Sequence Diversity of the Bacillus thuringiensis and B. cereus Sensu Lato Flagellin (H Antigen) Protein: Comparison with H Serotype Diversity

We set out to analyze the sequence diversity of the Bacillus thuringiensis flagellin (H antigen [Hag]) protein and compare it with H serotype diversity. Some other Bacillus cereus sensu lato species and strains were added for comparison. The internal sequences of the flagellin (hag) alleles from 80 Bacillus thuringiensis strains and 16 strains from the B. cereus sensu lato group were amplified and cloned, and their nucleotide sequences were determined and translated into amino acids. The flagellin allele nucleotide sequences for 10 additional strains were retrieved from GenBank for a total of 106 Bacillus species and strains used in this study. These included 82 B. thuringiensis strains from 67 H serotypes, 5 B. cereus strains, 3 Bacillus anthracis strains, 3 Bacillus mycoides strains, 11 Bacillus weihenstephanensis strains, 1 Bacillus halodurans strain, and 1 Bacillus subtilis strain. The first 111 and the last 66 amino acids were conserved. They were referred to as the C1 and C2 regions, respectively. The central region, however, was highly variable and is referred to as the V region. Two bootstrapped neighbor-joining trees were generated: a first one from the alignment of the translated amino acid sequences of the amplified internal sequences of the hag alleles and a second one from the alignment of the V region amino acid sequences, respectively. Of the eight clusters revealed in the tree inferred from the entire C1-V-C2 region amino acid sequences, seven were present in corresponding clusters in the tree inferred from the V region amino acid sequences. With regard to B. thuringiensis, in most cases, different serovars had different flagellin amino acid sequences, as might have been expected. Surprisingly, however, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. Likewise, serovars from the same H serotypes were most often found clustered together, with exceptions. Indeed, some serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. Species-wise, B. halodurans, B. subtilis, and B. anthracis formed specific branches, whereas the other four species, all in the B. cereus sensu lato group, B. mycoides, B. weihenstephanensis, B. cereus, and B. thuringiensis, did not form four specific clusters as might have been expected. Rather, strains from any of these four species were placed side by side with strains from the other species. In the B. cereus sensu lato group, B. anthracis excepted, the distribution of strains was not species specific.     

Genetic relationship in the 'Bacillus cereus group' by rep-PCR fingerprinting and sequencing of a Bacillus anthracis-specific rep-PCR fragment

AIMS: To evaluate the genetic relationship in the Bacillus cereus group by rep-PCR fingerprinting. METHODS AND RESULTS: A collection of 112 strains of the six species of the B. cereus group was analysed by rep-PCR fingerprinting using the BOX-A1R primer. A relative genetic distinctness was found among the species. Cluster analysis of the rep-PCR patterns showed clusters of B. thuringiensis strains quite separate from those of B. cereus strains. The B. anthracis strains represented an independent lineage in a B. cereus cluster. The B. mycoides, B. pseudomycoides and B. weihenstephanensis strains were clustered into three groups at some distance from the other species. Comparison of sequences of AC-390, a typical B. anthracis rep-PCR fragment, from 27 strains of B. anthracis, B. cereus, B. thuringiensis and B. weihenstephanensis, representative of different clusters identified by rep-PCR fingerprinting, confirmed that B. anthracis diverges from its related species. CONCLUSIONS: The genetic relationship deduced from the rep-PCR patterns indicates a relatively clear separation of the six species, suggesting that they can indeed be considered as separate units. SIGNIFICANCE AND IMPACT OF THE STUDY: rep-PCR fingerprinting can make a contribution in the clarification of the genetic relationships between the species of the B. cereus group.     

Population structure and evolution of the Bacillus cereus group

Representative strains of the Bacillus cereus group of bacteria, including Bacillus anthracis (11 isolates), B. cereus (38 isolates), Bacillus mycoides (1 isolate), Bacillus thuringiensis (53 isolates from 17 serovars), and Bacillus weihenstephanensis (2 isolates) were assigned to 59 sequence types (STs) derived from the nucleotide sequences of seven alleles, glpF, gmk, ilvD, pta, pur, pycA, and tpi. Comparisons of the maximum likelihood (ML) tree of the concatenated sequences with individual gene trees showed more congruence than expected by chance, indicating a generally clonal structure to the population. The STs followed two major lines of descent. Clade 1 comprised B. anthracis strains, numerous B. cereus strains, and rare B. thuringiensis strains, while clade 2 included the majority of the B. thuringiensis strains together with some B. cereus strains. Other species were allocated to a third, heterogeneous clade. The ML trees and split decomposition analysis were used to assign STs to eight lineages within clades 1 and 2. These lineages were defined by bootstrap analysis and by a preponderance of fixed differences over shared polymorphisms among the STs. Lineages were named with reference to existing designations: Anthracis, Cereus I, Cereus II, Cereus III, Kurstaki, Sotto, Thuringiensis, and Tolworthi. Strains from some B. thuringiensis serovars were wholly or largely assigned to a single ST, for example, serovar aizawai isolates were assigned to ST-15, serovar kenyae isolates were assigned to ST-13, and serovar tolworthi isolates were assigned to ST-23, while other serovars, such as serovar canadensis, were genetically heterogeneous. We suggest a revision of the nomenclature in which the lineage and clone are recognized through name and ST designations in accordance with the clonal structure of the population.     

Flagellin (FliC) protein sequence diversity among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> does not correlate with H serotype diversity

In Escherichia coli, the fliC gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Here, the presence of the flagellin fliC gene was studied in 86 Bacillus thuringiensis strains encompassing 67 H serotypes. Nineteen strains from four additional species in the B. cereus sensu lato group, B. cereus, B. anthracis, B. mycoides, and B. weihenstephanensis, were added for comparison purposes. The fliC genes were amplified, cloned and their nucleotide sequences determined and translated into amino acid sequences. A bootstrapped neighbor-joining tree was generated from the alignment of the translated amino acid sequences of the amplicons. Although most B. thuringiensis H serotypes had different flagellin amino acid sequences, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. In addition, although serovars from the same H serotype were sometimes found clustered together, several serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. No correlations could be established between flagellin (FliC) protein sequence diversity among B. thuringiensis H serotypes and H serotype diversity. These suggest that the B. thuringiensis fliC gene does not code for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. In a previous study, the authors have shown that the B. thuringiensis hag gene codes for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. It is proposed that the B. thuringiensis fliC gene studied here be renamed and that the so-called hag gene studied before be renamed fliC, both in accordance with the E. coli nomenclature. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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 L(1), L(2), 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.     

Homoduplex and heteroduplex polymorphisms of the amplified ribosomal 16S-23S internal transcribed spacers describe genetic relationships in the "Bacillus cereus group"

Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the "B. cereus group," advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as B. anthracis or serotypes of B. thuringiensis. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The second included B. cereus and B. thuringiensis, B. anthracis appeared as a lineage of B. cereus.     

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.     

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.     

Siderophores of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis

Three Bacillus anthracis Sterne strains (USAMRIID, 7702, and 34F2) and Bacillus cereus ATCC 14579 excrete two catecholate siderophores, petrobactin (which contains 3,4-dihydroxybenzoyl moieties) and bacillibactin (which contains 2,3-dihydroxybenzoyl moieties). However, the insecticidal organism Bacillus thuringiensis ATCC 33679 makes only bacillibactin. Analyses of siderophore production by previously isolated [Cendrowski et al., Mol. Microbiol. 52 (2004) 407-417] B. anthracis mutant strains revealed that the B. anthracis bacACEBF operon codes for bacillibactin production and the asbAB gene region is required for petrobactin assembly. The two catecholate moieties also were synthesized by separate routes. PCR amplification identified both asbA and asbB genes in the petrobactin producing strains whereas B. thuringiensis ATCC 33679 retained only asbA. Petrobactin synthesis is not limited to the cluster of B. anthracis strains within the B. cereus sensu lato group (in which B. cereus, B. anthracis, and B. thuringiensis are classified), although petrobactin might be prevalent in strains with pathogenic potential for vertebrates.     

Growth characteristics of Bacillus anthracis compared to other Bacillus spp. on the selective nutrient media Anthrax Blood Agar and Cereus Ident Agar

Anthrax Blood Agar (ABA) and Cereus Ident Agar (CEI) were evaluated as selective growth media for the isolation of Bacillus anthracis using 92 B. anthracis and 132 other Bacillus strains from 30 species. The positive predictive values for the identification of B. anthracis on ABA, CEI, and the combination of both were 72%, 71%, and 90%, respectively. Thus, less than 10% of all species were misidentified using both nutrient media. Species which might be misidentified as B. anthracis were B. cereus, B. mycoides, and B. thuringiensis. Particularly, 30% of B. weihenstephanensis strains were misidentified as B. anthracis.     

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.     

Sequence Diversity of Bacillus thuringiensis Flagellin (H Antigen) Protein at the Intra-H Serotype Level

In Bacillus thuringiensis, the hag gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Specific flagellin amino acid sequences have been correlated to specific B. thuringiensis H serotypes, H1 to H67. Ten H serotypes, however, contain three or more antigenic subfactors, labeled a, b, c, d, or e, and have been subdivided into 23 serovars. In the present study, we set out to analyze the sequence diversity of flagellins among serovars from the same H serotypes. We studied the hag genes in 39 B. thuringiensis strains representing the 23 serovars from the 10 H serotypes mentioned above. A serovar and a biovar from an 11th H serotype were also included. The hag genes were amplified and cloned and their nucleotide sequences were determined and translated into amino acid sequences, or the sequences were retrieved directly from GenBank when available. Strains of the H3 serotype contained two or three copies of the fla gene, an ortholog of the hag gene. Strains of the H6 serotype contained three copies. Strains of all other H serotypes each contained a single copy of the hag gene. Alignments of amino acid sequences from all copies in all strains of the H3 serotype revealed short signature sequences, GGAG and SGG, GPDPDDAVKNLT, and DITTTK, that appeared to be specific to the H3c, H3d, and H3e antigenic subfactors, respectively. Similar short signature sequences, GDIT, AFIK, TSAGKA, and SAPSKG, were revealed for H8b, H8c, H20b, and H20c, respectively. Amino acid sequences in the flagellin central variable region were highly conserved among serovars of the H3, H5, H11, and H20 serotypes and much more divergent among serovars of the H4, H10, H18, H24, and H28 serotypes. Two bootstrapped neighbor-joining trees were respectively generated from the alignments of the amino acid sequences translated from all copies of the hag genes in the B. thuringiensis strains of the H3 and H6 serotypes. Sequence identities and relationships were revealed. A third bootstrapped neighbor-joining tree was generated, this one from the alignment of the flagellin amino acid sequences from all the B. thuringiensis strains in the study. Eight clusters, I to VIII, were revealed. Although most clusters contained strains and serovars from the same H serotype, clusters VII and VIII contained serovars from different H serotypes.     

Development of real-time PCR assays for detection and quantification of Bacillus cereus group species: differentiation of B. weihenstephanensis and rhizoid B. pseudomycoides isolates from milk

Quantitative real-time PCR (qRT-PCR) offers an alternative method for the detection of bacterial contamination in food. This method provides the quantitation and determination of the number of gene copies. In our study, we established an RT-PCR assay using the LightCycler system to detect and quantify the Bacillus cereus group species, which includes B. cereus, B. anthracis, B. thuringiensis, B. weihenstephanensis, B. mycoides, and B. pseudomycoides. A TaqMan assay was designed to detect a 285-bp fragment of the motB gene encoding the flagellar motor protein, which was specific for the detection of the B. cereus group species, excluding B. pseudomycoides, and the detection of a 217-bp gene fragment of a hypothetical protein specific only for B. pseudomycoides strains. Based on three hydrolysis probes (MotB-FAM-1, MotB-FAM-2, and Bpm-FAM-1), it was possible to differentiate B. weihenstephanensis from the B. cereus group species with nonrhizoid growth and B. pseudomycoides from the whole B. cereus group. The specificity of the assay was confirmed with 119 strains belonging to the Bacillus cereus group species and was performed against 27 other Bacillus and non-Bacillus bacteria. A detection limit was determined for each assay. The assays performed well not only with purified DNA but also with DNA extracted from milk samples artificially contaminated with bacteria that belong to the B. cereus group species. This technique represents an alternative approach to traditional culture methods for the differentiation of B. cereus group species and differentiates B. weihenstephanensis and B. pseudomycoides in one reaction.     

Comparative analysis of the 16S to 23S ribosomal intergenic spacer sequences of Bacillus thuringiensis strains and subspecies and of closely related species.

Bacillus thuringiensis spacer regions between the 16S and 23S rRNAs were amplified with conserved primers, designated 19-mer and 23-mer primers. A spacer region of 144 bp was determined for all of 6 B. thuringiensis strains, 7 B. thuringiensis subspecies, and 11 B. thuringiensis field isolates, as well as for the closely related species Bacillus cereus and Bacillus anthracis. Computer analysis and alignment of nucleotide sequences identified three mutations and one deletion in the intergenic spacer region (ISR) of B. thuringiensis subsp. kurstaki HD-1 when compared with ISR sequences from other subspecies. The same differences were identified between the ISR of B. thuringiensis strains and the ISR of B. cereus and B. anthracis. These minor differences do not seem to be sufficient to allow the design of a species-specific oligonucleotide probe.     

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.     

A randomly amplified polymorphic DNA marker specific for the Bacillus cereus group is diagnostic for Bacillus anthracis

Aiming to develop a DNA marker specific for Bacillus anthracis and able to discriminate this species from Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides, we applied the randomly amplified polymorphic DNA (RAPD) fingerprinting technique to a collection of 101 strains of the genus Bacillus, including 61 strains of the B. cereus group. An 838-bp RAPD marker (SG-850) specific for B. cereus, B. thuringiensis, B. anthracis, and B. mycoides was identified. This fragment included a putative (366-nucleotide) open reading frame highly homologous to the ypuA gene of Bacillus subtilis. The restriction analysis of the SG-850 fragment with AluI distinguished B. anthracis from the other species of the B. cereus group.     

蜡状芽胞杆菌群菌株中部分病原相关因子的检测

对26株蜡状芽胞杆菌群菌株进行了肠毒素基因及其它病原相关因子的检测。PCR结果表明,17株蜡状芽胞杆菌群菌株中含有病原调控因子plcR的同源序列。采用3组溶血肠毒素hbl基因和3组非溶血肠毒素nhe基因特异性引物,分别可从73%的菌株中至少扩增出一个与预期DNA片段大小一致的片段,其中,苏云金芽胞杆菌菌株中溶血素hbl基因和非溶血素nhe基因的阳性检出率为83%。蜡状芽胞杆菌DBt248完全没有溶血活性,而且在溶血素hbl和非溶血素nhe基因的3个亚基以及病原调控因子plcR的PCR检测中均为阴性,有望作为宿主菌用于苏云金芽胞杆菌晶体蛋白的表达和应用。     

Genomic diversity and relationship of Bacillus thuringiensis and Bacillus cereus by multi-REP-PCR fingerprinting

The genomic diversity and relationship among 56 Bacillus thuringiensis and Bacillus cereus type strains were investigated by multi-REP-PCR fingerprinting consisting of three PCR reactions targeting the enterobacterial ERIC1 and ERIC2 and the streptococcal BOXA1R consensus sequences. A total of 113 polymorphic bands were generated in the REP-PCR profiles that allowed tracing of a single dendrogram with three major groups. Bacillus cereus strains clustered together in the A and B groups. Most of the B. thuringiensis strains clustered in group C, which included groups of serovars with a within-group similarity higher than 40% as follows: darmstadiensis, israelensis, and morrisoni; aizawai, kenyae, pakistani, and thompsoni; canadensis, entomocidus, galleriae, kurstaki, and tolworthi; alesti, dendrolimus, and kurstaki; and finitimus, sotto, and thuringiensis. Multi-REP-PCR fingerprinting clustered B. thuringiensis serovars in agreement with previously developed multilocus sequence typing schemes, indicating that it represents a rapid shortcut for addressing the genetic relationship of unknown strains with the major known serovars.