Susceptibility of extremophiles to far-UVC light for bioburden reduction in spacecraft assembly facilities.

The prevention and reduction of microbial species entering and leaving Earth's biosphere is a critical aspect of planetary protection research. While various decontamination methods exist and are currently utilized for planetary protection purposes, the use of far-UVC light (200-230 nm) as a means for microbial reduction remains underexplored. Unlike conventional germicidal ultraviolet at 254 nm, which can pose a health risk to humans even with small exposure doses, far-UVC light poses minimal health hazard making it a suitable candidate for implementation in occupied areas of spacecraft assembly facilities. This study investigates the efficacy of far-UVC 222-nm light to inactivate bacteria using microbial species which are relevant to planetary protection either in vegetative cell or spore form. All the tested vegetative cells demonstrated susceptibility to 222-nm exposure, although susceptibility varied among the tested species. Notably, Deinococcus radiodurans, a species highly tolerant to extreme environmental conditions, exhibited the most resistance to far-UVC exposure with a dose of 112 mJ/cm2 required for a 1-log reduction in survival. While spore susceptibility was similar across the species tested, Bacillus pumilus spores were the most resistant of the tested spores when analyzed with a bi-exponential cell killing model (D90 of 6.8 mJ/cm2). Overall, these results demonstrate the efficacy of far-UVC light for reducing microbial bioburden to help ensure the success and safety of future space exploration missions.

A lab-on-a-chip utilizing microwaves for bacterial spore disruption and detection.

Bacterial spores are problematic in agriculture, the food industry, and healthcare, with the fallout costs from spore-related contamination being very high. Spores are difficult to detect since they are resistant to many of the bacterial disruption techniques used to bring out the biomarkers necessary for detection. Because of this, effective and practical spore disruption methods are desirable. In this study, we demonstrate the efficiency of a compact microfluidic lab-on-chip built around a coplanar waveguide (CPW) operating at 2.45 GHz. We show that the CPW generates an electric field hotspot of ∼10 kV/m, comparable to that of a commercial microwave oven, while using only 1.2 W of input power and thus resulting in negligible sample heating. Spores passing through the microfluidic channel are disrupted by the electric field and release calcium dipicolinic acid (CaDPA), a biomarker molecule present alongside DNA in the spore core. We show that it is possible to detect this disruption in a bulk spore suspension using fluorescence spectroscopy. We then use laser tweezers Raman spectroscopy (LTRS) to show the loss of CaDPA on an individual spore level and that the loss increases with irradiation power. Only 22% of the spores contain CaDPA after exposure to 1.2 W input power, compared to 71% of the untreated control spores. Additionally, spores exposed to microwaves appear visibly disrupted when imaged using scanning electron microscopy (SEM). Overall, this study shows the advantages of using a CPW for disrupting spores for biomarker release and detection.

UV Disinfection sensitivity index of spores or protozoa: A model to predict the required fluence of spores or protozoa.

During UV disinfection, the required UV dose in terms of fluence depends upon the species of bacteria spore and protozoa. To rank their UV disinfection sensitivity, spore sensitivity index (SPSI) and protozoan sensitivity index (PSI) are defined. For spores, shoulder effect exists, therefore, SPSI is defined as the ratio between the ki of any spores for the linear portion of the dose response curve to the kir of Bacillus subtilis as the reference spore. After statistical analysis, the fluence of any spore can be predicted by SPSI through equation, H = (0.8358 ± 0.126)*LogI*SPSI + H0. PSI is defined as the ratio between the inactivation rate constants of a protozoa in reference to that of Cryptosporidium parvum. The equation predicting the fluence of any protozoa in reference to Cryptosporidium parvum is: H = 107.45*(3.86 ± 2.68)*LogI/PSI. Two regression equations suggest that protozoa require significantly higher UV dose than bacteria spores.

Effects of Desiccation and Freezing on Microbial Ionizing Radiation Survivability: Considerations for Mars Sample Return.

Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae); and vegetative cells and endospores of three Bacillus bacteria (B. subtilis, B. megaterium, B. thuringiensis). Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. Thus, the radiation survival threshold of polyploid D. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. Deinococcus radiodurans and S. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. radiodurans are joined by persistent Holliday junctions. We estimate ionizing radiation survival limits of polyploid DNA-based life-forms to be hundreds of millions of years of background radiation while buried in the martian subsurface. Our findings imply that forward contamination of Mars will essentially be permanent, and backward contamination is a possibility if life ever existed on Mars.

Uptake of UVc induced photoproducts of dipicolinic acid by Bacillus subtilis spores - Effects on the germination and UVc resistance of the spores.

Dipicolinic acid (DPA) is a specific molecule of bacterial spores which is essential to their resistance to various stresses such as ultraviolet (UV) exposure and to their germination. DPA has a particular photochemistry that remains imperfectly understood. In particular, due to its ability to absorb UVc radiation, it is likely to form in vitro a wide variety of photoproducts (DPAp) of which only about ten have been recently identified. The photochemical reactions resulting in DPAp, especially those inside the spores, are still poorly understood. Only one of these DPAp, which probably acts as a photosensitizer of DNA upon exposure to UVc, has been identified as having an impact on spores. However, as UVc is required to form DPAp, it is difficult to decouple the overall effect of UVc exposure from the possible effects of DPAp alone. In this study, DPAp were artificially introduced into the spores of the FB122 mutant strain of Bacillus subtilis, one that does not produce DPA. These experiments revealed that some DPAp may play a positive role for the spore. These benefits are visible in an improvement in spore germination rate and kinetics, as well as in an increase in their resistance to UVc exposure.

Microbial Protocols for Spacecraft: 1. Effects of Surface Texture, Low Pressure, and UV Irradiation on Recovery of Microorganisms from Surfaces.

Modeling risks for the forward contamination of planetary surfaces from endemic bioburdens on landed spacecraft requires precise data on the biocidal effects of space factors on microbial survival. Numerous studies have been published over the preceding 60 years on the survival of diverse microorganisms exposed to solar heating, solar ultraviolet (UV) irradiation, vacuum, ionizing radiation, desiccation, and many other planetary surface conditions. These data were generated with diverse protocols that can impair the interpretations of the results due to dynamic experimental errors inherent in all lab protocols. The current study (1) presents data on how metal surfaces can affect spore adhesion, (2) proposes doping and extraction protocols that can achieve very high recovery rates (close to 100%) from aluminum coupons with four Bacillus spp., (3) establishes a timeline in which dried spores on aluminum coupons should be used to minimize aging effects of spore monolayers, (4) confirms that vacuum alone does not dislodge spores dried on aluminum coupons, and (5) establishes that multiple UV irradiation sources yield similar results if properly cross-calibrated. The protocols are given to advance discussions in the planetary protection community on how to standardize lab protocols to align results from diverse labs into a coherent interpretation of how space conditions will degrade microbial survival over time.

The small acid-soluble proteins of Clostridioides difficile are important for UV resistance and serve as a check point for sporulation.

Clostridioides difficile is a nosocomial pathogen which causes severe diarrhea and colonic inflammation. C. difficile causes disease in susceptible patients when endospores germinate into the toxin-producing vegetative form. The action of these toxins results in diarrhea and the spread of spores into the hospital and healthcare environments. Thus, the destruction of spores is imperative to prevent disease transmission between patients. However, spores are resilient and survive extreme temperatures, chemical exposure, and UV treatment. This makes their elimination from the environment difficult and perpetuates their spread between patients. In the model spore-forming organism, Bacillus subtilis, the small acid-soluble proteins (SASPs) contribute to these resistances. The SASPs are a family of small proteins found in all endospore-forming organisms, C. difficile included. Although these proteins have high sequence similarity between organisms, the role(s) of the proteins differ. Here, we investigated the role of the main α/ß SASPs, SspA and SspB, and two annotated putative SASPs, CDR20291_1130 and CDR20291_3080, in protecting C. difficile spores from environmental insults. We found that SspA is necessary for conferring spore UV resistance, SspB minorly contributes, and the annotated putative SASPs do not contribute to UV resistance. In addition, the SASPs minorly contribute to the resistance of nitrous acid. Surprisingly, the combined deletion of sspA and sspB prevented spore formation. Overall, our data indicate that UV resistance of C. difficile spores is dependent on SspA and that SspA and SspB regulate/serve as a checkpoint for spore formation, a previously unreported function of SASPs.

What's new and notable in bacterial spore killing!

Spores of many species of the orders Bacillales and Clostridiales can be vectors for food spoilage, human diseases and intoxications, and biological warfare. Many agents are used for spore killing, including moist heat in an autoclave, dry heat at elevated temperatures, UV radiation at 254 and more recently 222 and 400 nm, ionizing radiation of various types, high hydrostatic pressures and a host of chemical decontaminants. An alternative strategy is to trigger spore germination, as germinated spores are much easier to kill than the highly resistant dormant spores-the so called "germinate to eradicate" strategy. Factors important to consider in choosing methods for spore killing include the: (1) cost; (2) killing efficacy and kinetics; (3) ability to decontaminate large areas in buildings or outside; and (4) compatibility of killing regimens with the: (i) presence of people; (ii) food quality; (iii) presence of significant amounts of organic matter; and (iv) minimal damage to equipment in the decontamination zone. This review will summarize research on spore killing and point out some common flaws which can make results from spore killing research questionable.

Ultraviolet-C inactivation and hydrophobicity of Bacillus subtilis and Bacillus velezensis spores isolated from extended shelf-life milk.

Bacterial spores are important in food processing due to their ubiquity, resistance to high temperature and chemical inactivation. This work aims to study the effect of ultraviolet C (UVC) on the spores of Bacillus subtilis and Bacillus velezensis at a molecular and individual level to guide in deciding on the right parameters that must be applied during the processing of liquid foods. The spores were treated with UVC using phosphate buffer saline (PBS) as a suspension medium and their lethality rate was determined for each sample. Purified spore samples of B. velezensis and B. subtilis were treated under one pass in a UVC reactor to inactivate the spores. The resistance pattern of the spores to UVC treatment was determined using dipicolinic acid (Ca-DPA) band of spectral analysis obtained from Raman spectroscopy. Flow cytometry analysis was also done to determine the effect of the UVC treatment on the spore samples at the molecular level. Samples were processed for SEM and the percentage spore surface hydrophobicity was also determined using the Microbial Adhesion to Hydrocarbon (MATH) assay to predict the adhesion strength to a stainless-steel surface. The result shows the maximum lethality rate to be 6.5 for B. subtilis strain SRCM103689 (B47) and highest percentage hydrophobicity was 54.9% from the sample B. velezensis strain LPL-K103 (B44). The difference in surface hydrophobicity for all isolates was statistically significant (P < 0.05). Flow cytometry analysis of UVC treated spore suspensions clarifies them further into sub-populations unaccounted for by plate counting on growth media. The Raman spectroscopy identified B4002 as the isolate possessing the highest concentration of Ca-DPA. The study justifies the critical role of Ca-DPA in spore resistance and the possible sub-populations after UVC treatment that may affect product shelf-life and safety. UVC shows a promising application in the inactivation of resistant spores though there is a need to understand the effects at the molecular level to design the best parameters during processing.

Assessment of saliva interference with UV-based disinfection technologies.

Worldwide shortages of personal protective equipment during COVID-19 pandemic has forced the implementation of methods for decontaminating face piece respirators such as N95 respirators. The use of UV irradiation to reduce bioburden of used respirators attracts attention, making proper testing protocols of uttermost importance. Currently artificial saliva is used but its comparison to human saliva from the UV disinfection perspective is lacking. Here we characterize UV spectra of human and artificial saliva, both fresh and after settling, to test for possible interference for UV-based disinfection. ASTM 2720 artificial saliva recipe (with either porcine or bovine mucin) showed many discrepancies from average (N = 18) human saliva, with different mucins demonstrating very different UV absorbance spectra, resulting in very different UV transmittance at different wavelength. Reducing porcine mucin concentration from 3 to 1.7 g/L brought UVA254 in the artificial saliva to that of average human saliva (although not for other wavelengths), allowing 254 nm disinfection experiments. Phosphate saline and modified artificial saliva were spiked with 8.6 log CFU/ml B. subtilis spores (ATCC 6633) and irradiated at dose of up to 100 mJ/cm2, resulting in 5.9 log inactivation for a saline suspension, and 2.8 and 1.1 log inactivation for ASTM-no mucin and ASTM-1.7 g/L porcine mucin 2 µL dried droplets, respectively. UVC irradiation of spores dried in human saliva resulted in 2.3 and 1.5 log inactivation, depending on the size of the droplets (2 vs 10 µL, respectively) dried on a glass surface. Our results suggest that in the presence of the current standard dried artificial saliva it is unlikely that UVC can achieve 6 log inactivation of B. subtilis spores using a realistic UV dose (e.g. less than 2 J/cm2) and the ATSM saliva recipe should be revised for UV decontamination studies.

Raman spectroscopy reveals alteration of spore compositions under different nutritional conditions in Lysinibacillus boronitolerans YS11.

Little is known about final spores components when bacteria undergo sporulation under different nutrient conditions. Different degrees of resistance and germination rates were observed in the three types of spores of Lysinibacillus boronitolerans YS11 (SD, Spores formed in Difco sporulation medium™; SC and SF, Spores formed in an agricultural byproduct medium with 10 mM CaCl2 and with 10 mM FeSO4, respectively). Stronger UV resistance was recorded for SF with 1.8-2.3-fold greater survival than SC and SD under UV treatment. The three spore types showed similar heat resistances at 80°C, but survival rates of SC and SD were much higher (∼1,000 times) than those of SF at 90°C. However, germination capacity of SF was 20% higher than those of SD and SC on Luria-Bertani agar plates for 24 h. SF germinated more rapidly in a liquid medium with high NaCl concentrations than SC and SD, but became slower under alkaline conditions. Raman spectroscopy was used to analyze the heterogeneities in the three types of vegetative cells and their spores under different nutritional conditions. Exponentially grown-each vegetative cells had different overall Raman peak values. Raman peaks of SC, SD, and SF also showed differences in adenine and amide III compositions and nucleic acid contents. Our data along with Raman spectroscopy provided the evidence that spores formed under under different growth conditions possess very different cellular components, which affected their survival and germination rates.

Effects of intense pulsed light and gamma irradiation on Bacillus cereus spores in mesquite pod flour.

This study was conducted to evaluate the inactivation of Bacillus cereus spore in mesquite flour with intense pulsed light (IPL) and gamma radiation. The physical, chemical, and toxicity of treated mesquite flour were also investigated. The results showed that up to 3.51 log10CFU/g B. cereus spore inactivation was achieved with 8 kGy of gamma radiation, and up to 1.69 log10CFU/g reductions could be achieved after 28s of catalytic IPL exposure. Although chemometric analysis showed 9-hydroxy-10,12-octadecadienoic acid was slightly increased after a 28s-catalytic IPL treatment, the concentration is within the acceptable range. No significant increase in acetic or propionic acids (typical off-flavor volatile compounds) was observed after either treatment. For cytotoxicity, the Caco-2 cell viability analysis revealed that these two technologies did not induce significant cytotoxicity to the treated mesquite flour. Overall, these two technologies exhibit strong potential for the decontamination of B. cereus in mesquite flour.

Antimicrobial photodynamic treatment as an alternative approach for Alicyclobacillus acidoterrestris inactivation.

Alicyclobacillus acidoterrestris is a cause of major concern for the orange juice industry due to its thermal and chemical resistance, as well as its spoilage potential. A. acidoterrestris spoilage of orange juice is due to off-flavor taints from guaiacol production and some halophenols. The present study aimed to evaluate the effectiveness of antimicrobial Photodynamic Treatment (aPDT) as an emerging technology to inactivate the spores of A. acidoterrestris. The aPDT efficiency towards A. acidoterrestris was evaluated using as photosensitizers the tetracationic porphyrin (Tetra-Py+-Me) and the phenothiazinium dye new methylene blue (NMB) in combination with white light-emitting diode (LED; 400-740 nm; 65-140 mW/cm2). The spores of A. acidoterrestris were cultured on YSG agar plates (pH 3.7 ± 0.1) at 45 °C for 28 days and submitted to the aPDT with Tetra-Py+-Me and NMB at 10 µM in phosphate-buffered saline (PBS) in combination with white light (140 mW/cm2). The use of Tetra-Py+-Me at 10 µM resulted in a 7.3 ± 0.04 log reduction of the viability of A. acidoterrestris spores. No reductions in the viability of this bacterium were observed with NMB at 10 µM. Then, the aPDT with Tetra-Py+-Me and NMB at 10 µM in orange juice (UHT; pH 3.9; 11°Brix) alone and combined with potassium iodide (KI) was evaluated. The presence of KI was able to potentiate the aPDT process in orange juice, promoting the inactivation of 5 log CFU/mL of A. acidoterrestris spores after 10 h of white light exposition (140 mW/cm2). However, in the absence of KI, both photosensitizers did not promote a significant reduction in the spore viability. The inactivation of A. acidoterrestris spores artificially inoculated in orange peels (105 spores/mL) was also assessed using Tetra-Py+-Me at 10 and 50 µM in the presence and absence of KI in combination with white light (65 mW/cm2). No significant reductions were observed (p < .05) when Tetra-Py+-Me was used at 10 µM, however at the highest concentration (50 µM) a significant spore reduction (≈ 2.8 log CFU/mL reductions) in orange peels was observed after 6 h of sunlight exposition (65 mW/cm2). Although the color, total phenolic content (TPC), and antioxidant capacity of orange juice and peel (only color evaluation) seem to have been affected by light exposition, the impact on the visual and nutritional characteristics of the products remains inconclusive so far. Besides that, the results found suggest that aPDT can be a potential method for the reduction of A. acidoterrestris spores on orange groves.

Clustering, morphology, and treatment resistance of Bacillus globigii spores recovered from a pilot-scale activated sludge system.

This study examines the organization and morphology of Bacillus globigii (BG) spores, a common surrogate for Bacillus anthracis, which were seeded and then recovered at various times from several points within a conventional, pilot-scale activated sludge system. Recovered BG spores were enumerated, microscopically examined, and tested for resistance to chemical (i.e. 5% H2O2 for 8 min), thermal (80 °C for 30 min), and ultraviolet light (8 W, 254 nm UV for 1 min) inactivation. Spores exposed to activated sludge germinated, sporulated, and exhibited unique multilayer clustering patterns and statistically significant changes (p < 0.005) in dimensional morphology. Spores collected in the later experimental stages (i.e., during weeks 6 and 7) were significantly more resistant (p ≤ 0.05) to inactivation than those collected on the first day of testing. These results have direct consequences for sludge treatment requirements at wastewater treatment plants that receive spore-containing waste streams.

Viability of Bacillus subtilis Spores Exposed to Ultraviolet Light at Ocean World Surface Temperatures.

This work investigated microorganism survival under temperature and ultraviolet (UV) radiation conditions found at the surface of ice-covered ocean worlds. These studies were motivated by a desire to understand the ability of resilient forms of life to survive under such conditions as a proxy for potential endogenic life and to inform planetary protection protocols for future missions. To accomplish this, we irradiated Bacillus subtilis spores with solar-like UV photons at temperatures ranging from room temperature down to 11 K and reported survival fractions with respect to fluence. We observed an increase in survival at low temperatures and found that the inactivation rate follows an Arrhenius-type behavior above 60 K. For solar-photon fluxes and surface temperatures at Europa and Enceladus, we found that Bacillus subtilis spores would be inactivated in less than an hour when in direct sunlight.

Inactivation of Escherichia coli, Salmonella enterica serovar Typhimurium, and Bacillus cereus in roasted grain powder by radio frequency heating.

AIMS: The objective of this study was to evaluate the antimicrobial effects of radio frequency (RF) heating and the combination treatment of RF heating with ultraviolet (UV) radiation against foodborne pathogens in roasted grain powder (RGP). METHODS AND RESULTS: Foodborne pathogens inoculated on RGP were subjected to RF heating or RF-UV combination treatments. After 120 s of RF heating, 4·68, 3·89 and 4·54 log reductions were observed for Escherichia coli, Salmonella Typhimurium and Bacillus cereus vegetative cells respectively. The combined RF-UV treatment showed synergistic effects of over 1 log unit compared to the sum of individual treatment for E. coli and S. Typhimurium, but not for B. cereus vegetative cells because of their high UV resistance. Germinated B. cereus cells were not significantly inactivated by RF heating (<1 log CFU per gram), and increased heat resistance compared to the vegetative cells was verified with mild heat treatment. The colour of RGP was not significantly affected by the RF or RF-UV treatments. CONCLUSIONS: Applying RF heating to grain-based food products has advantages for the inactivation of E. coli and S. Typhimurium in RGP. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study could be used as a basis for determining the treatment conditions for inactivating E. coli and other foodborne pathogens such as S. Typhimurium and B. cereus in RGP.

The spore coat is essential for Bacillus subtilis spore resistance to pulsed light, and pulsed light treatment eliminates some spore coat proteins.

Microbial surface contamination of equipment or of food contact material is a recurring problem in the food industry. Spore-forming bacteria are far more resistant to a wide variety of treatments than their vegetative forms. Understanding the mechanisms underlying decontamination processes is needed to improve surface decontamination strategies against endospores potentially at the source of foodborne diseases or food-spoilage. Pulsed light (PL) with xenon lamps delivers high-energy short-time pulses of light with wavelengths in the range 200 nm-1100 nm and a high UV-C fraction. Bacillus subtilis spores were exposed to either PL or to continuous UV-C. Gel electrophoresis and western blotting revealed elimination of various proteins of the spore coat, an essential outer structure that protects spores from a wide variety of environmental conditions and inactivation treatments. Proteomic analysis confirmed the elimination of some spore coat proteins after PL treatment. Transmission electron microscopy of PL treated spores revealed a gap between the lamellar inner spore coat and the outer spore coat. Overall, spores of mutant strains with defects in genes coding for spore coat proteins were more sensitive to PL than to continuous UV-C. This study demonstrates that radiations delivered by PL contribute to specific damage to the spore coat, and overall to spore inactivation.

Efficacy of pulsed xenon ultraviolet disinfection of multidrug-resistant bacteria and Clostridioides difficile spores.

BACKGROUND: Contamination of healthcare environments by multidrug-resistant organisms (MDRO) and Clostridioides difficile is a risk for healthcare-associated infections. The efficacy of pulsed xenon ultraviolet (PX-UV) disinfection in healthcare environments has been described previously. However, there are few reports about PX-UV disinfection in Japan. The aim of this study was to investigate in vitro the efficacy of PX-UV disinfection of MDRO and C. difficile spores commonly isolated in Japanese hospitals. METHODS: We investigated reductions in microbial counts after exposure to PX-UV of the following clinically-isolated organisms on seeding agar plates: methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium, carbapenemase-producing Klebsiella pneumoniae, extended spectrum ß-lactamase-producing Escherichia coli, multidrug resistant Acinetobacter baumannii, and C. difficile spores. We also visually assessed the attenuation of disinfection by shielding of MRSA and carbapenemase-producing K. pneumoniae from PX-UV exposure. RESULTS: PX-UV disinfection for 5 min induced >5-log colony-forming units (CFU)/cm2 growth inhibition of all the MDRO. PX-UV disinfection for 15 min induced >3-log CFU/cm2 growth inhibition of C. difficile spores. Where a plate was shielded from PX-UV exposure the bacteria showed confluent growth, but no colonies were observed on unshielded (exposed) parts of the plates. CONCLUSION: This study shows the efficacy of PX-UV disinfection against clinical MDROs. C. difficile spores were more resistant to PX-UV disinfection than vegetative bacteria. Further evaluation of the efficacy of PX-UV disinfection in reducing the contamination of real-world surfaces and the incidence of healthcare-associated infections is needed.

Application of a Krypton-Chlorine Excilamp To Control Alicyclobacillus acidoterrestris Spores in Apple Juice and Identification of Its Sporicidal Mechanism.

The aim of this study was to investigate the sporicidal effect of a krypton-chlorine (KrCl) excilamp against Alicyclobacillus acidoterrestris spores and to compare its inactivation mechanism to that of a conventional UV lamp containing mercury (Hg). The inactivation effect of the KrCl excilamp was not significantly different from that of the Hg UV lamp for A. acidoterrestris spores in apple juice despite the 222-nm wavelength of the KrCl excilamp having a higher absorption coefficient in apple juice than the 254-nm wavelength of the Hg UV lamp; this is because KrCl excilamps have a fundamentally greater inactivation effect than Hg UV lamps, which is confirmed under ideal conditions (phosphate-buffered saline). The inactivation mechanism analysis revealed that the DNA damage induced by the KrCl excilamp was not significantly different (P > 0.05) from that induced by the Hg UV lamp, while the KrCl excilamp caused significantly higher (P < 0.05) lipid peroxidation incidence and permeability change in the inner membrane of A. acidoterrestris spores than did the Hg UV lamp. Meanwhile, the KrCl excilamp did not generate significant (P > 0.05) intracellular reactive oxygen species, indicating that the KrCl excilamp causes damage only through the direct absorption of UV light. In addition, after KrCl excilamp treatment with a dose of 2,011 mJ/cm2 to reduce A. acidoterrestris spores in apple juice by 5 logs, there were no significant (P > 0.05) changes in quality parameters such as color (L*, a*, and b*), total phenolic compounds, and DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity.IMPORTANCEAlicyclobacillus acidoterrestris spores, which have high resistance to thermal treatment and can germinate even at low pH, are very troublesome in the juice industry. UV technology, a nonthermal treatment, can be an excellent means to control heat-resistant A. acidoterrestris spores in place of thermal treatment. However, the traditionally applied UV sources are lamps that contain mercury (Hg), which is harmful to humans and the environment; thus, there is a need to apply novel UV technology without the use of Hg. In response to this issue, excilamps, an Hg-free UV source, have been actively studied. However, no studies have been conducted applying this technique to control A. acidoterrestris spores. Therefore, the results of this study, which applied a KrCl excilamp for the control of A. acidoterrestris spores and elucidated the inactivation principle, are expected to be utilized as important basic data for application to actual industry or conducting further studies.

DNA Damage Kills Bacterial Spores and Cells Exposed to 222-Nanometer UV Radiation.

This study examined the microbicidal activity of 222-nm UV radiation (UV222), which is potentially a safer alternative to the 254-nm UV radiation (UV254) that is often used for surface decontamination. Spores and/or growing and stationary-phase cells of Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus aureus, and Clostridioides difficile and a herpesvirus were all killed or inactivated by UV222 and at lower fluences than with UV254B. subtilis spores and cells lacking the major DNA repair protein RecA were more sensitive to UV222, as were spores lacking their DNA-protective proteins, the α/ß-type small, acid-soluble spore proteins. The spore cores' large amount of Ca2+-dipicolinic acid (∼25% of the core dry weight) also protected B. subtilis and C. difficile spores against UV222, while spores' proteinaceous coat may have given some slight protection against UV222 Survivors among B. subtilis spores treated with UV222 acquired a large number of mutations, and this radiation generated known mutagenic photoproducts in spore and cell DNA, primarily cyclobutane-type pyrimidine dimers in growing cells and an α-thyminyl-thymine adduct termed the spore photoproduct (SP) in spores. Notably, the loss of a key SP repair protein markedly decreased spore UV222 resistance. UV222-treated B. subtilis spores germinated relatively normally, and the generation of colonies from these germinated spores was not salt sensitive. The latter two findings suggest that UV222 does not kill spores by general protein damage, and thus, the new results are consistent with the notion that DNA damage is responsible for the killing of spores and cells by UV222IMPORTANCE Spores of a variety of bacteria are resistant to common decontamination agents, and many of them are major causes of food spoilage and some serious human diseases, including anthrax caused by spores of Bacillus anthracis Consequently, there is an ongoing need for efficient methods for spore eradication, in particular methods that have minimal deleterious effects on people or the environment. UV radiation at 254 nm (UV254) is sporicidal and commonly used for surface decontamination but can cause deleterious effects in humans. Recent work, however, suggests that 222-nm UV (UV222) may be less harmful to people than UV254 yet may still kill bacteria and at lower fluences than UV254 The present work has identified the damage by UV222 that leads to the killing of growing cells and spores of some bacteria, many of which are human pathogens, and UV222 also inactivates a herpesvirus.