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.     

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.     

Draft Genome Sequence of Bacillus anthracis BF-1, Isolated from Bavarian Cattle

Bacillus anthracis BF-1 was isolated from a cow in Bavaria (Germany) that had succumbed to anthrax. Here, we report the draft genome sequence of this strain, which belongs to the European B2 subclade of B. anthracis. The closest phylogenetic neighbor of strain BF-1 is a strain isolated from cattle in France.     

Draft Genome Sequence of Bacillus anthracis UR-1, Isolated from a German Heroin User

We report the draft genome sequence of Bacillus anthracis UR-1, isolated from a fatal case of injectional anthrax in a German heroin user. Analysis of the genome sequence of strain UR-1 may aid in describing phylogenetic relationships between virulent heroin-associated isolates of B. anthracis isolated in the United Kingdom, Germany, and other European countries.     

Novel and Unique Diagnostic Biomarkers for Bacillus anthracis Infection

A search for bacterium-specific biomarkers in peripheral blood following infection with Bacillus anthracis was carried out with rabbits, using a battery of specific antibodies generated by DNA vaccination against 10 preselected highly immunogenic bacterial antigens which were identified previously by a genomic/proteomic/serologic screen of the B. anthracis secretome. Detection of infection biomarkers in the circulation of infected rabbits could be achieved only after removal of highly abundant serum proteins by chromatography using a random-ligand affinity column. Besides the toxin component protective antigen, the following three secreted proteins were detected in the circulation of infected animals: the chaperone and protease HtrA (BA3660), an NlpC/P60 endopeptidase (BA1952), and a protein of unknown function harboring two SH3 (Src homology 3) domains (BA0796). The three proteins could be detected in plasma samples from infected animals exhibiting 10³ to 10⁵ CFU/ml blood and also in standard blood cultures at 3 to 6 h post-bacterial inoculation at a bacteremic level as low as 10³ CFU/ml. Furthermore, the three biomarkers appear to be present only in the secretome of B. anthracis, not in those of the related pathogens B. thuringiensis and B. cereus. To the best of our knowledge, this is the first report of direct detection of B. anthracis-specific proteins, other than the toxin components, in the circulation of infected animals.The gram-positive spore-forming bacterium Bacillus anthracis is the causative agent of anthrax, a rare fatal disease which is initiated, in its most severe form, by inhalation of spores. Due to the severity of the disease, the ease of respiratory infection, and the extreme resistance of the spores to unfavorable environmental conditions, B. anthracis is considered a potential biological warfare agent (for a review, see references 8, 10, 35, 56, and 62), and in recent years, the need for novel reliable diagnostic approaches, improved vaccination strategies, novel therapeutic targets, and a better understanding of the pathogenesis of anthrax has been widely acknowledged.Inhaled B. anthracis spores are taken up by alveolar macrophages and germinate into vegetative bacilli which eventually invade the bloodstream, where they multiply massively and secrete toxins and virulence factors. Anthrax is toxinogenic in the sense that the bacterial binary exotoxin is necessary for the onset of the disease (54), yet other factors may be required for the colonization and expansion of bacteria in the host (15, 18, 31, 32, 37, 46, 65, 66, 70, 83). The toxin is composed of the following three proteins: protective antigen (PA), which mediates binding to the receptor on target cells and internalization of the toxin components (14, 74); lethal factor, a zinc protease targeting several mitogen-activated protein kinases (52); and edema factor (EF), a calmodulin-dependent adenylate cyclase (55, 57). The genes encoding the three exotoxin components are located on the native virulence plasmid pXO1. Genes encoding proteins with functions involved in the synthesis of the second major B. anthracis virulence determinant, an immunologically inert polyglutamyl capsule that protects bacteria from phagocytosis, are located on a second native virulence plasmid, pXO2 (56).In humans, the initial symptoms of inhalation anthrax are nonspecific and reminiscent of influenza-like upper respiratory tract infections. The second stage is characterized by high fever, respiratory failure, dyspnea, and shock. Unless patients are treated promptly, death occurs within 24 h of the onset of the second stage, preceded by massive bacteremia (27, 34, 73, 76). The mandatory treatment for anthrax is based on administration of antibiotics (17, 76), yet study of the disease in animal models has clearly established that the time of antibiotic administration postinfection is crucial for the effectiveness of the treatment, strongly supporting the importance of rapid diagnosis (2, 47, 48). At present, due to the severity of the disease and its rapid progression, treatment is administered to each person with confirmed contact with contaminated areas (76).Early accurate diagnosis of anthrax is complicated by the rarity of the disease and the nonspecific nature of the symptoms. Microbiologic identification of anthrax is based on the nonhemolytic nature of the typically white-gray colonies and the rod morphology of the gram-positive nonmotile bacilli detected in clinical samples or blood cultures (16, 19, 30, 73, 78). Immunofluorescence and immunohistochemistry targeted to bacterial proteins are not routinely conducted. Later in the course of the disease, seroconversion in response to the various components of the toxin may serve only as a retrospective confirmation of initial exposure. With the advent of genetic methodologies, B. anthracis in cultures inoculated with clinical and forensic samples can be detected specifically and accurately by PCR, usually designed to amplify genes located on the native virulence plasmids (30). Recently, the use of PA as a disease biomarker was suggested, owing to the presence of this protein in detectable amounts in the circulation of infected animals (53, 71). EF and lethal factor can be detected in the circulation only at later stages of infection (30).In recent years, the search for novel biomarkers of disease, including bacterial infections, has exploited the approach of global biological inspection based on functional genomic or proteomic studies (64, 85). We previously documented such global surveys, combined with a serological study of B. anthracis (5, 6, 20, 21, 22, 38, 39), for identification of vaccine and diagnostic marker candidates among extracellular (secreted or membranal) proteins. These studies indeed revealed a list of proteins that can serve as potential biomarkers, based on their immunogenicity (which probes their in vivo expression), abundance under various culture conditions, and functional relatedness to infection. In the present study, the search was extended by directly addressing the presence of bacterial secreted proteins in the circulation of B. anthracis-infected rabbits, using specific antibodies generated by DNA vaccination against the previously selected immunogenic proteins. Visualization of bacterial proteins in the circulation of infected animals was achieved only following depletion of highly abundant serum proteins by an affinity chromatography protocol. The search enabled the successful detection, in addition to PA, of three secreted proteins uniquely expressed by B. anthracis, i.e., HtrA (BA3660), the BA1952 endopeptidase, and a protein of unknown function (BA0796). All of these proteins are potential virulence-related factors. This is the first communication of the presence of B. anthracis secreted proteins other than the bacterial toxin in the circulation of infected animals, and their identification strongly supports the validity of the reductional screening approach for selection of disease biomarkers.     

Three Bacillus anthracis bacteriophages from topsoil

Three Bacillus anthracis bacteriophages from Iowa topsoil are characterized as to latent period, morphology, structural proteins, DNA size, and restriction endonuclease digestion. Electron micrographs indicate that the three isolates include two members of the Myoviridae and one smaller phage belonging to the Podoviridae. Phages Nk and DB resemble Myoviridae phage SP50 in morphology, but host range studies, protein, and DNA analysis indicate that both differ from SP50. Phage MH is very similar to phage phi 29, but differs in terms of host range, structural protein, and DNA characteristics.     

A Novel Multiplex PCR Discriminates Bacillus anthracis and Its Genetically Related Strains from Other Bacillus cereus Group Species

Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.     

Glyconanobiotics: Novel carbohydrated nanoparticle antibiotics for MRSA and Bacillus anthracis

This report describes the synthesis and evaluation of glycosylated polyacrylate nanoparticles that have covalently-bound antibiotics within their framework. The requisite glycosylated drug monomers were prepared from one of three known antibiotics, an N-sec-butylthio beta-lactam, ciprofloxacin, and a penicillin, by acylation with 3-O-acryloyl-1,2-O-isopropylidene-5,6 bis((chlorosuccinyl)oxy)-d-glucofuranose (7) or 6-O-acetyl-3-O-acryloyl-1,2-O-isopropylidene-5-(chlorosuccinyl)oxy-alpha-d-glucofuranose (10). These acrylated monomers were subjected to emulsion polymerization in a 7:3 (w:w) mixture of butyl acrylate-styrene in the presence of sodium dodecyl sulfate as surfactant (3 weight %) and potassium persulfate as a radical initiator (1 weight %). The resulting nanoparticle emulsions were characterized by dynamic light scattering and found to have similar diameters ( approximately 40 nm) and size distributions to those of our previously studied systems. Microbiological testing showed that the N-sec-butylthio beta-lactam and ciprofloxacin nanoparticles both have powerful in vitro activities against methicillin-resistant Staphylococcus aureus and Bacillus anthracis, while the penicillin-bound nanoparticles have no antimicrobial activity. This indicates the need for matching a suitable antibiotic with the nanoparticle carrier. Overall, the study shows that even relatively large, polar acrylate monomers (MW>1000 amu) can be efficiently incorporated into the nanoparticle matrix by emulsion polymerization, providing opportunities for further advances in nanomedicine.     

Structure of two iron-binding proteins from Bacillus anthracis

Bacillus anthracis is currently under intense investigation due to its primary importance as a human pathogen. Particularly important is the development of novel anti-anthrax vaccines, devoid of the current side effects. A novel class of immunogenic bacterial proteins consists of dodecamers homologous to the DNA-binding protein of Escherichia coli (Dps). Two Dps homologous genes are present in the B. anthracis genome. The crystal structures of these two proteins (Dlp-1 and Dlp-2) have been determined and are presented here. They are sphere-like proteins with an internal cavity. We also show that they act as ferritins and are thus involved in iron uptake and regulation, a fundamental function during bacterial growth.     

Novel Strategies for Enhanced Removal of Persistent Bacillus anthracis Surrogates and Clostridium difficile Spores from Skin

Background

Removing spores of Clostridium difficile and Bacillus anthracis from skin is challenging because they are resistant to commonly used antimicrobials and soap and water washing provides only modest efficacy. We hypothesized that hygiene interventions incorporating a sporicidal electrochemically generated hypochlorous acid solution (Vashe^®) would reduce the burden of spores on skin.

Methods

Hands of volunteers were inoculated with non-toxigenic C. difficile spores or B. anthracis spore surrogates to assess the effectiveness of Vashe solution for reducing spores on skin. Reduction in spores was compared for Vashe hygiene interventions versus soap and water (control). To determine the effectiveness of Vashe solution for removal of C. difficile spores from the skin of patients with C. difficile infection (CDI), reductions in levels of spores on skin were compared for soap and water versus Vashe bed baths.

Results

Spore removal from hands was enhanced with Vashe soak (>2.5 log₁₀ reduction) versus soap and water wash or soak (~2.0 log₁₀ reduction; P <0.05) and Vashe wipes versus alcohol wipes (P <0.01). A combined approach of soap and water wash followed by soaking in Vashe removed >3.5 log₁₀ spores from hands (P <0.01 compared to washing or soaking alone). Bed baths using soap and water (N =26 patients) did not reduce the percentage of positive skin cultures for CDI patients (64% before versus 57% after bathing; P =0.5), whereas bathing with Vashe solution (N =21 patients) significantly reduced skin contamination (54% before versus 8% after bathing; P =0.0001). Vashe was well-tolerated with no evidence of adverse effects on skin.

Conclusions

Vashe was safe and effective for reducing the burden of B. anthracis surrogates and C. difficile spores on hands. Bed baths with Vashe were effective for reducing C. difficile on skin. These findings suggest a novel strategy to reduce the burden of spores on skin.     

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.     

Cathelicidin administration protects mice from Bacillus anthracis spore challenge

Cathelicidins are a family of cationic peptides expressed in mammals that possess numerous bactericidal and immunomodulatory properties. In vitro analyses showed that human, mouse, and pig cathelicidins inhibited Bacillus anthracis bacterial growth at micromolar concentrations in the presence or absence of capsule. Combined in vitro analyses of the effects of each peptide on spore germination and vegetative outgrowth by time lapse phase contrast microscopy, transmission electron microscopy, and flow cytometric analysis showed that only the pig cathelicidin was capable of directly arresting vegetative outgrowth and killing the developing bacilli within the confines of the exosporium. C57BL/6 mice were protected from spore-induced death by each cathelicidin in a time- and dose-dependent manner. Protection afforded by the porcine cathelicidin was due to its bactericidal effects, whereas the human and mouse cathelicidins appeared to mediate protection through increased recruitment of neutrophils to the site of infection. These findings suggest that cathelicidins might be utilized to augment the initial innate immune response to B. anthracis spore exposure and prevent the development of anthrax.     

A modeled structure for amidase-03 from Bacillus anthracis

Homology models of amidase-03 from Bacillus anthracis were constructed using Modeller (9v2). Modeller constructs protein models using an automated approach for comparative protein structure modeling by the satisfaction of spatial restraints. A template structure of Listeria monocytogenes bacteriophage PSA endolysin PlyPSA (PDB ID: 1XOV) was selected from protein databank (PDB) using BLASTp with BLOSUM62 sequence alignment scoring matrix. We generated five models using the Modeller default routine in which initial coordinates are randomized and evaluated by pseudo-energy parameters. The protein models were validated using PROCHECK and energy minimized using the steepest descent method in GROMACS 3.2 (flexible SPC water model in cubic box of size 1 Å instead of rigid SPC model). We used G43a1 force field in GROMACS for energy calculations and the generated structure was subsequently analyzed using the VMD software for stereo-chemistry, atomic clash and misfolding. A detailed analysis of the amidase-03 model structure from Bacillus anthracis will provide insight to the molecular design of suitable inhibitors as drug candidates.