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.     

Characterization of an Environmental Strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> from a Hot Spring in Western Himalayas

Bacillus anthracis, the etiological agent of anthrax, is responsible for a serious and often fatal disease of mammalian livestock and humans and is an important biological warfare agent. Bacillus sp. AKG was isolated from a hot spring in western Himalayas and species-specific primers targeting gyrB gene identified the strain as B. anthracis within cereus-group. Cloning, sequencing, and phylogenetic analysis of the partial gyrB sequence from strain AKG indicated a close affiliation with B. anthracis and a few recently isolated strains of B. thuringiensis (e.g., strain Al Hakam and serovar konkukian). Phylogenetic analysis of two other housekeeping genes, clpC and gdpD yielded similar results. This observation is further substantiated by phylogenetic reconstruction using concatenated sequences (1680 bases) of the three genes (gyrB, clpC, and gdpD). Phenotypic features indicated a non-anthracis affiliation for the strain AKG. A novel strategy to distinguish among strains of B. anthracis, B. cereus, and B. thuringiensis based on whole proteome comparison was developed and tested for the identification of this environmental strain. Proteome comparison was used to establish the identity of this unknown environmental strain. Group of replicate 2DE gels for whole cell proteome were generated for each of the three species and strain AKG. Protein spots unique to each group and those showing match between the groups, in a pair-wise comparison, indicated strain AKG as a member of B. thuringiensis. This strategy can be used to assign strains of B. cereus group to their respective species.     

Effect of the Mammalian Arginase Inhibitor 2(<Emphasis Type="Italic">S</Emphasis>)-Amino-6-Boronohexanoic Acid on <Emphasis Type="Italic">Bacillus anthracis</Emphasis> Arginase

Macrophages, upon phagocytosing endospores of Bacillus anthracis, up-regulate the expression of the immunological isoform of nitric oxide synthase, NOS 2, concomitant with production of nitric oxide (NO•) from metabolism of l-arginine. We have previously demonstrated that macrophages that secrete NO• kill the bacilli of B. anthracis. To circumvent this microbicidal activity of NO•, B. anthracis has evolved pathways that include the enzyme arginase, which metabolizes l-arginine to ornithine and urea. Compounds that inhibit arginase might, therefore, offer a therapeutic approach to controlling B. anthracis infection. 2(S)-Amino-6-boronohexanoic acid (ABH) has been reported to be an inhibitor of mammalian arginase. In this study, we explore the inhibitory effect of ABH against B. anthracis arginase and its potential for future development, as an effective therapeutic agent against microbial infection. We found that ABH is an inhibitor of bacterial arginase in several different endospore strains of B. anthracis. Further, ABH inhibits neither the phagocytosis of these endospores nor the up-regulation of NOS 2 concomitant with secretion of NO•. These findings set the stage to determine how efficacious ABH will be in promoting NO•-mediating killing of B. anthracis.     

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.     

Comparative proteome analysis of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> with pXO1 plasmid content

Bacillus anthracis the causative agent of anthrax, is an important pathogen among the Bacillus cereus group of species because of its physiological characteristics and its importance as a biological warfare agent. Tripartite anthrax toxin proteins and a poly-D-glutamic acid capsule are produced by B. anthracis vegetative cells during mammalian hosts infection and when cultured in conditions that are thought to mimic the host environment. To identify the factors regulating virulence in B. anthracis the whole cell proteins were extracted from two B. anthracis strains and separated by narrow range immobilized pH gradient (IPG) strips (pH 4–7), followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins that were differentially expressed were identified by the peptide fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 23 proteins were identified as being either upregulated or downregulated in the presence or absence of the virulence plasmid pXO1. Two plasmid encoded proteins and 12 cellular proteins were identified and documented as potential virulence factors.     

Loop-mediated isothermal amplification for rapid detection of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> spores

A loop-mediated isothermal amplification (LAMP) assay system was employed for detecting Bacillus anthracis spores in pure cultures as well as in various simulated powder samples. The specificity of the designed LAMP primer sets was validated by assaying 13 B. anthracis strains and 33 non-B. anthracis species. The detection limits of the LAMP assay were 10 spores/tube for pure cultures and 100 spores/2 mg powder for simulated powder samples. The results show that the LAMP protocol is a promising method for detecting B. anthracis.     

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.     

A simple and sensitive method for detection of Bacillus anthracis by loop‐mediated isothermal amplification

Aims: To develop a rapid and simple system for detection of Bacillus anthracis using a loop‐mediated isothermal amplification (LAMP) method and determine the suitability of LAMP for rapid identification of B. anthracis infection. Methods and Results: A specific LAMP assay targeting unique gene sequences in the bacterial chromosome and two virulence plasmids, pXO1 and pXO2, was designed. With this assay, it was possible to detect more than 10 fg of bacterial DNA per reaction and obtain results within 30–40 min under isothermal conditions at 63°C. No cross‐reactivity was observed among Bacillus cereus group and other Bacillus species. Furthermore, in tests using blood specimens from mice inoculated intranasally with B. anthracis spores, the sensitivity of the LAMP assay following DNA extraction methods using a Qiagen DNeasy kit or boiling protocol was examined. Samples prepared by both methods showed almost equivalent sensitivities in LAMP assay. The detection limit was 3·6 CFU per test. Conclusions: The LAMP assay is a simple, rapid and sensitive method for detecting B. anthracis. Significance and Impact of the Study: The LAMP assay combined with boiling extraction could be used as a simple diagnostic method for identification of B. anthracis infection.     

Petrobactin is the Primary Siderophore Synthesized by <Emphasis Type="BoldItalic">Bacillus anthracis</Emphasis> Str. <Emphasis Type="BoldItalic">Sterne</Emphasis> under Conditions of Iron Starvation

The siderophores of Bacillus anthracis are critical for the pathogen’s proliferation and may be necessary for its virulence. Bacillus anthracis str. Sterne cells were cultured in iron free media and the siderophores produced were isolated and purified using a combination of XAD-2 resin, reverse-phase FPLC, and size exclusion chromatography. A combination of ¹H and ¹³C NMR spectroscopy, UV spectroscopy and ESI-MS/MS fragmentation were used to identify the primary siderophore as petrobactin, a catecholate species containing unusual 3,4-dihydroxybenzoate moieties, previously only identified in extracts of Marinobacter hydrocarbonoclasticus. A secondary siderophore was observed and structural analysis of this species is consistent with that reported for bacillibactin, a siderophore observed in many species of bacilli. This is the first structural characterization of a siderophore from B. anthracis, as well as the first characterization of a 3,4-DHB containing catecholate in a pathogen.     

Multiplex PCR assay for the detection of enterotoxic <Emphasis Type="Italic">Bacillus cereus</Emphasis> group strains and its application in food matrices

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are the major concerns for the food safety in terms of frequency and/or seriousness of the disease. Being members of the same group and sharing DNA homology to a larger extent, they do create problems when their specific detection/identification is attempted from different food and environmental sources. Numerous individual polymerase chain reaction (PCR) and few multiplex PCR (mPCR) methods have been employed to detect these organisms by targeting toxin genes but with lack of internal amplification control (IAC). Therefore, we attempted a mPCR with IAC for the detection of enterotoxic B. cereus group strains by selecting hbl A, nhe A and cyt K genes from B. cereus, indicative of the diarrheal potential and cry I A and pag genes, the plasmid borne phenotypic markers specific to B. thuringiensis and B. anthracis strains, respectively. Multiplex PCR assay validation was performed by simultaneous comparison with the results of single-target PCR assays and correlated to the classical conventional and biochemical identification of the organisms. The mPCR was able to detect as low as 10¹–10² organisms per ml following overnight enrichment of spiked food samples (vegetable biriyani and milk) in buffered peptone water (BPW). The presence of these organisms could also be detected by mPCR in naturally contaminated samples of rice based dishes and milk. The high throughput and cost-effective mPCR method described could provide a powerful tool for simultaneous, rapid and reliable detection of enterotoxic B. cereus group organisms.     

BslA,the S‐layer adhesin of B. anthracis,is a virulence factor for anthrax pathogenesis

Microbial pathogens use adhesive surface proteins to bind to and interact with host tissues, events that are universal for the pathogenesis of infectious diseases. A surface adhesin of Bacillus anthracis, the causative agent of anthrax, required to mediate these steps has not been discovered. Previous work identified BslA, an S‐layer protein, to be necessary and sufficient for adhesion of the anthrax vaccine strain, Bacillus anthracis Sterne, to host cells. Here we asked whether encapsulated bacilli require BslA for anthrax pathogenesis in guinea pigs. Compared with the highly virulent parent strain B. anthracis Ames, bslA mutants displayed a dramatic increase in the lethal dose and in mean time‐to‐death. Whereas all tissues of animals infected with B. anthracis Ames contained high numbers of bacilli, only few vegetative forms could be recovered from internal organs of animals infected with the bslA mutant. Surface display of BslA occurred at the poles of encapsulated bacilli and enabled the binding of vegetative forms to host cells. Together these results suggest that BslA functions as the surface adhesin of the anthrax pathogen B. anthracis strain Ames.     

Gamma-phage lysin PlyG sequence-based synthetic peptides coupled with Qdot-nanocrystals are useful for developing detection methods for Bacillus anthracis by using its surrogates, B. anthracis-Sterne and B. cereus-4342

Background  

Previous reports of site-directed deletion analysis on gamma (γ)-phage lysin protein (PlyG) have demonstrated that removal of a short amino acid sequence in the C-terminal region encompassing a 10-amino acid motif (190LKMTADFILQ199) abrogates its binding activity specific to the cell wall of Bacillus anthracis. Whether short synthetic peptides representing the10-amino acid PlyG putative binding motif flanked by surrounding N- and C-terminal residues also selectively bind to the bacterial cell wall has not been evaluated. If such peptides do demonstrate selective binding to the cell wall, they could serve as bio-probes towards developing detection technologies for B. anthracis. Furthermore, by using B. anthracis (Sterne, 34F2), an animal vaccine and B. cereus-4342, a γ-phage susceptible rare strain as surrogates of B. anthracis, development of proof-of-concepts for B. anthracis are feasible.     

Temperature Control of a 3,4-Dihydroxybenzoate (Protocatechuate)-Based Siderophore in <Emphasis Type="Italic">Bacillus anthracis</Emphasis>

Bacillus anthracis Sterne produced a catecholate siderophore named anthrachelin that was based on 3,4-dihydroxybenzoic acid (3,4-DHB, or protocatechuic acid), a catechol moiety previously unreported as a siderophore component. During iron restriction, both anthrachelin and free 3,4-DHB were excreted. Growth at 37°C (as compared with 23°C) decreased excretion of anthrachelin but not its precursor 3,4-DHB, suggesting that anthrachelin assembly is temperature regulated. A plasmidless strain also produced anthrachelin in an iron- and temperature-regulated fashion, indicating that anthrachelin genes are chromosomal. In addition to anthrachelin-mediated iron delivery, B. anthracis also used heme, hemoproteins, iron-transferrin, and certain heterologous siderophores (xenosiderophores) produced by other microorganisms as iron sources. Downregulation of anthrachelin production at the temperature of the mammalian host (which triggers toxin production in this pathogen) may focus the B. anthracis iron acquisition systems to exploit the iron sources prevailing in the infected host.     

Bacillus Species Proteins Involved in Spore Formation and Degradation: From Identification in the Genome,to Sequence Analysis,and Determination of Function and Structure

The members of Bacillus species are Gram-positive, ubiquitous spore-forming bacilli. Several genomic sequences have been made available during recent years, including Bacillus subtilis, a model organism among this genus, Bacillus anthracis, and their analyses provided a wealth of information about spore-forming bacteria. Some members of this species can cause serious diseases in livestock and humans. An important pathogen in this group of organisms is B. anthracis, which is the causative agent of anthrax. A summary of the B. subtilis genome information, based on the publicly released sequence, that allowed for the identification and characterization of new and novel proteins of this organism as well as similar proteins from other members of Bacillus species is provided. The primary goal for this work is to present a review of the genome sequence-identified genes that encode proteins involved in the sporulation, germination, and outgrowth processes. These three processes are essential for spore development and later its transformation into a vegetative cell. Additionally, for a few selected examples of the protein products of the identified genes, the application of bioinformatics and modeling tools is illustrated in order to determine their likely structure and function. Two three-dimensional models of the structures of such proteins, PrfA endonuclease and phosphatase PhoE, are presented together with the structure-based functional conclusions. The review of such studies provides an example of how the genomic sequence can be utilized in order to elucidate the structure and function of proteins, in particular proteins of the Bacillus species. Because only a limited number of proteins of Bacillus species organisms are involved in the synthesis and degradation of spores and have been characterized to date, this genome-based analysis may provide new insights into the developmental processes of bacterial organism.     

Temporal production of the two <Emphasis Type="Italic">Bacillus anthracis</Emphasis> siderophores,petrobactin and bacillibactin

Bacillus anthracis secretes two siderophores, petrobactin (PB) and bacillibactin (BB). These siderophores were temporally produced during germination and outgrowth of spores (the usual infectious form of B. anthracis) in low-iron medium. The siderophore PB was made first while BB secretion began several hours later. Spore outgrowth early in an infection may require PB, whereas delayed BB production suggests a role for BB in the later stages of the infection. Incubation of cultures (inoculated as vegetative cells) at 37°C, as compared to 2°C, increased PB production and decreased secretion of BB, suggesting that the production of PB and BB responded to the host temperature signal. The dual siderophores of B. anthracis may fulfill independent roles in the life cycle of B. anthracis.     

Passive administration of monoclonal antibodies to Anthrolysin O prolong survival in mice lethally infected with <Emphasis Type="Italic">Bacillus anthracis</Emphasis>

Background  

Bacillus anthracis has two major virulence factors: a tripartite toxin that produces lethal and edema toxins and a polyglutamic acid capsule. A recent report suggested that a toxin belonging to the cholesterol dependant cytolysin (CDC) family, anthrolysin O (ALO) was a new virulence factor for B. anthracis but subsequent studies have questioned its relevance in pathogenesis. In this study, we examined the immunogenicity of recombinant anthrolysin O (rALO) in mice.     

Computer-Intensive Statistical Procedures

The overall goal of this review is to summarize the current body of knowledge about the structure and function of major proteins of Bacillus anthracis and/or similar spore-forming organisms. B. anthracis is a key spore-forming biological threat agent, as well as human and animal Gram-positive bacterial pathogen. The structural information described here is limited to approximately the last 5 years. This information is then related to the role of the selected proteins in pathogenesis and in the possible development of novel vaccine and/or other antimicrobial agents against spore-forming organisms, including anthrax, a disease caused by B. anthracis.

Among spore-forming bacteria, Bacillus and Clostridium species are the predominant spore-forming bacilli that cause serious diseases. The biochemical properties and mechanism of catalysis of the novel spore germination protease that degrades small, acid-soluble proteins protecting DNA against damage, a cofactor independent phosphoglycerate mutase, NAD⁺ synthetase, and the three know B. anthracis toxins, protective antigen, lethal factor, and edema factor are described. The studies described in this work review and unify selected information critical for the prevention of microbial diseases such as anthrax. A strategy for the structure-guided development of new prophylactic and therapeutic agents is discussed.     

Responding to the threat of bioterrorism: a microbial ecology perspective – the case of anthrax

Anthrax is a disease of herbivores caused by the gram-positive bacterium Bacillus anthracis. It can affect cattle, sheep, swine, horses and various species of wildlife. The routes for the spread among wildlife are reviewed. There are three kinds of human anthrax – inhalation, cutaneous, and intestinal anthrax – which differ in their routes of infection and outcomes. In the United States, confirmation of cases is made by the isolation of B. anthracis and by biochemical tests. Vaccination is not recommended for the general public; civilians who should be vaccinated include those who, in their work places, come in contact with products potentially contaminated with B. anthracis spores, and people engaged in research or diagnostic activities. After September 11, 2001, there were bioterrorism anthrax attacks in the United States: anthrax-laced letters sent to multiple locations were the source of infectious B. anthracis. The US Postal Service issued recommendations to prevent the danger of hazardous exposure to the bacterium. B. anthracis spores can spread easily and persist for very long times, which makes decontamination of buildings very difficult. Early detection, rapid diagnosis, and well-coordinated public health response are the key to minimizing casualties. The US Government is seeking new ways to deter bioterrorism, including a tighter control of research on infectious agents, even though pathogens such as B. anthracis are widely spread in nature and easy to grow. It is necessary to define the boundary between defensive and offensive biological weapons research. Deterring bioterrorism should not restrict critical scientific research. Electronic Publication     

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.