Strategy for identification of Bacillus cereus and Bacillus thuringiensis strains closely related to Bacillus anthracis

Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these near neighbors are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.     

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.     

Comparison of PCR-RFLP, ribotyping and ERIC-PCR for typing Bacillus anthracis and Bacillus cereus strains

PCR-RFLP analysis of the vrrA gene and cerAB gene was used to investigate the genomic diversity in 21 strains of Bacillus anthracis and 28 strains of Bacillus cereus, and was compared with results obtained by ribotyping and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) analysis. VrrA-typing divided the B. anthracis into four groups. Except for one Pasteur vaccine strain, the vrrA PCR-RFLP profiles of the B. anthracis were separated into three groups, which were different from those of the B. cereus strains. Ribotyping separated the B. anthracis isolates into seven ribotypes, and a common fragment of an approximately 850 bp band from the ERIC-PCR fingerprints separated most B. anthracis strains into two groups. VrrA/cerAB PCR-RFLP, ribotyping and ERIC-PCR generated 18, 22 and 23 types, respectively, from B. cereus strains. The results suggest that a combination of all three methods provides a high resolution typing method for B. anthracis and B. cereus. Compared with ribotyping and ERIC-PCR, PCR-RFLP is simple to perform and has potential as a rapid method for typing and discriminating B. anthracis strains from other B. cereus group bacteria.     

Determination of the most closely related bacillus isolates to Bacillus anthracis by multilocus sequence typing

There have been many efforts to develop Bacillus anthracis detection assays, but the problem of false-positive results has often been encountered. Therefore, to validate an assay for B. anthracis detection, it is critical to examine its specificity with the most closely related Bacillus isolates that are available. To define the most closely related Bacillus isolates to B. anthracis in our Bacillus collections, we analyzed by multilocus sequence typing (MLST) the phylogeny of 77 closely related Bacillus isolates selected from 264 Bacillus isolates. The selection includes all the Bacillus isolates that have been shown in our previous studies to produce false-positive results by some anthrax-detection assays. The MLST phylogenetic analyses revealed that 27 of the non-B. anthracis isolates clustered within the B. anthracis clade, and four of them (three sequence types, STs) had the highest degree of genetic relatedness with B. anthracis, 18 (11 STs) had the second highest, and five (five STs) had the third highest. We anticipate that the inclusion of the 19 ST isolates when analyzing B. anthracis detection assays will prove to be useful for screening for their specificity to detect B. anthracis.     

The use of plasmid screening for the differentiation of Bacillus anthracis strains from closely related species of soil bacilli

B. anthracis virulent and vaccine strains differ from the strains of species closely related to B. anthracis, such as B. cereus and B. thuringiensis, in their plasmid spectrum. The use of their plasmid spectrum. The use of the plasmid analysis of the strain is recommended for laboratory practice as the main differential diagnostic test.     

Immuno-detection of anthrose containing tetrasaccharide in the exosporium of Bacillus anthracis and Bacillus cereus strains

Aims:  Bacillus anthracis strains of various origins were analysed with the view to describe intrinsic and persistent structural components of the Bacillus collagen-like protein of anthracis glycoprotein associated anthrose containing tetrasaccharide in the exosporium.
Methods and Results:  The tetrasaccharide consists of three rhamnose residues and an unique monosaccharide – anthrose. As anthrose was not found in spores of related strains of bacteria, we envisioned the detection of B. anthracis spores based on antibodies against anthrose-containing polysaccharides. Carbohydrate–protein conjugates containing the synthetic tetrasaccharide, an anthrose–rhamnose disaccharide or anthrose alone were employed to immunize mice. All three formulations were immunogenic and elicited IgG responses with different fine specificities. All sera and monoclonal antibodies derived from tetrasaccharide immunized mice cross-reacted not only with spore lysates of a panel of virulent B. anthracis strains, but also with some of the B. cereus strains tested.
Conclusions:  Our results demonstrate that antibodies to synthetic carbohydrates are useful tools for epitope analyses of complex carbohydrate antigens and for the detection of particular target structures in biological specimens.
Significance and Impact of the Study:  Although not strictly specific for B. anthracis spores, antibodies against the tetrasaccharide may have potential as immuno-capturing components for a highly sensitive spore detection system.     

Unconventional GIY-YIG homing endonuclease encoded in group I introns in closely related strains of the Bacillus cereus group

Several group I introns have been previously found in strains of the Bacillus cereus group at three different insertion sites in the nrdE gene of the essential nrdIEF operon coding for ribonucleotide reductase. Here, we identify an uncharacterized group IA intron in the nrdF gene in 12 strains of the B. cereus group and show that the pre-mRNA is efficiently spliced. The Bacillus thuringiensis ssp. pakistani nrdF intron encodes a homing endonuclease, denoted I-BthII, with an unconventional GIY-(X)₈-YIG motif that cleaves an intronless nrdF gene 7 nt upstream of the intron insertion site, producing 2-nt 3′ extensions. We also found four additional occurrences of two of the previously reported group I introns in the nrdE gene of 25 sequenced B. thuringiensis and one B. cereus strains, and one non-annotated group I intron at a fourth nrdE insertion site in the B. thuringiensis ssp. Al Hakam sequenced genome. Two strains contain introns in both the nrdE and the nrdF genes. Phylogenetic studies of the nrdIEF operon from 39 strains of the B. cereus group suggest several events of horizontal gene transfer for two of the introns found in this operon.     

Development of a PCR assay for identification of the Bacillus cereus group species

Aims:  A PCR technique was developed as a reliable and rapid identification method for the Bacillus cereus group species, based on a unique conserved sequence of the motB gene (encoding flagellar motor protein) from B. cereus , Bacillus thuringiensis and Bacillus anthracis .
Methods and Results:  Primer locations were identified against eight strains of the B. cereus group spp. from nucleotide sequences available in the National Centre for Biotechnology Information database. The PCR assay was applied for the identification of 117 strains of the B. cereus group spp. and 19 strains from other microbial species, with special emphasis on foodborne pathogens.
Conclusion:  The designed cross-species primers are group specific and did not react with DNA from other Bacillus and non- Bacillus species either motile or not. The primers system enabled us to detect 10³ CFU of B. cereus cells per millilitre of sample.
Significance and Impact of the Study:  Bacillus cereus group spp. belongs to one of the most prevalent foodborne pathogens. Bacterial growth results in production of different toxins; therefore, consumption of food containing >10⁶ bacteria per gram may result in emetic and diarrhoeal syndromes. A rapid and sensitive bacterial detection method is significant for food safety.     

Strategy for Identification of Bacillus cereus and Bacillus thuringiensis Strains Closely Related to Bacillus anthracis

Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these “near neighbors” are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.     

Comparison of Minisatellite Polymorphisms in the Bacillus cereus Complex: a Simple Assay for Large-Scale Screening and Identification of Strains Most Closely Related to Bacillus anthracis

Polymorphism of five tandem repeats that are monomorphic in Bacillus anthracis was investigated in 230 isolates of the B. cereus group and in 5 sequenced B. cereus genomes in search for markers allowing identification of B. cereus and B. thuringiensis strains most closely related to B. anthracis. Using this multiple-locus variable number of tandem repeat analysis (MLVA), a cluster of 30 strains was selected for further characterization. Eventually, six of these were characterized by multilocus sequence type analysis. One of the strains is only six point mutations (of almost 3,000 bp) away from B. anthracis and was also proposed to be closest to B. anthracis by MLVA analysis. However, this strain remains separated from B. anthracis by a number of significant genetic events observed in B. anthracis, including the loss of the hemolysin activity, the presence of four prophages, and the presence of the two virulence plasmids, pXO1 and pXO2. One particular minisatellite marker provides an efficient assay to identify the subset of B. cereus and B. thuringiensis strains closely related to B. anthracis. Based on these results, a very simple assay is proposed that allows the screening of hundreds of strains from the B. cereus complex, with modest equipment and at a low cost, to eventually fill the gap with B. anthracis and better understand the origin and making of this dangerous pathogen.     

The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1

We sequenced the complete genome of Bacillus cereus ATCC 10987, a non-lethal dairy isolate in the same genetic subgroup as Bacillus anthracis. Comparison of the chromosomes demonstrated that B.cereus ATCC 10987 was more similar to B.anthracis Ames than B.cereus ATCC 14579, while containing a number of unique metabolic capabilities such as urease and xylose utilization and lacking the ability to utilize nitrate and nitrite. Additionally, genetic mechanisms for variation of capsule carbohydrate and flagella surface structures were identified. Bacillus cereus ATCC 10987 contains a single large plasmid (pBc10987), of ~208 kb, that is similar in gene content and organization to B.anthracis pXO1 but is lacking the pathogenicity-associated island containing the anthrax lethal and edema toxin complex genes. The chromosomal similarity of B.cereus ATCC 10987 to B.anthracis Ames, as well as the fact that it contains a large pXO1-like plasmid, may make it a possible model for studying B.anthracis plasmid biology and regulatory cross-talk.     

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.     

Identification of a region of genetic variability among Bacillus anthracis strains and related species.

The identification of a region of sequence variability among individual isolates of Bacillus anthracis as well as the two closely related species, Bacillus cereus and Bacillus mycoides, has made a sequence-based approach for the rapid differentiation among members of this group possible. We have identified this region of sequence divergence by comparison of arbitrarily primed (AP)-PCR "fingerprints" generated by an M13 bacteriophage-derived primer and sequencing the respective forms of the only polymorphic fragment observed. The 1,480-bp fragment derived from genomic DNA of the Sterne strain of B. anthracis contained four consecutive repeats of CAATATCAACAA. The same fragment from the Vollum strain was identical except that two of these repeats were deleted. The Ames strain of B. anthracis differed from the Sterne strain by a single-nucleotide deletion. More than 150 nucleotide differences separated B. cereus and B. mycoides from B. anthracis in pairwise comparisons. The nucleotide sequence of the variable fragment from each species contained one complete open reading frame (ORF) (designated vrrA, for variable region with repetitive sequence), encoding a potential 30-kDa protein located between the carboxy terminus of an upstream ORF (designated orf1) and the amino terminus of a downstream ORF (designated lytB). The sequence variation was primarily in vrrA, which was glutamine- and proline-rich (30% of total) and contained repetitive regions. A large proportion of the nucleotide substitutions between species were synonymous. vrrA has 35% identity with the microfilarial sheath protein shp2 of the parasitic worm Litomosoides carinii.     

Cereulide synthetase gene cluster from emetic Bacillus cereus: Structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1

Background  

Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS), but its exact genetic organization and biochemical synthesis is unknown.     

Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains

Several human pathogens (e.g., Bacillus anthracis, Yersinia pestis, Bordetella pertussis, Plasmodium falciparum, and Mycobacterium tuberculosis) have very restricted unselected allelic variation in structural genes, which hinders study of the genetic relationships among strains and strain-trait correlations. To address this problem in a representative pathogen, 432 M. tuberculosis complex strains from global sources were genotyped on the basis of 230 synonymous (silent) single nucleotide polymorphisms (sSNPs) identified by comparison of four genome sequences. Eight major clusters of related genotypes were identified in M. tuberculosis sensu stricto, including a single cluster representing organisms responsible for several large outbreaks in the United States and Asia. All M. tuberculosis sensu stricto isolates of previously unknown phylogenetic position could be rapidly and unambiguously assigned to one of the eight major clusters, thus providing a facile strategy for identifying organisms that are clonally related by descent. Common clones of M. tuberculosis sensu stricto and M. bovis are distinct, deeply branching genotypic complexes whose extant members did not emerge directly from one another in the recent past. sSNP genotyping rapidly delineates relationships among closely related strains of pathogenic microbes and allows construction of genetic frameworks for examining the distribution of biomedically relevant traits such as virulence, transmissibility, and host range.     

Examination of intrinsic sulfonamide resistance in Bacillus anthracis: a novel assay for dihydropteroate synthase

Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides. A continuous spectrophotometric assay for measuring the enzymatic activity of DHPS was developed and used to examine the effects of sulfonamides on the enzyme. The new assay couples the production of MgPPi to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/alpha-glycerophosphate dehydrogenase reactions and monitors the disappearance of NADH at 340nm. The coupled enzyme assay demonstrates that resistance of the B. anthracis DHPS results in part from the use of the sulfonamides as alternative substrates, resulting in the formation of sulfonamide-pterin adducts, and not necessarily due to an inability to bind them.