Atrophaeus Spores Research Articles

Fate and transport of viable Bacillus anthracis simulant spores in ambient air during a large outdoor decontamination field exercise

ABSTRACT The Wide Area Demonstration (WAD) was a field exercise conducted under the U.S. EPA’s Analysis of Coastal Operational Resiliency program, in conjunction with the U.S. Department of Homeland Security and the U.S. Coast Guard. The purpose of the WAD was to operationalize at field scale aspects of remediation activities that would occur following an outdoor release of Bacillus anthracis spores, including sampling and analysis, decontamination, data management, and waste management. The WAD was conducted in May 2022 at Fort Walker (formerly known as Fort A.P. Hill) and utilized Bacillus atrophaeus as a benign simulant for B. anthracis. B. atrophaeus spores were inoculated onto the study area at the beginning of the study, and air samples were collected daily during each of the different phases of the WAD using Dry Filter Units (DFUs). Ten DFU air samplers were placed at the perimeter of the study area to collect bioaerosols onto two parallel 47-mm diameter polyester felt filters, which were then subsequently analyzed in a microbiological laboratory for the quantification of B. atrophaeus. The study demonstrated the use of DFUs as a rugged and robust bioaerosol collection device. The results indicated that the highest B. atrophaeus spore air concentrations (up to ~ 5 colony forming units/m3) occurred at the beginning of the demonstration (e.g. during inoculation and characterization sampling phases) and generally downwind from the test site, suggesting transport of the spores was occurring from the study area. Very few B. atrophaeus spores were detected in the air after several weeks and following decontamination of exterior surfaces, thus providing an indication of the site decontamination procedures’ effectiveness. No B. atrophaeus spores were detected in any of the blank or background samples. Implications: Following an incident involving a release of Bacillus anthracis spores or other biological threat agent into the outdoor environment, understanding the factors that may affect the bioagent’s fate and transport can help predict viable contaminant spread via the ambient air. This paper provides scientific data for the first time on ambient air concentrations of bacterial spores over time and location during different phases of a field test in which Bacillus atrophaeus (surrogate for B. anthracis) spores were released outdoors as part of a full-scale study on sampling and decontamination in an urban environment. This study advances the knowledge related to the fate and transport of bacterial spores (such as those causing anthrax disease) as an aerosol in the outdoor environment over the course of three weeks in a mock urban environment and has exposure and health risk implications. The highest spore air concentrations occurred at the beginning of the study (e.g. during inoculation of surfaces and characterization sampling), and in the downwind direction, but diminished over time; few B. atrophaeus spores were detected in the air after several weeks and following decontamination. Therefore, in an actual incident, potential reaerosolization of the microorganism and subsequent transport in the air during surface sampling and remediation efforts should be considered for determining exclusion zone locations and estimating potential risk to neighboring communities. The data also provide evidence suggesting that the large-scale decontamination of outdoor surfaces may reduce air concentrations of the bioagent, which is important since exposure of B. anthracis via inhalation is a primary concern.

Bacterial and fungal bioburden reduction on material surfaces using various sterilization techniques suitable for spacecraft decontamination.

Planetary protection is a guiding principle aiming to prevent microbial contamination of the solar system by spacecraft (forward contamination) and extraterrestrial contamination of the Earth (backward contamination). Bioburden reduction on spacecraft, including cruise and landing systems, is required to prevent microbial contamination from Earth during space exploration missions. Several sterilization methods are available; however, selecting appropriate methods is essential to eliminate a broad spectrum of microorganisms without damaging spacecraft components during manufacturing and assembly. Here, we compared the effects of different bioburden reduction techniques, including dry heat, UV light, isopropyl alcohol (IPA), hydrogen peroxide (H2O2), vaporized hydrogen peroxide (VHP), and oxygen and argon plasma on microorganisms with different resistance capacities. These microorganisms included Bacillus atrophaeus spores and Aspergillus niger spores, Deinococcus radiodurans, and Brevundimonas diminuta, all important microorganisms for considering planetary protection. Bacillus atrophaeus spores showed the highest resistance to dry heat but could be reliably sterilized (i.e., under detection limit) through extended time or increased temperature. Aspergillus niger spores and D. radiodurans were highly resistant to UV light. Seventy percent of IPA and 7.5% of H2O2 treatments effectively sterilized D. radiodurans and B. diminuta but showed no immediate bactericidal effect against B. atrophaeus spores. IPA immediately sterilized A. niger spores, but H2O2 did not. During VHP treatment under reduced pressure, viable B. atrophaeus spores and A. niger spores were quickly reduced by approximately two log orders. Oxygen plasma sterilized D. radiodurans but did not eliminate B. atrophaeus spores. In contrast, argon plasma sterilized B. atrophaeus but not D. radiodurans. Therefore, dry heat could be used for heat-resistant component bioburden reduction, and VHP or plasma for non-heat-resistant components in bulk bioburden reduction. Furthermore, IPA, H2O2, or UV could be used for additional surface bioburden reduction during assembly and testing. The systemic comparison of sterilization efficiencies under identical experimental conditions in this study provides basic criteria for determining which sterilization techniques should be selected during bioburden reduction for forward planetary protection.

Real-Time Monitoring of H2O2 Sterilization on Individual Bacillus atrophaeus Spores by Optical Sensing with Trapping Raman Spectroscopy

Hydrogen peroxide (H2O2), a strong oxidizer, is a commonly used sterilization agent employed during aseptic food processing and medical applications. To assess the sterilization efficiency with H2O2, bacterial spores are common microbial systems due to their remarkable robustness against a wide variety of decontamination strategies. Despite their widespread use, there is, however, only little information about the detailed time-resolved mechanism underlying the oxidative spore death by H2O2. In this work, we investigate chemical and morphological changes of individual Bacillus atrophaeus spores undergoing oxidative damage using optical sensing with trapping Raman microscopy in real-time. The time-resolved experiments reveal that spore death involves two distinct phases: (i) an initial phase dominated by the fast release of dipicolinic acid (DPA), a major spore biomarker, which indicates the rupture of the spore’s core; and (ii) the oxidation of the remaining spore material resulting in the subsequent fragmentation of the spores’ coat. Simultaneous observation of the spore morphology by optical microscopy corroborates these mechanisms. The dependence of the onset of DPA release and the time constant of spore fragmentation on H2O2 shows that the formation of reactive oxygen species from H2O2 is the rate-limiting factor of oxidative spore death.

Biosynthesis of Copper Oxide and Silver Nanoparticles by Bacillus Spores and Evaluation of the Feasibility of Their Use in Antimicrobial Paints.

Modification of paint with nanoparticles (NPs) provides self-cleaning, water/dirt-repellent, and other properties. Therefore, the aim of the present study was to biosynthesize silver (Ag) and copper oxide (CuO) NPs and to prepare NP-modified paint. To this end, AgNPs and CuONPs were biosynthesized using Bacillus atrophaeus spores and commercial and crude dipicolinic acid (DPA) extracted from the spore of this bacterium. The synthesized NPs were characterized using electron microscopy, Fourier-transform infrared (FTIR), X-ray diffraction analysis (XRD), and energy-dispersive X-ray spectroscopy (EDS) methods. A minimum inhibitory concentration (MIC) assay of NPs against Escherichia coli ATCC8739 and Staphylococcus aureus ATCC6538 was carried out. The antibacterial effects of prepared NP-paint complexes were assessed using an optical density (OD) comparison before and after adding metal sheets coated with NP-paint complexes into the nutrient broth medium. Four different types of NPs were synthesized in this research: AgNPs synthesized by spore (A), AgNPs synthesized by commercial DPA (B), AgNPs synthesized by crude DPA (C), and CuONPs synthesized by spore (D). SEM analysis confirmed the spherical shape of NPs. According to the results, NPs A, B, and D showed higher antibacterial activity against S. aureus compared to E. coli. Furthermore, the analysis of the antibacterial effects of NP-paint complexes suggested that paint-NPs A, B, and C displayed higher activity on E. coli compared to S. aureus. Moreover, the antibacterial effect of paint-NP D was significantly lower than other NPs. According to this robust antibacterial effect on pathogenic bacteria, it seems that these NP-paint complexes could be useful in public places such as hospitals, airports, dormitories, schools, and office buildings, where the rate of transmission of infection is high.

Bromine and Chlorine Disinfection of Cryptosporidium parvum Oocysts, Bacillus atrophaeus Spores, and MS2 Coliphage in Water.

Conventional water treatment practices utilizing chemical disinfection, especially chlorination, are considered generally effective in producing microbiologically safe drinking water. However, protozoan pathogens such as oocysts of Cryptosporidium parvum are very resistant to chlorine, which has led to consideration of alternative disinfectants for their control. Free bromine, HOBr, has not been evaluated extensively as an alternative halogen disinfectant for inactivation of Cryptosporidium parvum in drinking water or reclaimed water for non-potable uses. Bromine is a versatile disinfectant consisting of different chemical forms with persistent microbicidal efficacy under varied water quality conditions and is effective against a range of waterborne microbes of health concern. The objectives of this study are to (1) compare the efficacy of free bromine to free chlorine at similar concentrations (as milligrams per liter) for disinfection of Cryptosporidium parvum oocysts, Bacillus atrophaeus spores, and MS2 coliphage in a model buffered water and (2) evaluate the kinetics of inactivation of these microorganisms using appropriate disinfection models. Overall, at a target concentration of ∼5 mg/L, bromine averaged 0.6 log (73.8%) reductions of C. parvum oocyst infectivity after 300 min (CT: 1166 min·mg/L) and produced up to a 0.8 log reduction disinfectant activity. An ∼5.0 mg/L chlorine dose increased oocyst infectivity by only 0.4 log (64%) after 300 min (CT: 895 min·mg/L). Bacillus atrophaeus spores and MS2 coliphage treated with bromine and chlorine were reduced by 4 log10 (99.99%) for both disinfectants over the duration of the experiments.

Passenger vehicle interior decontamination by low concentration hydrogen peroxide vapor following a wide area biological contamination incident.

To assess low concentration hydrogen peroxide (LCHP) (H2O2) vapor dispersed with a home humidifier for its ability to decontaminate vehicle interiors contaminated with Bacillus anthracis surrogate Bacillus atrophaeus spores. Efficacy of a vaporized 3% H2O2 solution was evaluated for liquid volumes, on/off vehicle heating, ventilation, and air conditioning (HVAC) system operations, and temperatures that ranged from 5 to 27°C. Survival of the spores was assessed by quantification of remaining viable spores with efficacy quantified in terms of mean log10 reduction. Decontamination efficacy after the 6-day dwell time increased when the 3% H2O2 liquid volume was doubled, increasing from 4-of-10 to 10-of-10 nondetects (zero colonies counted using standard dilution and filter plating) inside the vehicle cabin. Recirculating cabin air through the HVAC system during decontamination decreased efficacy to 6-of-10 non-detects. While no 6-log10 reduction in viable spores was observed on the cabin filter with the cabin filter kept in place, a 6-log10 reduction was achieved after its removal and placement in the cabin during treatment. Results from this study allow for informed decisions on the use of LCHP vapor as an effective decontamination approach for vehicle interiors.

Digital Twin for biological safety cabinet to reproduce performance and field certification test specified by NSF/ANSI 49 - 2019

The airflow balance test to confirm the physical performance of biological safety cabinets (BSC) is standardized by JIS K 3800 in accordance with NSF/ANSI 49 in Japan. In the test, Bacillus atrophaeus spores are used as an evaluation index, which poses a few limitations, such as their dependence on the environmental conditions to be tested. The aim of this study is to develop BSC digital twins and discuss reasonable alternatives for performance evaluation tests while maintaining the accuracy of the bacterial test performance. Herein, we provide the outline of a digital twin model for BSCs and the results of a numerical analysis, where a passive scalar is used as an alternative pollutant in the evaluation index.

Growth, development and thermal resistance of Bacillus atrophaeus spores using whey culture media

Whey represents an important environmental problem for the dairy industry due to its high volume and organic load. Studies that evaluate new forms of use of these residues such as growth medium for microorganisms, can help to develop a better destination providing an added value bioproduct. This work aims to evaluate the development of Bacillus atrophaeus spores ATCC 9372 growing in whey culture media to determine the reuse of this dairy residue. Whey at a concentration varying from 1.0g. L-1 to 20.0g. L-1 was dissolved into water and Bacillus atrophaeus spores were incubated for 6 days. Thermal treatments were performed at 102oC and 104oC and in all conditions evaluated, the decay of spores population was linear. In solid media, D-values at 102oC ranged from 1.24 min to 2.98 min and at 104oC ranged from 1.27 min to 2.35 min. The culture media containing whey can enable the growth and sporulation of Bacillus atrophaeus. This study confirms the possibility of using this dairy industry residue as an alternative to obtain bacterial spores, which may be used in validation of thermal.

Dry heat sterilization modelling for spacecraft applications.

Inactivation processes using heat are widely used for disinfection and sterilization. Dry heat sterilization of spacecraft equipment has been the preferred microbial inactivation method as part of interplanetary travel protection strategies. An antimicrobial model, based on temperature and exposure time based on experimental data, was developed to provide reliable sterilization processes to be used for interplanetary applications. Bacillus atrophaeus spores, traditionally used to challenge dry heat sterilization processes, were tested over a range of temperatures in comparison with spores of Bacillus canaveralius that have been shown to have a higher heat resistance profile. D-value and Z-values were determined and used to develop a mathematical model for parametric sterilization applications. The impact of the presence of a contaminating soil, representative of Mars dust, was also tested to verify the practical application of the model to reduce the risk of microbial contamination in such environments. The sterilization model developed can be used as an intrinsic part of risk reduction strategies for interplanetary protection. Forward and backward planetary protection strategies to reduce the risks of microbial contamination during interplanetary exploration and research is an important consideration. The development of a modern sterilization model, with consideration of microorganisms identified with higher levels of heat resistance than traditionally deployed in terrestrial applications, allows for the consideration of optimal inactivation processes to define minimum criteria for engineering design. The ability to inactivate living microorganisms, as well as to degrade biomolecules, provides a reliable method to reduce the risk of known and potentially unknown contaminants in future applications.

Direct Observation of the Sporicidal Action of Hydrogen Peroxide on Bacillus atrophaeus Spores By Optical Trapping Raman Microscopy

Hydrogen peroxide (H2O2), a strong oxidizer, is frequently used for chemical sterilization in aseptic food processing. Due to their inherent robustness, bacterial spores, i.e. spores of Bacillus atrophaeus, are commonly employed to evaluate the efficacy of the sterilization procedure [1]. Despite its widespread use, however, the details of the sporicidal action of H2O2 are not yet fully understood, with various mechanisms being discussed in the literature [2,3]. Here, we employ optical trapping Raman microscopy to elucidate the sporicidal mechanism of H2O2. Optical trapping Raman microscopy allows us to isolate, investigate, and manipulate individual spores from a sample (Fig. 1A). Combining optical microscopy with the real-time, label-free chemical information obtained from Raman spectroscopy directly reveals the sequence of steps that lead to oxidative spore death after H2O2 exposure (Fig. 1B).We find that spore degradation begins with the fast, cooperative release of dipicolinic acid (DPA), a key spore biomarker and a major component of spore’s core. This indicates a breach of the inner membrane surrounding the core. The onset of DPA release depends on the concentration of H2O2. DPA outflux is accompanied by the formation of oxidation products (mostly carbonyl compounds) and oxidation continues after exchange of DPA with the surrounding medium is complete. The remaining spore shell then slowly disintegrates and continues to shrink on a timescale of hundreds of seconds. Simultaneous observation of spore morphology and size by optical microscopy and particle tracking corroborates this three-step mechanism.The presented assay allows on-line monitoring of spore composition and morphology, which directly reveals the mechanism underlying the sporicidal action of H2O2. In addition, trapped spores can be exposed to different chemical or physical stimuli (temperature, light, chemical composition of the surrounding medium, etc.) to investigate a variety of industrially relevant sterilization conditions. Jildeh, Z. B., Wagner, P. H. & Schöning, M. J. Sterilization of Objects, Products, and Packaging Surfaces and Their Characterization in Different Fields of Industry: The Status in 2020. physica status solidi (a) 218, 2000732 (2021). Setlow, P. & Christie, G. What’s new and notable in bacterial spore killing. World J Microbiol Biotechnol 37, 144 (2021). Setlow, P. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol 101, 514-525 (2006). Figure 1

Effective inactivation of Bacillus atrophaeus spores and Escherichia coli on disposable face masks using ultraviolet laser irradiation

Due to the widespread emergence of COVID-19, face masks have become a common tool for reducing transmission risk between people, increasing the need for sterilization methods against mask-contaminated microorganisms. In this study, we measured the efficacy of ultraviolet (UV) laser irradiation (266 nm) as a sterilization technique against Bacillus atrophaeus spores and Escherichia coli on three different types of face mask. The UV laser source demonstrated high penetration of inner mask layers, inactivating microorganisms in a short time while maintaining the particle filtration efficiency of the masks. This study demonstrates that UV laser irradiation is an efficient sterilization method for removing pathogens from face masks.

Large-scale decontamination of disposable FFP2 and FFP3 respirators by hydrogen peroxide vapour, Finland, April to June 2020.

BackgroundThe shortage of FFP2 and FFP3 respirators posed a serious threat to the operation of the healthcare system at the onset of the COVID-19 pandemic.AimOur aim was to develop and validate a large-scale facility that uses hydrogen peroxide vapour for the decontamination of used respirators.MethodsA multidisciplinary and multisectoral ad hoc group of experts representing various organisations was assembled to implement the collection and transport of used FFP2 and FFP3 respirators from hospitals covering 86% of the Finnish population. A large-scale decontamination facility using hydrogen peroxide vapour was designed and constructed. Microbiological tests were used to confirm efficacy of hydrogen peroxide vapour decontamination together with a test to assess the effect of decontamination on the filtering efficacy and fit of respirators. Bacterial and fungal growth in stored respirators was determined by standard methods.ResultsLarge-scale hydrogen peroxide vapour decontamination of a range of FFP2 and FFP3 respirator models effectively reduced the recovery of biological indicators: Geobacillus stearothermophilus and Bacillus atrophaeus spores, as well as model virus bacteriophage MS2. The filtering efficacy and facial fit after hydrogen peroxide vapour decontamination were not affected by the process. Microbial growth in the hydrogen peroxide vapour-treated respirators indicated appropriate microbial cleanliness.ConclusionsLarge-scale hydrogen peroxide vapour decontamination was validated. After effective decontamination, no significant changes in the key properties of the respirators were detected. European Union regulations should incorporate a facilitated pathway to allow reuse of appropriately decontaminated respirators in a severe pandemic when unused respirators are not available.

Effect of oxygen on the germination and culturability of Bacillus atrophaeus spores

The effect of oxygen on the germination and culturability of aerobic Bacillus atrophaeus spores was investigated in this study. Under oxic or anoxic conditions, various nutritional and non-nutritional germinants were utilized to induce germination. Tb3+-dipicolinic acid fluorescence assay and phase-contrast microscopy were used to track the germination process. The final germination level, germination half time, and germination speed were used to define germination kinetics. Colony-forming unit enumeration was used to assess the culturability of germinated spores germinated with or without oxygen. The results show that in the absence of oxygen, the final germination level was unaffected, germination half time decreased by up to 35.0%, germination speed increased by up to 27.4%, and culturability decreased by up to 95.1%. It is suggested that oxygen affects some germinant receptor-dependent germination pathways, implying that biomolecules engaged in these pathways may be oxygen-sensitive. Furthermore, spores that have completed the germination process in either anoxic or oxic conditions may have different culturability. This research contributed to a better understanding of the fundamental mechanism of germination.

UV laser sterilization of Bacillus atrophaeus spores on ceramic tiles

Ultraviolet (UV) radiation is one of the most common methods for sterilizing materials with suspected biological warfare agent contamination. In this study, UV laser sterilization of ceramic tiles was investigated as a method for destroying Bacillus atrophaeus spores. The effects of UV irradiation were evaluated as a function of water absorption (porous and vitrified), tile color (black and white), and surface brightness (matte and glossy). Spores were loaded onto the surface of each tile and irradiated with a 266-nm UV laser. The irradiated spores were then removed by scraping with a razor blade and cultured in an agar plate for 24 h. The sterilizing effects of the UV laser were greater on the vitrified (group BI b ) tiles than on the porous (group BIII) tiles. In addition, sterilization was more effective on white and matte tiles than on black and glossy tiles, respectively.

Cold plasma aseptic packaging

Cold plasma aseptic packaging

Evaluation of a modified rapid viability-polymerase chain reaction method for Bacillus atrophaeus spores in water matrices

A rapid method that provides information on the viability of organisms is needed to protect public health and ensure that remediation efforts following a release of a biological agent are effective. The rapid viability-polymerase chain reaction (RV-PCR) method combines broth culture and molecular methods to provide results on whether viable organisms are present in less than 15 h. In this study, a modified RV-PCR (mRV-PCR) method was compared to a membrane-filtration culture method for the detection of viable Bacillus spores in water matrices. Samples included small and large volumes of chlorine and non‑chlorine treated tap water. Large volume water samples (up to 100 L), were processed by ultrafiltration using a semi-automated waterborne pathogen concentrator, followed by centrifugation as a secondary concentration technique. The concentrated samples were analyzed by mRV-PCR and culture methods. The overall agreement between the mRV-PCR and culture methods when seed concentrations were greater than 10 spores per sample volume analyzed was 96%. The total time from the start of sample processing to the final sample result for the mRV-PCR method was decreased by approximately 2 h, in comparison to the previously published RV-PCR method because of the incorporation of shorter, more efficient primary and secondary concentration steps and a shorter DNA extraction technique. Overall, this study confirmed that RV-PCR is a promising approach for identifying viable Bacillus spores in small- and large-volume water samples and for producing results in less time than traditional culture methods.

Toward a Test Protocol for Surface Decontamination Using a Mobile Whole-room UVGI Device†.

Mobile whole‐room UVGI devices are used in healthcare settings to control surface‐borne pathogens. Unfortunately, no standard method comparing the efficacy of these devices is available. We accessed the effect of shadows on UVC 254 nm inactivation. The evaluation of a mobile whole‐room UVGI device used spores of Bacillus atrophaeus as a surrogate for Clostridium difficile and Staphylococcus aureus as a surrogate for MSRA. Inactivation after 10 min of exposure varied significantly depending on whether the spores received direct UV exposure (4.3 log reduction), both direct and reflected UV exposure (3.0–4.0 log reduction) or reflected UV exposure alone (<1.0 log reduction). The susceptibility (z‐value) for inactivation of B. atrophaeus spores on a glass surface was estimated to be 0.00312 m2 J−1. Staphylococcus aureus microbial log reductions were approximately 5.5 for direct UV exposure, 3.6–5.2 for both direct and reflected UV exposure and approximately 2.75 for only reflected UV exposure. Our measurement of reflected dose ranged from 0.46% to 1.47%. Based on our findings, B. atrophaeus spores should be considered as a model organism for testing the impact of shadows on mobile whole‐room UVGI device inactivation. Optimizing the reflected component of whole‐room UVGI is important, especially for UVC‐resistant organisms.

Managing the bacterial contamination risk in an axenic mice animal facility.

A gap exists between good laboratory practices with axenic animals and the procedures applied. This work examined the efficacy of sodium dichloroisocyanurate (MB-10) and potassium peroxymonosulfate (Virkon™) disinfectants, as well as the appropriate soaking time for materials used with the ISOcage Biosafety Station™. We also compared the microbial load in cage systems hosting mice over 2 weeks in axenic rooms (ARs) and in typical specific-pathogen-free (SPF) non-axenic rooms (NARs) to identify resistant microorganisms, targeted for longer soaking disinfection, and evaluated the necessary procedures for reducing the microbial load in AR. Staphylococcus was the most frequently isolated genus (in both ARs and NARs). An average of three spore-forming microorganisms per cage were counted from AR. The disinfection time to reach 1 log reduction for Bacillus atrophaeus spores varied from 138 s (100 ppm MB-10) to 290 (Virkon™) to <20 s for S. epidermidis (100 ppm MB-10). AR management protocols lead to a microbial load that is 1000 times lower than that found in NARs. Data comparing the microbial load in SPF and axenic facilities can be used to improve the effectiveness of their microbial control procedures.

Mechanisms of Sporicidal Activity Induced by Ionized Hydrogen Peroxide in the Spores of Bacillus atrophaeus.

Introduction: Ionized hydrogen peroxide (iHP) is a new technology used for the decontamination of surfaces or laboratory areas. It utilizes a low concentration of hydrogen peroxide (H2O2) mixed with air and ionized through a cold plasma arc. This technology generates reactive oxygen species as a means of decontamination. Objectives: The purpose of this study is to review the effects of iHP on the structure of the spores of Bacillus atrophaeus by observing its effects using transmission electron microscopy (TEM) and also by evaluating the existence of DNA damage by fluorescence-based quantitative polymerase chain reaction (qPCR). Methods: Spore samples of B. atrophaeus decontaminated using iHP at different exposure times (Control, 1, 2, 6, and 12 h) were fixed for TEM. In addition, DNA was extracted for evaluation of DNA damages using fluorescence-based qPCR assays. Results: Damages to the spore structures of B. atrophaeus caused by the decontamination process with iHP at different exposure times (Control, 1, 2, 6, and 12 h) can be observed in micrographs. The effects of the decontamination to short DNA segment (132 base pairs [bp]) of the yaaH gene using qPCR present a linear degradation, and for the long DNA segment (680 bp), it presents a biphasic mode. Conclusion: The results of the qPCR analysis show two initial stages of damage to DNA with very noticeable damage at 12 h contact time, which confirms the observations of the TEM micrographs for the B. atrophaeus spores. The study demonstrates damage to the spore core DNA.

Sterilization effects of UV laser irradiation on Bacillus atrophaeus spore viability, structure, and proteins.

Bacillus spores are highly resistant to toxic chemicals and extreme environments. Because some Bacillus species threaten public health, spore inactivation techniques have been intensively investigated. We exposed Bacillus atrophaeus spores to a 266 nm Nd:YVO4 laser at a laser power of 1 W and various numbers of scans. As a result, the UV laser reduced the viability of Bacillus atrophaeus spores. Although the outer coat of spores remained intact after UV laser irradiation of 720 scans, damage inside the spores was observed. Spore proteins were identified by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry during the course of UV laser irradiation. Photochemical and photothermal processes are believed to be involved in the UV laser sterilization of Bacillus spores. Our findings suggest that a UV laser is capable of sterilizing Bacillus atrophaeus spores.