Endospore surface properties of commonly used Bacillus anthracis surrogates vary in aqueous solution

The hydrophobic character and electrophoretic mobility (EPM) of microorganisms are vital aspects of understanding their interactions with the environment. These properties are fundamental in fate-and-transport, physiological, and virulence studies, and thus integral in surrogate selection. Hydrophobic and electrostatic forces are significant contributors to particle and microorganism mobility in the environment. Herein, the surface properties of commonly used Bacillus anthracis surrogate endospores were tested under comparable conditions with respect to culture, endospore purification, buffer type and strength. Additionally, data is presented of endospores suspended in dechlorinated tap water to evaluate the surrogates in regard to a breach of water infrastructure security. The surface properties of B. anthracis were found to be the most hydrophobic and least electronegative among the six Bacillus species tested across buffer strength. The effect of EPM on hydrophobicity varies in a species-specific manner. This study demonstrates that surrogate surface properties differ and care must be taken when choosing the most suitable surrogate. Moreover, it is shown that Bacillus thuringensis best represents Bacillus anthracis-Sterne with respect to both EPM and hydrophobicity across all test buffers.     

Inactivation of Spores of Bacillus anthracis Sterne, Bacillus cereus, and Bacillus thuringiensis subsp. israelensis by Chlorination

Three species of Bacillus were evaluated as potential surrogates for Bacillus anthracis for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Spores of Bacillus thuringiensis subsp. israelensis were found to be an appropriate surrogate for spores of B. anthracis for use in chlorine inactivation studies.     

Decontamination Efficacy and Skin Toxicity of Two Decontaminants against Bacillus anthracis

Decontamination of bacterial endospores such as Bacillus anthracis has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing Bacillus anthracis spores in solution and on a human skin simulant (porcine cadaver skin), compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in human skin keratinocyte primary cultures. The chlorine dioxide decontaminant was similarly effective to sodium hypochlorite in reducing spore numbers of Bacillus anthracis Ames in liquid suspension after a 10 minute exposure. After five minutes, the chlorine dioxide product was significantly more efficacious. Decontamination of isolated swine skin contaminated with Bacillus anthracis Sterne with the chlorine dioxide product resulted in no viable spores sampled. The toxicity of the chlorine dioxide decontaminant was up to two orders of magnitude less than that of sodium hypochlorite in human skin keratinocyte cultures. In summary, the chlorine dioxide based decontaminant efficiently killed Bacillus anthracis spores in liquid suspension, as well as on isolated swine skin, and was less toxic than sodium hypochlorite in cultures of human skin keratinocytes.     

Genotyping of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> and Closely Related Microorganisms

Bacillus cereus sensu lato group, which includes the causative agent of anthrax Bacillus anthracis, has a high degree of genetic similarity. However, in recent years, the development of molecular genetics has made it possible to find some molecular markers for identifying Bacillus anthracis in order to differentiate it from the closely related bacilli, get the clustering of the collection of strains, and describe the phylogenetic relationships between the clusters. This article deals with up-to-date methods of genotyping of Bacillus anthracis; it also evaluates its discrimination ability, informational value, validity of the results, efficiency, simplicity, and ease of use. In addition, international online resources and the possibility of their use for comparing collections of the B. anthracis strains isolated from various parts of the world are discussed.     

Bacillus anthracis Multiplication, Persistence, and Genetic Exchange in the Rhizosphere of Grass Plants

Bacillus anthracis, the causative agent of anthrax, is known for its rapid proliferation and dissemination in mammalian hosts. In contrast, little information exists regarding the lifestyle of this important pathogen outside of the host. Considering that Bacillus species, including close relatives of B. anthracis, are saprophytic soil organisms, we investigated the capacity of B. anthracis spores to germinate in the rhizosphere and to establish populations of vegetative cells that could support horizontal gene transfer in the soil. Using a simple grass plant-soil model system, we show that B. anthracis strains germinate on and around roots, growing in characteristic long filaments. From 2 to 4 days postinoculation, approximately one-half of the B. anthracis CFU recovered from soil containing grass seedlings arose from heat-sensitive organisms, while B. anthracis CFU retrieved from soil without plants consisted of primarily heat-resistant spores. Coinoculation of the plant-soil system with spores of a fertile B. anthracis strain carrying the tetracycline resistance plasmid pBC16 and a selectable B. anthracis recipient strain resulted in transfer of pBC16 from the donor to the recipient as early as 3 days postinoculation. Our findings demonstrate that B. anthracis can survive as a saprophyte outside of the host. The data suggest that horizontal gene transfer in the rhizosphere of grass plants may play a role in the evolution of the Bacillus cereus group species.     

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.     

Characterization of AmiBA2446, a Novel Bacteriolytic Enzyme Active against Bacillus Species

There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis. We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature.     

UV Resistance of Bacillus anthracis Spores Revisited: Validation of Bacillus subtilis Spores as UV Surrogates for Spores of B. anthracis Sterne

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.     

Genetic distribution of 295 Bacillus cereus group members based on adk-screening in combination with MLST (Multilocus Sequence Typing) used for validating a primer targeting a chromosomal locus in B. anthracis

The genetic distribution of 295 Bacillus cereus group members has been investigated by using a modified Multilocus Sequence Typing method (MLST). By comparing the nucleic acid sequence of the adk gene fragment, isolates of B. cereus group members most related to B. anthracis may be easily identified. The genetic distribution, with focus on the B. anthracis close neighbours, was used to evaluate a new primer set for specific identification of B. anthracis. This primer set, BA5510-1/2, targeted the putative B. anthracis specific gene BA5510. Real-time PCR using BA5510-1/2 amplified the target fragment from all B. anthracis strains tested and only two (of 289) non-B. anthracis strains analysed. This is one of the most thoroughly validated chromosomal B. anthracis markers for real-time PCR identification, in which the screened collection contained several very closely related B. anthracis strains.     

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.     

Proteomic Profiling and Identification of Immunodominant Spore Antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Δ-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.     

Use of Long-Range Repetitive Element Polymorphism-PCR To Differentiate Bacillus anthracis Strains

The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105 B. anthracis strains of diverse geographical origins. All B. anthracis strains produced fingerprints comprising seven to eight bands, referred to as “skeleton” bands, while one to three “diagnostic” bands differentiated between B. anthracis strains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracis strains.     

Homoduplex and Heteroduplex Polymorphisms of the Amplified Ribosomal 16S-23S Internal Transcribed Spacers Describe Genetic Relationships in the “Bacillus cereus Group”

Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the “B. cereus group,” advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as B. anthracis or serotypes of B. thuringiensis. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The second included B. cereus and B. thuringiensis, B. anthracis appeared as a lineage of B. cereus.     

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.     

Multigeneration Cross-Contamination of Mail with Bacillus anthracis Spores

The release of biological agents, including those which could be used in biowarfare or bioterrorism in large urban areas, has been a concern for governments for nearly three decades. Previous incidents from Sverdlosk and the postal anthrax attack of 2001 have raised questions on the mechanism of spread of Bacillus anthracis spores as an aerosol or contaminant. Prior studies have demonstrated that Bacillus atrophaeus is easily transferred through simulated mail handing, but no reports have demonstrated this ability with Bacillus anthracis spores, which have morphological differences that may affect adhesion properties between spore and formite. In this study, equipment developed to simulate interactions across three generations of envelopes subjected to tumbling and mixing was used to evaluate the potential for cross-contamination of B. anthracis spores in simulated mail handling. In these experiments, we found that the potential for cross-contamination through letter tumbling from one generation to the next varied between generations while the presence of a fluidizer had no statistical impact on the transfer of material. Likewise, the presence or absence of a fluidizer had no statistically significant impact on cross-contamination levels or reaerosolization from letter opening.     

Bacillus anthracis physiology and genetics

Bacillus anthracis is a member of the Bacillus cereus group species (also known as the “group 1 bacilli”), a collection of Gram-positive spore-forming soil bacteria that are non-fastidious facultative anaerobes with very similar growth characteristics and natural genetic exchange systems. Despite their close physiology and genetics, the B. cereus group species exhibit certain species-specific phenotypes, some of which are related to pathogenicity. B. anthracis is the etiologic agent of anthrax. Vegetative cells of B. anthracis produce anthrax toxin proteins and a poly-d-glutamic acid capsule during infection of mammalian hosts and when cultured in conditions considered to mimic the host environment. The genes associated with toxin and capsule synthesis are located on the B. anthracis plasmids, pXO1 and pXO2, respectively. Although plasmid content is considered a defining feature of the species, pXO1- and pXO2-like plasmids have been identified in strains that more closely resemble other members of the B. cereus group. The developmental nature of B. anthracis and its pathogenic (mammalian host) and environmental (soil) lifestyles of make it an interesting model for study of niche-specific bacterial gene expression and physiology.     

Differentiation of Bacillus anthracis, B. cereus, and B. thuringiensis by Using Pulsed-Field Gel Electrophoresis

A pulsed-field gel electrophoresis (PFGE) method was developed for discriminating Bacillus anthracis from B. cereus and B. thuringiensis. A worldwide collection of 25 B. anthracis isolates showed high-profile homology, and these isolates were unambiguously distinguished from B. cereus and B. thuringiensis isolates by cluster analysis of the whole-genome macrorestriction enzyme digestion patterns generated by NotI.     

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.     

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.     

Detection technologies for Bacillus anthracis: Prospects and challenges

Bacillus anthracis is a Gram-positive, spore-forming bacterium representing the etiological agent of acute infectious disease anthrax, a lethal but rare disease of animals and humans in nature. With recent use of anthrax as a bioweapon, a number of techniques have been recently developed and evaluated to facilitate its rapid detection of B. anthracis in the environment as well as in point-of-care settings for humans suspected of exposure to the pathogen. Complex laboratory methods for B. anthracis identification are required since B. anthracis has similarities with other Bacillus species and its existence in both spore and vegetative forms. This review discusses current challenges and various improvements associated with anthrax agent detection.