Spore Decontamination Research Articles

Decontamination of Bacterial Spores Using a Bio-reaction System

This report describes the test results of decontamination of Bacillus atrophaeus spores. The spore solution at 109 cfu/mL concentration was treated with chemical decontaminants, hydrogen peroxide, DF-200, sodium dichloroisocyanurate(NaDCC), and perasasfe. DF-200 was not suitable for decontamination of the spore solution. Among them, the optimal decontamination reaction conditions of perasafe and NaDCC were established from test tube experiments in a mL-scale, and they were validated in a bio-reaction system in a L-scale. The optimal conditions of perasafe and NaDCC were 1.0 % of concentration with 5 minutes of reaction time and 0.3 % of concentration with 10 minutes of reaction time, respectively. Also, both decontaminants required adequate agitation for decontamination. The results in the bio-reaction system were similar with the results from the smaller scale using test tubes, showing that the optimal conditions from the smaller scale experiments can be applied to the larger scale.

UV-Induced Spectral and Morphological Changes in Bacterial Spores for Inactivation Assessment.

The ability to detect and inactivate spore-forming bacteria is of significance within, for example, industrial, healthcare, and defense sectors. Not only are stringent protocols necessary for the inactivation of spores but robust procedures are also required to detect viable spores after an inactivation assay to evaluate the procedure's success. UV radiation is a standard procedure to inactivate spores. However, there is limited understanding regarding its impact on spores' spectral and morphological characteristics. A further insight into these UV-induced changes can significantly improve the design of spore decontamination procedures and verification assays. This work investigates the spectral and morphological changes to Bacillus thuringiensis spores after UV exposure. Using absorbance and fluorescence spectroscopy, we observe an exponential decay in the spectral intensity of amino acids and protein structures, as well as a logistic increase in dimerized DPA with increased UV exposure on bulk spore suspensions. Additionally, using micro-Raman spectroscopy, we observe DPA release and protein degradation with increased UV exposure. More specifically, the protein backbone's 1600-1700 cm-1 amide I band decays slower than other amino acid-based structures. Last, using electron microscopy and light scattering measurements, we observe shriveling of the spore bodies with increased UV radiation, alongside the leaking of core content and disruption of proteinaceous coat and exosporium layers. Overall, this work utilized spectroscopy and electron microscopy techniques to gain new understanding of UV-induced spore inactivation relating to spore degradation and CaDPA release. The study also identified spectroscopic indicators that can be used to determine spore viability after inactivation. These findings have practical applications in the development of new spore decontamination and inactivation validation methods.

Ultrastable and Responsive Foams Based on 10-Hydroxystearic Acid Soap for Spore Decontamination.

Currently, there is renewed interest in using fatty acid soaps as surfactants. Hydroxylated fatty acids are specific fatty acids with a hydroxyl group in the alkyl chain, giving rise to chirality and specific surfactant properties. The most famous hydroxylated fatty acid is 12-hydroxystearic acid (12-HSA), which is widely used in industry and comes from castor oil. A very similar and new hydroxylated fatty acid, 10-hydroxystearic acid (10-HSA), can be easily obtained from oleic acid by using microorganisms. Here, we studied for the first time the self-assembly and foaming properties of R-10-HSA soap in an aqueous solution. A multiscale approach was used by combining microscopy techniques, small-angle neutron scattering, wide-angle X-ray scattering, rheology experiments, and surface tension measurements as a function of temperature. The behavior of R-10-HSA was systematically compared with that of 12-HSA soap. Although multilamellar micron-sized tubes were observed for both R-10-HSA and 12-HSA, the structure of the self-assemblies at the nanoscale was different, which is probably due to the fact that the 12-HSA solutions were racemic mixtures, while the 10-HSA solutions were obtained from a pure R enantiomer. We also demonstrated that stable foams based on R-10-HSA soap can be used for cleaning applications, by studying spore removal on model surfaces in static conditions via foam imbibition.

Decontamination of Spores on Model Stainless-Steel Surface by Using Foams Based on Alkyl Polyglucosides.

In the food industry, the surfaces of processing equipment are considered to be major factors in the risk of food contamination. The cleaning process of solid surfaces is essential, but it requires a significant amount of water and chemicals. Herein, we report the use of foam flows based on alkyl polyglucosides (APGs) to remove spores of Bacillus subtilis on stainless-steel surfaces as the model-contaminated surface. Sodium dodecyl sulfate (SDS) was also studied as an anionic surfactant. Foams were characterized during flows by measuring the foam stability and the bubble size. The efficiency of spores' removal was assessed by enumerations. We showed that foams based on APGs could remove efficiently the spores from the surfaces, but slightly less than foams based on SDS due to an effect of SDS itself on spores removal. The destabilization of the foams at the end of the process and the recovery of surfactant solutions were also evaluated by using filtration. Following a life cycle assessment (LCA) approach, we evaluated the impact of the foam flow on the global environmental footprint of the process. We showed significant environmental impact benefits with a reduction in water and energy consumption for foam cleaning. APGs are a good choice as surfactants as they decrease further the environmental impacts.

Dodecylamine rapidly kills of spores of multiple Firmicute species: properties of the killed spores and the mechanism of the killing.

Previous work showed that Bacillus subtilis dormant spore killing and germination by dodecylamine take place by different mechanisms. This new work aimed to optimize killing of B. subtilis and other Firmicutes spores and to determine the mechanism of the killing. Spores of seven Firmicute species were killed rapidly by dodecylamine under optimal conditions and more slowly by decylamine or tetradecylamine. The killed spores were not recovered by additions to recovery media, and some of the killed spores subsequently germinated, all indicating that dodecylamine-killed spores truly are dead. Spores of two species treated with dodecylamine were more sensitive to killing by a subsequent heat treatment, and spore killing of at least one species was faster with chemically decoated spores. The cores of dodecylamine-killed spores were stained by the nucleic acid stain propidium iodide, and dodecylamine-killed wild-type and germination-deficient spores released their stores of phosphate-containing small molecules. This work indicates that dodecylamine is likely a universal sporicide for Firmicute species, and it kills spores by damaging their inner membrane, with attendant loss of this membrane as a permeability barrier. There is a significant need for agents that can effectively kill spores of a number of Firmicute species, especially in wide area decontamination. Dodecylamine appears to be a universal sporicide with a novel mechanism of action, and this or some comparable molecule could be useful in wide area spore decontamination.

Supercritical carbon dioxide-based cleaning and sterilization treatments for the reuse of filtering facepiece respirators FFP2 in the context of COVID-19 pandemic

Supercritical Carbon Dioxide treatment of clean and soiled Filtering Face-Piece respirators is shown to meet mandatory requirements for safe reuse of FFP2. Preliminary tests enabled us to select optimal conditions for Supercritical Carbon Dioxide treatment of FFP2, for one hour at 343 K under 7.5 MPa, with a biocide solution. FFP2s from Paul Boyé ® were then tested before and after Supercritical Carbon Dioxide treatment with a soiling solution and a biological indicator (i.e. spores of GeoBacillus stearothermophilus) with respect to three objectives: (i) washing of organic deposits, (ii) sterilization (i.e. spores inactivation) and (iii) preservation of the filtration performances. The proposed Supercritical Carbon Dioxide treatment fulfils these three criterions, with effective decontamination of spores and promising washing preserving FFP2 requirements for filtration efficiency of aerosol (> 94%) with acceptable pressure drop (< 240 Pa). As a simple and low-cost one-step recycling method, Supercritical Carbon Dioxide fits to economical and sustainable development standpoints but also allows one to come back to good practices of face protective equipment use, lowering eventual shortage and tensions for most countries importing these respirators.

The application of plasma-activated water combined with mild heat for the decontamination of Bacillus cereus spores in rice (Oryza sativa L. ssp. japonica)

This study applied the combination of plasma-activated water (PAW) and mild heat (40 °C and 55 °C) for the decontamination of Bacillus cereus spores in rice (Oryza sativa L. ssp. japonica). In this study, PAW combined with 40 °C and 55 °C (PAW-40 and PAW-55) achieved 1.54 and 2.12 log10 CFU g−1 reductions of B. cereus spores in rice after 60 min exposure, which was significantly higher than the inactivation level (0.72 log10 CFU g−1) induced by PAW treatment alone. The assays for the leakage of intracellular contents (nucleic acids, proteins, dipicolinic acid-DPA) and the propidium iodide (PI) fluorescence probe confirmed that the combination of PAW and mild heat caused significant damages on the intact multilayered structure of B. cereus spores than the individual PAW did. The application of scanning electron microscope (SEM) and transmission electron microscope (TEM) provided a direct observation for the visible disruption of external structure and leakage of intracellular components caused by the combination of PAW and mild heat. Besides, the combination of PAW and mild heat caused no adverse effect on the texture and sensory qualities of the rice after cooking. These findings provide some knowledge for the application of PAW combined with mild heat for the decontamination of spores in rice.

Combining spore germination and heat inactivation to decontaminate materials contaminated with Bacillus anthracis spores.

To add a spore germination step in order to reduce decontamination temperature and time requirements compared to the current hot, humid air decontamination parameters, which are 75-80°C, ≥72h, 70-90% RH, down to ≤60°C and ≤24h total decontamination time. Bacillus anthracis spore germination with l-alanine+inosine+calcium dipicolinate (CaDPA) was quantified at 0-40°C, several time points and spore concentrations of 5-9 log10 per ml. Germination efficiency at 0-40°C was >99% at <8 log10 spores per ml. The temperature optimum was 20°C. Germination efficiency was significantly higher but slower at 0°C compared to ≥30°C at ≥8 log10 spores per ml. A single germinant application followed by 60°C, 1-h treatment consistently inactivated >2 log10 (>99%) of spores. However, a repeat application of germinant was needed to achieve the objective of ≥6 log10 spore inactivation out of a 7 log10 challenge (≥99·9999%) for ≤24h total decontamination time for nylon and aircraft performance coating. l-alanine+inosine+CaDPA stimulated germination across wide temperature and spore concentration ranges. Germination expands the scope of spore decontamination to include materials from any industry sector that can be sprayed with an aqueous germinant solution.

YpeB dimerization may be required to stabilize SleB for effective germination of Bacillus anthracis spores

BackgroundBacillus cells faced with unfavorable environmental conditions undergo an asymmetric division process ultimately leading to the formation of the bacterial spore. In some instances the spore serves as an infectious agent; such is the case with the spore of Bacillus anthracis and the disease anthrax. Spores are resistant to a variety of environment conditions including traditional decontamination techniques due to the formation of specialized cellular structures. One such structure, the spore cortex, is a thick layer of modified peptidoglycan that contributes to spore dormancy through maintenance of the dehydrated state of the spore core. During spore germination, degradation of the cortex is required to facilitate complete hydration of the core and a return to vegetative growth. Degradation of the cortex is accomplished through the action of germination-specific lytic enzymes. One of these enzymes, SleB, has been previously shown to require the presence of the YpeB protein for its stable incorporation and subsequent function in spores of B. anthracis. The focus of the present study is to identify protein interactions of YpeB through in vivo chemical cross-linking and two-hybrid analysis.ResultsConserved residues within YpeB PepSY domains were altered to facilitate implementation of a site-specific chemical cross-linker, 4-Azidophenacyl bromide. Analyses of crosslinked-spore extracts suggests that YpeB exists as a dimer or larger multimer within the spore, potentially mediated through interactions of the C-terminal domains. Spores expressing stable truncated forms of YpeB were crosslinked and corresponding truncated dimers were detected. Further characterization of individual YpeB domains using bacterial two-hybrid analysis indicated a possible role for both N-and C-terminal domains in YpeB oligomerization.ConclusionsThe YpeB protein likely exists as dimer or higher-order multimer in the dormant spore. Both the N- and C-terminal YpeB domains contribute to multimerization. SleB likely also exists as an oligomer, and SleB and YpeB may be found together within a protein complex. Disassembly of this complex during spore germination likely allows SleB to become active in spore cortex degradation. Further study of this protein complex may contribute to the development of methods to inhibit or stimulate germination, allowing more effective spore decontamination.

Identification of L-Valine-initiated-germination-active genes in Bacillus subtilis using Tn-seq.

Bacterial endospores can survive harsh environmental conditions and long-term dormancy in the absence of nutrients, but can rapidly germinate under favorable conditions. In the present study, we employed transposon sequencing (Tn-seq) to identify genes with previously uncharacterized roles in spore germination. Identified genes that encoded spore inner membrane proteins were chosen for study of defined mutants, which exhibited delayed germination in several assays in response to varying germinants. Significantly slowed release of DPA indicated that mutants were affected in Stage I of germination. Several mutants exhibited phenotypic traits consistent with failure of a GerA germinant receptor-mediated response, while others appeared to have a more general loss of response to varied germinants. Use of a gerA-lacZ transcriptional fusion and quantitative western blotting of GerAC allowed mutants to be classified based upon normal or decreased gerA transcription and normal or reduced GerA accumulation. Fourteen genes were identified to have newly described roles within Bacillus spore germination. A more complete understanding of this process can contribute to the development of better spore decontamination procedures.

Killing of spores of Bacillus species by cetyltrimethylammonium bromide.

To investigate effects of the cationic surfactant cetyltrimethylammonium bromide (CTAB), a disinfectant, on spores of Bacillus species. The ability of CTAB to trigger release of Bacillus spores' large depot of dipicolinic acid (DPA) in a 1:1 chelate with Ca2+ (CaDPA), and to kill spores was investigated. CTAB-triggered CaDPA release from spores of Bacillus subtilis, Bacillus cereus and Bacillus megaterium, but was not followed by completion of germination. CaDPA release triggered by CTAB increased at higher temperatures, and was optimal for B. subtilis spores at pH 9·4 and 30μgml-1 CTAB. CTAB also killed Bacillus spores as shown by plate counts and vital staining of treated dormant spores, and after their germination. However, B. cereus and B. megaterium spores were more CTAB-sensitive than were B. subtilis spores. CaDPA release from and killing of CTAB-treated spores of isogenic B. subtilis mutants lacking germination proteins was also examined, and compared with effects of the well-known germinant dodecylamine on spores, to determine how CTAB exerts its effects on spores. The results of this investigation showed that CTAB kills spores of three Bacillus species, perhaps by damaging the spore inner membrane, although it is also possible that some killing by this agent follows its triggering of spore germination. The results of this work indicate that CTAB is also a disinfectant, but also a sporicide, and may be a useful adjunct in spore decontamination, especially at higher temperatures.

Review of Decontamination Techniques for the Inactivation of Bacillus anthracis and Other Spore-Forming Bacteria Associated with Building or Outdoor Materials.

Since the intentional release of Bacillus anthracis spores through the U.S. Postal Service in the fall of 2001, research and development related to decontamination for this biological agent have increased substantially. This review synthesizes the advances made relative to B. anthracis spore decontamination science and technology since approximately 2002, referencing the open scientific literature and publicly available, well-documented scientific reports. In the process of conducting this review, scientific knowledge gaps have also been identified. This review focuses primarily on techniques that are commercially available and that could potentially be used in the large-scale decontamination of buildings and other structures, as well as outdoor environments. Since 2002, the body of scientific data related to decontamination and microbial sterilization has grown substantially, especially in terms of quantifying decontamination efficacy as a function of several factors. Specifically, progress has been made in understanding how decontaminant chemistry, the materials the microorganisms are associated with, environmental factors, and microbiological methods quantitatively impact spore inactivation. While advancement has been made in the past 15 years to further the state of the science in the inactivation of bacterial spores in a decontamination scenario, further research is warranted to close the scientific gaps that remain.

Fighting Ebola with novel spore decontamination technologies for the military.

Recently, global public health organizations such as Doctors without Borders (MSF), the World Health Organization (WHO), Public Health Canada, National Institutes of Health (NIH), and the U.S. government developed and deployed Field Decontamination Kits (FDKs), a novel, lightweight, compact, reusable decontamination technology to sterilize Ebola-contaminated medical devices at remote clinical sites lacking infra-structure in crisis-stricken regions of West Africa (medical waste materials are placed in bags and burned). The basis for effectuating sterilization with FDKs is chlorine dioxide (ClO2) produced from a patented invention developed by researchers at the US Army Natick Soldier RD&E Center (NSRDEC) and commercialized as a dry mixed-chemical for bacterial spore decontamination. In fact, the NSRDEC research scientists developed an ensemble of ClO2 technologies designed for different applications in decontaminating fresh produce; food contact and handling surfaces; personal protective equipment; textiles used in clothing, uniforms, tents, and shelters; graywater recycling; airplanes; surgical instruments; and hard surfaces in latrines, laundries, and deployable medical facilities. These examples demonstrate the far-reaching impact, adaptability, and versatility of these innovative technologies. We present herein the unique attributes of NSRDEC’s novel decontamination technologies and a Case Study of the development of FDKs that were deployed in West Africa by international public health organizations to sterilize Ebola-contaminated medical equipment. FDKs use bacterial spores as indicators of sterility. We review the properties and structures of spores and the mechanisms of bacterial spore inactivation by ClO2. We also review mechanisms of bacterial spore inactivation by novel, emerging, and established non-thermal technologies for food preservation, such as high pressure processing, irradiation, cold plasma, and chemical sanitizers, using an array of Bacillus subtilis mutants to probe mechanisms of spore germination and inactivation. We employ techniques of high-resolution atomic force microscopy and phase contrast microscopy to examine the effects of γ-irradiation on bacterial spores of Bacillus anthracis, Bacillus thuringiensis, and Bacillus atrophaeus spp. and of ClO2 on B. subtilis spores, and present in detail assays using spore bio-indicators to ensure sterility when decontaminating with ClO2.

Optimization of sporicidal activity and environmental Bacillus endospores decontamination by biogenic silver nanoparticle.

The intent of this study is to decontaminate Bacillus endospores and to determine the D-values using silver nanoparticles (AgNPs) synthesized from Streptomyces sp. cell filtrate. AgNPs synthesis was performed extracellularly followed by characterization using spectrophotometer, High Resolution Transmission Electron Microscope and x-ray diffraction pattern analysis. Subsequently, the optimized conditions for the decontamination and D-value estimation of Bacillus endospores were determined using the response surface methodology. The environmental spore decontamination study was performed in mice model. AgNPs were visibly and spectroscopically identified which were spherical with the size range of less than 20 nm. The synthesized AgNPs destroyed 1log10 CFU Bacillus endospores at around 20 min. The adherence of AgNPs to the surface of spore coat, pit formation and its complete structural loss was detected under field emission scanning electron microscopy. All the mice exposed to AgNP-treated spores showed no sign of pathological lesions. The results of our study strongly suggest that the application of AgNPs as a sporicidal agent could be a new approach in consistently eliminating the hazardous Bacillus spores.

Role of YpeB in cortex hydrolysis during germination of Bacillus anthracis spores.

The infectious agent of the disease anthrax is the spore of Bacillus anthracis. Bacterial spores are extremely resistant to environmental stresses, which greatly hinders spore decontamination efforts. The spore cortex, a thick layer of modified peptidoglycan, contributes to spore dormancy and resistance by maintaining the low water content of the spore core. The cortex is degraded by germination-specific lytic enzymes (GSLEs) during spore germination, rendering the cells vulnerable to common disinfection techniques. This study investigates the relationship between SleB, a GSLE in B. anthracis, and YpeB, a protein necessary for SleB stability and function. The results indicate that ΔsleB and ΔypeB spores exhibit similar germination phenotypes and that the two proteins have a strict codependency for their incorporation into the dormant spore. In the absence of its partner protein, SleB or YpeB is proteolytically degraded soon after expression during sporulation, rather than escaping the developing spore. The three PepSY domains of YpeB were examined for their roles in the interaction with SleB. YpeB truncation mutants illustrate the necessity of a region beyond the first PepSY domain for SleB stability. Furthermore, site-directed mutagenesis of highly conserved residues within the PepSY domains resulted in germination defects corresponding to reduced levels of both SleB and YpeB in the mutant spores. These results identify residues involved in the stability of both proteins and reiterate their codependent relationship. It is hoped that the study of GSLEs and interacting proteins will lead to the use of GSLEs as targets for efficient activation of spore germination and facilitation of spore cleanup.

Decontamination efficacy of three commercial-off-the-shelf (COTS) sporicidal disinfectants on medium-sized panels contaminated with surrogate spores of Bacillus anthracis.

In the event of a wide area release and contamination of a biological agent in an outdoor environment and to building exteriors, decontamination is likely to consume the Nation’s remediation capacity, requiring years to cleanup, and leading to incalculable economic losses. This is in part due to scant body of efficacy data on surface areas larger than those studied in a typical laboratory (5×10-cm), resulting in low confidence for operational considerations in sampling and quantitative measurements of prospective technologies recruited in effective cleanup and restoration response. In addition to well-documented fumigation-based cleanup efforts, agencies responsible for mitigation of contaminated sites are exploring alternative methods for decontamination including combinations of disposal of contaminated items, source reduction by vacuuming, mechanical scrubbing, and low-technology alternatives such as pH-adjusted bleach pressure wash. If proven effective, a pressure wash-based removal of Bacillus anthracis spores from building surfaces with readily available equipment will significantly increase the readiness of Federal agencies to meet the daunting challenge of restoration and cleanup effort following a wide-area biological release. In this inter-agency study, the efficacy of commercial-of-the-shelf sporicidal disinfectants applied using backpack sprayers was evaluated in decontamination of spores on the surfaces of medium-sized (∼1.2 m2) panels of steel, pressure-treated (PT) lumber, and brick veneer. Of the three disinfectants, pH-amended bleach, Peridox, and CASCAD evaluated; CASCAD was found to be the most effective in decontamination of spores from all three panel surface types.

Levels of germination proteins in Bacillus subtilis dormant, superdormant, and germinating spores.

Bacterial endospores exhibit extreme resistance to most conditions that rapidly kill other life forms, remaining viable in this dormant state for centuries or longer. While the majority of Bacillus subtilis dormant spores germinate rapidly in response to nutrient germinants, a small subpopulation termed superdormant spores are resistant to germination, potentially evading antibiotic and/or decontamination strategies. In an effort to better understand the underlying mechanisms of superdormancy, membrane-associated proteins were isolated from populations of B. subtilis dormant, superdormant, and germinated spores, and the relative abundance of 11 germination-related proteins was determined using multiple-reaction-monitoring liquid chromatography-mass spectrometry assays. GerAC, GerKC, and GerD were significantly less abundant in the membrane fractions obtained from superdormant spores than those derived from dormant spores. The amounts of YpeB, GerD, PrkC, GerAC, and GerKC recovered in membrane fractions decreased significantly during germination. Lipoproteins, as a protein class, decreased during spore germination, while YpeB appeared to be specifically degraded. Some protein abundance differences between membrane fractions of dormant and superdormant spores resemble protein changes that take place during germination, suggesting that the superdormant spore isolation procedure may have resulted in early, non-committal germination-associated changes. In addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Understanding the reasons for superdormancy may allow for better spore decontamination procedures.

In vitro and in vivo analyses of the Bacillus anthracis spore cortex lytic protein SleL

The bacterial endospore is the most resilient biological structure known. Multiple protective integument layers shield the spore core and promote spore dehydration and dormancy. Dormancy is broken when a spore germinates and becomes a metabolically active vegetative cell. Germination requires the breakdown of a modified layer of peptidoglycan (PG) known as the spore cortex. This study reports in vitro and in vivo analyses of the Bacillus anthracis SleL protein. SleL is a spore cortex lytic enzyme composed of three conserved domains: two N-terminal LysM domains and a C-terminal glycosyl hydrolase family 18 domain. Derivatives of SleL containing both, one or no LysM domains were purified and characterized. SleL is incapable of digesting intact cortical PG of either decoated spores or purified spore sacculi. However, SleL derivatives can hydrolyse fragmented PG substrates containing muramic-δ-lactam recognition determinants. The muropeptides that result from SleL hydrolysis are the products of N-acetylglucosaminidase activity. These muropeptide products are small and readily released from the cortex matrix. Loss of the LysM domain(s) decreases both PG binding and hydrolysis activity but these domains do not appear to determine specificity for muramic-δ-lactam. When the SleL derivatives are expressed in vivo, those proteins lacking one or both LysM domains do not associate with the spore. Instead, these proteins remain in the mother cell and are apparently degraded. SleL with both LysM domains localizes to the coat or cortex of the endospore. The information revealed by elucidating the role of SleL and its domains in B. anthracis sporulation and germination is important in designing new spore decontamination methods. By exploiting germination-specific lytic enzymes, eradication techniques may be greatly simplified.

Association and decontamination of Bacillus spores in a simulated drinking water system

The objective of this work was to elucidate the disinfectant susceptibility of Bacillus anthracis Sterne (BA) and a commercial preparation of Bacillus thuringiensis (BT) spores associated with a simulated drinking water system. Biofilms composed of indigenous water system bacteria were accumulated on copper and polyvinyl chloride (PVC) pipe material surfaces in a low-flow pipe loop and uniformly mixed tank reactor (CDC biofilm reactor). Application of a distributed shear during spore contact resulted in approximately a 1.0 and 1.6 log 10 increase in the number of spores associated with copper and PVC surfaces, respectively. Decontamination of spores in both free suspension and after association with biofilm-conditioned pipe materials was attempted using free chlorine and monochloramine. Associated spores required 5- to 10-fold higher disinfectant concentrations to observe the same reduction of viable spores as in suspension. High disinfectant concentrations (103 mg/L free chlorine and 49 mg/L monochloramine) yielded less than a 2-log 10 reduction in viable associated spores after 60 min. Spores associated with biofilms on copper surfaces consistently yielded higher Ct values than PVC.

Defensive and simultaneous actions of glycoconjugates during spore decontamination

An estimated $1 billion was lost in decontaminating areas exposed to anthrax in the 2001 attacks. To counter the threat of biological attacks, an effective ‘green’ decontaminant is vital to minimize the consequences of such attacks. The objective of our research was to study the ability of glycoconjugate ligands to decontaminate Bacillus cereus spores on hard surfaces. Polyvalent glycoconjugates (also known as neoglycoconjugates) were tested during decontamination of B. cereus spores. Resulting colony forming units (CFU) of viable spores were a direct evidence of glycoconjugate decontamination efficacy. Our results indicate a substantial, decreasing CFU count due to defensive and simultaneous actions of glycoconjugates compared to spores only used as controls. Decontamination of B. cereus spores was most efficiently and consistently achieved using Galα1→3GalNAcβ-PAA-flu glycoconjugate under both defensive and simultaneous conditions. Atomic force microscopy (AFM) allowed us to visualize decontamination at the nanoscale level using glycoconjugates. AFM reveals the size of glycoconjugate agglomerates (clusters) and a noticeably different morphology of glycoconjugate-treated spores during decontamination. Morphological features of untreated spores disappear under a thin layer of glycoconjugate solution. This thin layer is formed due to the defensive action of glycoconjugates. Simultaneous action has shown agglomeration of glycoconjugates in solution with B. cereus spores in glycoconjugate suspensions. Glycoconjugates might be useful for the development of an environment-friendly decontaminant of bacterial spores.