Surface Plasmon Resonance Biosensor for Detection of Bacillus anthracis, the Causative Agent of Anthrax from Soil Samples Targeting Protective Antigen

Bacillus anthracis, the causative agent of anthrax is one of the most important biological warfare agents. In this study, surface plasmon resonance (SPR) technology was used for indirect detection of B. anthracis by detecting protective antigen (PA), a common toxin produced by all live B. anthracis bacteria. For development of biosensor, a monoclonal antibody raised against B. anthracis PA was immobilized on carboxymethyldextran modified gold chip and its interaction with PA was characterized in situ by SPR and electrochemical impedance spectroscopy. By using kinetic evaluation software, K_D (equilibrium constant) and B_max (maximum binding capacity of analyte) were found to be 20 fM and 18.74, respectively. The change in Gibb’s free energy (∆G = −78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 12 fM purified PA. When anthrax spores spiked soil samples were enriched, PA produced in the sample containing even a single spore of B. anthracis could be detected by SPR. PA being produced only by the vegetative cells of B. anthracis, confirms indirectly the presence of B. anthracis in the samples. The proposed method can be a very useful tool for screening and confirmation of anthrax suspected environmental samples during a bio-warfare like situation.     

Preparedness for an anthrax attack

Bacillus anthracis is a long-known bacterial organism with a uniquely stable spore stage. Its stability and the lethal disease which results when the spore is inhaled made it a favorite of state-sponsored biological weapons programs throughout the Cold War era. It is also believed to be high on the list of candidate microbial agents which could be used by terrorist groups or lone actors. Its unique characteristics make protection of humans, especially civilians, from an intentional biological attack very difficult. The author argues that an all-hazards/public health approach – which would also be needed for any natural or deliberate outbreak, no matter the agent – should serve as a foundation of preparation for the specific anthrax countermeasures. Because B. anthracis is a unique organism, specific countermeasures for anthrax detection, diagnostics, prophylaxis and therapy, should be developed in nations or regions where the threat of biological attack is believed to warrant such preparation. Other considerations for a nation interested in anthrax preparedness are discussed.     

Rapid detection methods for <Emphasis Type="Italic">Bacillus anthracis</Emphasis> in environmental samples: a review

Bacillus anthracis is a Gram-positive, spore-forming bacterium, which causes anthrax, an often lethal disease of animals and humans. Although the disease has been well studied since the nineteenth century, it has witnessed a renewed interest during the past decade, due to its use as a bioterrorist agent in the fall of 2001 in the USA. A number of techniques aimed at rapidly detecting B. anthracis, in environmental samples as well as in point-of-care settings for humans suspected of exposure to the pathogen, are now available. These technologies range from culture-based methods to portable DNA amplification devices. Despite recent developments, specific identification of B. anthracis still remains difficult because of its phenotypic and genotypic similarities with other Bacillus species. Accordingly, many efforts are being made to improve the specificity of B. anthracis identification. This mini-review discusses the current challenges around B. anthracis identification, not only in reach-back laboratories but also in the field (in operational conditions).     

Detection of B. anthracis Spores and Vegetative Cells with the Same Monoclonal Antibodies

Bacillus anthracis, the causative agent of anthrax disease, could be used as a biothreat reagent. It is vital to develop a rapid, convenient method to detect B. anthracis. In the current study, three high affinity and specificity monoclonal antibodies (mAbs, designated 8G3, 10C6 and 12F6) have been obtained using fully washed B. anthracis spores as an immunogen. These mAbs, confirmed to direct against EA1 protein, can recognize the surface of B. anthracis spores and intact vegetative cells with high affinity and species-specificity. EA1 has been well known as a major S-layer component of B. anthracis vegetative cells, and it also persistently exists in the spore preparations and bind tightly to the spore surfaces even after rigorous washing. Therefore, these mAbs can be used to build a new and rapid immunoassay for detection of both life forms of B. anthracis, either vegetative cells or spores.     

Interactions between Bacillus anthracis and Plants May Promote Anthrax Transmission

Environmental reservoirs are essential in the maintenance and transmission of anthrax but are poorly characterized. The anthrax agent, Bacillus anthracis was long considered an obligate pathogen that is dormant and passively transmitted in the environment. However, a growing number of laboratory studies indicate that, like some of its close relatives, B. anthracis has some activity outside of its vertebrate hosts. Here we show in the field that B. anthracis has significant interactions with a grass that could promote anthrax spore transmission to grazing hosts. Using a local, virulent strain of B. anthracis, we performed a field experiment in an enclosure within a grassland savanna. We found that B. anthracis increased the rate of establishment of a native grass (Enneapogon desvauxii) by 50% and that grass seeds exposed to blood reached heights that were 45% taller than controls. Further we detected significant effects of E. desvauxii, B. anthracis, and their interaction on soil bacterial taxa richness and community composition. We did not find any evidence for multiplication or increased longevity of B. anthracis in bulk soil associated with grass compared to controls. Instead interactions between B. anthracis and plants may result in increased host grazing and subsequently increased transmission to hosts.     

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     

Bacillusanthracis Surface-Layer Proteins Assemble by Binding to the Secondary Cell Wall Polysaccharide in a Manner that Requires csaB and tagO

Bacillus anthracis, the causative agent of anthrax, requires surface (S)-layer proteins for the pathogenesis of infection. Previous work characterized S-layer protein binding via the surface layer homology domain to a pyruvylated carbohydrate in the envelope of vegetative forms. The molecular identity of this carbohydrate and the mechanism of its display in the bacterial envelope are still unknown. Analyzing acid-solubilized, purified carbohydrates by mass spectrometry and NMR spectroscopy, we identify secondary cell wall polysaccharide (SCWP) as the ligand of S-layer proteins. In agreement with the model that surface layer homology domains bind to pyruvylated carbohydrate, SCWP was observed to be linked to pyruvate in a manner requiring csaB, the only structural gene known to be required for S-layer assembly. B. anthracis does not elaborate wall teichoic acids; however, its genome harbors tagO and tagA, genes responsible for the synthesis of the linkage unit that tethers teichoic acids to the peptidoglycan layer. The tagO gene appears essential for B. anthracis growth and complements the tagO mutant phenotypes of staphylococci. Tunicamycin-mediated inhibition of TagO resulted in deformed, S-layer-deficient bacilli. Together, these results suggest that tagO-mediated assembly of linkage units tethers pyruvylated SCWP to the B. anthracis envelope, thereby enabling S-layer assembly and providing for the pathogenesis of anthrax infections.     

Evaluation of the House Fly Musca domestica as a Mechanical Vector for an Anthrax

Anthrax is a disease of human beings and animals caused by the encapsulated, spore-forming, Bacillus anthracis. The potential role of insects in the spread of B. anthracis to humans and domestic animals during an anthrax outbreak has been confirmed by many studies. Among insect vectors, the house fly Musca domestica is considered a potential agent for disease transmission. In this study, laboratory-bred specimens of Musca domestica were infected by feeding on anthrax-infected rabbit carcass or anthrax contaminated blood, and the presence of anthrax spores in their spots (faeces and vomitus) was microbiologically monitored. It was also evaluated if the anthrax spores were able to germinate and replicate in the gut content of insects. These results confirmed the role of insects in spreading anthrax infection. This role, although not major, given the huge size of fly populations often associated with anthrax epidemics in domestic animals, cannot be neglected from an epidemiological point of view and suggest that fly control should be considered as part of anthrax control programs.     

Bacillus Spore Inactivation Methods Affect Detection Assays

Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Assays for detection in the laboratory often employ inactivated preparations of spores or nonpathogenic simulants. This study uses several common biodetection platforms to detect B. anthracis spores that have been inactivated by two methods and compares those data to detection of spores that have not been inactivated. The data demonstrate that inactivation methods can affect the sensitivity of nucleic acid- and antibody-based assays for the detection of B. anthracis spores. These effects should be taken into consideration when comparing laboratory results to data collected and assayed during field deployment.     

In vivo murine and in vitro M-like cell models of gastrointestinal anthrax

Bacillus anthracis is the causative agent of anthrax and is acquired by three routes of infection: inhalational, gastrointestinal and cutaneous. Gastrointestinal (GI) anthrax is rare, but can rapidly result in severe, systemic disease that is fatal in 25%–60% of cases. Disease mechanisms of GI anthrax remain unclear due to limited numbers of clinical cases and the lack of experimental animal models. Here, we developed an in vivo murine model of GI anthrax where spore survival was maximized through the neutralization of stomach acid followed by an intragastric administration of a thiabendazole paste spore formulation. Infected mice showed a dose-dependent mortality rate and pathological features closely mimicking human GI anthrax. Since Peyer's patches in the murine intestine are the primary sites of B. anthracis growth, we developed a human M (microfold)-like-cell model using a Caco-2/Raji B-cell co-culturing system to study invasive mechanisms of GI anthrax across the intestinal epithelium. Translocation of B. anthracis spores was higher in M-like cells than Caco-2 monolayers, suggesting that M-like cells may serve as an initial entry site for spores. Here, we developed an in vivo murine model of GI anthrax and an in vitro M-like cell model that could be used to further our knowledge of GI anthrax pathogenesis.     

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.     

Human Transferrin Confers Serum Resistance against Bacillus anthracis

The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the identification of human transferrin as the inhibiting factor. Purified transferrin blocks growth of both the fully virulent encapsulated B. anthracis Ames and the non-encapsulated Sterne strain. Growth inhibition was also observed in serum of wild-type mice but not of hypotransferrinemic mice that only have ∼1% circulating transferrin levels. We were able to definitely assign the bacteriostatic activity of transferrin to its iron-binding function: neither iron-saturated transferrin nor a recombinant transferrin mutant unable to bind iron could inhibit growth of B. anthracis. Additional iron could restore bacterial growth in human serum. The observation that other important Gram-positive pathogens are not inhibited by transferrin suggests they have evolved effective mechanisms to circumvent serum iron deprivation. These findings provide a better understanding of human host defense mechanisms against anthrax and provide a mechanistic basis for the antimicrobial activity of human transferrin.     

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.     

Methylation-dependent DNA restriction in Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax, is poorly transformed with DNA that is methylated on adenine or cytosine. Here we characterize three genetic loci encoding type IV methylation-dependent restriction enzymes that target DNA containing C5-methylcytosine (m5C). Strains in which these genes were inactivated, either singly or collectively, showed increased transformation by methylated DNA. Additionally, a triple mutant with an ~ 30-kb genomic deletion could be transformed by DNA obtained from Dam⁺Dcm⁺E. coli, although at a low frequency of ~ 10^− 3 transformants/10⁶ cfu. This strain of B. anthracis can potentially serve as a preferred host for shuttle vectors that express recombinant proteins, including proteins to be used in vaccines. The gene(s) responsible for the restriction of m6A-containing DNA in B. anthracis remain unidentified, and we suggest that poor transformation by such DNA could in part be a consequence of the inefficient replication of hemimethylated DNA in B. anthracis.     

Germination and Amplification of Anthrax Spores by Soil-Dwelling Amoebas

While anthrax is typically associated with bioterrorism, in many parts of the world the anthrax bacillus (Bacillus anthracis) is endemic in soils, where it causes sporadic disease in livestock. These soils are typically rich in organic matter and calcium that promote survival of resilient B. anthracis spores. Outbreaks of anthrax tend to occur in warm weather following rains that are believed to concentrate spores in low-lying areas where runoff collects. It has been concluded that elevated spore concentrations are not the result of vegetative growth as B. anthracis competes poorly against indigenous bacteria. Here, we test an alternative hypothesis in which amoebas, common in moist soils and pools of standing water, serve as amplifiers of B. anthracis spores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show that B. anthracis germinates and multiplies within Acanthamoeba castellanii. The growth kinetics of a fully virulent B. anthracis Ames strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture with A. castellanii showed a nearly 50-fold increase in spore numbers after 72 h. In contrast, the plasmidless strain 9131 showed little growth, demonstrating that plasmid pX01 is essential for growth within A. castellanii. Electron and time-lapse fluorescence microscopy revealed that spores germinate within amoebal phagosomes, vegetative bacilli undergo multiplication, and, following demise of the amoebas, bacilli sporulate in the extracellular milieu. This analysis supports our hypothesis that amoebas contribute to the persistence and amplification of B. anthracis in natural environments.     

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.     

The Genome of a Bacillus Isolate Causing Anthrax in Chimpanzees Combines Chromosomal Properties of B. cereus with B. anthracis Virulence Plasmids

Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as “B. cereus variety (var.) anthracis”.     

Modulation of the Bacillus anthracis Secretome by the Immune Inhibitor A1 Protease

The Bacillus anthracis secretome includes protective antigen, lethal factor, and edema factor, which are the components of anthrax toxin, and other proteins with known or potential roles in anthrax disease. Immune inhibitor A1 (InhA1) is a secreted metalloprotease that is unique to pathogenic members of the Bacillus genus and has been associated with cleavage of host proteins during infection. Here, we report the effect of InhA1 on the B. anthracis secretome. Differential in-gel electrophoresis of proteins present in culture supernatants from a parent strain and an isogenic inhA1-null mutant revealed multiple differences. Of the 1,340 protein spots observed, approximately one-third were less abundant and one-third were more abundant in the inhA1 secretome than in the parent strain secretome. Proteases were strongly represented among those proteins exhibiting a 9-fold or greater change. InhA1 purified from a B. anthracis culture supernatant directly cleaved each of the anthrax toxin proteins as well as an additional secreted protease, Npr599. The conserved zinc binding motif HEXXH of InhA1 (HEYGH) was critical for its proteolytic activity. Our data reveal that InhA1 directly and indirectly modulates the form and/or abundance of over half of all the secreted proteins of B. anthracis. The proteolytic activity of InhA1 on established secreted virulence factors, additional proteases, and other secreted proteins suggests that this major protease plays an important role in virulence not only by cleaving mammalian substrates but also by modulating the B. anthracis secretome itself.     

LytR-CpsA-Psr Enzymes as Determinants of Bacillus anthracis Secondary Cell Wall Polysaccharide Assembly

Bacillus anthracis, the causative agent of anthrax, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and associated proteins (BSLs) function as chain length determinants and bind to the secondary cell wall polysaccharide (SCWP). In this study, we identified the B. anthracis lcpD mutant, which displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, the B. anthracis lcpB3 variant displayed reduced cell size and chain length, which could be attributed to increased deposition of BSLs. In other bacteria, LytR-CpsA-Psr (LCP) proteins attach wall teichoic acid (WTA) and polysaccharide capsule to peptidoglycan. B. anthracis does not synthesize these polymers, yet its genome encodes six LCP homologues, which, when expressed in S. aureus, promote WTA attachment. We propose a model whereby B. anthracis LCPs promote attachment of SCWP precursors to discrete locations in the peptidoglycan, enabling BSL assembly and regulated separation of septal peptidoglycan.