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Bacterial spores in materials and equipment pose significant biosecurity risks, making effective disinfection crucial. This study evaluated Ortho-phthalaldehyde (OPA) and a quaternary ammonia-glutaraldehyde solution (AG) for inactivating spores of Bacillus thuringiensis (BT), B. cereus (BC), and two strains of B. velezensis (BV1 and BV2). Spores of BV1 and BT were treated with 22.5 mg/m 3 OPA by dry fumigation or 1 mg/mL AG by spray for 20 min, according to the manufacturer's recommendation. As no sporicidal effect was observed, OPA was tested at 112.5 mg/m 3 for 40 min, showing effectiveness for BT but not for BV1. Minimum bactericidal concentration (MBC) tests revealed higher MBC values for glutaraldehyde, prompting an overnight test with 112.5 mg/m 3 OPA by dry fumigation and 50 mg/mL AG by spray, using formaldehyde as a control. AG reduced all Bacillus strains, but with limited sporicidal effect. OPA was sporicidal for BT and BV1 but not for BC and BV2, indicating a strain-dependent effect. Formaldehyde performed better overall but did not completely inactivate BV2 spores. Our findings suggest that OPA and AG have potential as formaldehyde replacements in wet disinfection procedures., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Chlorine dioxide (ClO 2 ) is a powerful disinfectant widely regarded as a safe and effective hygienic agent in pharmaceutical plants and other manufacturing facilities that require sterility. However, the efficacy of low concentrations of ClO 2 gas on sterilizing spore-forming bacteria remains uncertain. In this study, we investigated the optimal conditions for disinfection of spore-forming bacteria with low concentrations of ClO 2 gas using biological indicators. The results showed that 0.05 ppmv (0.13 mg/m 3 ) ClO 2 gas at 37±1℃, 86±2%RH sterilized Bacillus atrophaeus in 14 d and Geobacillus stearothermophilus in 28 d (>6 log 10 reductions）. Based on these results, we propose that low concentrations of ClO 2 gas are useful for controlling contamination by spore-forming bacteria in aseptic facilities.

Alkaline glutaraldehyde (GTA) is a high-level chemical disinfectant/sterilant and has a broad microbial kill spectrum. The precise antimicrobial mechanism of GTA remains debated. GTA kill times are extremely variable across different organisms, illustrating the need for a better understanding of GTA kill mechanisms related to different organisms. A commonly proposed GTA kill mechanism suggests that it works by cross-linking accessible primary amines on important surface proteins. If true, the antimicrobial activity of GTA may directly correlate to the number of these available functional groups. Bacillus species form highly resistant bacterial endospores that are commonly used as one of the most stringent test organisms for disinfection and sterilization. In this study, we compared the log reduction times of alkaline GTA on spores from 4 Bacillus species to fluorescent profiles generated using Alexa Fluor™ amine-reactive dyes. GTA kill times were also compared to mean spore coat thicknesses as measured with scanning electron microscopy (SEM). Fluorescence values generated from bound amine-reactive dye showed a strong, positive correlation to GTA susceptibility, as measured by GTA 6-log 10 reduction times. Spore coat thickness also showed a strong, positive correlation to reduction time values. Results support the hypothesis that GTA kill times are directly related to the number of available primary amines on bacterial endospores. Results also indicated that the killing efficacy of GTA may be influenced by its ability to penetrate the spore coat to reach additional targets, suggesting that damaging important biomolecules beyond surface proteins may be involved in GTA killing mechanisms., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The resistance formation of spores in general and of Bacillus atrophaeus in particular has long been the focus of science in the bio-defense, pharmaceutical and food industries. In the food industry, it is used as a biological indicator (BI) for the evaluation of the inactivation effects of hydrogen peroxide in processing and end packaging lines' sterilization. Defined BI resistances are critical to avoid false positive and negative tests, which are salient problems due to the variable resistance of currently available commercial BIs. Although spores for use as BIs have been produced for years, little is known about the influence of sporulation conditions on the resistance as a potential source of random variability. This study therefore examines the dependence of spore resistance on the temperature, pH and partial oxygen saturation during submerged production in a bioreactor. For this purpose, spores were produced under different sporulation conditions and their resistance, defined by the D-value, was determined using a count reduction test in tempered 35% liquid hydrogen peroxide. The statistical analysis of the test results shows a quadratic dependence of the resistance on the pH, with the highest D‑values at neutral pH. The sporulation temperature has a linear influence on the resistance. The higher the temperature, the higher the D‑value. However, these factors interact with each other, which means that the temperature only influences the resistance when the pH is within a certain range. The oxygen partial pressure during sporulation has no significant influence. Based on the data obtained, a model could be developed enabling the resistance of BIs to be calculated, predicted and standardized depending on the sporulation conditions. BI manufacturers could thus produce BIs with defined resistances for the validation of sterilization effects in aseptic packaging/filling lines for the reliable manufacture of shelf-stable and safe food products.

The aim of this study was to investigate the influence of surface hydrophobicity and roughness of carrier materials on the inactivation of bacterial spores with gaseous hydrogen peroxide whereas condensate formation is prevented. Spores of Bacillus subtilis and Bacillus atrophaeus were applied either as single spore culture or as a mixed spore population to simulate natural contamination with microorganisms of different characteristics. Inactivation with gaseous hydrogen peroxide was carried out at 5200 ppm hydrogen peroxide without condensate formation. The inactivation results of B. subtilis and B. atrophaeus spores on carrier materials with varying surface hydrophobicity differed significantly. However, inactivation of the mixed spore populations resulted in similar resistance compared to the single spore batches. The results of this study indicate that surface hydrophobicity most probably has an impact on the inactivation with gaseous hydrogen peroxide whereas surface roughness only plays a minor role., (© 2020 The Authors. Journal of Food Science published by Wiley Periodicals, Inc. on behalf of Institute of Food Technologists.)

Aims: In case of biological hazards and pandemics, personal protective equipment of rescue forces is currently manually decontaminated with harmful disinfectants, primarily peracetic acid. To overcome current drawbacks regarding supply, handling and disposal of chemicals, the use of plasma processed air (PPA) represents a promising alternative for surface decontamination on site. In this study, the sporicidal efficiency of a portable plasma system, designed for field applications, was evaluated., Methods and Results: The developed plasma device is based on a dielectric barrier discharge (DBD) and operated with ambient air as process gas. PPA from the plasma nozzle was flushed into a treatment chamber (volume: 300 l) and bacterial endospores (Bacillus subtilis and Bacillus atrophaeus) dried on different surfaces were treated under variable conditions. Reductions in spores by more than 4 log 10 were found within 3 min of PPA exposure. However, the presence of endospores in agglomerates or in an organic matrix as well as the complexity of the respective surface microstructure negatively affected the inactivation efficiency. When endospores were embedded in a dried protein matrix, mechanical wiping with swabs during exposure to PPA increased the inactivation effect significantly. Gaseous ozone alone did not provide a sporicidal effect. Significant spore inactivation was only obtained when water vapour was injected into the PPA stream., Conclusion: The results show that endospores dried on surfaces can be reduced by several orders of magnitude within few minutes in a treatment chamber which is flushed with PPA from of a DBD plasma nozzle., Significance and Impact of the Study: Plasma processed air generated on site by DBD plasma nozzles could be a suitable alternative for the disinfection of various surfaces in closed rooms., (© 2019 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Today, there is no effective non-thermal method to inactivate unwanted bacterial spores in foods. High-Pressure (HP) process has been shown to act synergistically with moderate heating and the bacteriocin nisin to inactivate spores but the mechanisms have not been elucidated. The purpose of the present work was to investigate in depth the synergy of HP and nisin on various foodborne spore species and to bring new elements of understandings. For this purpose, spores of Bacillus pumilus, B. sporothermodurans, B. licheniformis, B. weihenstephanensis, and Clostridium sp. were suspended in MES buffer, in skim milk or in a liquid medium simulating cooked ham brine and treated by HP at 500 MPa for 10 min at 50 °C or 20 °C. Nisin (20 or 50 IU/mL) was added at three different points during treatment: during HP, during and or in the plating medium of enumeration. In the latter two cases, a high synergy was observed with the inhibition of the spores of Bacillus spp. The evaluation of the germinated fraction of Bacillus spp. spores after HP revealed that this synergy was likely due to the action of nisin on HP-sensitized spores, rather than on HP-germinated spores. Thus, the combination of nisin and HP can lead to Bacillus spp. spore inhibition at 20 °C. And Nisin can act on HP-treated spores, even if they are not germinated. This paper provides new information about the inhibition of spores by the combination of HP and nisin. The high synergy observed at low temperature has not been reported yet and could allow food preservation without the use of any thermal process., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A combined calorimetric gas- and spore-based biosensor array is presented in this work to monitor and evaluate the sterilization efficacy of gaseous hydrogen peroxide in aseptic filling machines. H 2 O 2 has been successfully measured under industrial conditions. Furthermore, the effect of H 2 O 2 on three different spore strains , namely Bacillus atrophaeus, Bacillus subtilis and Geobacillus stearothermophilus, has been investigated by means of SEM, AFM and impedimetric measurements. In addition, the sterilization efficacy of a spore-based biosensor and the functioning principle are addressed and discussed: the sensor array is convenient to be used in aseptic food industry to guarantee sterile packages., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Growth/no growth boundary models for Bacillus spores that accounted for the effects of environmental pH, water activity (a w ), acetic acid, lactic acid, bacterial strain, and storage period were developed using conventional logistic regression and machine learning algorithms. Growth in tryptic soy broth at 317 conditions comprising nine levels of pH (4.0-6.5), six levels of a w (0.85-1.00), six levels of acetic acid concentrations (0-0.8%), and five levels of lactic acid concentrations (0-0.8%) was examined to confirm growth limit conditions. All models developed using logistic regression, neural network, and deep learning on the basis of obtained datasets successfully described growth/no growth boundaries of three Bacillus species. Although the logistic regression model failed to describe growth limits under some conditions, neural network and deep learning approaches enabled to determine them in such cases. The developed models were evaluated by independent experimental data of growth in tryptic soy broth and in clam soup. The deep learning model enabled better prediction of independent data with smaller probabilistic variability values than those of the logistic regression and neural network models. The deep learning procedure can be utilized for growth boundary modeling to control bacterial growth safely and flexibly., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Glycerol monolaurate is a broadly antimicrobial fatty acid monoester, killing bacteria, fungi, and enveloped viruses. The compound kills stationary-phase cultures of Bacillus anthracis , suggesting that the molecule may kill spores. In this study, we examined the ability of glycerol monolaurate alone or solubilized in a nonaqueous gel to kill vegetative cells and spores of aerobic B. anthracis , B. subtilis , and B. cereus and anaerobic Clostridium perfringens and Clostridium ( Clostridioides ) difficile. Glycerol monolaurate alone was bactericidal for all five organisms tested. Glycerol monolaurate alone was effective in killing spores. When solubilized in a nonaqueous gel, the glycerol monolaurate gel was bactericidal for all spores tested. The data suggest that glycerol monolaurate nonaqueous gel could be effective in decontaminating environmental and body surfaces, such as skin. IMPORTANCE Bacillus and Clostridium spores are known to be highly resistant to killing, persisting on environmental and human body surfaces for long periods of time. In favorable environments, these spores may germinate and cause human diseases. It is thus important to identify agents that can be used on both environmental and human skin and mucosal surfaces and that are effective in killing spores. We previously showed that the fatty acid monoester glycerol monolaurate (GML) kills stationary-phase cultures of Bacillus anthracis Since such cultures are likely to contain spores, it is possible that GML and a human-use-approved GML nonaqueous gel would kill Bacillus and Clostridium spores. The significance of our studies is that we have identified GML, and, to a greater extent, GML solubilized in a nonaqueous gel, as effective in killing spores from both bacterial genera., (Copyright © 2018 Schlievert et al.)

Aims: Four commercially available robotic vacuum cleaners were assessed for sampling efficiency of wet disseminated Bacillus atrophaeus spores on carpet, polyvinyl chloride (PVC) and laminate flooring. Furthermore, their operability was evaluated and decontamination efficiency of one robot was assessed, using a sodium hypochlorite solution., Methods and Results: In an environmental chamber, robots self-navigated around 4 m 2 of flooring containing a single contaminated 0·25 m 2 tile (c. 10 4 spores per cm 2 ). Contamination levels at predetermined locations were assessed by macrofoam swabs (PVC and laminate) or water soluble tape (carpet), before and after sampling. Robots were dismantled postsampling and spore recoveries assessed. Aerosol contamination was also measured during sampling. Robot sampling efficiencies were variable, however, robots recovered most spores from laminate (up to 17·1%), then PVC and lastly the carpet. All robots spread contamination from the 'hotspot' (all robots spread <0·6% of the contamination to other areas) and became surface contaminated. Spores were detected at low levels during air sampling (<5·6 spores per litre). Liquid decontamination inactivated 99·1% of spores from PVC., Conclusions: Robotic vacuum cleaners show promise for both sampling and initial decontamination of indoor flooring., Significance and Impact of the Study: In the event of a bioterror incident, e.g. deliberate release of Bacillus anthracis spores, areas require sampling to determine the magnitude and extent of contamination, and to establish decontamination efficacy. In this study, we investigate robotic sampling methods against high concentrations of bacterial spores applied by wet deposition to different floorings, contamination spread to other areas, potential transfer of spores to the operators and assessment of a wet vacuum robot for spore inactivation. The robots' usability was evaluated and how they can be employed in real life scenarios. This will help to reduce the economic cost of sampling and the risk to sampling/decontamination teams., (© 2018 Crown copyright. Journal of Applied Microbiology © 2018 The Society for Applied Microbiology. This article is published with the permission of the Controller of HMSO and the Queen’s Printer for Scotland.)

Bacterial spores resist environmental extremes and protect key spore macromolecules until more supportive conditions arise. Spores germinate upon sensing specific molecules, such as nutrients. Germination is regulated by specialized mechanisms or structural features of the spore that limit contact with germinants and enzymes that regulate germination. Importantly, germination renders spores more susceptible to inactivating processes such as heat, desiccation, and ultraviolet radiation, to which they are normally refractory. Thus, germination can be intentionally induced through a process called germination-induction and subsequent treatment of these germinated spores with common disinfectants or gentle heat will inactivate them. However, while the principle of germination-induction has been shown effective in the laboratory, this strategy has not yet been fully implemented in real-word scenarios. Here, we briefly review the mechanisms of bacterial spore germination and discuss the evolution of germination-induction as a decontamination strategy. Finally, we examine progress towards implementing germination-induction in three contexts: biodefense, hospital settings and food manufacture., Significance and Impact: This article reviews implementation of germination-induction as part of a decontamination strategy for the cleanup of bacterial spores. To our knowledge this is the first time that germination-induction studies have been reviewed in this context. This article will provide a resource which summarizes the mechanisms of germination in Clostridia and Bacillus species, challenges and successes in germination-induction, and potential areas where this strategy may be implemented., (Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

Contact angle measurement of microorganisms is often described in literature, either to investigate their hydrophobic characteristic or the adhesion behavior of cells. However, in some key aspects the preparation methods differ. Thus, it is difficult to compare results and to choose a procedure for repetition of measurements. The aim of this paper is to point out some critical points during microorganism film preparation that can alter the resulting contact angles. Depending on the moisturizing medium and equilibration time, contact angles differ significantly., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Decontamination of Bacillus spores adhered to common drinking water infrastructure surfaces was evaluated using a variety of disinfectants. Corroded iron and cement-mortar lined iron represented the infrastructure surfaces, and were conditioned in a 23 m long, 15 cm diameter (75 ft long, 6 in diameter) pilot-scale drinking water distribution pipe system. Decontamination was evaluated using increased water velocity (flushing) alone at 0.5 m s -1 (1.7 ft s -1 ), as well as free chlorine (5 and 25 mg L -1 ), monochloramine (25 mg L -1 ), chlorine dioxide (5 and 25 mg L -1 ), ozone (2.0 mg L -1 ), peracetic acid 25 mg L -1 ) and acidified nitrite (0.1 mol L -1 at pH 2 and 3), all followed by flushing at 0.3 m s -1 (1 ft s -1 ). Flushing alone reduced the adhered spores by 0.5 and 2.0 log 10 from iron and cement-mortar, respectively. Log 10 reduction on corroded iron pipe wall coupons ranged from 1.0 to 2.9 at respective chlorine dioxide concentrations of 5 and 25 mg L -1 , although spores were undetectable on the iron surface during disinfection at 25 mg L -1 . Acidified nitrite (pH 2, 0.1 mol L -1 ) yielded no detectable spores on the iron surface during the flushing phase after disinfection. Chlorine dioxide was the best performing disinfectant with >3.0 log 10 removal from cement-mortar at 5 and 25 mg L -1 . The data show that free chlorine, monochloramine, ozone and chlorine dioxide followed by flushing can reduce adhered spores by > 3.0 log 10 on cement-mortar., (Published by Elsevier Ltd.)

Aims: To determine the responses of spores of Bacillus subtilis and Bacillus anthracis surrogate Bacillus thuringiensis Al Hakam to I 2 treatment., Methods and Results: Spores of B. subtilis and B. thuringiensis killed by aqueous 30°C-I 2 could germinate, and their inner membrane (IM) was intact. Spore coats were important in I 2 resistance, DNA-protective proteins were not important, and survivors of I 2 treatment were not mutagenized. Viabilities of I 2 -treated, 90-98% killed spores were much lower on high-salinity media, and the treated spores were more heat sensitive than the untreated spores. Germinated I 2 -killed spores were dead as determined by staining with nucleic acid dyes, and many appeared to have been lysed., Conclusions: Aqueous I 2 appeared to kill B. subtilis and B. thuringiensis spores such that spores lyse soon after they germinate, and not by causing DNA damage or rupture of spores' IM. I 2 treatment also generated many damaged spores that could only be recovered under nonstressful conditions., Significance and Impact of the Study: This work shows that spores of the model organism B. subtilis, and B. thuringiensis, a surrogate for B. anthracis spores, exhibit similar mechanisms of resistance to and killing by I 2 . Generation by I 2 treatment of conditionally dead spores indicates that appropriate media are essential to efficiently enumerate viable I 2 -treated spores., (© 2016 The Society for Applied Microbiology.)

Destruction of bioweapon facilities due to explosion or fire could aerosolize highly pathogenic microorganisms. The post-event air quality assessment is conducted through air sampling. A bioaerosol sample (often collected on a filter for further culture-based analysis) also contains combustion products, which may influence the microbial culturability and, thus, impact the outcome. We have examined the interaction between spores deposited on collection filters using two simulants of Bacillus anthracis [B. thuringiensis (Bt) and B. atrophaeus (referred to as BG)] and incoming combustion products of Al as well as Mg and B·Ti (common ingredient of metalized explosives). Spores extracted from Teflon, polycarbonate, mixed cellulose ester (MCE), and gelatin filters (most common filter media for bioaerosol sampling), which were exposed to combustion products during a short-term sampling, were analyzed by cultivation. Surprisingly, we observed that aluminum combustion products enhanced the culturability of Bt (but not BG) spores on Teflon filters increasing the culturable count by more than an order of magnitude. Testing polycarbonate and MCE filter materials also revealed a moderate increase of culturability although gelatin did not. No effect was observed with either of the two species interacting on either filter media with products originated by combustion of Mg and B·Ti. Sample contamination, spore agglomeration, effect of a filter material on the spore survival, changes in the spore wall ultrastructure and germination, as well as other factors were explored to interpret the findings. The study raises a question about the reliability of certain filter materials for collecting airborne bio-threat agents in combustion environments., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bacterial spores can survive for long periods without nutrients and in harsh environmental conditions. This survival is influenced by the structure of the spore, the presence of protective compounds, and water retention. These compounds, and the physical state of water in particular, allow some species of bacterial spores to survive sterilization schemes with hydrogen peroxide and UV light. The chemical nature of the spore core and its water has been a subject of some contention and the chemical environment of the water impacts resistance paradigms. Either the spore has a glassy core, where water is immobilized along with other core components, or the core is gel-like with mobile water diffusion. These properties affect the movement of peroxide and radical species, and hence resistance. Deuterium solid-state NMR experiments are useful for examining the nature of the water inside the spore. Previous work in our lab with spores of Bacillus subtilis indicate that, for spores, the core water is in a more immobilized state than expected for the gel-like core theory, suggesting a glassy core environment. Here, we report deuterium solid-state NMR observations of the water within UV- and peroxide-resistant spores from Bacillus pumilus SAFR-032. Variable-temperature NMR experiments indicate no change in the line shape after heating to 50 °C, but an overall decrease in signal after heating to 100 °C. These results show glass-like core dynamics within B. pumilus SAFR-032 that may be the potential source of its known UV-resistance properties. The observed NMR traits can be attributed to the presence of an exosporium containing additional labile deuterons that can aid in the deactivation of sterilizing agents.

Members of the genus Bacillus include important food-borne pathogen and spoilage microorganisms for food industry. Essential oils are natural products extracted from herbs and spices, which can be used as natural preservatives in many foods because of their antibacterial, antifungal, antioxidant and anti-carcinogenic properties. The aim of this research was to explore the effect of the addition of different concentrations of thymol to the heating and recovery media on the thermal resistance of spores of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis at different temperatures. While the heat resistance was hardly reduced when thymol was present in the heating medium, the effect in the recovery medium was greater, reducing the D100 °C values down to one third for B. subtilis and B. cereus when 0.5 mM thymol was added. This effect was dose dependent and was also observed at other heating temperatures., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A wide range of oligo-p-phenylene ethynylenes has been shown to exhibit good biocidal activity against both Gram-negative and Gram-positive bacteria. While cell death may occur in the dark, these biocidal compounds are far more effective in the light as a result of their ability to sensitize the production of cell-damaging reactive oxygen species. In these studies, the interactions of a specific cationic oligo-p-phenylene ethynylene with spore-forming Bacillus atrophaeus and Bacillus anthracis Sterne have been investigated. Flow cytometry assays are used to rapidly monitor cell death as well as spore germination. This compound effectively killed Bacillus anthracis Sterne vegetative cells (over 4 log reduction), presumably by severe perturbations of the bacterial cell wall and cytoplasmic membrane, while also acting as an effective spore germinant in the dark. While 2 log reduction of B. anthracis Sterne spores was observed, it is hypothesized that further killing could be achieved through enhanced germination.

The inhibitory activities of nisin or/and potassium sorbate on spores and vegetative cells of Bacillus sporothermodurans LTIS27, which are known to be a contaminant of dairy products and to be extremely heat-resistant, were investigated. First, the tested concentrations of nisin or potassium sorbate inhibited vegetative cell growth; with the minimum inhibitory concentrations were 5 × 10(3) IU/ml and 2% (w/v), respectively. Then, the behaviour of vegetative cells and spores in presence of sub-lethal concentrations of nisin (50 UI/ml) or/and potassium sorbate (0.2%), in milk at 37 °C for 5 days, were evaluated. In the absence of inhibitors, strain grew and sporulated at the end of the exponential phase. Nisin (50 UI/ml) was able to inhibit spore outgrowth but didn't affect their germination. It induced an immediate and transitory reduction (1.6log(10) after 1 h and 2.8log(10) after 6 h of incubation) of vegetative cell growth which reappeared between 10 h and 24 h. Potassium sorbate (0.2%) had a durable bacteriostatic effect (1.1log(10) after 6 h), on vegetative cells, followed by a slower regrowth. It was able to inhibit both germination and outgrowth of spores. Association of nisin and potassium sorbate, at sub-lethal concentrations, showed a synergistic effect and resulted in a total inhibition of cells growth after 5 days. The results illustrate the efficacy of nisin and potassium sorbate in combination, and the commercial potential of applying such treatment to decontaminate any product that has a problem with persistence of bacterial spores., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Aim: 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., Materials & Methods: 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., Results: 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., Conclusion: 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.

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.

Bacillus strains are often isolated from biofilms in the food industries. Previous works have demonstrated that sporulation could occur in biofilms, suggesting that biofilms would be a significant source of food contamination with spores. In this study, we investigated the properties of mono-species and mixed Bacillus biofilms and the ability of Bacillus strains to sporulate inside biofilms. Bacillus strains were able to form mono-species biofilms on stainless steel coupons, with up to 90% spores after a 48 h-incubation. These spores were highly resistant to cleaning but were easily transferred to agar, mimicking the cross-contamination of food, thereby suggesting that biofilms would be of particular concern due to a potential for Bacillus spore food contamination. This hypothesis was strengthened by the fact that Bacillus strains were able to form mixed biofilms with resident strains and that sporulation still occurred easily in these complex structures., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: L-alanine, acting through the GerA receptor, was recently found to be an efficient germinant in Bacillus licheniformis ATCC14580/DSM13., Results: In this study, we show that several of 46 examined B. licheniformis strains germinate remarkably slower than the type strain when exposed to L-alanine. These strains are not necessarily closely related, as determined by MLST (multi-locus sequence typing). Three of the slow-germinating strains were further examined in order to see whether nucleotide substitutions in the gerA sequences were responsible for the slow L-alanine germination. This was performed by complementing the transformable type strain derivate MW3ΔgerAA with gerA variants from the three slow-germinating strains; NVH1032, NVH1112 and NVH800., Conclusions: A wide selection of B. licheniformis strains was evaluated for L-alanine-induced germination efficiency. Our results show that gerA substitutions could only partially explain why spores of some B. licheniformis strains responded slower than others in the presence of L-alanine.

Spores of Bacillus sporothermodurans are known to be a contaminant of dairy products and to be extremely heat-resistant. A central composite experimental design with three factors using response surface methodology was used to evaluate the effect of nisin (50-150 UI/mL), temperature (80-100 °C), and temperature-holding time (10-20 min) on the inactivation of B. sporothermodurans LTIS27 spores in distilled water, in skim milk and in chocolate milk. The experimental values were shown to be significantly in good agreement with the values predicted by the quadratic equation since the adjusted determination coefficients (Radj(2)) were around 0.97. By analyzing the response surfaces plots, the inactivation was shown to be higher in distilled water than in skim milk under all the conditions tested. Five-log cycle reductions of B. sporothermodurans spores were obtained after a treatment at 95 °C for 12 min in presence of 125 UI of nisin/mL in distilled water or at 100 °C for 13 min in presence of 134 UI of nisin/mL in skim milk or at 100 °C for 15 min in presence of 135 UI of nisin/mL in chocolate milk. This study showed the efficiency of nisin (15-184 UI/mL) in combination with temperature (73-106 °C) to inactivate spores of B. sporothermodurans in milk., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bacterial endospores are resistant to many environmental factors from temperature extremes to ultraviolet irradiation and are generally more difficult to inactivate or kill than vegetative bacterial cells. It is often considered necessary to treat spores or samples containing spores with chemical fixative solutions for prolonged periods of time (e.g., 1-21 days) to achieve fixation/inactivation to enable electron microscopy (EM) examination outside of containment laboratories. Prolonged exposure to chemical fixatives, however, can alter the ultrastructure of spores for EM analyses. This study was undertaken to determine the minimum amount of time required to inactivate/sterilize and fix spore preparations from several bacterial species using a universal fixative solution for EM that maintains the ultrastructural integrity of the spores. We show that a solution of 4% paraformaldehyde with 1% glutaraldehyde inactivated spore preparations of Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis, and Clostridium perfringens in 30 min, and Bacillus subtilis in 240 min. These results suggest that this fixative solution can be used to inactivate and fix spores from several major groups of bacterial spore formers after 240 min, enabling the fixed preparations to be removed from biocontainment and safely analyzed by EM outside of biocontainment.

The development of new value-added applications for glycerol is of worldwide interest because of the environmental and economic problems that may be caused by an excess of glycerol generated from biodiesel production. A novel use of glycerol as a major substrate for production of a low-cost sterilization biological indicator system (BIS; spores on a carrier plus a recovery medium) was investigated. A sequential experimental design strategy was applied for product development and optimization. The proposed recovery medium enables germination and outgrowth of heat-damaged spores, promoting a D (160 °C) value of 6.6 ± 0.1 min. Bacillus atrophaeus spores production by solid-state fermentation reached a 2.3 ± 1.2 × 10(8) CFU/g dry matter. Sporulation kinetics results allowed this process to be restricted in 48 h. Germination kinetics demonstrated the visual identification of nonsterile BIS within 24 h. Performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with the regulatory requirements. Cost breakdowns were from 41.8 (quality control) up to 72.8 % (feedstock). This is the first report on sterilization BIS production that uses glycerol as a sole carbon source, with significant cost reduction and the profitable use of a biodiesel byproduct.

The aims of this work were to (a) evaluate the susceptibility of endospores of Bacillus cereus, B. licheniformis, B. sphaericus and B. subtilis to photodynamic inactivation using a tricationic porphyrin as photosensitizer, (b) assess the efficiency of adsorption of the photosensitizer in endospore material as a determinant of the susceptibility of endospores of different Bacillus species to photo-inactivation, (c) determine the value of B. cereus as a model organism for studies of antimicrobial photodynamic inactivation of bacterial endospores. The results of irradiation experiments with endospores of four species of Bacillus showed that B. cereus was the only species for which efficient endospore photo-inactivation (> 3 log reduction) could be achieved. Endospores of B. licheniformis, B. sphaericus and B. subtilis were virtually resistant to photo-inactivation with tricationic porphyrin. The amount of porphyrin bound to endospore material was not significantly different between species, regardless of the presence of an exosporium or exosporium-like outer layer. The sensitivity of endospores to photodynamic inactivation with a tricationic porphyrin is highly variable among different species of the genus Bacillus. The presence of an exosporium in endospores of B. cereus and B. sphaericus, or an exosporium-like glycoprotein layer in endospores of B. subtilis, did not affect the amount of bound photosensitizer and did not explain the inter-species variability in susceptibility to photodynamic inactivation. The results imply that the use of B. cereus as a more amenable surrogate of the exosporium-producing B. anthracis must be carefully considered when testing new photosensitizers for their antimicrobial photo-inactivation properties., (© 2012 The Societies and Wiley Publishing Asia Pty Ltd.)

Objectives: Ozone has been used as an alternative method for the decontamination of water, food, equipment and instruments. The objective of this study was to evaluate the antimicrobial effects of ozonated water on the sanitization of dental instruments that were contaminated by Escherichia coli, Staphylococcus aureus, Candida albicans and the spores of Bacillus atrophaeus., Methods: A total of one hundred and twenty standardized samples of diamond dental burs were experimentally contaminated with E. coli (ATCC 25922), S. aureus (ATCC 6538) and C. albicans (ATCC 18804) and the spores of B. atrophaeus (ATCC 6633) for 30 min. After the contamination, the samples were exposed to ozonated water (10mg/L O(3)) for 10 or 30min. The control group was composed of samples that were exposed to distilled water for 30 min. After the exposure to the ozonated water, 0.1 mL aliquots were seeded onto BHI agar to count the colony-forming units per milliliter (CFU/mL) of E. coli, S. aureus, and B. atrophaeus. Sabouraud dextrose agar was used to count the CFU/mL of C. albicans. The results were subjected to an analysis of variance and the Tukey test., Results: For all of the microorganisms studied, the ozonated water reduced the number of CFU/mL after 10 and 30 min of sanitization, and this microbial reduction was dependent on the duration of the exposure to the ozonated water. E. coli exhibited the greatest reduction in CFU/mL (2.72-3.78 log) followed by S. aureus (2.14-3.19 log), C. albicans (1.44-2.14 log) and the spores of B. atrophaeus (1.01-1.98 log)., Conclusion: The ozonated water was effective in reducing the CFU of E. coli, S. aureus, C. albicans and B. atrophaeus spores, suggesting that ozonated water can be used for the sanitization of dental instruments., (Copyright © 2012 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.)

Accidental release of biological agents from a bioweapon facility may contaminate large areas, possibly causing disastrous environmental consequences. To address this issue, novel halogen-containing reactive materials are being designed with the added capability to inactivate viable airborne microorganisms. This study determined the efficiency of combustion products of such materials to inactivate aerosolized bacteria and viruses. Spores of Bacillus atrophaeus and MS2 viruses dispersed in dry air were exposed for subsecond time intervals to hydrocarbon flames seeded with different reactive powders so that bioaerosol particles interacted with the combustion products in a controlled high-temperature environment. The experiments were designed to quantify differences in the biocidal effects of different reactive material powders including Al and Mg, a B•Ti nanocomposite, an 8Al•MoO(3) nanothermite, and a novel Al•I(2) nanocomposite. Compared to pure hydrocarbon flame, powder-seeded flame (with no iodine) produced about an order of magnitude greater inactivation of bacterial spores. The iodine-containing material increased the spore inactivation by additional 2 orders of magnitude. The aerosolized MS2 viruses (generally not as stress-resistant as spores) were fully inactivated when exposed to combustion of either the iodinated or noniodinated powders. Overall, the study suggests a great biocidal potential of combustion products generated by novel iodine-containing nanocomposite materials.

A new method for rapidly eliminating pathogenic microorganisms in large air space using spraying *OH radicals is presented in this paper With a physical method of strong electric-field discharge, large numbers of *OH radicals were produced by the oxygen activated particles of O2+, O(1D), O(3P), etc., and the introducing reagent HO2-. The gram-positive bacteria Bacillus subtilis, the gram-negative bacteria Serratia marcescens, and Bacillus spores were used for the eliminating experiments. Results show that the different microorganisms were rapidly killed by *OH radicals with a concentration of 0.8 mg/L and spraying density of 21 microL/m2 within 4 sec. Cell morphological changes were also observed under microscope. The cells of B. subtilis and Bacillus spores in their cellular wall, cellular membrane, or cell protoplasm were greatly destroyed when being exposed to a killing dosage of *OH radicals.

This study was taken up with a view to generate basic information on spore hardiness to ethanol in various Bacillus species and related genera, and to assess the effectiveness of different levels of ethanol as a bacterial disinfectant. Predominantly spore-bearing cultures of five Bacillus spp. (B. pumilus, B. subtilis, B. megaterium, B. fusiformis and B. flexus) that were isolated from the spent-alcohol used during plant tissue culture work were challenged with aqueous ethanol (25, 50, 60, 70, 80 and 90% v/v) in 1 ml volumes at 10¹⁰⁻¹¹ CFU ml⁻¹. Monitoring the spore endurance through spotting and plating revealed prolonged tolerance (>12 months) at different alcohol levels depending on the organism except in 90% where no survival was observed beyond 2-12 months. Spores of related genera like Paenibacillus and Lysinibacillus also showed long-term ethanol survival. Alcohol tolerance of spore-forming organisms depended on the extent of spores and spore hardiness, which in turn varied with the organism, strain, age of culture, growing conditions and other factors as authenticated with ATCC strains of B. pumilus and B. subtilis. Aqueous 90% ethanol caused instant inactivation of vegetative cells in different spore formers and twelve other non-sporulating Gram-positive and Gram-negative organisms tested. Taking into account both vegetative cells and spores, the appropriate concentration of ethanol as a disinfectant emerged to be 90% followed by absolute ethanol compared with the generally recommended 70-80% level.

Bacillus pumilus SAFR-032 spores originally isolated from the Jet Propulsion Laboratory spacecraft assembly facility clean room are extremely resistant to UV radiation, H(2)O(2), desiccation, chemical disinfection and starvation compared to spores of other Bacillus species. The resistance of B. pumilus SAFR-032 spores to standard industrial clean room sterilization practices is not only a major concern for medical, pharmaceutical and food industries, but also a threat to the extraterrestrial environment during search for life via spacecraft. The objective of the present study was to investigate the potential of Alexa-FISH (fluorescence in situ hybridization with Alexa Fluor® 488 labeled oligonucleotide) method as a molecular diagnostic tool for enumeration of multiple sterilant-resistant B. pumilus SAFR-032 spores artificially encapsulated in, and released via organic solvent from, a model polymeric material: poly(methylmethacrylate) (Lucite, Plexiglas). Plexiglas is used extensively in various aerospace applications and in medical, pharmaceutical and food industries. Alexa-FISH signals were not detected from spores via standard methods for vegetative bacterial cells. Optimization of a spore permeabilization protocol capitalizing on the synergistic action of proteinase-K, lysozyme, mutanolysin and Triton X-100 facilitated efficient spore detection by Alexa-FISH microscopy. Neither of the Alexa-probes tested gave rise to considerable levels of Lucite- or solvent-associated background autofluorescence, demonstrating the immense potential of Alexa-FISH for rapid quantification of encapsulated B. pumilus SAFR-032 spores released from poly(methylmethacrylate)., (© 2012 The Societies and Blackwell Publishing Asia Pty Ltd.)

Release of dipicolinic acid (DPA) and its fluorescence with terbium (Tb(3+)) allow rapid measurement of the germination and viability of spores of Bacillus and Clostridium species. However, germination of coat-deficient Bacillus spores was strongly inhibited by Tb(3+) and some other multivalent cations. Tb(3+) also inhibited germination of coat-deficient Clostridium perfringens spores.

Bacillus spores are a known cause of food spoilage and their increased resistance poses a major challenge in efficient elimination. Recent studies on bacterial cultures at the single cell level have revealed how minor differences in essential spore properties, such as core water content or germinant receptor levels, can cause the observed differences in spore germination and outgrowth behavior. Moreover, heterogeneous behavior is influenced by commonly accepted food preservation techniques, such as heating or the usage of weak organic acids. Understanding the underlying molecular mechanisms and key players involved in phenotypic heterogeneity of spores, while taking the spore's history into account, will improve predictability of the spore's behavior to various treatments and triggers., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Bacterial spores are frequently intrinsically resistant to biocides and only a number of alkylating and oxidising biocides are sporicidal under certain conditions. Activity against spores is affected by several key factors such as concentration, exposure time, soiling, and the types of surface to be treated. Sporicidal efficacy is usually achieved after an exposure time of several minutes with a high concentration of a biocide. Failure to understand these factors will result in decreased sporicide activity and spore survival. Sporicides in healthcare settings are used for surface disinfection and for the high level disinfection of certain medical devices (e.g. endoscopes). With efficacy data in mind, sporicidal activity should be achieved for the disinfection of medical devices where both high concentration and long exposure time occur. However, for the disinfection of environmental surfaces, high concentration is not recommended, nor is long exposure time achievable. In this case, sporicidal activity is severely reduced and spore survival following treatment is to be expected and contributes to the explanation of spore persistence on surfaces., (Copyright © 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.)

The simultaneous nutrient germination of hundreds of individual wild-type spores of three Bacillus species and a number of Bacillus subtilis strains has been measured by two new methods, and rates of release of the great majority of the large pool of dipicolinic acid (DPA) from individual spores of B. subtilis strains has been measured by Raman spectroscopy with laser tweezers. The results from these analyses and published data have allowed a number of significant conclusions about the germination of spores of Bacillus species as follows. (i) The time needed for release of the great majority of a Bacillus spore's DPA once rapid DPA release had begun (DeltaT(release)) during nutrient germination was independent of the concentration of nutrient germinant used, the level of the germinant receptors (GRs) that recognize nutrient germinants used and heat activation prior to germination. Values for DeltaT(release) were generally 0.5 to 3 min at 25 to 37 degrees C for individual wild-type spores. (ii) Despite the conclusion above, germination of individual spores in populations was very heterogeneous, with some spores in wild-type populations completing germination > or = 15-fold slower than others. (iii) The major factor in the heterogeneity in germination of individual spores in populations was the highly variable lag time, T(lag), between mixing spores with nutrient germinants and the beginning of DeltaT(release). (iv) A number of factors decrease spores' T(lag) values including heat activation, increased levels of GRs/spore, and higher levels of nutrient germinants. These latter factors appear to affect the level of activated GRs/spore during nutrient germination. (v) The conclusions above lead to the simple prediction that a major factor causing heterogeneity in Bacillus spore germination is the number of functional GRs in individual spores, a number that presumably varies significantly between spores in populations.

Aims: Electrochemically activated solutions (ECAS) are generated from halide salt solutions via specially designed electrolytic cells. The active solutions are known to possess high biocidal activity against a wide range of target microbial species, however, literature revealing the kill-kinetics of these solutions is limited. The aim of the study was to identify the kill-rate and extent of population kill for a range of target species (including endospores) using ECAS generated at the anode (anolyte)., Methods and Results: Standard suspensions of methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus atrophaeus spores and Clostridium difficile spores were treated with anolyte in a quantitative suspension assay. For vegetative cells, all concentrations of anolyte tested reduced the viable population to below the detection limit within 10 s. At a concentration of 99%, anolyte produced a log(10) reduction factor of greater than five in viable B. atrophaeus endospores within 90 s and reduced numbers of C. difficile endospores to below the experimental detection limit within 20 s at concentrations of 5% or greater., Conclusions: Anolyte was highly effective in killing test-bacteria and spores. The bactericidal efficacy was retained against vegetative cells at dilutions as low as 1% and against C. difficile spores as low as 5%., Significance and Impact of Study: The results of this study demonstrate that ECAS are effective at lower concentrations and act more rapidly than previously reported. Potent bactericidal and sporicidal activity coupled with point-of-use generation, low production-costs and environmental compatibility suggest that acidic ECAS has the potential to be a useful addition to the current armoury of disinfectants.

Bacterial spores such as Bacillus atrophaeus are one of the most resistant life forms known and are extremely resistant to chemical and environmental factors in the dormant state. During germination, as bacterial spores progress towards the vegetative state, they become susceptible to anti-sporal agents. B. atrophaeus spores were exposed to the non-nutritive germinant dodecylamine (DDA), a cationic surfactant that can also be used as a killing agent, for up to 60 min, or to the nutrient germinant L-alanine. In kinetic studies, 99% of the spores were killed within 5 min of exposure to DDA. Atomic force microscopy (AFM) can be used as a sensitive tool to assess how the structure of the spore coat changes upon exposure to germinants or killing agents. Changes in cell height and roughness over time of exposure to DDA were examined using AFM. DDA caused the spore height to decrease by >50%, which may have been due to a partial breakdown of the spore coat. Treatment of B. atrophaeus with the nutrient germinant resulted in a decrease in height of spores after 2 h of incubation, from 0.7 +/- 0.1 microm to 0.3 +/- 0.2 microm. However, treatment with L-alanine did not change the surface roughness of the spores, indicating that the changes that occur during germination take place underneath the spore coat. We propose that exposure to DDA at high concentrations causes pores to form in the coat layer, killing B. atrophaeus without the need to fully germinate spores., (Published 2009 by John Wiley & Sons, Ltd.)

The susceptibility of spores of Bacillus subtilis, Bacillus cereus, and avirulent Bacillus anthracis to treatment with hydrogen peroxide, peroxyacetic acid, a peroxy-fatty acid mixture, sodium hypochlorite, and acidified sodium chlorite was investigated. Results indicated that B. cereus spores may be reasonable predictors of B. anthracis spore inactivation by peroxyacetic acid-based biocides. However, B. cereus was not a reliable predictor of B. anthracis inactivation by the other biocides. In studies comparing B. cereus and B. subtilis, B. cereus spores were more resistant (by 1.5 to 2.5 log CFU) than B. subtilis spores to peroxyacetic acid, the peroxy-fatty acid mixture, and acidified sodium chlorite. Conversely, B. subtilis spores were more resistant than B. cereus spores to hydrogen peroxide. These findings indicated the relevance of side-by-side testing of target organisms and potential surrogates against categories of biocides to determine whether both have similar properties and to validate the use of the surrogate microorganisms.

Manganese oxides are widespread in the environment and their surface reactivity has the potential to modifythe geochemical behavior of uranium. We have investigated the effect of different concentrations of U and Mn on the coupled biogeochemical oxidation-reduction reactions of U and Mn. Experiments conducted in the presence of Mn(II)-oxidizing spores from Bacillus sp. strain SG-1 and 5% headspace oxygen show that the Mn oxides produced by these spores can rapidly oxidize UO2. Thirty to fifty times more UO2 is oxidized in the presence of Mn oxides compared to Mn oxide free controls. As a consequence of this U02 oxidation, Mn oxides are reduced to soluble Mn(II) that can be reoxidized by SG-1 spores. SG-1 spores cannot directly oxidize U02, but U02 oxidation proceeds rapidly with Mn(II) concentrations of <5 microM. The rate of UO2 oxidation is equal to the rate of MnO2 reduction with UO2 oxidation controlled by the initial concentrations of UO2, dissolved Mn(II) (in systems with spores), or Mn(IV) oxides (in systems containing preformed MnO2). U(VI) and UO2 decrease the Mn(II) oxidation rate in different ways by inhibiting the Mn(II)-oxidizing enzyme or decreasing the available Mn(II). These results emphasize the need to consider the impact of Mn(II)-oxidizing bacteria when predicting the potential for U02 oxidation in the subsurface.

The quaternary ammonium compound (QUAT) lauryl dimethyl benzyl ammonium bromide (LDBAB) is commercialized in the Caribbean, Central, and South America under label claims to sterilize medical and dental instruments in 30 minutes or less. But QUAT formulations are classified as low-level disinfectants with no sporicidal activity or tuberculocidal efficacy. This study evaluated the presumptive sporicidal and tuberculocidal activities of 2 LDBAB formulations with label claims of high-level disinfectant action used widely in the Americas. In this study, sporicidal activity was evaluated against a challenge inoculum of 10(6)Bacillus atrophaeus spores. Tuberculocidal activity was evaluated against Mycobacterium tuberculosis H37Rv using the quantitative suspension test described in European Standard EN14348:2005. Tested at the recommended product concentrations indicated on their respective labels, both LDBAB solutions failed to demonstrate sporicidal activity and tuberculocidal efficacy. These findings underscore the need for public health authorities, as well as medical and dental professionals, to correctly identify LDBAB as a low-level disinfectant and avoid its use in instrument processing, a practice that may endanger lives.

Identification of microorganisms, specifically of vegetative cells and spores, by intact cell mass spectrometry (ICMS) is an emerging new technology. The technique provides specific biomarker profiles which can be employed for bacterial identification at the genus, species, or even at the subspecies level holding the potential to serve as a rapid and sensitive identification technique in clinical or food microbiology and also for sensitive detection of biosafety level (BSL) 3 microorganisms. However, the development of ICMS as an identification technique for BSL-3 level microorganisms is hampered by the fact that no MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) compatible inactivation procedure for microorganisms, and particularly for bacterial endospores, has been evaluated so far. In this report we describe a new methodology for effective inactivation of microorganisms which is compatible with the analysis of microbial protein patterns by MALDI-TOF mass spectrometry. The main challenge of this work was to define the conditions that ensure microbial inactivation and permit at the same time comprehensive analysis of microbial protein patterns. Among several physical, chemical, and mechanical inactivation procedures, inactivation by trifluoroacetic acid (TFA) proved to be the best method in terms of bactericidal capacity and information content of the mass spectra. Treatment of vegetative cells by 80% TFA alone for 30 min assured complete inactivation of microbial cells under all conditions tested. For spore inactivation, the "TFA inactivation protocol" was developed which is a combination of TFA treatment with basic laboratory routines such as centrifugation and filtering. This MALDI-TOF/ICMS compatible sample preparation protocol is simple and rapid (30 min) and assures reliable inactivation of vegetative cells and spores of highly pathogenic (BSL-3) microorganisms.

Although commercial sanitizers can inactivate bacterial spores in food processing environments, relatively little data exist as to the decontamination of products and surfaces by consumers using commercial household products. Should a large scale bioterrorism incident occur in which consumer food products were contaminated with a pathogenic sporeformer such as Bacillus anthracis, there may be a need to decontaminate these products before disposal as liquid or solid waste. Studies were conducted to test the efficacy of commercial household products for inactivating spores of Bacillus cereus (used as a surrogate for B. anthracis) in vitro and in fluid milk. Validation of the resistance of the B. cereus spores was confirmed with B. anthracis spores. Fifteen commercial products, designed as either disinfectants or sanitizers or as potential sanitizers, were purchased from retail markets. Products selected had one of the following active compounds: NaOCl, HCl, H2O2, acetic acid, quaternary ammonium compounds, ammonium hydroxide, citric acid, isopropanol, NaOH, or pine oil. Compounds were diluted in water (in vitro) or in 2% fat fluid milk, and spores were exposed for up to 6 h. Products containing hypochlorite were most effective against B. cereus spores. Products containing HCl or H2O2 also reduced significant numbers of spores but at a slower rate. The resistance of spores of surrogate B. cereus strains to chlorine-containing compounds was similar to that of B. anthracis spores. Therefore, several household products on the market may be used to decontaminate fluid milk or similar food products contaminated by spores of B. anthracis.

The spores of six strains of Bacillus anthracis (four virulent and two avirulent) were compared with those of four other types of spore-forming bacteria for their resistance to four liquid chemical sporicides (sodium hypochlorite at 5,000 ppm available chlorine, 70,000 ppm accelerated H2O2, 1,000 ppm chlorine dioxide, and 3,000 ppm peracetic acid). All test bacteria were grown in a 1:10 dilution of Columbia broth (with manganese) incubated at 37 degrees C for 72 h. The spore suspensions, heat treated at 80 degrees C for 10 min to rid them of any viable vegetative cells, contained 1 x 10(8) to 3 x 10(8) CFU/ml. The second tier of the quantitative carrier test (QCT-2), a standard of ASTM International, was used to assess for sporicidal activity, with disks (1 cm in diameter) of brushed and magnetized stainless steel as spore carriers. Each carrier, with 10 microl (> or = 10(6) CFU) of the test spore suspension in a soil load, was dried and then overlaid with 50 microl of the sporicide being evaluated. The contact time at room temperature ranged from 5 to 20 min, and the arbitrarily set criterion for acceptable sporicidal activity was a reduction of > or = 10(6) in viable spore count. Each test was repeated at least three times. In the final analysis, the spores of Bacillus licheniformis (ATCC 14580(T)) and Bacillus subtilis (ATCC 6051(T)) proved to be generally more resistant than the spores of the strains of B. anthracis tested. The use of one or both of the safe and easy-to-handle surrogates identified here should help in developing safer and more-effective sporicides and also in evaluating the field effectiveness of existing and newer formulations in the decontamination of objects and surfaces suspected of B. anthracis contamination.

Persistence of Bacillus atrophaeus subsp. globigii spores on corroded iron coupons in drinking water was studied using a biofilm annular reactor. Spores were inoculated at 10(6) CFU/ml in the dechlorinated reactor bulk water. The dechlorination allowed for observation of the effects of hydraulic shear and biofilm sloughing on persistence. Approximately 50% of the spores initially adhered to the corroded iron surface were not detected after 1 month. Addition of a stable 10 mg/liter free chlorine residual after 1 month led to a 2-log(10) reduction of adhered B. atrophaeus subsp. globigii, but levels on the coupons quickly stabilized thereafter. Increasing the free chlorine concentration to 25 or 70 mg/liter had no additional effect on inactivation. B. atrophaeus subsp. globigii spores injected in the presence of a typical distribution system chlorine residual (approximately 0.75 mg/liter) resulted in a steady reduction of adhered B. atrophaeus subsp. globigii over 1 month, but levels on the coupons eventually stabilized. Adding elevated chlorine levels (10, 25, and 70 mg/liter) after 1 month had no effect on the rate of inactivation. Decontamination with elevated free chlorine levels immediately after spore injection resulted in a 3-log(10) reduction within 2 weeks, but the rate of inactivation leveled off afterward. This indicates that free chlorine did not reach portions of the corroded iron surface where B. atrophaeus subsp. globigii spores had adhered. B. atrophaeus subsp. globigii spores are capable of persisting for an extended time in the presence of high levels of free chlorine.

It was hypothesized that supercritical carbon dioxide (SC-CO(2)) treatment could serve as an alternative sterilization method at various temperatures (40-105 degrees C), CO(2) pressures (200-680 atm), and treatment times (25 min to 6 h), and with or without the use of a passive additive (distilled water, dH(2)O) or an active additive (hydrogen peroxide, H(2)O(2)). While previous researchers have shown that SC-CO(2) possesses antimicrobial properties, sterilization effectiveness has not been shown at sufficiently low treatment temperatures and cycle times, using resistant bacterial spores. Experiments were conducted using Geobacillus stearothermophilus and Bacillus atrophaeus spores. Spore strips were exposed to SC-CO(2) in commercially available supercritical fluid extraction and reaction systems, at varying temperatures, pressures, treatment times, and with or without the use of a passive additive, such as dH(2)O, or an active additive, such as H(2)O(2). Treatment parameters were varied from 40 to 105 degrees C, 200-680 atm, and from 25 min to 6 h. At 105 degrees C without H(2)O(2), both spore types were completely deactivated at 300 atm in 25 min, a shorter treatment cycle than is obtained with methods in use today. On the other hand, with added H(2)O(2) (<100 ppm), 6 log populations of both spore types were completely deactivated using SC-CO(2) in 1 h at 40 degrees C. It was concluded from the data that large populations of resistant bacterial spores can be deactivated with SC-CO(2) with added H(2)O(2)at lower temperatures and potentially shorter treatment cycles than in most sterilization methods in use today., ((c) 2006 Wiley Periodicals, Inc.)

Daptomycin inhibited 67 of 70 clinical isolates of Bacillus species at < or =1 microg/ml and 100% of them at < or =2 microg/ml. It showed bactericidal activity similar to that of ciprofloxacin against vegetative cells but not against spores. For 2 strains, the ciprofloxacin MICs were >4 g/ml, and 10 strains were resistant to erythromycin.