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.     

Differential analysis of Bacillus anthracis after pX01 plasmid curing and comprehensive data on Bacillus anthracis infection in macrophages and glial cells

Bacillus anthracis is a gram-positive bacterial organism responsible for anthrax. This organism has two pathogenic plasmids: pX01 and pX02. The genetic function of pX01, which comprises about 198 kb, is not known, except for a region called the pathogenic island, which contains three genes-pag, lef, and cya-that code for three toxic proteins. A 2-D difference gel electrophoresis (2-D DIGE) system was used to verify the existence of proteins controlled by the pX01 plasmid, and protein regulation data were obtained using DeCyder software. A total of 1728 proteins were identified in the wild-type strain of this organism and 1684 in the pX01 plasmid. Twenty-seven of these proteins disappeared and eight appeared when the pX01 plasmid was removed. An additional 52 proteins were downregulated and 15 were upregulated when this plasmid was removed. A total of 102 proteins have been identified using the MALDI-TOF method of analysis, including 49 whose functions are unknown. Among these, 31 participate in metabolic processes, two in cellular processes, 15 in the processing of genetic information, and five in the processing of extracellular information. Another seven proteins participate in bacterial virulence and pathogenesis. We investigated the functions of these proteins in other bacteria, particularly the B. anthracis derivative H9041. Bacterial growth differed between pX01+/pX02+ B. anthracis and its pX01-/pX02+ derivative as did the cytotoxicity of macrophages infected by pX01+/pX02+ B. anthracis and the pX01-pX02+ derivative. We also found that S100B protein levels increased in the host infected with pX01+/pX02+ B. anthracis or its pX01-/pX02+ derivative. These data suggest that the pX01 plasmid plays a key role in the regulation of protein functions in B. anthracis.     

制备炭疽芽胞杆菌检测基因芯片的初步研究

建立制备炭疽芽胞杆菌检测基因芯片的技术,并探讨研制检测炭疽芽胞杆菌基因芯片的方法。酶切炭疽芽胞杆菌的毒素质粒和荚膜质粒,通过建立质粒DNA文库的方法获取探针,并打印在经过氨基化修饰的玻片上,制成用于炭疽芽胞杆菌检测的基因芯片。收集了290个阳性克隆探针,制备了检测炭疽芽胞杆菌的基因芯片。提取炭疽芽胞杆菌质粒DNA与基因芯片杂交,经ScanArray Lite芯片阅读仪扫描得到初步的杂交荧光图像。通过分析探针的杂交信号初步筛选出273个基因片段作为芯片下一步研究的探针。     

study of the virulence of a recombinant strain of Bacillus anthracis, obtained as a result of transductive transfer of chromosomal genes from a strain of Bacillus cereus

Auxotrophic markers of B. anthracis strains differing them from other Bacillus representatives have been determined. Chromosome genes from prototrophic B. cereus strain were transduced into auxotrophic B. anthracis strain. The properties of transductants were studied in order to establish common transfer of chromosomal determinants responsible for realization of various signs. Transduction mating between species resulted in construction of prototroph B. anthracis strains (pX01- pX02+), whose derivatives are characterized by decreased virulence for laboratory animals.     

Multiplex amplification test system for the identification and differentiation of Bacillus anthracis

The multiplex amplification test system for the identification of Bacillus anthracis with primers to plasmid cya (pX01), capC (pX02) genes and chromosomal sap gene were developed. The primers to sap gene were selected by the authors and, after being tested on 72 microbial strains of the genus Bacillus, proposed as more specific in comparison with the known primers to chromosomal locus Ba 813. The proposed test system permitted the simultaneous identification of B. anthracis of all plasmid variants, the evaluation of their potential virulence and the differentiation of B. anthracis nonplasmid strains from bacilli of the group Bacillus cereus.     

炭疽芽胞杆菌基因芯片探针文库的构建

为制备炭疽芽胞杆菌的基因芯片探针文库,以炭疽芽胞杆菌毒素质粒pX01和荚膜质粒pX02为原材料,用Sau3A I酶切pX01和pX02质粒DNA,Taq DNA聚合酶72℃补平加A,经AT克隆,PCR初步鉴定筛选出炭疽质粒片段的阳性克隆．DNA自动分析仪对克隆片段进行序列测定;用生物信息学方法对其片段进行同源性分析;并将克隆的探针打印于玻片上,制备成炭疽芽胞杆茵基因芯片,与炭疽杆茵质粒DNA样品进行初步芯片杂交的实验,杂交实验的阳性率达到了90％以上,证明大部分克隆探针属于炭疽芽胞杆菌．炭疽芽胞杆菌基因芯片探针文库的构建为基因芯片探针的制备摸索出一条简便、高效、可行的方法．     

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.     

Species-specific peptide ligands for the detection of Bacillus anthracis spores

Currently available detectors for spores of Bacillus anthracis, the causative agent of anthrax, are inadequate for frontline use and general monitoring. There is a critical need for simple, rugged, and inexpensive detectors capable of accurate and direct identification of B. anthracis spores. Necessary components in such detectors are stable ligands that bind tightly and specifically to target spores. By screening a phage display peptide library, we identified a family of peptides, with the consensus sequence TYPXPXR, that bind selectively to B. anthracis spores. We extended this work by identifying a peptide variant, ATYPLPIR, with enhanced ability to bind to B. anthracis spores and an additional peptide, SLLPGLP, that preferentially binds to spores of species phylogenetically similar to, but distinct from, B. anthracis. These two peptides were used in tandem in simple assays to rapidly and unambiguously identify B. anthracis spores. We envision that these peptides can be used as sensors in economical and portable B. anthracis spore detectors that are essentially free of false-positive signals due to other environmental Bacillus spores.     

Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysis

AIMS: To determine whether microarray analysis could be employed for the differential identification of a range of environmental Bacillus sp. from four strains of Bacillus anthracis. METHODS AND RESULTS: Oligonucleotide probes were designed that were specific to virulence factor genes of B. anthracis (pag, lef and cap), the variable number tandem repeat region of the B. anthracis vrrA gene and to the 16S-23S rRNA intergenic transcribed spacer region (ITS) and pleiotropic regulator (plcR) regions of the Bacillus cereus subgroup species. Generic probes were also designed to hybridize with conserved regions of the 16S rRNA genes of Bacillus (as a positive control), Neisseria sp., Pseudomonas sp., Streptococcus sp., Mycobacterium sp. and to all members of the Enterobacteriaceae to allow simultaneous detection of these bacteria. Identification of B. anthracis was found to rely entirely on hybridization of DNA specific to regions of the pag, lef and cap genes. Cross-reaction was observed between B. anthracis and other Bacillus species with all the other Bacillus probes tested. Results obtained using microarray hybridizations were confirmed using conventional microbiological techniques and found to have very high comparability. CONCLUSIONS: Microarray-based assays are an effective method for the identification of B. anthracis from mixed-culture environmental samples without problems of false-positivity that have been observed with conventional PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of environmental Bacillus sp. by conventional PCR is prone to potential for reporting false-positives. This study provides a method for the exclusion of such isolates.     

Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR

Bacillus anthracis spores have been shown to be an efficient biological weapon and their recent use in bioterrorist attacks has demonstrated the need for rapid and specific diagnostics. A TaqMan real-time PCR for identification of B. anthracis was developed, based on the two plasmids, pX01 and pX02, both of which are necessary for pathogenicity, as well as on the chromosomally encoded rpoB gene. Bacteria picked from colonies or pelleted from liquid cultures were directly inoculated into the PCR mix, thus avoiding time-consuming DNA preparation and minimizing handling risks. B. anthracis spores were cultivated for a few hours in enrichment broth before PCR analysis, or used directly for real-time PCR, thus allowing to confirm or exclude potential attacks approximately 2-3 h after the material has arrived in the laboratory.     

突发疫情中炭疽芽孢杆菌的分离及鉴定

对疑似炭疽感染病牛牛肉标本和牛血污染土壤标本进行了病原菌分离,经菌落形态和菌体形态观察、血清学实验和生化鉴定,证明分离到的细菌为炭疽芽孢杆菌。为进一步了解其特性,分别用保护性抗原、水肿因子和荚膜基因特异性引物对2株菌进行PCR扩增。结果显示,这两株菌有两个毒力相关质粒pX01和pX02,为有毒株。序列测定表明,这两株菌基因间同源性达99%,这两株菌与GenBank中炭疽芽孢杆菌A2012株、Ames Ancestor株和A16R疫苗株同源性达99%。     

Investigation of spore surface antigens in the genus Bacillus by the use of polyclonal antibodies in immunofluorescence tests

Fluorescein-conjugated rabbit antibodies to formalized spores of Bacillus anthracis were tested against strains of B. anthracis and other Bacillus species in a subjective immunofluorescence test. The lack of reaction of B. anthracis Vollum spores with conjugated antibody raised against B. anthracis Sterne spores indicated that spores of the Vollum strain lacked a major surface antigen present in most of the other anthrax strains tested, including the non-encapsulated strains Sterne and the Soviet ST1, variants cured of the pX01 plasmid that codes for the toxin, and several virulent strains. Four other antibody preparations, raised against B. anthracis Vollum, New Hampshire, Ames and Strain 15, reacted to an approximately similar degree with spores of all four strains and of Sterne, indicating that Vollum has at least one spore antigen in common with these other strains. The anti-Sterne and anti-Vollum conjugates both displayed cross-reactions with spores of strains of B. cereus, B. coagulans, B. subtilis, B. megaterium, B. polymyxa, B. pumilus and B. thuringiensis. Absorption of the anti-anthrax conjugates with B. cereus NCTC 8035 and NCTC 10320 removed all these cross-reactions, demonstrating the existence of spore antigens specific for anthrax.     

Investigation of spore surface antigens in the genus Bacillus by the use of polyclonal antibodies in immunofluorescence tests

Fluorescein-conjugated rabbit antibodies to formalized spores of Bacillus anthracis were tested against strains of B. anthracis and other Bacillus species in a subjective immunofluorescence test. The lack of reaction of B. anthracis Vollum spores with conjugated antibody raised against B. anthracis Sterne spores indicated that spores of the Vollum strain lacked a major surface antigen present in most of the other anthrax strains tested, including the non-encapsulated strains Sterne and the Soviet ST1, variants cured of the pX01 plasmid that codes for the toxin, and several virulent strains. Four other antibody preparations, raised against B, anthracis Vollum, New Hampshire, Ames and Strain 15, reacted to an approximately similar degree with spores of all four strains and of Sterne, indicating that Vollum has at least one spore antigen in common with these other strains. The anti-Sterne and anti-Vollum conjugates both displayed cross-reactions with spores of strains of B. cereus, B. coagulans, B. subtilis, B. megaterium, B. polymyxa, B. pumilus and B. thuringiensis. Absorption of the anti-anthrax conjugates with B. cereus NCTC 8035 and NCTC 10320 removed all these cross-reactions, demonstrating the existence of spore antigens specific for anthrax.     

The native virulence plasmid combination affects the segregational stability of a theta-replicating shuttle vector in Bacillus anthracis var. New Hampshire

The segregational stability of a small, theta-replicating, non-mobilizable shuttle plasmid (pAEX-5E) was determined in fully virulent (pX01+/pX02+), partially cured (pX01+/pX02- and pX01-/pX02+) and fully cured (pX01-/pX02-) derivatives of Bacillus anthracis var. New Hampshire. Under the growth conditions used (L-broth, 37 degrees C, aerobic, batch culture), pAEX-5E remained segregationally stable in the pX01-/pX02+ and pX01-/pX02- derivatives for in excess of 100 culture generations, but was expelled from the pX01+/pX02+ and pX01+/pX02- derivatives (100% loss occurred after 101+/-3.8 and 54+/-6.0 culture generations, respectively). In the presence of antibiotic selection pressure to maintain pAEX-5E (5 microg erythromycin ml-1) no comparable loss of pX01 or pX02 was observed over 100 generations of growth in any of the derivatives of B. anthracis. Under these conditions the pX01+/pX02- derivative had an extended culture doubling time (td+/-S. E. of the mean) of 75.3 +/- 1.4 min compared with 47.3 +/- 1.1, 46.2 +/- 0.86 and 43.2 +/- 1.2 min for the pX01+/pX02+, pX01-/pX02+ and pX01-/pX02- derivatives, respectively. That antibiotic resistance was pAEX-5E-mediated was confirmed using a second antibiotic marker (kanamycin). After100 generations of growth in the presence of erythromycin, colonies were shown to have retained kanamycin resistance. Southern blot analysis, in conjunction with plasmid rescue to Escherichia coli confirmed that, after 100 culture generations in the presence of antibiotic selection pressure, pAEX-5E had remained structurally stable and had not integrated into the B. anthracis genome.     

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.     

Glycine-induced cryotransformation of plasmids into Bacillus anthracis

Different cloning vectors (pC194, pBC16, pUB110, pBD10, pBD8, pAM beta 1) and Bacillus anthracis plasmid pX02 were introduced into B. anthracis by a transformation method. To induce an artificial competence state for uptake of isolated plasmid DNA, the cultures were treated with glycine, to reduce cross-linking of peptidoglycan, followed by freezing and thawing. The procedure is extremely rapid and relatively efficient (maximum transformation efficiency about 10(3) c.f.u. per micrograms DNA) and allows different cloning vectors with molecular masses ranging from 1.8 to 17.7 MDa to be introduced into B. anthracis.     

Fate of germinated Bacillus anthracis spores in primary murine macrophages

We investigated the fate of germinated Bacillus anthracis spores after their germination in Swiss murine peritoneal macrophages and in the cell line RAW264.7. We found that the lethal toxin and the oedema toxin are germ-associated factors that are essential for the survival of the vegetative form in host cells. We also found that pX02 is not involved in this complex pathogenic process. By transmission electron microscopy, we showed the tight interaction between the exosporium of the spore and the phagosomal membrane of the macrophage. Our data strongly suggest that the B. anthracis toxinogenic, unencapsulated Sterne strain (7702) does not multiply within macrophages. These results contributed to reveal the strategies used by B. anthracis to survive within the host and to reach the external medium where they proliferate.     

Fluorescent detection techniques for real-time multiplex strand specific detection of Bacillus anthracis using rapid PCR

Speed is a key area in our development of PCR assays for Bacillus anthracis. We believe that the strand specific detection of amplicons within 10 min is a realistic goal and that this will be achieved through fluorescent in-tube assays. We have used the Idaho LightCycler to study and develop candidate assays for B. anthracis. New strand specific fluorescent methods have been developed and a number of formats have been studied for speed and sensitivity. Internal controls have been developed as a method of improving our assay confidence. In this communication we will introduce the field of rapid PCR whilst discussing previous work in the areas described above, the development of our own rapid assay and a novel internal control system for B. anthracis. This work used PCR assays and hardware that are either commercially available, or have been previously described in open literature publications.     

Quantitative immunofluorescence studies of the serology of Bacillus anthracis spores.

A fluorescein-conjugated antibody against formalin-inactivated spores of Bacillus anthracis Vollum reacted only weakly with a variety of Bacillus species in microfluorometric immunofluorescence assays. A conjugated antibody against spores of B. anthracis Sterne showed little affinity for spores of several B. anthracis isolates including B. anthracis Vollum, indicating that more than one anthrax spore serotype exists.