Virulotypes of Bacillus anthracis strains isolated in Poland

Bacillus anthracis--the causative agent of anthrax--possesses several virulence genes located in the chromosome as well as in two B. anthracis virulence plasmids: pXO1 and pXO2. In the presented study, we determined occurrence of six virulence markers located in the virulence plasmids (capA, capB, capC, pagA, lef and cya) for capsule and toxin production together with virulence-associated gene gerXA and chromosomal gene sap, which are responsible for germination and S-layer biosynthesis respectively. Fourteen strains of B. anthracis isolated in Poland and belonging to five different MLVA genotypes were analyzed by PCR for presence of the aforementioned genes. Two virulotypes were found in tested strains. The only variation was absence of capA, capB and capC due to a lack of pXO2.     

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.     

Genotype diversity of the Bacillus anthracis strains isolated from the Caucasus region

The study of the genotypes of Bacillus anthracis strains isolated from the Caucasus region was performed using MLVA. Among 149 strains, 32 distinctive MLVA-8 genotypes belonging to Ala, A3a, A4 and B1 molecular diversity groups were identified. 9 genotypes were not described previously; 6 genotypes were not found in other geographic regions and could be considered as endemic for Caucasus. The majority of the identified genotypes are widespread not only in Caucasus, but also throughout Eurasia, Africa, and America. Molecular diversity of Caucasian isolates is comparable to the worldwide diversity. This represents historical relations of this region, proximity to ancient trade routes and intensity of the anthrax epizootic in Caucasus. The obtained results are of interest from the theoretical point of view, as well as for the application in epidemiological research of the anthrax outbreaks.     

Identification of Bacillus anthracis strains in China

As part of a project to establish a reference strain collection, phenotypic, biochemical and genetic analyses were carried out on 84 strains of Bacillus anthracis, 81 of them of Chinese origin from various sources. Particular differences from reports on isolates of other origins were the possession of fimbriae and single polar flagella with consequent motility in 77, self-agglutination by 64 and failure to ferment maltose in 60 of the Chinese strains. The findings were considered to be of significance to identification, classification and evolution of B. anthracis.     

Occurrence and genetic diversity of Bacillus anthracis strains isolated in an active wool-cleaning factory

Culturable microorganisms from various samples taken at an active factory performing wool and goat hair cleaning were isolated and analyzed. Bacillus anthracis was found in air filter dust, wastewater, and goat hairs, where it accounted for approximately 1% of the total counts of viable bacteria. Consistent with the countries of origin of the processed material (South Caucasian and Middle Eastern), all B. anthracis isolates belonged to the same phylogenetic cluster, as determined by variable-number tandem repeat (VNTR) typing at eight loci. Within this cluster, five closely related VNTR subtypes could be identified, of which two were previously unreported. Additional diversity was observed when more sensitive genetic markers were assayed, demonstrating the multifocal nature of goat hair contamination. Goat hair originating from areas where anthrax is endemic remains a material with high biological risk for modern woolworkers.     

Gene typing of infectious tularemia strains isolated from the Stavropol and Krasnodar Territories

The typing of F. tularensis strains by four variable number of tandem repeats (VNTR) loci has been carried out. Among the strains isolated in the Stavropol and Krasnodar Territories seven genotypes have been detected and their spread in different natural foci has been analyzed. The data thus obtained suggest that the VNTR analysis may become an important instrument for studying the structure of the natural foci of tularemia and evolutionary relationships between individual areas of these foci.     

Characterization of Bacillus anthracis strains used for vaccination

Three Bacillus anthracis strains, formerly used as anti-anthrax vaccine strains in Argentina, were characterized from genetic and pathogenic perspectives. Southern blotting and PCR with pXO1 and pXO2 probes and primers, as well as pathogenicity and protection tests in guinea pigs and mice, were performed. Two of the B. anthracis strains contained both pXO1 and pXO2 plasmids, as did the fully virulent strains, while the third was a Sterne-type strain (pXO1+, pXO2-). The three strains were, however, markedly less pathogenic than a wild-type virulent strain. The methodology applied here may be used to characterize other B. anthracis strains.     

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.     

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.     

两株炭疽芽胞杆菌MLST 新序列型(ST)

【目的】2株炭疽芽胞杆菌(Bacillus anthracis)17003-14和17003-32的多位点序列分型(Multilocussequence typing,MLST)研究。【方法】选取B.anthracis基因组7个常见管家基因位点glpF、gmk、ilvD、pta、pur、pycA和tpi进行PCR扩增、测序,与MLST数据库中的等位基因序列进行比对,确定菌株的序列型(sequence type,ST)。【结果】B.anthracis 17003-14和17003-32的等位基因编号分别为113、31、1、43、1、53、7和113、31、1、43、1、53、37,比对结果显示这2株细菌的等位基因编号组合未见报道。【结论】17003-14和17003-32为新ST菌株,已被MLST数据库确认,注册号(pubMLST id)分别为id-1053和id-1054。     

Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from fermented soybean foods manufactured in Asian countries

Natto-like fermented soybean products are manufactured and consumed in many Asian countries. In this study, we isolated thirty-four Bacillus strains capable of producing gamma-polyglutamic acid (PGA) from natto in mountainous areas of South Asia and Southeast Asia and from soils in Japan. To elucidate the phylogeny of these PGA-producing strains, phylogenetic trees based on sequences of 16S rDNA, housekeeping genes of rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) were constructed. A phylogenetic tree based on 16S rDNA sequences showed that twenty-one isolates were clustered in the same group of B. subtilis. The other thirteen isolates were located in the cluster of B. amyloliquefaciens. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. The results of the present study indicate that PGA-producing strains isolated from local natto in Asian countries and soil in Japan can be divided into two species, B. subtilis and B. amyloliquefaciens.     

Heterogeneity of Bacillus anthracis strains in terms of their adhesive capacity

The homogeneity of colonies of two B. anthracis vaccine strains in R- and RS- forms (100 colonies of each strain) in terms of their adhesive capacity was studied. B. anthracis strain 228/8 showed more heterogeneous composition than B. anthracis strain 71/12, moderately and highly adhesive colonies prevailing in both forms and nonadhesive colonies being absent. The prevalence of highly adhesive clones was established in the RS- form of B. anthracis strain 72/12 in comparison with R- form. By the average value of the adhesion index the RS- form colonies of this strain were classified as highly adhesive, while the colonies in the R- form were characterized as moderately adhesive.     

Diversity of Bacillus anthracis strains in Georgia and of vaccine strains from the former Soviet Union

Despite the increased number of anthrax outbreaks in Georgia and the other Caucasian republics of the former Soviet Union, no data are available on the diversity of the Bacillus anthracis strains involved. There is also little data available on strains from the former Soviet Union, including the strains previously used for vaccine preparation. In this study we used eight-locus variable-number tandem repeat analyses to genotype 18 strains isolated from infected animals and humans at different sites across Georgia, where anthrax outbreaks have occurred in the last 10 years, and 5 strains widely used for preparation of human and veterinary vaccines in the former Soviet Union. Three different genotypes affiliated with the A3.a cluster were detected for the Georgian isolates. Two genotypes were previously shown to include Turkish isolates, indicating that there is a regional strain pattern in the South Caucasian-Turkish region. Four of the vaccine strains were polymorphic, exhibiting three different patterns of the cluster A1.a genotype and the cluster A3.b genotype. The genotype of vaccine strain 71/12, which is considered an attenuated strain in spite of the presence of both of the virulence pXO plasmids, appeared to be a novel genotype in the A1.a cluster.     

Immunobiological properties of 100 years old Bacillus anthracis vaccine strains Lange

The results of the study of B. anthracis vaccine strains Lange 1 and 2 in the form of spores after prolonged storage in 30% glycerine solution at room temperature are presented. The study revealed that spores stored for 100 years germinated and bacterial cells proved to be viable when cultivated in artificial nutrient media and in vivo. They exhibited typical cultural, morphological, biochemical, virulent, antigenic and immunogenic properties.     

Oligonucleotide microarray identification of Bacillus anthracis strains using support vector machines

The capability of a custom microarray to discriminate between closely related DNA samples is demonstrated using a set of Bacillus anthracis strains. The microarray was developed as a universal fingerprint device consisting of 390 genome-independent 9mer probes. The genomes of B. anthracis strains are monomorphic and therefore, typically difficult to distinguish using conventional molecular biology tools or microarray data clustering techniques. Using support vector machines (SVMs) as a supervised learning technique, we show that a low-density fingerprint microarray contains enough information to discriminate between B. anthracis strains with 90% sensitivity using a reference library constructed from six replicate arrays and three replicates for new isolates.     

Multilocus sequence typing reveals that Bacillus cereus strains isolated from clinical infections have distinct phylogenetic origins

Eight strains of Bacillus cereus isolated from bacteremia and soft tissue infections were assigned to seven sequence types (STs) by multilocus sequence typing (MLST). Two strains from different locations had identical STs. The concatenated sequences of the seven STs were aligned with 65 concatenated sequences from reference STs and a neighbor-joining tree was constructed. Two strains were distantly related to all reference STs. Three strains were recovered in a clade that included Bacillus anthracis, B. cereus and rare Bacillus thuringiensis strains while the other three strains were assigned to two STs that were more closely affiliated to most of the B. thuringiensis STs. We conclude that invasive B. cereus strains do not form a single clone or clonal complex of highly virulent strains.     

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.     

Immunogenicity of the recombinant Bacillus strains with cloned gene of biosynthesis of protective antigen against Bacillus anthracis

Microbe Russian Anti-Plague Research Institute, Saratov A hybrid plasmid pUB110PA-1 demonstrating stable functioning in the cells of Bacillus strains and containing the gene of biosynthesis of Bacillus anthracis protective antigen was constructed. The recombinant strains surpassing the anthrax vaccinal cultures in the secreted synthesis of the protective antigen were obtained and their immunological efficacy was assessed. A single inoculation of Guinea pigs with the dose of 5 x 107 spores of the recombinant strains imparted efficient protection against B. anthracis challenge. Immune responses were characterized by high indices of immunity and titers of antibodies to the protective antigen. In contrast to the anthrax vaccinal preparations, the gene-engineering strains imposed no residual virulence for BALB/n mice and Guinea pigs.