Investigation of spore surface antigens in the genus Bacillus by the use of polyclonal antibodies in immunofluorescence tests

Fluorescein-conjugated rabbit antibodies to formalized spores of Bacillus anthracis were tested against strains of B. anthracis and other Bacillus species in a subjective immunofluorescence test. The lack of reaction of B. anthracis Vollum spores with conjugated antibody raised against B. anthracis Sterne spores indicated that spores of the Vollum strain lacked a major surface antigen present in most of the other anthrax strains tested, including the non-encapsulated strains Sterne and the Soviet ST1, variants cured of the pX01 plasmid that codes for the toxin, and several virulent strains. Four other antibody preparations, raised against B. anthracis Vollum, New Hampshire, Ames and Strain 15, reacted to an approximately similar degree with spores of all four strains and of Sterne, indicating that Vollum has at least one spore antigen in common with these other strains. The anti-Sterne and anti-Vollum conjugates both displayed cross-reactions with spores of strains of B. cereus, B. coagulans, B. subtilis, B. megaterium, B. polymyxa, B. pumilus and B. thuringiensis. Absorption of the anti-anthrax conjugates with B. cereus NCTC 8035 and NCTC 10320 removed all these cross-reactions, demonstrating the existence of spore antigens specific for anthrax.     

Investigation of spore surface antigens in the genus Bacillus by the use of polyclonal antibodies in immunofluorescence tests

Fluorescein-conjugated rabbit antibodies to formalized spores of Bacillus anthracis were tested against strains of B. anthracis and other Bacillus species in a subjective immunofluorescence test. The lack of reaction of B. anthracis Vollum spores with conjugated antibody raised against B. anthracis Sterne spores indicated that spores of the Vollum strain lacked a major surface antigen present in most of the other anthrax strains tested, including the non-encapsulated strains Sterne and the Soviet ST1, variants cured of the pX01 plasmid that codes for the toxin, and several virulent strains. Four other antibody preparations, raised against B, anthracis Vollum, New Hampshire, Ames and Strain 15, reacted to an approximately similar degree with spores of all four strains and of Sterne, indicating that Vollum has at least one spore antigen in common with these other strains. The anti-Sterne and anti-Vollum conjugates both displayed cross-reactions with spores of strains of B. cereus, B. coagulans, B. subtilis, B. megaterium, B. polymyxa, B. pumilus and B. thuringiensis. Absorption of the anti-anthrax conjugates with B. cereus NCTC 8035 and NCTC 10320 removed all these cross-reactions, demonstrating the existence of spore antigens specific for anthrax.     

Comparison of direct and indirect immunoradiometric assays (IRMA) for Bacillus anthracis spores immobilised on multispot microscope slides

A solid phase immunoradiometric assay (IRMA) is described in which Bacillus anthracis spores were heat fixed to the wells of glass multispot microscope slides. Assays for spores of B. anthracis Vollum and Sterne strains with ³H labels were evaluated in the direct and indirect versions. Neither signal nor signal-to-noise characteristics of indirect assays were greatly improved by the use of immunopurified antibody (IPAB) or IgG anti-bacterial reagents rather than antiserum. However, the specificity of the direct and indirect assays for B. anthracis strains and B. cereus NCTC 8035 was altered by immunopurification of the anti-bacterial reagent. Although the signal-to-noise ratio was sometimes higher in indirect than in direct assays, signal values were usually no better. Evidence was produced that the overall ratio of the indirect: direct antibody molecules bound by preparations of B. anthracis spores rarely exceeded two but the antibody-molecular ratio for antigens on extracellular material in spore preparations was much higher than the ratio for antigens on the spores themselves.     

A preliminary assessment of Bacillus anthracis spore inactivation using an electrochemically activated solution (ECASOL)

AIM: To evaluate the efficacy of electrochemically activated solution (ECASOL) in decontaminating Bacillus anthracis Ames and Vollum 1B spores, with and without changing the source water hardness and final ECASOL pH. METHODS AND RESULTS: Five different ECASOL formulations were generated, in which the source water hardness and final ECASOL pH were varied, resulting in cases where significant changes in free available chlorine (FAC) and oxidative-reduction potential (ORP) were observed. B. anthracis Ames and Vollum 1B spores were suspended in the various ECASOL formulations for 30 min, and decontamination efficacy was determined; calcium hypochlorite [5% high-test hypochlorite (HTH)] was used as a positive control. The five different ECASOL formulations yielded mean FAC levels ranging from 305 to 464 ppm, and mean ORP levels ranging from +826 to +1000 mV. Exposure to all the ECASOL formulations and 5% HTH resulted in >or=7.0 log reductions in both B. anthracis Ames and Vollum 1B spores. CONCLUSIONS: The present testing demonstrated that ECASOL with a minimum of c. 300-ppm FAC levels and +800-mV ORP inactivated the B. anthracis spores in suspension, similar to 5% HTH. Significance and Impact of the Study: These results provide information for decontaminating B. anthracis Ames and Vollum 1B spores in suspension using ECASOL.     

Dual-parameter scatter-flow immunofluorescence analysis of Bacillus spores

Using a commercial flow cytometer (Cyto-fluorograf), narrow-forward-angle (NFA) light-scatter signals were detected for spore preparations of Bacillus anthracis Vollum, B. anthracis Sterne, B. cereus NCTC 8035, and B. subtilis var niger. In the flow immunofluorescence (FIF) analysis of spores stained with fluorescein-conjugated hyperimmune antibody to B. anthracis Vollum spores, fluorescence histograms could be acquired by selecting on NFA scatter. Fluorescence data selected on ninety degree scatter were rather noisier. Fluorescence analysis by dual parameter NFA scatter-FIF techniques was shown to have several advantages over the subtraction FIF method reported earlier. The implication from FIF analysis of spore suspensions and corresponding cell-free supernatants that the peak in the fluorescence histogram was caused by signals from fluorescing spores, was confirmed by use of the cell sorter and subsequent microscopy of the sorted samples. Although a proportion of spore aggregates was present in samples sorted from the right-hand tail of the fluorescence histogram, it was demonstrated that the majority of the observed distribution of fluorescence was not due to the formation of aggregates but was rather an expression of variation in the degree of staining of individual spores.     

HtrA is a major virulence determinant of Bacillus anthracis

We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1(+) , pXO2(+) ), or in the ΔVollum (pXO1(-), pXO2(-), nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ΔhtrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation-associated neutral protease A (NprA). VollumΔhtrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 10(8) spores, in the guinea pig model of anthrax), as compared with the parental wild-type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans-complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ΔhtrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ΔhtrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.     

Monoclonal antibodies against spore antigens of Bacillus anthracis

Abstract A murine monoclonal antibody produced against heat inactivated spores of Bacillus anthracis Ames, reacted with live or inactivated spores of several anthrax strains in indirect immunofluorescence (IF) tests. The reactive anthrax strain gave only a moderate degree of reaction. No staining of anthrax vegetative cells was observed. The monoclonal did not react with spores of non-anthrax Bacillus strains that gave cross reactions with mouse hyperimmune antiserum raised against Ames spores. The staining of individual spores in B. anthracis preparations was more heterogeneous with the monoclonal antibody than with the hyperimmune serum. Evidence is produced that the epitope for this monoclonal is not stable during long-term storage of inactivated spore preparations, and is not fully available for reaction with antibody until late in spore maturation. The monoclonal did not react by immunoblotting (Western blotting) of spore extracts.     

Monoclonal antibodies against spore antigens of Bacillus anthracis

A murine monoclonal antibody produced against heat inactivated spores of Bacillus anthracis Ames, reacted with live or inactivated spores of several anthrax strains in indirect immunofluorescence (IF) tests. The reactive anthrax strain gave only a moderate degree of reaction. No staining of anthrax vegetative cells was observed. The monoclonal did not react with spores of non-anthrax Bacillus strains that gave cross reactions with mouse hyperimmune antiserum raised against Ames spores. The staining of individual spores in B. anthracis preparations was more heterogeneous with the monoclonal antibody than with the hyperimmune serum. Evidence is produced that the epitope for this monoclonal is not stable during long-term storage of inactivated spore preparations, and is not fully available for reaction with antibody until late in spore maturation. The monoclonal did not react by immunoblotting (Western blotting) of spore extracts. A monoclonal antibody produced against Ames spore extracts reacted with about 1% of Ames spores in IF tests, but not reproducible reactions with other anthrax strains were recorded. This monoclonal interacted with three bands in Western blots of anthrax spore extracts.     

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.     

Construction, crystal structure and application of a recombinant protein that lacks the collagen-like region of BclA from Bacillus anthracis spores

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called BclA, which comprises a central collagen-like region (CLR) and a globular C-terminal domain. Here, the entire CLR coding sequence of BclA was removed, and the resulting protein (tBclA) produced in Escherichia coli. The crystallographic structure of tBclA was determined to 1.35 A resolution, and consists of an all-beta structure with a TNF-like jelly fold topology (12 beta-strands which form 2 beta-sheets of five strands each) consistent with previous studies on wild-type BclA. These globular domains are tightly packed into trimeric structures (surface shape complementarity; S (c) = 0.83), demonstrating that formation of the core structure of BclA is independent of the anchoring collagen-like region. A polyclonal antibody raised against tBclA recognized B. anthracis spores directly, and showed little cross-reactivity (<10%) with the spores of the closely related species Bacillus cereus and Bacillus thuringiensis, when compared to two other polyclonal antibodies raised against B. anthracis spore extracts and inactivated spores. The tBclA protein was used to purify a pool of specific antibodies from bovine colostrum whey samples from cows inoculated with the Sterne strain anthrax vaccine, which also showed reactivity with B. anthracis spores. Together, these results demonstrate that tBclA provides a safer and more effective way to the production and purification of antibodies with high binding affinity for B. anthracis spores. Biotechnol. Bioeng. 2008;99: 774-782. (c) 2007 Wiley Periodicals, Inc.     

Use of superabsorbent polymer gels for surface decontamination of Bacillus anthracis spores

Aims:  This study evaluated the inactivation of Bacillus anthracis Vollum spores dried on a nonporous surface using a superabsorbent polymer (SAP) gel containing commercially available liquid decontaminants.
Methods and Results:  The first phase determining the availability of the liquid decontaminant within the SAP showed that the SAP gel containing pH-adjusted sodium hypochlorite (NaOCl) inhibited B. anthracis growth while the water control SAP gel had no affect on growth. For testing surface decontamination, B. anthracis spores were dried onto steel coupons painted with chemical agent resistant coating and exposed to SAP containing either pH-adjusted NaOCl, chlorine dioxide (ClO₂) or hydrogen peroxide/peracetic acid (H₂O₂/PA) for 5 and 30 min. At contact times of both 5 and 30 min, all of the SAP gels containing pH-adjusted NaOCl, ClO₂ or H₂O₂/PA inactivated B. anthracis spores at levels ranging from 2·2 to ≥7·6 log reductions.
Conclusions:  Incorporation of three commercially available decontaminant technologies into a SAP gel promotes inactivation of B. anthracis spores without observable physical damage to the test surface.
Significance and Impact of the Study:  This work provides preliminary data for the feasibility of using SAP in inactivating B. anthracis spores on a nonporous surface, supporting the potential use of SAP in surface decontamination.     

Difference between the spore sizes of Bacillus anthracis and other Bacillus species

AIMS: To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions. METHODS AND RESULTS: Spores from several Bacillus species, including seven strains of B. anthracis and six close neighbours, were prepared and studied using identical media, protocols and instruments. Here, we report the spore length and diameter distributions, as determined by transmission electron microscopy (TEM). We calculated the aspect ratio and volume of each spore. All the studied strains of B. anthracis had similar diameter (mean range between 0.81 +/- 0.08 microm and 0.86 +/- 0.08 microm). The mean lengths of the spores from different B. anthracis strains fell into two significantly different groups: one with mean spore lengths 1.26 +/- 0.13 microm or shorter, and another group of strains with mean spore lengths between 1.49 and 1.67 microm. The strains of B. anthracis that were significantly shorter also sporulated with higher yield at relatively lower temperature. The grouping of B. anthracis strains by size and sporulation temperature did not correlate with their respective virulence. CONCLUSIONS: The spores of Bacillus subtilis and Bacillus atrophaeus (previously named Bacillus globigii), two commonly used simulants of B. anthracis, were considerably smaller in length, diameter and volume than all the B. anthracis spores studied. Although rarely used as simulants, the spores of Bacillus cereus and Bacillus thuringiensis had dimensions similar to those of B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefence against B. anthracis. The data presented here should help in the selection of simulants that better resemble the properties of B. anthracis, and thus, more accurately represent the performance of collectors, detectors and other countermeasures against this threat agent.     

Rapid detection of Bacillus anthracis using monoclonal antibody functionalized QCM sensor

Since the anthrax spore bioterrorism attacks in America in 2001, the early detection of Bacillus anthracis spores and vegetative cells has gained significant interest. At present, many polyclonal antibody-based quartz crystal microbalance (QCM) sensors have been developed to detect B. anthracis simulates. To achieve a simultaneous rapid detection of B. anthracis spores and vegetative cells, this paper presents a biosensor that utilizes an anti-B. anthracis monoclonal antibody designated to 8G3 (mAb 8G3, IgG) functionalized QCM sensor. Having compared four kinds of antibody immobilizations on Au surface, an optimized mAb 8G3 was immobilized onto the Au electrode with protein A on a mixed self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA) and 6-mercaptohexan-1-ol (6-MHO) as adhesive layer. The detection of B. anthracis was investigated under three conditions: dip-and-dry, static addition and flow through procedure. The results indicated that the sensor yielded a distinct response to B. anthracis spores or vegetative cells but had no significant response to Bacillus thuringiensis species. The functionalized sensor recognized B. anthracis spores and vegetative cells specifically from its homophylic ones, and the limit of detection (LOD) reached 10(3)CFU or spores/ml of B. anthracis in less than 30 min. Cyclic voltammogram (CV) and scanning electronic microscopy (SEM) were performed to characterize the surface of the sensor in variable steps during the modification and after the detection. The mAb functionalized QCM biosensor will be helpful in the fabrication of a similar biosensor that may be available in anti-bioterrorism in the future.     

Isolation, kinetic analysis, and structural characterization of an antibody targeting the Bacillus anthracis major spore surface protein BclA

One method of laboratory- or field-based testing for anthrax is detection of Bacillus anthracis spores by high-affinity, high specificity binding reagents. From a pool of monoclonal antibodies, we selected one such candidate (A4D11) with high affinity for tBclA, a truncated version of the B. anthracis exosporium protein BclA. Kinetic analysis utilising both standard and kinetic titration on a Biacore biosensor indicated antibody affinities in the 300 pM range for recombinant tBclA, and the A4D11 antibody was also re-formatted into scFv configuration with no loss of affinity. However, assays against B. anthracis and related Bacilli species showed limited binding of intact spores as well as significant cross-reactivity between species. These results were rationalized by determination of the three-dimensional crystallographic structure of the scFv-tBclA complex. A4D11 binds the side of the tBclA trimer, contacting a face of the antigen normally packed against adjacent trimers within the exosporium structure; this inter-spore interface is highly conserved between Bacilli species. Our results indicate the difficulty of generating a high-affinity antibody to differentiate between the highly conserved spore structures of closely related species, but suggest the possibility of future structure-based antibody design for this difficult target.     

Monoclonal antibodies for Bacillus anthracis spore detection and functional analyses of spore germination and outgrowth

All members of the Bacillus genus produce endospores as part of their life cycle; however, it is not possible to determine the identity of spores by casual or morphological examination. The 2001 anthrax attacks demonstrated a need for fast, dependable methods for detecting Bacillus anthracis spores in vitro and in vivo. We have developed a variety of isotypes and specificities of mAbs that were able to distinguish B. anthracis spores from other Bacillus spores. The majority of Abs were directed toward BclA, a major component of the exosporium, although other components were also distinguished. These Abs did not react with vegetative forms. Some Abs distinguished B. anthracis spores from spores of distantly related species in a highly specific manner, whereas others discriminated among strains that are the closest relatives of B. anthracis. These Abs provide a rapid and reliable means of identifying B. anthracis spores, for probing the structure and function of the exosporium, and in the analysis of the life cycle of B. anthracis.     

Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants

AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.     

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.     

3种细菌芽胞遗传同源性及抗力对比研究

比较细菌芽胞的遗传同源性、结构和抗力差异,为炭疽芽胞的替代试验菌的可靠评价提供依据。采用资料检索、生物信息分析、显微镜观察和微生物学技术分析不同芽胞的遗传同源性、超微结构和抗力差异。炭疽芽胞与腊样芽胞的结构和大小相似,生物遗传同源性最近,对热力、UVC和有效氯的抗力相近。腊样芽胞对炭疽芽胞的代表性最好,可以倾向性选用其替代炭疽芽胞进行试验研究。     

Immuno-detection of anthrose containing tetrasaccharide in the exosporium of Bacillus anthracis and Bacillus cereus strains

Aims:  Bacillus anthracis strains of various origins were analysed with the view to describe intrinsic and persistent structural components of the Bacillus collagen-like protein of anthracis glycoprotein associated anthrose containing tetrasaccharide in the exosporium.
Methods and Results:  The tetrasaccharide consists of three rhamnose residues and an unique monosaccharide – anthrose. As anthrose was not found in spores of related strains of bacteria, we envisioned the detection of B. anthracis spores based on antibodies against anthrose-containing polysaccharides. Carbohydrate–protein conjugates containing the synthetic tetrasaccharide, an anthrose–rhamnose disaccharide or anthrose alone were employed to immunize mice. All three formulations were immunogenic and elicited IgG responses with different fine specificities. All sera and monoclonal antibodies derived from tetrasaccharide immunized mice cross-reacted not only with spore lysates of a panel of virulent B. anthracis strains, but also with some of the B. cereus strains tested.
Conclusions:  Our results demonstrate that antibodies to synthetic carbohydrates are useful tools for epitope analyses of complex carbohydrate antigens and for the detection of particular target structures in biological specimens.
Significance and Impact of the Study:  Although not strictly specific for B. anthracis spores, antibodies against the tetrasaccharide may have potential as immuno-capturing components for a highly sensitive spore detection system.     

Decontamination assessment of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using a hydrogen peroxide gas generator

AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. METHODS AND RESULTS: Bacillus anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to > or =1000 ppm hydrogen peroxide gas for 20 min. Hydrogen peroxide exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials except G. stearothermophilus on industrial carpet. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with both surrogates. The effectiveness of gaseous hydrogen peroxide on the growth of biological indicators and spore strips was evaluated in parallel as a qualitative assessment of decontamination. At 1 and 7 days postexposure, decontaminated biological indicators and spore strips exhibited no growth, while the nondecontaminated samples displayed growth. CONCLUSIONS: Significant differences in decontamination efficacy of hydrogen peroxide gas on porous and nonporous surfaces were observed when comparing the mean log reduction in B. anthracis spores with B. subtilis and G. stearothermophilus spores. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using hydrogen peroxide gas.