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Combinatorial application of different dye removal methods with specific features can lead to a novel and robust decolorizing system. In this study the bacterial spore laccase and TiO2 nanoparticles were co-entrapped to enhance dye degradation. The optimum entrapment conditions were achieved in the presence of alginate 2% (w/v) and Ca2+ (0.2M), Cu2+ (0.05M) and Zn2+ (0.25M) as matric polymer and counterions, respectively. Immobilized laccase showed a wide range of pH and temperature stability in comparison to the free spores. The entrapped degradation systems include single laccase, single TiO2, laccase + TiO2 (one-step remediation), TiO2/laccase (two-step remediation), and laccase/TiO2 (two-step remediation) that result to the 22%, 26% 45.6%, 47.6%, and 69.3% indigo carmine decolorization in 60 min. In the kinetic studies, the half-life of indigo carmine (25 mg/l) in the remediation processes containing laccase, TiO2, laccase + TiO2, TiO2/laccase, and laccase/TiO2 was calculated as 173, 138, 161, 115, and 57 min, respectively. The degradation products by co-entrapped system were not toxic against Sorghum vulgare. The results showed two-step decolorization by co-entrapped spore laccase and TiO2 nanoparticles, including the pretreatment of dye by laccase, and then, treatment by TiO2 has potential for degradation of indigo carmine.

In line with the recent promotion of biocementation as an environmentally friendly ground improvement method, this study presents an investigation into microbially induced calcite precipitation (MICP) as a method of improving the engineering properties of soft clay. Bacillus pasteurii bacterium in vegetative cell and bacterial spore forms were used to induce MICP in clay specimens. Untreated and treated clay specimens were tested for their mechanical properties and microstructures through unconfined compression (UC) tests, free–free resonance (FFR) tests, X-ray diffraction (XRD) tests, and scanning electron microscopy with energy dispersive X-ray (SEM/EDX) tests. Results showed that both vegetative cells and bacterial spores can effectively enhance the strength and modulus of clays by inducing MICP to generate calcite crystals. Clays treated with vegetative cells exhibited earlier improvements in their strength than clays treated with bacterial spores due to earlier activity availability; however, the clays treated with bacterial spores exhibited greater strength improvements in the long term. Bacterial spores may also prove more convenient to use in geotechnical engineering practice.

The germination kinetics of Bacillus subtilis, Bacillus cereus, and Bacillus megaterium spores were comprehensively studied using nutrients, dodecylamine, exogenous dipicolinic acid (DPA), and the nonthermal technology of High Pressure Processing (HPP) as germinants at water activities aw = 0.995–0.698 and DPA loss measured for spore populations and single spores. Primary findings were: i) germinant receptor (GR)-dependent germination was less sensitive to changes in aw than were other germination mechanisms; ii) HPP germination was less sensitive to changes in aw than was germination by other germinants, and iii) the irreversible commitment step was more sensitive to inhibition by lowering aw than were DPA release or cortex peptidoglycan hydrolysis. Presently, we use the three-step Quasi-chemical Germination Kinetics (QCGK) model, Transition State Theory, and Extrathermodynamics' Bunnett equation to determine the effects of humectants on spore germination rates. The QCGK model is an ordinary differential equation system that postulates spore germination occurs as a sequence of two productive steps with associated rate constants (k1 is irreversible and k2) and one nonproductive step (k3). The QCGK model fits the germination kinetics data well for changes in the observed dynamics with decreasing aw. Transition State Theory calculated negative activation volumes (ΔV∗) from the pressure-dependences of k1 and k2, and Bunnett equation calculations established linear relationships for the model's rate parameters, including rate constants and maximum germination rates, with variations in aw. Taken together, these results suggest that the irreversible step (k1) is rate-limiting and the inverse dependence of k1 and k2 on humectant concentration could occur through shielding of the activated complex by the solvated nonelectrolytes at low aw, which prevents the germination intermediate from linking to the aqueous environment. Practical applications High pressure processing is a food safety method that provides the least alteration of food appearance, texture and flavor. This research shows that food additives such as sugar can diminish the efficiency of high pressure food processing.

Contamination of toxic spore-forming bacteria is problematic since spores can survive a plethora of disinfection chemicals and it is hard to rapidly detect if the disinfection chemical has inactivated the spores. Thus, robust decontamination strategies and reliable detection methods to identify dead from viable spores are critical. In this work, we investigate the chemical changes of Bacillus thuringiensis spores treated with sporicidal agents such as chlorine dioxide, peracetic acid, and sodium hypochlorite using laser tweezers Raman spectroscopy. We also image treated spores using SEM and TEM to verify if we can correlate structural changes in the spores with changes to their Raman spectra. We found that over 30 min, chlorine dioxide did not change the Raman spectrum or the spore structure, peracetic acid showed a time-dependent decrease in the characteristic DNA/DPA peaks and similar to 20% of the spores were degraded and collapsed, and spores treated with sodium hypochlorite showed an abrupt drop in DNA and DPA peaks within 20 min and some structural damage to the exosporium. Structural changes appeared in spores after 10 min, compared to the inactivation time of the spores, which is less than a minute. We conclude that vibrational spectroscopy provides powerful means to detect changes in spores but it might be problematic to identify if spores are live or dead after a decontamination procedure.

Bacteria belonging to the orders Bacillales and Clostridiales form spores in response to nutrient starvation. From a simplified morphological perspective, the spore can be considered as comprising a central protoplast or core, that is, enveloped sequentially by an inner membrane (IM), a peptidoglycan cortex, an outer membrane, and a proteinaceous coat. All of these structures are characterized by unique morphological and/or structural features, which collectively confer metabolic dormancy and properties of environmental resistance to the quiescent spore. These properties are maintained until the spore is stimulated to germinate, outgrow and form a new vegetative cell. Spore germination comprises a series of partially overlapping biochemical and biophysical events – efflux of ions from the core, rehydration and IM reorganization, disassembly of cortex and coat – all of which appear to take place in the absence of de novo ATP and protein synthesis. If the latter points are correct, why then do spores of all species examined to date contain a diverse range of mRNA molecules deposited within the spore core? Are some of these molecules “functional,” serving as translationally active units that are required for efficient spore germination and outgrowth, or are they just remnants from sporulation whose sole purpose is to provide a reservoir of ribonucleotides for the newly outgrowing cell? What is the fate of these molecules during spore senescence, and indeed, are conditions within the spore core likely to provide any opportunity for changes in the transcriptional profile of the spore during dormancy? This review encompasses a historical perspective of spore ribonucleotide biology, from the earliest biochemical led analyses – some of which in hindsight have proved to be remarkably prescient – through the transcriptomic era at the turn of this century, to the latest next generation sequencing derived insights. We provide an overview of the key literature to facilitate reasoned responses to the aforementioned questions, and many others, prior to concluding by identifying the major outstanding issues in this crucial area of spore biology.

Contamination of toxic spore-forming bacteria is problematic since spores can survive a plethora of disinfection chemicals. It is also problematic to rapidly detect if the disinfection chemical was active, leaving spores dead. Robust decontamination strategies, as well as reliable detection methods to identify dead from viable spores, are thus critical. Vibrational detection methods such as Raman spectroscopy has been suggested for rapid diagnostics and differentiation of live and dead spores. We investigate in this work, using laser tweezers Raman spectroscopy, the changes in Raman spectra of Bacillus thuringiensis spores treated with sporicidal agents such as chlorine dioxide, peracetic acid, and sodium hypochlorite. We also imaged treated spores using SEM and TEM to verify if any changes to the spore structure can be correlated to the Raman spectra. We found that chlorine dioxide did not change the Raman spectrum or the spore structure; peracetic acid shows a time-dependent decrease in the characteristic DNA/DPA peaks and ∼20 % of the spores were degraded and collapsed; spores treated with sodium hypochlorite show an abrupt drop in DNA and DPA peaks within 20 minutes all though the spore structure was overall intact, however, the exosporium layer was reduced. Structural changes appeared over several minutes, compared to the inactivation time of the spores, which is less than a minute. We conclude that vibrational spectroscopy provides powerful means to detect changes in spores but it might be problematic to identify if spores are live or dead after a decontamination procedure.

Removing arsenic from aquatic environments has become an urgent problem worldwide. In this study, Ti 4+ - loaded bacterial spore were adopted as a novel adsorbent (spore@Ti 4+ microspheres) for the adsorption efficient removal of arsenite (As(III)) and arsenate (As(V)). The developed adsorbents were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectra (FTIR) and X-ray photoelectron spectroscopy (XPS). Results indicated that the adsorption kinetics was well described by a pseudo-second-order kinetic model, and the adsorption process rapidly achieved equilibrium within 15 min at pH 7.0. The adsorption mechanism was also investigated. The maximum adsorption capacities for As(III) and As(V) were 97.26 mg g −1 and 137.01 mg g −1 , respectively, based on the isothermal studies. These properties suggest that spore@Ti 4+ microspheres can be potentially applied in water treatment.

One of the prerequisites of successful address delivery is controlling the release of encapsulated drugs. The new method of bacterial spore encapsulation in polyelectrolyte microcapsules allows for degrading the nanoscale membrane shell of microcapsules. The possibility of encapsulating spore forms of Bacillus subtilis in polystyrenesulfonate sodium/ polyallylamine hydrochloride (PSS/PAH) polyelectrolyte microcapsules was demonstrated. The activation and growth on a nutrient medium of encapsulated bacterial spores led to 60% degradation of the microcapsules nanoscale membrane shell. As a result, 18.5% of Fluorescein isothiocyanatedextran was encapsulated into polyelectrolyte microcapsules, and 28.6% of the encapsulated concentration of FITC-dextran was released into the solution.

Exposure to specific germinant can induce germination in dormant bacterial spores converting them into vegetative cells which are metabolically active and fragile. This phenomenon of conversion of spores from one phase to another could be a keynote potential strategy for development of different type of techniques ranging from spore detection to their eradication and spore-based biosensing. To extend this biphasic spore germination-based approach in order to facilitate development of detection systems, mechanistic details of markers that signals the process of germination initiation in bacterial spores are required. These markers possess a high level of specificity for differentiation in germinating and dormant spores. In this line, present review underscores the structural properties, sporulation and germination in bacterial spores, and covers a detailed description on various biomarkers namely absorbance (600 nm), dipicolinic acid (DPA), refractility of spores, nucleic acid, ATP, spore’s heat resistance, and enzymes which could be valuable in perceiving germination in bacterial spores. Assaying germination using these markers can further explore the efficient use of spores in development of detection devices for food and environment safety.

Since the 2001 anthrax attacks, researchers have focused on the development of a bacterial spore biomarker detector with rapid response time and high sensitivity and selectivity. Hence, a ratiometric fluorescent nanosensor based on lanthanide-functionalized attapulgite (Atta) was designed and prepared using dye-doped Atta as an internal reference. Atta, which is famous because of Maya Blue, is a hydrated magnesium aluminum silicate present in nature, with pronounced adsorption and a large specific surface area rich in silicon hydroxyl. The adsorption of dye molecules benefits the formation of an internal reference with orange fluorescence. Meanwhile, silicon hydroxyl would be available for terbium (Tb 3+ ) complex grafting, which favors the sensitization of the bacterial spore biomarker. With increasing dipicolinic acid (DPA) concentrations, energy transfer from DPA to Tb 3+ gradually enhanced, thereby resulting in strong and predominant green fluorescence. The ratiometric fluorescence intensity is highly sensitive. It has a limit of detection of 9.8 nM, which is much lower than the infectious dosage of Bacillus anthracis spores (60 μM) for humans. The rapid response time (10s), high selectivity, and reliable and practical performances in actual applications make this ratiometric fluorescent nanosensor useful in the detection of biomarkers for various bacterial spores. Moreover, the reported construction of visual ratiometric detection system follows the sustainable development idea of ‘‘from nature, for nature, into the nature’’ and promotes the use of naturally renewable resources to produce promising and eco-friendly functional materials for various applications.

The advantage of using high pressure (HP) cycling treatment compared with continuous HP treatment was investigated for the inactivation of bacterial spores. The effects of parameters such as pulse number, pressure level, treatment temperature, compression and decompression rates, and time between pulses were evaluated. For this purpose, Bacillus subtilis and B. cereus spores (108 and 106 CFU/mL respectively) were suspended in 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution, tryptone salt (TS) buffer solution, or infant milk and treated by HP cycling at 300–400 MPa, at 38–60 °C, for 1–5 pulses. Pressure cycling reduced the number of viable spores by 1.8 and 5.9 log respectively for B. subtilis and B. cereus species. Continuous HP treatments were performed at the same pressure and temperature for similar treatment durations. Our results showed that the spore inactivation ratio was correlated with the cumulative exposure time to pressure rather than to effects of the cycling process. Greater spore inactivation caused by HP cycling was observed only when faster compression and decompression rates were applied, probably due to adiabatic heating. A three-step kinetic model was developed, which seemed to support our hypothesis regarding the mechanisms of inactivation by pressure cycling and continuous HP treatments. Industrial relevance The resistance of bacterial spores to HP limits the industrial applications to refrigerated food products. In this study, we investigated the use of pressure cycling as a means to improve spore baroinactivation at moderate temperatures (T

We report an extremely sensitive and ultra-low-cost PVDF-based SERS substrate using an easy fabrication process that can be conducted in a laboratory and scaled up for industrial applications. Hydrophilic PVDF membranes were sequentially dipped in ethanol and aqueous sodium chloride solution, following which multi-shaped Ag nanoparticles were deposited on the surface of the membranes by suction filtration. The simple solvating pretreatment led to an approximately 188-fold enhancement in the SERS signal intensity of R6G. Using the hydrophilic PVDF-based SERS substrate, subnanomolar sensitivity for R6G was achieved using 3 μL of sample volume on a 3×3 mm2 SERS substrate within 1 min detection time and without the need for concentration or drying processes. To demonstrate the feasibility of the developed SERS substrate as a sensor for bacterial spore detection, it was applied for the detection of the representative biomarker dipicolinic acid (DPA). The limits of detection of DPA and Bacillus spores were measured as 1 ppm and 5×103 spores/mL, respectively. The optimized configuration of low substrate area and wet micro-analyte volume makes the PVDF-based SERS substrate a simple and low-cost tool for the trace-level detection of small molecules in a rapid, sensitive, and high-throughput manner.

cited By 3; International audience; In this work, our goal was to study the capability of a single metallic oxide to neutralize a chemical agent and to exhibit an antibacterial effect. We tested two types of magnesium oxides, MgO. The first MgO sample tested, which commercial data size characteristic was −325 mesh (MgO-1) destroyed in 3 h, 89.7% of paraoxon and 93.2% of 4-nitrophenol, the first degradation product. The second MgO sample, which commercial data size was

Numerous cases have been reported globally showing outbreaks, dissemination of infections and/or spoilage of medicinal products, food and other consumables that affects human life and may lead to death in some cases. One of the most critical measures of microbial contamination that should be taken into consideration is the use of proper disinfectant depending on the type of activities and the work load of the healthcare institution, subjects or media for microbial transfer and the affected population. The current study shows significant difference in sporicidal activity between two types of commercial peroxygen/Silver based disinfectants obtained from same manufacturer and delivered through same distributor at the same time. The peroxygen component in one of the disinfectant products is Hydrogen peroxide while in the other product is Peroxyacetic acid/Hydrogen Peroxide mixture. Preliminary rapid assessment of the disinfectants activity was required using the most resistance microbial form (bacterial spore) as a reference microorganism to challenge the biocidal products. Bacillus pumilus and B. subtilis spores were exposed to both disinfectants at three different concentrations levels covering the range recommended by the manufacturer: 1, 3 and 5% (v/v). The first formula did not exceed 0.5 logarithmic reduction(LR) even after 30 minutes. While the other product achieved more than two folds LR (more than 100 times reduction in microbial population) after ten minutes contact time. Appropriate initial screening of biocidal activity in commercial disinfectants market is critical step that should be performed by the healthcare facilities before practical application. Otherwise, inefficient control on bioburden may lead to devastating consequences on human health. An initial, non-laborious, time-saving and non-expensive screening test using the most resistance microorganisms is encouraged to be performed by healthcare facilities prior to practical application of disinfectant rather than reliance solely on random selection of biocidal agents using informational data without confirmatory experiments.

Concrete cracks must be repaired promptly in order to prevent structural damage and to prolong the structural life of the building (or other such construction). Biological self-healing concrete is a recent alternative technology involving the biochemical reaction of microbial induced calcium carbonate precipitation (MICP). This study determined the most appropriate technique to encapsulate spores of Bacillus sphaericus LMG 22257 with sodium alginate so as to protect the bacterial spores during the concrete mixing and hardening period. Three techniques (extrusion, spray drying and freeze drying) to encapsulate the bacterial spores with sodium alginate were evaluated. The freeze-drying process provided the highest bacterial spore survival rate (100%), while the extruded and spray-dried processes had a lower spore survival rate of 93.8% and 79.9%, respectively. To investigate the viability of microencapsulated spores after being mixed with mortar, the decomposed urea analysis was conducted. The results revealed that the freeze-dried spores also showed the highest level of urea decomposition (metabolic activity assay used as a surrogate marker of spore germination and vegetative cell viability). Thus, the self-healing performance of concrete mixed with freeze-dried spores was evaluated. The results showed that the crack healing ratio observed from the mortar specimens with freeze-dried microencapsulated spores were significantly higher than those without bacteria. This study revealed that freeze drying has a high potential as a microencapsulation technique for application to self-healing concrete technology.

Background Bacillus 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. Results Conserved 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. Conclusions The 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. Electronic supplementary material The online version of this article (10.1186/s12866-019-1544-1) contains supplementary material, which is available to authorized users.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo Alkalization is a step of cocoa processing and consists of the use of alkali and high temperature to improve the sensorial and technological qualities of cocoa. Intense food processing can select spores, which can compromise safety and quality of the final product. Thus, the aim of this study was to evaluate the fate of B. cereus and G. stearothermophilus spores during the alkalization of pre-roasted (Pr) nibs (held at 120 °C) and unroasted (Ur) nibs (held at 90 °C) using potassium carbonate (0, 2, 4 and 6% w/w). In all conditions, log-linear inactivation kinetics with a tail was observed. The inactivation rate (kmax) for B. cereus varied from 0.065 to 1.67 min−1, whereas the kmax for G. stearothermophilus varied from 0.012 to 0.063 min−1. For both microorganisms, the lowest kmax values were observed during Ur nibs alkalization. The carbonate concentration increase promoted kmax values reduction. The highest tail values were observed for G. stearothermophilus in Ur nibs alkalization, reaching 3.04 log spores/g. Tail formation and low kmax values indicated that cocoa alkalization does not cause significant reductions on bacterial spore population. Therefore, the microbiological control should be primarily ensured by the raw material quality and by avoiding recontamination in the cocoa

UNLABELLED Previous work reported that decoated Bacillus cereus spores incubated in 4 mol l(-1) CaCl2 are killed at lower temperatures than spores in water. This wet heat sensitization was suggested to support a role for an osmoregulatory peptidoglycan cortex in spore cores' low water content, and their wet heat resistance. Current work has replicated this finding with spores of B. cereus, Bacillus megaterium and Bacillus subtilis. However, this work found that decoated spores apparently killed at 80°C in 4 mol l(-1) CaCl2 : (i) were recovered on plates containing lysozyme; (ii) lost no dipicolinic acid (DPA) and their inner membrane remained impermeable; (iii) released no DPA upon stimulation with nutrient germinants and could not complete germination; and (iv) released DPA relatively normally upon stimulation with dodecylamine. These results indicate that decoated spores treated with 80°C- 4 mol l(-1) CaCl2 are not dead, but some protein(s) essential for spore germination, most likely germinant receptors, are inactivated by this treatment. Thus, the original finding does not support a role for an osmoregulatory cortex in spore wet heat resistance. SIGNIFICANCE AND IMPACT OF THE STUDY Bacillus spores' low core water content is a major factor in their wet heat resistance. One suggested mechanism for achieving low spore core water content is osmoregulated expansion of spores' peptidoglycan cortex. Evidence for this mechanism includes a report that decoated Bacillus cereus spores incubated in 4 mol l(-1) CaCl2 exhibit drastically reduced heat resistance. The current work shows that this heat sensitization of decoated spores of three Bacillus species is most likely due to inactivation of some crucial spore germination protein(s), since while treated spores appear dead, their apparent low viability is rescued by triggering spore germination with lysozyme.

This theoretical effort is the first to explore the possible hypothesis that terahertz optical activity of Bacillus spores arises from normal vibrational modes of spore coat subcomponents in the terahertz frequency range. Bacterial strains like Bacillus and Clostridium form spores with a hardened coating made of peptidoglycan to protect its genetic material in harsh conditions. In recent years, electron microscopy and atomic force microscopy has revealed that bacterial spore surfaces are decorated with nanocylinders and honeycomb nanostructures. In this article, a simple elastic continuum model is used to describe the vibration of these nanocylinders mainly in Bacillus subtilis, which also leads to the conclusion that the terahertz signature of these spores arises from the vibration of these nanostructures. Three vibrating modes: radial/longitudinal, torsional and flexural, have been identified and discussed for the nanocylinders. The effect of bound water, which shifts the vibration frequency, is also discussed. The peptidoglycan molecule consists of polar and charged amino acids; hence, the sporal surface local vibrations interact strongly with the terahertz radiation.

International audience; In foodstuffs, physico-chemical interactions and/or physical constraints between spores, inhibitors and food components may exist. Thus, the objective of this study was to investigate such interactions using a model emulsion as a microbial medium in order to improve bacterial spore control with better knowledge of the interactions in the formulation.Emulsions were prepared with hexadecane mixed with nutrient broth using sonication and were stabilized by Tween 80 and Span 80. The hexadecane ratio was either 35% (v/v) or 50% (v/v) and each emulsion was studied in the presence of organic acid (acetic, lactic or hexanoic) at two pH levels (5.5 and 6). Self-diffusion coefficients of emulsion components and the organic acids were measured by Pulsed Field Gradient-Nuclear Magnetic Resonance (PFG-NMR). The inhibition effect on the spore germination and cell growth of Bacillus weihenstephanensis KBAB4 was characterized by the measure of the probability of growth using the most probable number methodology, and the measure of the time taken for the cells to germinate and grow using a single cell Bioscreen® method and using flow cytometry. The inhibition of spore germination and growth in the model emulsion depended on the dispersed phase volume fraction and the pH value. The effect of the dispersed phase volume fraction was due to a combination of (i) the lipophilicity of the biocide, hexanoic acid, that may have had an impact on the distribution of organic acid between hexadecane and the aqueous phases and (ii) the antimicrobial activity of the emulsifier Tween 80 detected at the acidic pH value. The interface phenomena seemed to have a major influence. Future work will focus on the exploration of these phenomena at the interface.

This report summarizes the current state of knowledge on the persistence of biological agents on drinking water infrastructure (such as pipes) along with information on decontamination should persistence occur. Decontamination options for drinking water infrastructure have been explored for some biological agents, but data gaps remain. Data on bacterial spore persistence on common water infrastructure materials such as iron and cement-mortar lined iron show that spores can be persistent for weeks after contamination. Decontamination data show that common disinfectants such as free chlorine have limited effectiveness. Decontamination results with germinant and alternate disinfectants such as chlorine dioxide are more promising. Persistence and decontamination data were collected on vegetative bacteria, such as coliforms, Legionella and Salmonella. Vegetative bacteria are less persistent than spores and more susceptible to disinfection, but the surfaces and water quality conditions in many studies were only marginally related to drinking water systems. However, results of real-world case studies on accidental contamination of water systems with E. coli and Salmonella contamination show that flushing and chlorination can help return a water system to service. Some viral persistence data were found, but decontamination data were lacking. Future research suggestions focus on expanding the available biological persistence data to other common infrastructure materials. Further exploration of non-traditional drinking water disinfectants is recommended for future studies.

Clostridia in the milk can lead to late blowing, a cheese defect. Clostridia are ubiquitous, deriving from both the farm environment and the feed ingested by the cows, and are transferred into the milk through faecal contamination. Our aim was to investigate the effect of different in-parlour practices on the content of anaerobic spore-forming bacteria in milk, and to monitor the variation in spore content in the feed and environment. The experiment, conducted in an experimental dairy during autumn, was repeated in exactly the same way for two consecutive years. The experimental design applied three different milking routines in three consecutive 7-d periods: forestripping alone (F); forestripping and post-dipping (F+Post); pre-dipping, wiping, forestripping and post-dipping (Pre+F+Post). Teat skin swabs and samples of feed, faeces, bedding materials and milk were collected for microbiological analyses. The dietary forage of the lactating cows included maize silage, which, in both years, was found to have the highest level of clostridial spore contamination. Pre-dipping with a detergent/emollient solution, and drying with a disposable paper towel, proved much more efficient in reducing spore contamination than forestripping alone, both on the teats (1·30 vs. 2·20 log10 MPN/swab; P < 0·001) and in the milk (1·82 vs. 2·47 log10 MPN/L, P < 0·02), while post-dipping had little influence on spore count. The standard plate count in milk was significantly lower with Pre+F+Post treatment than with F (3·80 vs. 4·51 log10 CFU/mL, P < 0·01). The teat preparation procedure did not influence the lactic acid bacterial levels in the milk, which is very positive in that decreased lactic acid bacterial content can lessen raw milk cheese quality.

Outgrowth heterogeneity of bacterial spore populations complicates both prediction and efficient control of spore outgrowth. In this study, the impact of mild preservation stresses on outgrowth of Bacillus cereus ATCC 14579 spores was quantified during the first stages of outgrowth. Heterogeneity in outgrowth of heat-treated (90 °C for 10 min) and non-heat-treated germinated single spores to the maximum micro-colony stage of 256 cells was assessed by direct imaging on Anopore strips, placed on BHI plates at pH 7 and pH 5.5, without and with added NaCl or sorbic acid (HSA). At pH 7 non-heated and heat-treated germinated spores required 6 h to reach the maximum microcolony stage with limited heterogeneity, and these parameters were only slightly affected with both types of spores when incubated at pH 7 with added NaCl. Notably, the most pronounced effects were observed during outgrowth of spores at pH 5.5 without and with added NaCl or HSA. Non-heat-treated germinated spores showed again efficient outgrowth with limited heterogeneity reaching the maximum microcolony stage after 6 h at pH 5.5, which increased to 12 h and 16 h with added NaCl and HSA, respectively. In contrast, heat-treated spores displayed a strong delay between initial germination and swelling and further outgrowth at pH 5.5, resulting in large heterogeneity and low numbers of fastest growers reaching the maximum microcolony stage after 10, 12 and 24 h, without and with added NaCl or HSA, respectively. This work shows that Anopore technology provides quantitative information on the impact of combined preservation stresses on outgrowth of single spores, showing that outgrowth of germinated heat-treated spores is significantly affected at pH 5.5 with a large fraction of spores arrested in the early outgrowth stage, and with outgrowing cells showing large heterogeneity with only a small fraction committed to relatively fast outgrowth. Keywords Bacillus cereus; Spores; Outgrowth; Heterogeneity; Anopore; Stress

In multiple operational scenarios, explosive charges are used to neutralize confined or unconfined stores of bacterial spores. The spore destruction is achieved by post-detonation combustion and mixing of hot detonation product gases with the ambient flow and spore clouds. In this work, blast wave interaction with bacterial spore clouds and the effect of post-detonation combustion on spore neutralization are investigated using numerical simulations. Spherical explosive charges (radius, $$R_\mathrm{C}$$ = 5.9 cm) comprising of nitromethane are modeled in the vicinity of a spore cloud, and the spore kill in the post-detonation flow is quantified. The effect of the mass of the spores and the initial distance, $$d^0$$ , of the spore cloud from the explosive charge on the percentage of spores neutralized is investigated. When the spores are initially placed within a distance of 3.0 $$R_\mathrm{C}$$ , within 0.1 ms after detonation of the charge, all the spores are neutralized by the blast wave and the hot detonation product gases. In contrast, almost all the spores survived the explosion when $$d^0$$ is greater than 8.0 $$R_\mathrm{C}$$ . The percentage of intact spores varied from 0 to 100 for 3.0 $$R_\mathrm{C}$$ $$

This work reports the activity (at various conditions) of conessine isolated from methanolic extract of Holarrhena floribunda, on the inhibition of the germination of two Bacillus spores species. This activity was studied by treating spores of Bacillus cereus T and Bacillus stearothermophilus CNCH 5781 with effective concentrations of conessine at various temperature, pH and treatment times. The inhibition of germination was evaluated by the culture of treated spores on agar medium and the number of colony obtained was compared with that of control culture (not treated with conessine). We found that conessine used at 50 and 100 µg/ml for 20 min each decreased considerably the germination of spore of B. cereus T and B. stearothermophilus CNCH 5781. The maximum temperature of conessine activity for B. cereus T was at 30 and 60°C for B. stearothermophilus CNCH 5781 spores. Furthermore, the activity of conessine was sensitive to pH change and was more effective at pH 6 on both bacterial spore strains. The treatment of spores with conessine at various lengths of time demonstrated that, the activity of the compound on both bacterial spores was strongly related to the bacterial species. This study suggested that the activity of conessine on the inhibition of germination of Bacillus spore depends on physico-chemical factors and the bacterial species.

Classically, dormant endospores are defined by their resistance properties, particularly their resistance to heat. Much of the heat resistance is due to the large amount of dipicolinic acid (DPA) stored within the spore core. During spore germination, DPA is released and allows for rehydration of the otherwise-dehydrated core. In Bacillus subtilis, 7 proteins are encoded by the spoVA operon and are important for DPA release. These proteins receive a signal from the activated germinant receptor and release DPA. This DPA activates the cortex lytic enzyme CwlJ, and cortex degradation begins. In Clostridium difficile, spore germination is initiated in response to certain bile acids and amino acids. These bile acids interact with the CspC germinant receptor, which then transfers the signal to the CspB protease. Activated CspB cleaves the cortex lytic enzyme, pro-SleC, to its active form. Subsequently, DPA is released from the core. C. difficile encodes orthologues of spoVAC , spoVAD , and spoVAE . Of these, the B. subtilis SpoVAC protein was shown to be capable of mechanosensing. Because cortex degradation precedes DPA release during C. difficile spore germination (opposite of what occurs in B. subtilis), we hypothesized that cortex degradation would relieve the osmotic constraints placed on the inner spore membrane and permit DPA release. Here, we assayed germination in the presence of osmolytes, and we found that they can delay DPA release from germinating C. difficile spores while still permitting cortex degradation. Together, our results suggest that DPA release during C. difficile spore germination occurs though a mechanosensing mechanism. IMPORTANCE Clostridium difficile is transmitted between hosts in the form of a dormant spore, and germination by C. difficile spores is required to initiate infection, because the toxins that are necessary for disease are not deposited on the spore form. Importantly, the C. difficile spore germination pathway represents a novel pathway for bacterial spore germination. Prior work has shown that the order of events during C. difficile spore germination (cortex degradation and DPA release) is flipped compared to the events during B. subtilis spore germination, a model organism. Here, we further characterize the C. difficile spore germination pathway and summarize our findings indicating that DPA release by germinating C. difficile spores occurs through a mechanosensing mechanism in response to the degradation of the spore cortex.

Summary Spores of Bacillus species can remain dormant and resistant for years, but can rapidly ‘come back to life’ in germination triggered by agents, such as specific nutrients, and non-nutrients, such as CaDPA, dodecylamine and hydrostatic pressure. Major events in germination include release of spore core monovalent cations and CaDPA, hydrolysis of the spore cortex peptidoglycan (PG) and expansion of the spore core. This leads to a well-hydrated spore protoplast in which metabolism and macromolecular synthesis begin. Proteins essential for germination include the GerP proteins that facilitate germinant access to spores' inner layers, germinant receptors (GRs) that recognize and respond to nutrient germinants, GerD important in rapid GR-dependent germination, SpoVA proteins important in CaDPA release and cortex-lytic enzymes that degrade cortex PG. Rates of germination of individuals in spore populations are heterogeneous, and methods have been developed recently to simultaneously analyse the germination of multiple individual spores. Spore germination heterogeneity is due primarily to large variations in GR levels among individual spores, with spores that germinate extremely slowly and termed superdormant having very low GR levels. These and other aspects of spore germination will be discussed in this review, and major unanswered questions will also be discussed.

A previous report indicated that a formic acid chemical extraction method for the preparation of protein extracts for matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) identification, with filtration of extracts through 0.2 μm regenerated cellulose (RC) filters, would not reliably inactivate or exclude Bacillus anthracis Vollum cells or spores when tested under high stringency conditions. B. anthracis was recovered from 13/36 extracts (3/18 from vegetative cell extracts and 10/18 from bacterial spore extracts). In this paper we report the repetition of this study but with the substitution of the 0.2 μm, regenerated cellulose, filters with 0.1 μm polyvinylidene fluoride (PVDF) filters. Experiments were conducted under the same high stringency post-treatment viability test methods (100% of resulting protein content; 7 days Luria (L)-broth and a further 7 days L-agar plate incubation; or 7 days L-agar plate only incubation). B. anthracis was not recovered from any of 18 replicates generated from high concentrations of vegetative cells (107 to 108 cfu), but a single B. anthracis colony was recovered from one of 18 replicates generated from high concentrations of bacterial spores (108 cfu), using a post-treatment viability culture method of 7 days on L-agar plate only. We discuss our results in the context of other similar studies and also a requirement to develop standardised post-treatment viability test methods.

Since bacterial spores, such as Bacillus anthracis spores, are extremely hazardous to human beings and animals, efforts have focused on the development of bacterial spore detector with rapid response and high selectivity and sensitivity. Therefore, we reported a facile one-step chelating-reagent-assisted hydrothermal synthesis of lanthanide-doped fluorapatite (FA) nanoprobes for detecting the biomarker of bacterial spores. In FA synthesis, ethylenediaminetetraacetic acid (EDTA) can serve not only as a shape controller and a stabilizer but also as a chelating reagent for lanthanide ions. After the fabrication, terbium (or europium) ions were not only embedded into lattice models of FA nanocrystals, but also chelated by EDTA on the surface of the FA nanocrystals, which can be available for detecting the biomarker (i.e., dipicolinic acid) of bacterial spores. The obtained FA: Ln–EDTA nanoprobes can provide reference fluorescence which play crucial roles in the calibration and correction of concentration. The fluorescence intensities of FA: Tb–EDTA or FA: Eu–EDTA from Tb3+ or Eu3+ emission were highly sensitive and increased gradually with increasing DPA concentrations. The limit of detection of FA: Tb–EDTA was 8.2 nM, which is lower than that of FA: Eu–EDTA (20.9 nM). It can be attributed to the fact that DPA can achieve a more effective energy transfer to Tb3+ than to Eu3+. Additionally, the nanoprobes were successfully applied for the determination of DPA in fetal calf serum samples. Given their rapid response and high sensitivity and selectivity, these ratiometric fluorescent nanoprobes are promising tools for the detection of bacterial spores.

Aims To determine the mechanism of autoclave killing of Geobacillus stearothermophilus spores used in biological indicators (BIs) for steam autoclave sterilization, and rates of loss of spore viability and a spore enzyme used in BIs. Methods and Results Spore viability, dipicolinic acid release, nucleic acid staining, α-glucosidase activity, protein structure and mutagenesis were measured during autoclaving of G. stearothermophilus spores. Loss of dipicolinic acid and increases in spore core nucleic acid staining were slower than loss of spore viability. Spore core α-glucosidase was also lost more slowly than spore viability, although soluble α-glucosidase in spore preparations was lost more rapidly. However, spores exposed to an effective autoclave sterilization lost all viability and α-glucosidase activity. Apparently killed autoclaved spores were not recovered by artificial germination in supportive media, much spore protein was denatured during autoclaving, and partially killed autoclave-treated spore preparations did not acquire mutations. Conclusions These results indicate that autoclave-killed spores cannot be revived, spore killing by autoclaving is likely by protein damage, and spore core α-glucosidase activity is lost more slowly than spore viability. Significance and Impact of Study This work provides insight into the mechanism of autoclave killing of spores of an organism used in BIs, and that a spore enzyme in a BI is more stable to autoclaving than spore viability. This article is protected by copyright. All rights reserved.

The sporicidal effect of hydrogen peroxide (H2O2) in the sterilization of low density polyethylene film (LDPE) was evaluated using a Central Composite Design (CCD). The effects of contact time (5-19s), bath temperature (23–70 °C) and concentration of H2O2 (0–35%) in an immersion bath were investigated. A 16 cm2 film surface was evenly inoculated with 100 μL of the test microorganism Bacillus subtilis var. globigii ATCC9372 spores. The effective H2O2 sporicidal activity was demonstrated at concentrations from 18 to 35% and in a temperature range from 46 to 70 °C, resulting in 2–7 decimal reductions of B. subtilis spores. A quadratic mathematical model representative of the action of H2O2 on the B. subtilis spores was developed as a function of concentration, time and temperature. Test specimens sanitized with 28% H2O2 at 60 °C for 8 s showed 4 decimal reductions. In the same sterilization procedure, but extending the time to 16 s, this value increased to 7 decimal reductions, demonstrating the efficiency of H2O2 as a function of contact time. The sterilization system tested showed satisfactory performance in the sterilization of LDPE films, being capable of reaching up to 7 decimal reductions of the bacterial spore population.

A basic approach was optimized for the synthesis of highly selective and sensitive in situ mesoporous (MCM) type imprinted silica polymers for the detection of dipicolinic acid (DPA) using europium as a reporter. DPA is a ubiquitous biochemical marker available during the germination event of endospore-forming bacteria such as Bacillus . Additionally, an MCM-MIP (molecularly imprinted polymeric phenomena) detector and a companion MCM-non-surface-MIP detector were synthesized using europium reporters for the sensing of DPA under optimized laboratory conditions. Our results showed that the in situ molecular imprinting process enabled rapid, selective detection of DPA with high sensitivity compared to MCM-MIP (imprinted for DPA; no DPA present), MCM-Non-MIP (no imprint present), and MCM-SR-MIP (imprinted with DPA present) detectors. The lower detection limit observed for DPA concentration is 5.49 × 10(-10) mol dm(-3) for MCM-MIP. The performance of the sensor in high-salt-water conditions, under photo-bleaching, and its reusability were also evaluated. The synthesized in situ MCM-MIP material should permit the detection of DPA for field assays related to suspect bacterial sporulation events.

Carbohydrate-protein interaction is one of the most important molecular events deemed critical for numerous biological processes. Therefore, understanding this interaction is essential. In this study, we used bacterial spore display techniques to present multiple copies of streptavidin on the surface of spores to explore carbohydrate-protein interaction in solution and on chips. By applying bacterial spores displaying streptavidin, we developed a new method which allows sensitive, versatile, and passive detection of carbohydrate-protein interactions with a 10-fold increase in sensitivity. The linear relationship of interactions between carbohydrates and labeled concanavalin A (con A) in solution and on functionalized bacterial spore chips has also been confirmed. To the best of our knowledge, this is the first example of development and characterization of binding behavior in carbohydrateprotein interactions using bacterial spore-displayed streptavidin. We believe this strategy may enable new high-throughput screening of carbohydrate interactions as well as establish a basis for monitoring inhibitors of carbohydrate-binding proteins when developing new drugs.

Spores of Geobacillus stearothermophilus and Bacillus subtilis adhering to the surface of stainless steel (SS) and polypropylene (PP) were subjected to rinsing in water or NaOH solution to study their removability. The spores were made adherent through deposition from suspension under humid and dry conditions. The humid condition kept the surface wet throughout the deposition, whereas the dry condition made them dry up finally. Experimental results demonstrated that drying significantly enhanced both quantity and strength of the spore adhesion for both types of surface. No removal of the spores adhering to surfaces exposed to dry environment was observed through water rinsing with shear force application. Even with 0.2% NaOH, they were hardly removed irrespective of temperature. Although 1% NaOH removed or inactivated most of spores on SS surface, it left considerable viable spores on PP surface. Thus enhanced alkali tolerance was indicated for spores adhering to surfaces, especially made of PP, exposed to dry environment.

The mineralization of dipicolinic acid (DPA) molecules in bacterial spore cores with Ca2+ ions to form Ca-DPA is critical to the wet-heat resistance of spores. This resistance to “wet-heat” also depends on the physical properties of water and DPA in the hydrated Ca-DPA-rich protoplasm. Using reactive molecular dynamics simulations, we have determined the phase diagram of hydrated Ca-DPA as a function of temperature and water concentration, which shows the existence of a gel phase along with distinct solid-gel and gel-liquid phase transitions. Simulations reveal monotonically decreasing solid-gel-liquid transition temperatures with increasing hydration, which explains the experimental trend of wet-heat resistance of bacterial spores. Our observation of different phases of water also reconciles previous conflicting experimental findings on the state of water in bacterial spores. Further comparison with an unmineralized hydrated DPA system allows us to quantify the importance of Ca mineralization in decreasi...

Protein A chromatography media require sanitization between batches as well as prior to long-term storage. While sodium hydroxide (NaOH) is probably one of the most widely used sanitants within the bioprocess industry, it cannot be used with silica- or controlled pore glass (CPG)-based adsorbents due to the instability of the base matrix at high pH. Benzyl alcohol is commonly used for sanitizing such adsorbents, though extended contact times may be required to meet desired microbial log reduction values, especially for fungal and bacterial spore formers. With the rising market need for monoclonal antibody therapeutics, higher manufacturing throughput may be required. In such cases, a shorter sanitization cycle would be extremely beneficial to maximize manufacturing throughput and productivity. This paper describes the development of a new synergistic sanitant solution, designated PAB (120 mM phosphoric acid, 167 mM acetic acid, 2.2% benzyl alcohol) that delivers improved microbial kill kinetics, enabling sanitization times of 2-3h at room temperature, while maintaining acceptable adsorbent stability. Both the approaches taken to establish the effectiveness of the improved solution as well as confirmation of its process compatibility are covered here.

To test the suitability of DNA stable isotope probing (DNA-SIP) for characterizing bacterial spore populations in soils, the properties of Bacillus subtilis cells and spores intensely labeled with [(13)C]glucose were characterized. Spore germination, vegetative growth rates, and sporulation efficiency were indistinguishable on glucose versus [(13)C]glucose, as were spore wet heat and UV resistance. Unlabeled and (13)C-labeled spores contained 1.0989 and 74.336 at.% (13)C, and exhibited wet densities of 1.356 and 1.365 g/ml, respectively. Chromosomal DNAs containing (12)C versus (13)C were readily separated by their different buoyant densities in cesium chloride/ethidium bromide gradients.

Strains of Clostridium perfringens that cause acute food poisoning have been shown to produce spores that are significantly more heat resistant than those of other strains. Previous studies demonstrated that the spore core density and the ratio of spore cortex peptidoglycan relative to the germ cell wall were factors that correlated with the heat resistance of a C. perfringens spore. To further evaluate these relationships, mutant strains of C. perfringens SM101 were constructed with null mutations in dacF, encoding a D,D-carboxypeptidase, and in the spmA-spmB operon, which is involved in spore core dehydration. The dacF mutant was shown to produce less spore cortex peptidoglycan and had a corresponding decrease in spore heat resistance. The spmA-spmB strain produced highly unstable spores with significantly lower core densities and increased heat sensitivity, which were easily destroyed during treatments affecting the spore coat layers. These results support the previous assertion that a threshold core density as well as a high ratio of cortex peptidoglycan relative to the germ cell wall contribute to the formation of a more heat-resistant spore in this species.

Bacterial spores are resistant to a wide range of chemical and physical insults that are normally lethal for the vegetative form of the bacterium. While the integrity of the protein coat of the spore is crucial for spore survival in vitro, far less is known about how the coat provides protection in vivo against predation by ecologically relevant hosts. In particular, assays had characterized the in vitro resistance of spores to peptidoglycan-hydrolyzing enzymes like lysozyme that are also important effectors of innate immunity in a wide variety of hosts. Here, we use the bacteriovorous nematode Caenorhabditis elegans , a likely predator of Bacillus spores in the wild, to characterize the role of the spore coat in an ecologically relevant spore-host interaction. We found that ingested wild-type Bacillus subtilis spores were resistant to worm digestion, whereas vegetative forms of the bacterium were efficiently digested by the nematode. Using B. subtilis strains carrying mutations in spore coat genes, we observed a correlation between the degree of alteration of the spore coat assembly and the susceptibility to the worm degradation. Surprisingly, we found that the spores that were resistant to lysozyme in vitro can be sensitive to C. elegans digestion depending on the extent of the spore coat structure modifications.

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 Nacetylglucosamine and muramic--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. Bacterial endospores are metabolically dormant and are highly resistant to many treatments that rapidly kill vegetative cells. The resistance properties are dependent on the relative dehydration of the spore core or cytoplasm and the high concentrations of calcium dipicolinic acid (DPA) and other core solutes (26). When exposed to a nutrient-rich environment, spores initiate germination and outgrowth into vegetative cells. Nutrient germinant molecules bind to Ger protein family receptors in the inner forespore membrane surrounding the spore core and trigger biophysical changes (20). The spore becomes partially rehydrated as water enters the spore core, and the calcium DPA that was in the core is released into the environment. To complete the germination process and resume metabolism (11, 24), the spore must go through a second stage, which is more biochemical (20). This involves activation of spore cortex peptidoglycan (PG) lytic enzymes, which are present in an inactive state within the dormant spore. As the cortex is degraded, the cell expands to its full size and regains full metabolic activity. Due to the key role that cortex lytic enzymes play in germination, they are potential targets for spore decontamination methods that could involve either irreversible inactivation or gratuitous activation of the enzymes. The latter would lead to premature spore germination into vegetative forms that are much more easily killed. PG consists of glycan strands with alternating residues of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) that are cross-linked by peptide side chains located on the NAM residues (Fig. 1). While the PG structure in vegetative cells can vary between species, primarily in the amino acid composition of the peptide side chains, spore PG structure has been found to be highly conserved across species (3, 30). The peptide side chains contain L-alanine, D-glutamate, meso-diaminopimelic acid (Dpm), and D-alanine. The cross-linking occurs between the Dpm on one strand and the D-alanine of a tetrapeptide on an adjacent strand. The spore PG is composed of the inner, germ cell wall layer (10 to 20% of the PG), which resembles the PG of vegetative cells (6, 19), and the cortex, which has important and unique chemical modifications (2, 3, 5, 23, 28, 30). In the cortex, approximately 50% of the muramic acid residues (the residues on every alternate disaccharide) have their peptide side chains completely removed, and these NAM residues are converted to muramic--lactam. In the Bacillus species previously examined, 25% of the cortex muramic acid residues have tetrapeptide side chains, while the majority of the remaining residues have their peptides cleaved to single L-alanine residues. Both the shortening and the removal of peptide side chains cause a decrease in cross-linking

Bacteria living in soil collected from a rice paddy in Fukuoka, Japan, were examined by electron microscopy using a freeze-substitution fixation method. Most of the observed bacteria could be categorized, based on the structure of the cell envelope and overall morphology, into one of five groups: (i) bacterial spore; (ii) Gram-positive type; (iii) Gram-negative type; (iv) Mycobacterium like; and (v) Archaea like. However, a few of the bacteria could not be readily categorized into one of these groups because they had unique cell wall structures, basically resembling those of Gram-negative bacteria, but with the layer corresponding to the peptidoglycan layer in Gram-negative bacteria being extremely thick, like that of the cortex of a bacterial spore. The characteristic morphological features found in many of these uncultured, soil-dwelling cells were the nucleoid being in a condensed state and the cytoplasm being shrunken. We were able to produce similar morphologies in vitro using a Salmonella sp. by culturing under low-temperature, low-nutrient conditions, similar to those found in some natural environments. These unusual morphologies are therefore hypothesized to be characteristic of bacteria in resting or dormant stages.

Holographic sensors for the detection of Bacillus species spore germination and vegetative growth are described. Reflection holograms were fabricated using a diffusion method for the distribution of ultra-fine silver bromide grains into pre-formed polymer films, followed by holographic recording using a frequency doubled Nd:YAG (532 nm) laser. Changes in holographic replay wavelength or diffraction intensity were used to characterise the swelling behaviour or structural integrity of a range of holographic matrices in response to various extracellular products of bacterial spore germination and vegetative metabolism. Divalent metal ion-sensitive holograms containing a methacrylated analogue of nitrilotriacetic acid (NTA) as the chelating monomer were successfully used to monitor Ca2+ ions released during B. subtilis spore germination in real-time, which was within minutes of sample addition; the holographic response manifested as a 16 nm blue-shift in diffraction wavelength over the progress of germination. Similarly, pH-sensitive holograms comprising methacrylic acid (MAA) as the ionisable monomer were responsive to changes in pH associated with early vegetative metabolism following germination of B. megaterium spores; a visually perceptible blue-shift in holographic replay wavelength of 75 nm was observed. Casein and starch-based holographic matrices, prepared by co-polymerisation of the appropriate substrate with acrylamide, were used to detect exo-enzymes released during later stages of B. megaterium and B. subtilis vegetative cell growth; holographic responses of both matrices were visible as a reduction in diffraction intensity due to progressive fringe disruption caused by enzymatic cleavage. The combined monitoring of various germination and growth events using the range of aforementioned holographic sensors provides a novel, comprehensive means for the detection of viable bacterial spores.

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) in a 1:1 chelate with calcium ion (Ca-DPA) comprises 5 to 15% of the dry weight of spores of Bacillus species. Ca-DPA is important in spore resistance to many environmental stresses and in spore stability, and Ca-DPA levels in spore populations can vary with spore species/strains, as well as with sporulation conditions. We have measured levels of Ca-DPA in large numbers of individual spores in populations of a variety of Bacillus species and strains by using microfluidic Raman tweezers, in which a single spore is trapped in a focused laser beam and its Ca-DPA is quantitated from the intensity of the Ca-DPA-specific band at 1,017 cm 1 in Raman spectroscopy. Conclusions from these measurements include the following: (i) Ca-DPA concentrations in the spore core are >800 mM, well above Ca-DPA solubility; (ii) SpoVA proteins may be involved in Ca-DPA uptake in sporulation; and (iii) Ca-DPA levels differ significantly among individual spores in a population, but much of this variation could be due to variations in the sizes of individual spores. Spores of Bacillus species are metabolically dormant cells formed in sporulation, a process that is normally triggered by starvation (25, 30). These spores are extremely resistant to a variety of harsh treatments, including radiation, heat, desiccation, and toxic chemicals (20, 29). As a consequence, spores can survive for many years in their dormant state (15). At least some of the novel spore properties are due to the large amount (5 to 15% of dry weight) of the spore-specific molecule pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) that is located in the spore’s central region or core as a 1:1 chelate with

Dormant spores of Bacillus , Clostridium and related species can survive for years, largely because spore DNA is well protected against damage by many different agents. This DNA protection is partly a result of the high level of Ca 2+ -dipicolinic acid in spores and DNA repair during spore outgrowth, but is primarily caused by the saturation of spore DNA with a group of small, acid-soluble spore proteins (SASP), which are synthesized in the developing spore and then degraded after completion of spore germination. The structure of both DNA and SASP alters upon their association and this causes major changes in the chemical and photochemical reactivity of DNA.

Endospores (i.e., bacterial spores) embedded in polar ices present an opportunity to investigate the most durable form of life in an ideal medium for maintaining long-term viability. However, little is known about the endospore distribution and viability in polar ices. We have determined germinable endospore concentrations of bacterial spores capable of germination in a Greenland ice core (GISP2 94 m, ID# G2-271) using two complementary endospore viability assays (EVA), recently developed in our laboratory. These assays are based on bulk spectroscopic analysis (i.e., spectroEVA), and direct microscopic enumeration (i.e., microEVA) of ice core concentrates. Both assays detect dipicolinic acid (DPA) release during l-alanine induced germination via terbium ion (Tb3+)-DPA luminescence. Using spectroEVA, the germinable and total bacterial spore concentrations were found to be 295+/-19 spores mL(-1) and 369+/-36 spores mL(-1), respectively, (i.e., 80% of the endospores were capable of germination). Using microEVA, the germinating endospore concentration was found to be 27+/-2 spores mL(-1). The total cell concentration, as determined by DAPI stain fluorescence microscopy, was 7.0 x 10(3)+/-6.7 x 10(2) cells mL(-1). Culturing attempts yielded 2 CFU mL(-1) (4 degrees C). We conclude that endospores capable of germination in the GISP2 ice cores are readily determined using novel endospore viability assays.

Ultra high pressure homogenization (UHPH) opens up new areas for dynamic high pressure assisted thermal sterilization of liquids. Bacillus amyloliquefaciens spores are resistant to high isostatic pressure and temperature and were suggested as potential surrogate for high pressure thermal sterilization validation. B. amyloliquefaciens spores suspended in PBS buffer (0.01 M, pH 7.0), low fat milk (1.5%, pH 6.7), and whole milk (3.5%, pH 6.7) at initial concentration of ~106 CFU/mL were subjected to UHPH treatments at 200, 300, and 350 MPa with an inlet temperature at ~80°C. Thermal inactivation kinetics of B. amyloliquefaciens spores in PBS and milk were assessed with thin wall glass capillaries and modeled using first-order and Weibull models. The residence time during UHPH treatments was estimated to determine the contribution of temperature to spore inactivation by UHPH. No sublethal injury was detected after UHPH treatments using sodium chloride as selective component in the nutrient agar medium. The inactivation profiles of spores in PBS buffer and milk were compared and fat provided no clear protective effect for spores against treatments. Treatment at 200 MPa with valve temperatures lower than 125°C caused no reduction of spores. A reduction of 3.5 log10CFU/mL of B. amyloliquefaciens spores was achieved by treatment at 350 MPa with a valve temperature higher than 150°C. The modeled thermal inactivation and observed inactivation during UHPH treatments suggest that temperature could be the main lethal effect driving inactivation., Frontiers in Microbiology, 6, ISSN:1664-302X

Spores of various Bacillus and Clostridium species are among the most resistant life forms known. Since the spores of some species are causative agents of much food spoilage, food poisoning, and human disease, and the spores of Bacillus anthracis are a major bioweapon, there is much interest in the mechanisms of spore resistance and how these spores can be killed. This article will discuss the factors involved in spore resistance to agents such as wet and dry heat, desiccation, UV and γ-radiation, enzymes that hydrolyze bacterial cell walls, and a variety of toxic chemicals, including genotoxic agents, oxidizing agents, aldehydes, acid, and alkali. These resistance factors include the outer layers of the spore, such as the thick proteinaceous coat that detoxifies reactive chemicals; the relatively impermeable inner spore membrane that restricts access of toxic chemicals to the spore core containing the spore's DNA and most enzymes; the low water content and high level of dipicolinic acid in the spore core that protect core macromolecules from the effects of heat and desiccation; the saturation of spore DNA with a novel group of proteins that protect the DNA against heat, genotoxic chemicals, and radiation; and the repair of radiation damage to DNA when spores germinate and return to life. Despite their extreme resistance, spores can be killed, including by damage to DNA, crucial spore proteins, the spore's inner membrane, and one or more components of the spore germination apparatus.

A newly marketed anthrax detector that originated in the US space program promises to reduce the detection time of the system currently used by the US Postal Service. Devised by the CA-based Universal Detection Technology Inc., BSM-2000 continuously samples surrounding air for high concentrations of bacterial spores and triggers an alarm only on registering a bacterial spore count of 50 or more per liter, on the assumption that a bioterrorist attack will release spores in much greater quantities than normally found. It features a fiber optic system that delivers a microwave beam that heats the outer shell of any bacterial spore and causes it to explode and release a particular chemical, dipicolinic acid (DPA), which is characteristic of all bacteria, including anthrax. The liberated DPA adheres to a tape strip within the device, and if enough collects on the strip, ultraviolet light causes a bright green luminescence to appear. BSM-2000 requires little maintenance and does not require cartridge replacement.

The subcellular localization of a germination-specific cortex-lytic enzyme, SleB, of Bacillus subtilis during sporulation was observed by using fusions of N-terminal region of SleB to the green fluorescent protein (GFP). A fusion with a putative peptidoglycan-binding motif (SleB1-108-GFP) formed a fluorescent ring around the forespore of the wild type strain, as expected from the known location of the intact SleB in the dormant spore. SleB1-108-GFP formed a similar fluorescent ring around the forespore of the gerE mutant which has a severe defect in the coat structure, and of the cwlD mutant which lacks a muramic delta-lactam unique to the spore peptidoglycan (cortex), whereas the fusion could not attach to the spore of the cwlDgerE mutant. By contrast, a fusion without the motif (SleB1-45-GFP) could not be recruited around the forespore of the gerE mutant though it appeared to be accumulated on the outside of the spore of the wild type strain. Since SleB was shown to degrade only the cortex with muramic delta-lactam, these results suggested that a proper localization of SleB requires a strict interaction between the motif of the enzyme and the delta-lactam structure of the cortex, not the formation of normal coat layer.