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.     

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.     

Formaldehyde gas inactivation of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials

AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using formaldehyde gas. METHODS AND RESULTS: B. anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to approx. 1100 ppm formaldehyde gas for 10 h. Formaldehyde exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with B. subtilis (galvanized metal and painted wallboard paper) and G. stearothermophilus (industrial carpet and painted wallboard paper). Formaldehyde gas inactivated>or=50% of the biological indicators and spore strips (approx. 1x10(6) CFU) when analyzed after 1 and 7 days. CONCLUSIONS: Formaldehyde gas significantly reduced the number of viable spores on both porous and nonporous materials in which the two surrogates exhibited similar log reductions to that of B. anthracis on most test materials. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using formaldehyde gas.     

Bacillus anthracis contamination and inhalational anthrax in a mail processing and distribution center

AIMS: Four inhalational anthrax cases occurred in a large mail processing and distribution center in Washington, DC, after envelopes containing Bacillus anthracis spores were processed. This report describes the results of sampling for B. anthracis spores during investigations conducted in October and December 2001. METHODS AND RESULTS: Wet swabs, wet wipes, vacuum sock, and air-filter samples were collected throughout the facility to characterize the extent of building contamination. The results showed widespread contamination of B. anthracis spores, particularly associated with one delivery bar code sorter (DBCS) machine that had sorted the spore-containing envelopes and an area where the envelopes were handled by postal workers. Spore concentrations decreased as distance from the DBCS machine increased, but spores were widely dispersed into surrounding areas. CONCLUSION: The spatial distribution of culture positive samples was closely related to the work areas of the inhalational anthrax cases and supported epidemiological evidence that the workers became ill from exposure to B. anthracis spores in areas where the contaminated envelopes had travelled. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this investigation were used to guide decontamination efforts and provided baseline spore concentrations for follow-up measurements after the building had been cleaned. Implementing methods to reduce aerosolization and dispersion of dust within the facility would reduce postal workers' potential exposures to bioterrorism agents.     

A biological decontamination process for small, privately owned buildings

An urban wide-area recovery and restoration effort following a large-scale biological release will require extensive resources and tax the capabilities of government authorities. Further, the number of private decontamination contractors available may not be sufficient to respond to the needs. These resource limitations could create the need for decontamination by the building owner/occupant. This article provides owners/occupants with a simple method to decontaminate a building or area following a wide-area release of Bacillus anthracis using liquid sporicidal decontamination materials, such as pH-amended bleach or activated peroxide; simple application devices; and high-efficiency particulate air-filtered vacuums. Owner/occupant decontamination would be recommended only after those charged with overseeing decontamination-the Unified Command/Incident Command-identify buildings and areas appropriate for owner/occupant decontamination based on modeling and environmental sampling and conduct health and safety training for cleanup workers.     

Carbohydrates and glycoproteins of Bacillus anthracis and related bacilli: targets for biodetection

The spore is the form released in a bioterrorism attack. There is a real need for definition of new targets for Bacillus anthracis that might be incorporated into emerging biodetection technologies. Particularly of interest are macromolecules found in B. anthracis that are (1) spore-specific, (2) readily accessible on the spore surface and (3) distinct from those present in related organisms. One of the few biochemical methods to identify the spores of B. anthracis is based on the presence of rhamnose and 3-O-methyl rhamnose as determined by gas chromatography-mass spectrometry. Related organisms additionally contain 2-O-methyl rhamnose and fucose. Carbohydrates and glycoproteins of the B. cereus group of organisms and the related B. subilis group are reviewed here. It is hypothesized that the spore-specific carbohydrate is a component of the newly described glycoprotein of the exosporium of B. anthracis. Further work to define the protein and carbohydrate components of the glycoprotein of B. anthracis could be highly useful in developing new technologies for rapid biodetection.     

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.     

Determination of the efficacy of two building decontamination strategies by surface sampling with culture and quantitative PCR analysis

The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus ("Bacillus subtilis subsp. niger," also referred to as BG), a Bacillus anthracis surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 10(5) to 10(6) CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable B. atrophaeus spores were detected. After decontamination with chlorine dioxide gas, no culturable B. atrophaeus was detected in 24 of 27 samples (89%). However, QPCR analysis showed that B. atrophaeus DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies.     

In vitro studies of peptidoglycan binding and hydrolysis by the Bacillus anthracis germination-specific lytic enzyme SleB

The Bacillus anthracis endospore loses resistance properties during germination when its cortex peptidoglycan is degraded by germination-specific lytic enzymes (GSLEs). Although this event normally employs several GSLEs for complete cortex removal, the SleB protein alone can facilitate enough cortex hydrolysis to produce vulnerable spores. As a means to better understand its enzymatic function, SleB was overexpressed, purified, and tested in vitro for depolymerization of cortex by measurement of optical density loss and the solubilization of substrate. Its ability to bind peptidoglycan was also investigated. SleB functions independently as a lytic transglycosylase on both intact and fragmented cortex. Most of the muropeptide products that SleB generates are large and are potential substrates for other GSLEs present in the spore. Study of a truncated protein revealed that SleB has two domains. The N-terminal domain is required for stable peptidoglycan binding, while the C-terminal domain is the region of peptidoglycan hydrolytic activity. The C-terminal domain also exhibits dependence on cortex containing muramic-δ-lactam in order to carry out hydrolysis. As the conditions and limitations for SleB activity are further elucidated, they will enable the development of treatments that stimulate premature germination of B. anthracis spores, greatly simplifying decontamination measures.     

Cortex peptidoglycan lytic activity in germinating Bacillus anthracis spores

Bacterial endospore dormancy and resistance properties depend on the relative dehydration of the spore core, which is maintained by the spore membrane and its surrounding cortex peptidoglycan wall. During spore germination, the cortex peptidoglycan is rapidly hydrolyzed by lytic enzymes packaged into the dormant spore. The peptidoglycan structures in both dormant and germinating Bacillus anthracis Sterne spores were analyzed. The B. anthracis dormant spore peptidoglycan was similar to that found in other species. During germination, B. anthracis released peptidoglycan fragments into the surrounding medium more quickly than some other species. A major lytic enzymatic activity was a glucosaminidase, probably YaaH, that cleaved between N-acetylglucosamine and muramic-delta-lactam. An epimerase activity previously proposed to function on spore peptidoglycan was not detected, and it is proposed that glucosaminidase products were previously misidentified as epimerase products. Spore cortex lytic enzymes and their regulators are attractive targets for development of germination inhibitors to kill spores and for development of activators to cause loss of resistance properties for decontamination of spore release sites.     

A systematic methodology for selecting decontamination strategies following a biocontamination event

Decontamination and recovery of a facility or outdoor area after a wide-area biological incident involving a highly persistent agent (eg, Bacillus anthracis spores) is a complex process that requires extensive information and significant resources, which are likely to be limited, particularly if multiple facilities or areas are affected. This article proposes a systematic methodology for evaluating information to select the decontamination or alternative treatments that optimize use of resources if decontamination is required for the facility or area. The methodology covers a wide range of approaches, including volumetric and surface decontamination, monitored natural attenuation, and seal and abandon strategies. A proposed trade-off analysis can help decision makers understand the relative appropriateness, efficacy, and labor, skill, and cost requirements of the various decontamination methods for the particular facility or area needing treatment--whether alone or as part of a larger decontamination effort. Because the state of decontamination knowledge and technology continues to evolve rapidly, the methodology presented here is designed to accommodate new strategies and materials and changing information.     

Decreased time for detection and quantification of virulent Bacillus anthracis and Yersinia pestis using a BioNanoPore (BNPTM) membrane technology

Many aspects of biodefense research require quantitative growth assessments of the test agent. This study evaluated the BioNanoPore (BNP™) technology to quantitate Bacillus anthracis and Yersinia pestis faster than traditional plate counting methods. The BNP™ technology enabled quantification of B. anthracis and Y. pestis in phosphate-buffered saline and naïve rabbit blood at 6 and 24 h, respectively. After 6 h of growth, counts for B. anthracis ranged from 6·19–6·45 log₁₀ CFU ml⁻¹ on BNP™, while counts after 24 h on tryptic soy agar (TSA) ranged from 6·51–6·58 log₁₀ CFU ml⁻¹. For Y. pestis , counts on BNP™ at 24 h ranged from 6·31–6·41 log₁₀ CFU ml⁻¹ on BNP™ and ranged from 6·44–6·89 log₁₀ CFU ml⁻¹ on TSA at 48 h. This study demonstrates that the BNP™ technology provides a more rapid detection of B. anthracis and Y. pestis , which could aid in the evaluation of potential medical countermeasures and treatments as well as other biological defense applications such as surface sampling or decontamination efficacy.     

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.     

The national framework and consequence management guidance following a biological attack

Consequence management following a release of aerosolized Bacillus anthracis spores requires a high level of technical understanding and direction. National policies and regulations address the topics of preparedness goals and organizational structure, but they do not tell responders how to perform remediation. Essential considerations include determining what must be cleaned, evaluating health risks, ascertaining the priority of cleanup, and selecting appropriate decontamination technologies to meet consensus and risk-derived clearance goals. This article highlights key features of a national-level framework that has been developed to guide a risk-based decision process and inform technical personnel of the best practices to follow during each activity leading to the restoration of functions at affected facilities or areas. The framework and associated guidance follows the scheme of 6 phases for response and recovery arrived at through interagency consensus and approval. Each phase is elaborated in a series of detailed decision flowcharts identifying key questions that must be addressed and answered from the time that first indications of a credible biological attack are received to final reoccupancy of affected areas and a return to normal daily functions.     

Development of an aerosol surface inoculation method for bacillus spores

A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 10(7) CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies.     

Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility

The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures) can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.     

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.     

The development and assessment of DNA and oligonucleotide probes for the specific detection of Bacillus anthracis

Two DNA probes and a number of oligonucleotide probes were designed from the virulence factor genes of Bacillus anthracis. These probes were tested for specificity against 52 B. anthracis strains and 233 Bacillus strains encompassing 23 other species. A rapid slot blotting technique was used for screening the large numbers of isolates involved. All probes tested appeared to be specific for B. anthracis under high stringency conditions. These probes could differentiate between virulent and avirulent strains. The probes were also applied to the detection of B. anthracis in routine environmental and clinical samples. A non-radioactive hybridization and detection system based on digoxigenin-11-dUTP was developed.     

Inactivation of Bacillus anthracis spores by liquid biocides in the presence of food residue

Biocide inactivation of Bacillus anthracis spores in the presence of food residues after a 10-min treatment time was investigated. Spores of nonvirulent Bacillus anthracis strains 7702, ANR-1, and 9131 were mixed with water, flour paste, whole milk, or egg yolk emulsion and dried onto stainless-steel carriers. The carriers were exposed to various concentrations of peroxyacetic acid, sodium hypochlorite (NaOCl), or hydrogen peroxide (H(2)O(2)) for 10 min at 10, 20, or 30 degrees C, after which time the survivors were quantified. The relationship between peroxyacetic acid concentration, H(2)O(2) concentration, and spore inactivation followed a sigmoid curve that was accurately described using a four-parameter logistic model. At 20 degrees C, the minimum concentrations of peroxyacetic acid, H(2)O(2), and NaOCl (as total available chlorine) predicted to inactivate 6 log(10) CFU of B. anthracis spores with no food residue present were 1.05, 23.0, and 0.78%, respectively. At 10 degrees C, sodium hypochlorite at 5% total available chlorine did not inactivate more than 4 log(10) CFU. The presence of the food residues had only a minimal effect on peroxyacetic acid and H(2)O(2) sporicidal efficacy, but the efficacy of sodium hypochlorite was markedly inhibited by whole-milk and egg yolk residues. Sodium hypochlorite at 5% total available chlorine provided no greater than a 2-log(10) CFU reduction when spores were in the presence of egg yolk residue. This research provides new information regarding the usefulness of peroxygen biocides for B. anthracis spore inactivation when food residue is present. This work also provides guidance for adjusting decontamination procedures for food-soiled and cold surfaces.     

Molecular characterization of Bacillus anthracis using multiplex PCR, ERIC-PCR and RAPD

AIMS: To investigate the molecular characterization of Bacillus anthracis strains by multiplex PCR, enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and random amplification of polymorphic DNA (RAPD). METHODS AND RESULTS: Three primers were used to amplify the cya, cap and cereolysinAB genes in the multiplex PCR. Two distinct ERIC-PCR and RAPD fragments, which separated B. anthracis into two groups, were used as probes in Southern hybridization experiments. The probes hybridized only to the cya+ B. anthracis strains identified by the multiplex PCR. Nucleotide sequence analysis of the two cloned fragments showed they were from the pXO1 plasmid of B. anthracis. CONCLUSION: Multiplex PCR simultaneously identified isolates of the Bacillus cereus group and the B. anthracis virulence factors. ERIC-PCR and RAPD, combined with the Southern hybridization analyses, differentiated B. anthracis strains and separated them from the closely related B. cereus group bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: ERIC-PCR and RAPD assay could be effective in differentiating virulent from avirulent B. anthracis. Our results also show that the amplification of the large plasmids was allowed in the ERIC-PCR and RAPD assay.