Differentiation of Bacillus anthracis and other 'Bacillus cereus group' bacteria using IS231-derived sequences

Abstract Sequences based on the conserved 20 bp inverted repeat of IS 231 variants were used as polymerase chain reaction-based fingerprinting primers of the member species of the Bacillus cereus group ( B. anthracis, B. cereus, B. thuringiensis and B. mycoides ), because of their close association with transposons, principally Tn 4430 in B. thuringiensis . Fingerprints of B. anthracis were simple, and specifically allowed its identification and sub-differentiation from other members of the group. Fingerprints for B. cereus were strain-specific; those for B. thuringensis gave a 1650 bp product, characteristic of 1S 231 variants A-F. The same reaction conditions gave one or two bands for both B. anthracis and B. cereus that differed by restriction endonuclease mapping from the B. thuringiensis PCR product and established IS 231 restriction maps; this does not preclude some kind of relationship between these products and IS 231 .     

Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-direct sequencing

The primary structures of the 23S ribosomal RNA genes of Bacillus anthracis and an emetic strain of Bacillus cereus were determined by direct sequencing of enzymatically amplified chromosomal DNA. The 23S rRNA gene sequences of B. anthracis and B. cereus were found to be almost identical and showed only two differences (a single nucleotide change, and a single base insertion in B. cereus). The feasibility of using PCR-direct sequencing for the rapid sequence determination of large-subunit rRNA genes is demonstrated.     

Genetic relationships of Bacillus anthracis and closely related species based on variable-number tandem repeat analysis and BOX-PCR genomic fingerprinting

Variable-number tandem repeats (VNTR) analysis and BOX-repeat-based PCR (BOX-PCR) genomic fingerprinting were performed on 25 Bacillus strains to investigate the genetic relatedness of Bacillus anthracis to the closely related species. Based on VNTR analysis, all B. anthracis strains could be assigned to (VNTR)(4), which is the most commonly found type in the world. Interestingly, a (VNTR)(2) was also observed in Bacillus cereus KCTC 1661 and with an exact match to the tandem repeats found in B. anthracis. This finding has never been reported before in the closely related species. According to the BOX-PCR, B. anthracis strains clustered together and separated reliably from the closely related species. However, B. cereus KCTC 1661 was linked to the B. anthracis cluster and showed close relationships with B. anthracis strains. These results indicated that there was a strong correlation between VNTR analysis and BOX-PCR genomic fingerprinting.     

Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis

Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.     

Phenotypic and genotypic comparisons of 23 strains from the Bacillus cereus complex for a selection of known and putative B. thuringiensis virulence factors

Sixteen Bacillus thuringiensis, four Bacillus cereus and three Bacillus anthracis isolates were screened for a selection of known and putative B. thuringiensis virulence factors. PCR primers were designed to detect genes for phosphatidylcholine specific phospholipase C, phosphatidylinositol specific phospholipase C, immune inhibitor A, vegetative insecticidal protein 3A, a protein proposed to be involved in capsule synthesis, a newly identified Ser/Thr kinase homologue and enterotoxin entS. Motility, the presence of flagella, haemolysis, chitinase and lecithinase production were also evaluated. The widely varying profiles of the 23 strains from the complex provide a pool of different genotypes that can help to identify factors involved in pathogenicity.     

Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group

Aims:  To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores.
Methods and Results:  Characterized strains and bacteria from environmental samples were analysed by microbiological and molecular methods (PCR and restriction analysis). Environmental isolates often shared several microbiological features with B. anthracis , e.g. lack of β -haemolysis and phospholipase C activity, and only the gamma phage assay was specific for B. anthracis . PCR assays targeting markers from the virulence plasmids exclusively detected B. anthracis , but other PCR targets were also detected in nonanthrax isolates. Additionally, the restriction pattern in an Alu I restriction analysis of the SG-749 fragment is not 100% specific. The loci used for multiple-locus variable-number tandem repeat analysis of B. anthracis are also present in other members of the B. cereus group, but amplicon sizes are usually different.
Conclusions:  Environmental samples often contain borderline isolates closely related to B. anthracis both on microbiological and genetic levels. Real-time PCR targeting plasmidal and chromosomal markers should be used for rapid and definitive exclusion of a virulent strain of B. anthracis in such samples.
Significance and Impact of the Study:  This study gives an overview of the current microbiological and molecular methods used for identification of B. anthracis and shows that most assays have limits when borderline isolates present in environmental samples are analysed.     

利用扩增片段长度多态性分析鉴别炭疽和蜡样芽孢杆菌

目的:利用扩增片段长度多态性(AFLP)分析建立鉴别炭疽芽孢杆菌和蜡样芽孢杆菌的分子生物学方法。方法:3株炭疽芽孢杆菌和3株蜡样芽孢杆菌基因组经限制性内切酶EcoRⅠ和MseⅠ酶切后与对应接头连接,通过预扩增和选择性扩增获得特异性DNA片段,将片段进行毛细管电泳,并利用GeneScan和BioNumerics软件对电泳数据进行分析。结果:选择性扩增最佳引物组合为EcoRⅠ-G/MseⅠ-A,其扩增片段在100~500 bp范围内的有效数量为40~50条;比较炭疽芽孢杆菌和蜡样芽孢杆菌的AFLP特征峰值图和DNA指纹图谱,确定了5个有明显差异的片段区。结论:利用AFLP分析可对芽孢杆菌属中相近的炭疽芽孢杆菌和蜡样芽孢杆菌进行鉴别,该方法可作为炭疽芽孢杆菌传统鉴定方法的补充。     

Identity of hemolysins produced by Bacillus thuringiensis and Bacillus cereus

A hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis var. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.     

利用ERIC-PCR对苏云金芽胞杆菌与蜡状芽胞杆菌进行鉴定的研究

为了探索ERIC-PCR技术在苏云金芽胞杆菌和蜡状芽胞杆菌的鉴定及分型中的应用价值,本研究采用PCR方法初步检测苏云金芽胞杆菌杀虫晶体蛋白基因的组成,并对苏云金芽胞杆菌和蜡状芽胞杆菌的总DNA进行ERIC-PCR扩增,分析ERIC-PCR指纹图谱的特点并采用NTSYS2.10软件对其进行聚类。结果显示,各菌株的ERIC指纹图谱表现出不同程度的多态性,但图谱与菌株所含cry基因的类型存在一定的相关性。聚类分析结果显示,含有相同或相近cry基因类型的Bt菌株在进化树上趋向聚为一类,而不含cry基因的蜡状芽胞杆菌趋向于与不含cry基因的Bt菌株聚为一类或单独聚类。若在多种模式菌株的参考下,该方法可用于苏云金芽胞杆菌的初步鉴定和分型。     

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.     

Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis

Genome features of the Bacillus cereus group genomes (representative strains of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis sub spp. israelensis) were analyzed and compared with the Bacillus subtilis genome. A core set of 1381 protein families among the four Bacillus genomes, with an additional set of 933 families common to the B. cereus group, was identified. Differences in signal transduction pathways, membrane transporters, cell surface structures, cell wall, and S-layer proteins suggesting differences in their phenotype were identified. The B. cereus group has signal transduction systems including a tyrosine kinase related to two-component system histidine kinases from B. subtilis. A model for regulation of the stress responsive sigma factor sigmaB in the B. cereus group different from the well studied regulation in B. subtilis has been proposed. Despite a high degree of chromosomal synteny among these genomes, significant differences in cell wall and spore coat proteins that contribute to the survival and adaptation in specific hosts has been identified.     

Purification and sequencing of cerein 7B, a novel bacteriocin produced by Bacillus cereus Bc7

Cerein 7B is a new bacteriocin produced simultaneously with cerein 7A by Bacillus cereus Bc7 in liquid brain heart infusion cultures. Both bacteriocins are not synergistic. The two peptides have been purified to homogeneity by hydrophobic interaction, cation exchange and reverse-phase liquid chromatography. They can be distinguished by their N-terminal amino acid sequences N-Gly-Trp-Gly-Asp-Val-Leu (7A) and N-Gly-Trp-Trp-Asn-Ser-Trp-Gly-Lys (7B). Pre-cerein 7B is 74 amino acids long and contains an 18 aminoacid double-glycine type leader sequence that is removed to produce the mature bacteriocin. The leader peptide sequence is related to that of sec-independent secretion signals suggesting that cerein 7B belongs to class II sec-independent bacteriocins.     

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。     

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.     

The autolytic phenotype of the Bacillus cereus group

AIM: To determine the autolytic phenotype of five species in the Bacillus cereus group. METHODS AND RESULTS: The autolytic rate of 96 strains belonging to five species in the B. cereus group was examined under starvation conditions at pH 6, 6.5 and 8.5 in different buffers. The autolytic rate was strain-dependent with a wide variability at pH 6, but higher and more uniform at pH 6.5. At pH 8.5, and respect to the extent of autolysis at pH 6.5, it was relatively low for most of the strains with the lowest values between 13 and 52% in Bacillus mycoides and Bacillus pseudomycoides. Peptidoglycan hydrolase patterns evaluated by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cells of Bacillus thuringiensis ssp. tolworthi HD125 as an indicator, revealed complex profiles with lytic bands of about 90, 63, 46, 41, 38, 32, 28 and 25 kDa in B. cereus, B. thuringiensis and Bacillus weihenstephanensis. Bacillus mycoides and B. pseudomycoides had simpler profiles with lytic bands of 63, 46 and 38 kDa. Changes in the autolytic pattern were observed for cells harvested at the stationary phase of growth (72 h) showing an increase in the intensity of the 25 kDa band in the case of B. cereus, B. thuringiensis and B. weihenstephanensis, while no changes were observed for B. mycoides. Using Micrococcus lysodeicticus and Listeria monocytogenes as indicators lytic activity was retained by proteins of 63, 46, 38, 32 and 25 kDa and a new one of about 20 kDa in B. mycoides. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases except for those of B. mycoides and B. weihenstephanensis. Lytic activity was retained in the presence of MgCl(2), MnCl(2) and EDTA and increased at basic pH. CONCLUSIONS: Bacillus cereus/B. thuringiensis/B. weihenstephanensis showed a high extent of autolysis around neutral pH, even though they presented relatively complex autolysin profiles at alkaline pH. Bacillus mycoides/B. pseudomycoides had a higher extent of autolysis at acidic pH and a simpler autolysin pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the autolytic phenotype expand the phenotypic characterization of the different species in the B. cereus group.     

Genetic relationships between sympatric populations of Bacillus cereus and Bacillus thuringiensis, as revealed by rep-PCR genomic fingerprinting

The bacterial strain Bacillus cereus is closely related to Bacillus thuringiensis, although any genetic relationship between the two strains is still in debate. Using rep-PCR genomic fingerprinting, we established the genetic relationships between Brazilian sympatric populations of B. cereus and B. thuringiensis simultaneously collected from two geographically separate sites. We observed the formation of both B. thuringiensis and B. cereus clusters, as well as strains of B. cereus that are more closely related to B. thuringiensis than to other B. cereus strains. In addition, lower genetic variability was observed among B. thuringiensis clusters compared to B. cereus clusters, indicating that either the two species should be categorized as separate or that B. thuringiensis may represent a clone from a B. cereus background.     

野生Bc菌株产黑色素的研究

Bc58是一株野生蜡状芽抱杆菌菌株,经L-酪氨酸诱导后可产生红棕色色素。通过红外光谱及各种化学测定证明该色素与Sigma公司标准黑色素（Melanin）的性质相似。生测结果显示添加Bc58黑色素的Bt制剂经紫外照射5h后的LC50为16．1μg／mL,与未经紫外照射的Bt制剂的LC50 15．2μg／mL基本相同,而比未添加黑色素的Bt制剂经紫外照射后的杀虫毒力高出近1倍。经SDS—PAGE检测表明该黑色素可保护苏云金杆菌晶体蛋白在紫外光下基本不降解,表明Bc58黑色素是一种优良的紫外保护剂。     

Characterization of melanin produced by a wild-type strain of Bacillus cereus

Bacillus cereus 58 (Bc58)is a UV-resistant wild type strain that has an ability to produce a sorrel pigment induced by L-tyrosine.The Fourier-transform infrared (FT-IR)spectra and chemical tests of its pigment are similar to that of the standard melanin (Sigma).A bioassay shows that the LC50 of a Bacillus thuringiensis (Bt)formulation added with the melanin of Bc58 and exposed to UV for 5 h is 16.1 μg/ml,which is similar to that of the Bt formulation without UV treatment,however,it is almost double that of the Bt formulation exposed to UV without the melanin of Bc58.The result of SDS-PAGE indicates that the melanin of Bc58 can protect the insecticidal crystal proteins from degradation.This suggests that it is an excellent UV protective agent for the insecticidal crystal proteins of the Bt formulation.