work, the genotypic similarity of arthropod-borne B. cereus s.l. isolates, and their symbiotic relationship with the host are assessed. Bacilli of this group were recovered from the digestive tracts of sow bugs (Porcellio scaber) collected in three closely located sites. Their genotypic diversity was investigated using pulse-field gel electrophoresis (PFGE) following the whole-genome DNA digestions with NotI and AscI, and PCR amplification of virulence genes. The majority of the sow-bug Bacillus cereus sensu stricto isolates originating from the same but also from different sites displayed identical PFGE patterns, virulence gene content and enterotoxicity, indicating strong genetic and genomic relationships. The sow-bug Bacillus mycoides/Bacillus pseudomycoides strains displayed a higher diversity. The isopod-B. cereus s.l. relationship was also evaluated using antibiotic-resistant derivatives of B. cereus s.s., B. mycoides/B. pseudomycoides and Bacillus thuringiensis reintroduced into sow bugs. Both spores and vegetative cells of B. cereus s.l. were recovered from sow bugs over a 30-day period, strongly suggesting that these bacteria are natural residents of terrestrial isopods.     

A DNA microarray facilitates the diagnosis of Bacillus anthracis in environmental samples

Aims:  In order to improve the diagnosis of Bacillus anthracis in environmental samples, we established a DNA microarray based on the ArrayTube technology of Clondiag.
Methods and Results:  Total DNA of a bacterial colony is randomly biotinylated and hybridized to the array. The probes on the array target the virulence genes, the genomic marker gene rpoB , as well as the selective 16S rDNA sequence regions of B. anthracis , of the Bacillus cereus group and of Bacillus subtilis . Eight B. anthracis reference strains were tested and correctly identified. Among the analysed environmental Bacillus isolates, no virulent B. anthracis strain was detected.
Conclusions:  This array clearly differentiates B. anthracis from members of the B. cereus group and other Bacillus species in environmental samples by chromosomal ( rpoB ) and plasmid markers. Additionally, recognition of B. cereus strains harbouring the toxin genes or atypical B. anthracis strains that have lost the virulence plasmids is feasible.
Significance and Impact of the Study:  The array is applicable to the complex diagnostics for B. anthracis detection in environmental samples. Because of low costs, high security and easy handling, the microarray is applicable to routine diagnostics.     

The hidden lifestyles of Bacillus cereus and relatives

Bacillus cereus sensu lato, the species group comprising Bacillus anthracis, Bacillus thuringiensis and B. cereus (sensu stricto), has previously been scrutinized regarding interspecies genetic correlation and pathogenic characteristics. So far, little attention has been paid to analysing the biological and ecological properties of the three species in their natural environments. In this review, we describe the B. cereus sensu lato living in a world on its own; all B. cereus sensu lato can grow saprophytically under nutrient-rich conditions, which are only occasionally found in the environment, except where nutrients are actively collected. As such, members of the B. cereus group have recently been discovered as common inhabitants of the invertebrate gut. We speculate that all members disclose symbiotic relationships with appropriate invertebrate hosts and only occasionally enter a pathogenic life cycle in which the individual species infects suitable hosts and multiplies almost unrestrained.     

Polymerase chain reaction assay for the detection of Bacillus cereus group cells

Recent investigations have shown that members of the Bacillus cereus group carry genes which have the potential to cause gastrointestinal and somatic diseases. Although most cases of diseases caused by the B. cereus group bacteria are relatively mild, it is desirable to be able to detect members of the B. cereus group in food and in the environment. Using 16S rDNA as target, a PCR assay for the detection of B. cereus group cells has been developed. Primers specific for the 16S rDNA of the B. cereus group bacteria were selected and used in combination with consensus primers for 16S rDNA as internal PCR procedure control. The PCR procedure was optimized with respect to annealing temperature. When DNA from the B. cereus group bacteria was present, the PCR assay yielded a B. cereus specific fragment, while when non-B. cereus prokaryotic DNA was present, the consensus 16S rDNA primers directed synthesis of the PCR products. The PCR analyses with DNA from a number of non-B. cereus confirmed the specificity of the PCR assay.     

Substrate analysis of homoserine acyltransferase from Bacillus cereus

Substrate specificity within the family of enzymes designated as homoserine transsuccinylases is variable, with some organisms utilizing succinyl-CoA and other organisms utilizing acetyl-CoA. In this study it is shown that the enzyme from Bacillus cereus uses acetyl-CoA as its acyl donor, but its catalytic rate is significantly lower than other HTS family members. BcHTS is inactivated by both iodoacetamide and diethyl pyrocarbonate and the enzyme can be partially protected from inactivation by the presence of succinyl-CoA. This leads to the conclusion that BcHTS can bind both acetyl-CoA and succinyl-CoA and suggests that it may represent an intermediate between the succinate-transferring HTS family members and the acetate-transferring HTS family members. The B. cereus enzyme was unable to rescue growth of an Escherichia coli strain lacking a functional transsuccinylase, however.     

Comparative analysis of the diversity of aerobic spore-forming bacteria in raw milk from organic and conventional dairy farms

Bacterial contamination of raw milk can originate from different sources: air, milking equipment, feed, soil, faeces and grass. It is hypothesized that differences in feeding and housing strategies of cows may influence the microbial quality of milk. This assumption was investigated through comparison of the aerobic spore-forming flora in milk from organic and conventional dairy farms. Laboratory pasteurized milk samples from five conventional and five organic dairy farms, sampled in late summer/autumn and in winter, were plated on a standard medium and two differential media, one screening for phospholipolytic and the other for proteolytic activity of bacteria. Almost 930 isolates were obtained of which 898 could be screened via fatty acid methyl ester analysis. Representative isolates were further analysed using 16S rRNA gene sequencing and (GTG)(5)-PCR. The majority of aerobic spore-formers in milk belonged to the genus Bacillus and showed at least 97% 16S rRNA gene sequence similarity with type strains of Bacillus licheniformis, Bacillus pumilus, Bacillus circulans, Bacillus subtilis and with type strains of species belonging to the Bacillus cereus group. About 7% of all isolates may belong to possibly new spore-forming taxa. Although the overall diversity of aerobic spore-forming bacteria in milk from organic vs. conventional dairy farms was highly similar, some differences between both were observed: (i) a relatively higher number of thermotolerant organisms in milk from conventional dairy farms compared to organic farms (41.2% vs. 25.9%), and (ii) a relatively higher number of B. cereus group organisms in milk from organic (81.3%) and Ureibacillus thermosphaericus in milk from conventional (85.7%) dairy farms. One of these differences, the higher occurrence of B. cereus group organisms in milk from organic dairy farms, may be linked to differences in housing strategy between the two types of dairy farming. However, no plausible clarification was found for the relatively higher number of thermotolerant organisms and the higher occurrence of U. thermosphaericus in milk from conventional dairy farms. Possibly this is due to differences in feeding strategy but no decisive indications were found to support this assumption.     

Detection of large plasmids from the Bacillus cereus group

The members of the Bacillus cereus group, Bacillus anthracis, Bacillus thuringiensis, and B. cereus senso stricto, are largely defined by their content of large plasmids, which encode major virulence factors. Here we offer an easy, fast, and reliable protocol for the isolation and detection of large plasmids up to the size of at least 350kb. Furthermore, using this method, we report that Bacillus mycoides contain large plasmids.     

Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray

Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect differences in both the genotypes and phenotypes of the B. cereus group organisms.     

Bacillus species proteins involved in spore formation and degradation: from identification in the genome,to sequence analysis,and determination of function and structure

The members of Bacillus species are Gram-positive, ubiquitous spore-forming bacilli. Several genomic sequences have been made available during recent years, including Bacillus subtilis, a model organism among this genus, Bacillus anthracis, and their analyses provided a wealth of information about spore-forming bacteria. Some members of this species can cause serious diseases in livestock and humans. An important pathogen in this group of organisms is B. anthracis, which is the causative agent of anthrax. A summary of the B. subtilis genome information, based on the publicly released sequence, that allowed for the identification and characterization of new and novel proteins of this organism as well as similar proteins from other members of Bacillus species is provided. The primary goal for this work is to present a review of the genome sequence-identified genes that encode proteins involved in the sporulation, germination, and outgrowth processes. These three processes are essential for spore development and later its transformation into a vegetative cell. Additionally, for a few selected examples of the protein products of the identified genes, the application of bioinformatics and modeling tools is illustrated in order to determine their likely structure and function. Two three-dimensional models of the structures of such proteins, PrfA endonuclease and phosphatase PhoE, are presented together with the structure-based functional conclusions. The review of such studies provides an example of how the genomic sequence can be utilized in order to elucidate the structure and function of proteins, in particular proteins of the Bacillus species. Because only a limited number of proteins of Bacillus species organisms are involved in the synthesis and degradation of spores and have been characterized to date, this genome-based analysis may provide new insights into the developmental processes of bacterial organism.     

多重PCR技术检测微生物肥料中巨大芽孢杆菌和蜡样群芽孢杆菌的研究与应用

巨大芽孢杆菌是微生物肥料生产中的常用菌种, 与之形态上相似的蜡样群芽孢杆菌(蜡样芽孢杆菌、苏云金芽孢杆菌、蕈状芽孢杆菌)则是产品中常见的污染菌, 传统方法区分两者费时费力, 有必要建立检测这两类芽孢杆菌的PCR方法。本文利用已登录的spoOA基因序列分别设计和筛选了上述两个种(群)的特异引物, 并建立了多重PCR检测技术。使用该方法对巨大芽孢杆菌、蜡样群芽孢杆菌和其他芽孢菌共3属13种24株标准菌株的基因组DNA进行扩增, 以检验其特异性。结果显示, 巨大芽孢杆菌、蜡样群芽孢杆菌基因组DNA分别产生大小不同的唯一产物, 其他芽孢杆菌均为阴性。该多重PCR检测方法的灵敏度经测定为105 CFU/mL。同时对10株待测菌株和8个微生物肥料产品进行检测, 其鉴定结果与常规鉴定结果一致。以上结果表明, 本文建立的多重PCR方法具有较高的特异性和灵敏度, 可快速、准确鉴定巨大芽孢杆菌和蜡样群芽孢杆菌, 在微生物肥料检测方面有良好的实用前景。     

Strain discrimination among B. anthracis and related organisms by characterization of bclA polymorphisms using PCR coupled with agarose gel or microchannel fluidics electrophoresis

The bclA gene codes for the protein backbone of the exosporium glycoprotein BclA of B. anthracis. BclA has a central collagen-like region formed by polymorphic GXX repeats and conserved amino- and carboxy-termini. It is noted here that the bclA gene is also present in the genome of Bacillus cereus and Bacillus thuringiensis. There is considerable size heterogeneity among the BclA proteins, both for species and strains, due to different numbers of GPT repeats and [GPT]5GDTGTT repeats (BclA repeats). PCR products that included the entire variable region were analyzed by conventional agarose gel electrophoresis and by micro-channel fluidics (MCF) LabChip to assess differences in molecular weight (MW). Both methods provided discrimination at the strain level for B. cereus group organisms. Results obtained by MCF electrophoresis were superior to conventional agarose gel analysis demonstrating improved reproducibility and much faster analysis time. The expression of a carbohydrate-rich exosporium (corresponding to BclA) in other members of the B. cereus group, in addition to B. anthracis, was also demonstrated ultra-structurally. Analysis of sequence variability within the bclA gene CLR revealed even greater potential for strain and species identification.     

The bcr1 DNA repeat element is specific to the Bacillus cereus group and exhibits mobile element characteristics

Bacillus cereus strains ATCC 10987 and ATCC 14579 harbor an approximately 155-bp repeated element, bcr1, which is conserved in B. cereus, B. anthracis, B. thuringiensis, and B. mycoides but not in B. subtilis and B. licheniformis. In this study, we show by Southern blot hybridizations that bcr1 is present in all 54 B. cereus group strains tested but absent in 11 Bacillus strains outside the group, suggesting that bcr1 may be specific and ubiquitous to the B. cereus group. By comparative analysis of the complete genome sequences of B. cereus ATCC 10987, B. cereus ATCC 14579, and B. anthracis Ames, we show that bcr1 is exclusively present in the chromosome but absent from large plasmids carried by these strains and that the numbers of full-length bcr1 repeats for these strains are 79, 54, and 12, respectively. Numerous copies of partial bcr1 elements are also present in the three genomes (91, 128, and 53, respectively). Furthermore, the genomic localization of bcr1 is not conserved between strains with respect to chromosomal position or organization of gene neighbors, as only six full-length bcr1 loci are common to at least two of the three strains. However, the intergenic sequence surrounding a specific bcr1 repeat in one of the three strains is generally strongly conserved in the other two, even in loci where bcr1 is found exclusively in one strain. This finding indicates that bcr1 either has evolved by differential deletion from a very high number of repeats in a common ancestor to the B. cereus group or is moving around the chromosome. The identification of bcr1 repeats interrupting genes in B. cereus ATCC 10987 and ATCC 14579 and the presence of a flanking TTTAT motif in each end show that bcr1 exhibits features characteristic of a mobile element.     

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.     

Correlation of 16S ribosomal DNA signature sequences with temperature-dependent growth rates of mesophilic and psychrotolerant strains of the Bacillus cereus group

Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the Bacillus cereus group revealed signatures which were specific for these two thermal groups of bacteria. Further analysis of the genomic DNA from a wide range of food and soil isolates showed that B. cereus group strains have between 6 and 10 copies of 16S rDNA. Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures. The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance.     

Extending the Bacillus cereus group genomics to putative food-borne pathogens of different toxicity

The Bacillus cereus group represents sporulating soil bacteria containing pathogenic strains which may cause diarrheic or emetic food poisoning outbreaks. Multiple locus sequence typing revealed a presence in natural samples of these bacteria of about 30 clonal complexes. Application of genomic methods to this group was however biased due to the major interest for representatives closely related to Bacillus anthracis. Albeit the most important food-borne pathogens were not yet defined, existing data indicate that they are scattered all over the phylogenetic tree. The preliminary analysis of the sequences of three genomes discussed in this paper narrows down the gaps in our knowledge of the B. cereus group. The strain NVH391-98 is a rare but particularly severe food-borne pathogen. Sequencing revealed that the strain should be a representative of a novel bacterial species, for which the name Bacillus cytotoxis or Bacillus cytotoxicus is proposed. This strain has a reduced genome size compared to other B. cereus group strains. Genome analysis revealed absence of sigma B factor and the presence of genes encoding diarrheic Nhe toxin, not detected earlier. The strain B. cereus F837/76 represents a clonal complex close to that of B. anthracis. Including F837/76, three such B. cereus strains had been sequenced. Alignment of genomes suggests that B. anthracis is their common ancestor. Since such strains often emerge from clinical cases, they merit a special attention. The third strain, KBAB4, is a typical facultative psychrophile generally found in soil. Phylogenic studies show that in nature it is the most active group in terms of gene exchange. Genomic sequence revealed high presence of extra-chromosomal genetic material (about 530kb) that may account for this phenomenon. Genes coding Nhe-like toxin were found on a big plasmid in this strain. This may indicate a potential mechanism of toxicity spread from the psychrophile strain community. The results of this genomic work and ecological compartments of different strains incite to consider a necessity of creating prophylactic vaccines against bacteria closely related to NVH391-98 and F837/76. Presumably developing of such vaccines can be based on the properties of non-pathogenic strains such as KBAB4 or ATCC14579 reported here or earlier. By comparing the protein coding genes of strains being sequenced in this project to others we estimate the shared proteome, or core genome, in the B. cereus group to be 3000+/-200 genes and the total proteome, or pan-genome, to be 20-25,000 genes.