Mechanistic insight into roles of α/ß-type small acid-soluble proteins, RecA, and inner membrane proteins during bacterial spore inactivation by ohmic heating.

AIM: Ohmic heating (OH) (i.e. heating by electric field) more effectively kills bacterial spores than traditional wet heating, yet its mechanism remains poorly understood. This study investigates the accelerated spore inactivation mechanism using genetically modified spores. METHODS AND RESULTS: We investigated the effects of OH and conventional heating (CH) on various genetically modified strains of Bacillus subtilis: isogenic PS533 (wild type_1), PS578 [lacking spores' α/ß-type small acid-soluble proteins (SASP)], PS2318 (lacking recA, encoding a DNA repair protein), isogenic PS4461 (wild type_2), and PS4462 (having the 2Duf protein in spores, which increases spore wet heat resistance and decreases spore inner membrane fluidity). Removal of SASP brought the inactivation profiles of OH and CH closer, suggesting the interaction of these proteins with the field. However, the reemergence of a difference between CH and OH killing for SASP-deficient spores at the highest tested field strength suggested there is also interaction of the field with another spore core component. Additionally, RecA-deficient spores yielded results like those with the wild-type spores for CH, while the OH resistance of this mutant increased at the lower tested temperatures, implying that RecA or DNA are a possible additional target for the electric field. Addition of the 2Duf protein markedly increased spore resistance both to CH and OH, although some acceleration of killing was observed with OH at 50 V/cm. CONCLUSIONS: In summary, both membrane fluidity and interaction of the spore core proteins with electric field are key factors in enhanced spore killing with electric field-heat combinations.

Oral administration of probiotic spore ghosts for efficient attenuation of radiation-induced intestinal injury.

Radiation-induced intestinal injury is the most common side effect during radiotherapy of abdominal or pelvic solid tumors, significantly impacting patients' quality of life and even resulting in poor prognosis. Until now, oral application of conventional formulations for intestinal radioprotection remains challenging with no preferred method available to mitigate radiation toxicity in small intestine. Our previous study revealed that nanomaterials derived from spore coat of probiotics exhibit superior anti-inflammatory effect and even prevent the progression of cancer. The aim of this work is to determine the radioprotective effect of spore coat (denoted as spore ghosts, SGs) from three clinically approved probiotics (B.coagulans, B.subtilis and B.licheniformis). All the three SGs exhibit outstanding reactive oxygen species (ROS) scavenging ability and excellent anti-inflammatory effect. Moreover, these SGs can reverse the balance of intestinal flora by inhibiting harmful bacteria and increasing the abundance of Lactobacillus. Consequently, administration of SGs significantly reduce radiation-induced intestinal injury by alleviating diarrhea, preventing X-ray induced apoptosis of small intestinal epithelial cells and promoting restoration of barrier integrity in a prophylactic study. Notably, SGs markedly improve weight gain and survival of mice received total abdominal X-ray radiation. This work may provide promising radioprotectants for efficiently attenuating radiation-induced gastrointestinal syndrome and promote the development of new intestinal predilection.

The small acid-soluble proteins of spore-forming organisms: similarities and differences in function.

The small acid-soluble proteins are found in all endospore-forming organisms and are a major component of spores. Through their DNA binding capabilities, the SASPs shield the DNA from outside insults (e.g., UV and genotoxic chemicals). The absence of the major SASPs results in spores with reduced viability when exposed to UV light and, in at least one case, the inability to complete sporulation. While the SASPs have been characterized for decades, some evidence suggests that using newer technologies to revisit the roles of the SASPs could reveal novel functions in spore regulation.

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.

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.

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.

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.

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.

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.

Role of DNA repair in Bacillus subtilis spore resistance to high energy and low energy electron beam treatments.

Bacillus subtilis spore inactivation mechanisms under low energy electron beam (LEEB) and high energy electron beam (HEEB) treatment were investigated using seven mutants lacking specific DNA repair mechanisms. The results showed that most of the DNA repair-deficient mutants, including ΔrecA, ΔKu ΔligD, Δexo Δnfo, ΔuvrAB and ΔsbcDC, had reduced resistances towards electron beam (EB) treatments at all investigated energy levels (80 keV, 200 keV and 10 MeV) compared to their wild type. This result suggested DNA damage was induced during EB treatments. The mutant lacking recA showed the lowest resistance, followed by the mutant lacking Ku and ligD. These findings indicated that recA, Ku and ligD and their associated DNA repair mechanisms, namely, homologous recombination and non-homologous end joining, play important roles in spore survival under EB treatment. Furthermore, exoA, nfo, uvrAB, splB, polY1 and polY2, which are involved in nucleotide damage repair/removal, showed different levels of effects on spore resistance under EB treatment. Finally, the results suggested that HEEB and LEEB inactivate B. subtilis spores through similar mechanisms. This research will provide a better understanding of how EB technologies inactivate B. subtilis spores and will contribute to the application of these technologies as a non-thermal, gentle spore control approach.

Properties of Aged Spores of Bacillus subtilis.

Bacillus spores incubated on plates for 2 to 98 days at 37°C had identical Ca-dipicolinic acid contents, exhibited identical viability on rich- or poor-medium plates, germinated identically in liquid with all germinants tested, identically returned to vegetative growth in rich or minimal medium, and exhibited essentially identical resistance to dry heat and similar resistance to UV radiation. However, the oldest spores had a lower core water content and significantly higher wet heat and NaOCl resistance. In addition, 47- and 98-day spores had lost >98% of intact 16S and 23S rRNA and 97 to 99% of almost all mRNAs, although minimal amounts of mononucleotides were generated in 91 days. Levels of 3-phosphoglyceric acid (3PGA) also fell 30 to 60% in the oldest spores, but how the 3PGA was lost is not clear. These results indicate that (i) translation of dormant spore mRNA is not essential for completion of spore germination, nor is protein synthesis from any mRNA; (ii) in sporulation for up to 91 days at 37°C, the RNA broken down generates minimal levels of mononucleotides; and (iii) the lengths of time that spores are incubated in sporulation medium should be considered when determining conditions for spore inactivation by wet heat, in particular, in using spores to test for the efficacy of sterilization regimens.IMPORTANCE We show that spores incubated at 37°C on sporulation plates for up to 98 days have lost almost all mRNAs and rRNAs, yet the aged spores germinated and outgrew as well as 2-day spores, and all these spores had identical viability. Thus, it is unlikely that spore mRNA, rRNA, or protein synthesis is important in spore germination. Spores incubated for 47 to 98 days also had much higher wet heat resistance than 2-day spores, suggesting that spore "age" should be considered in generating spores for tests of sterilization assurance. These data are the first to show complete survival of hydrated spores for ∼100 days, complementing published data showing dry-spore survival for years.

Photocatalytic inactivation of bioaerosols in a fixed-bed reactor with TiO2-coated glass rings.

The photocatalytic inactivation of Bacillus subtilis spores in air was evaluated employing a fixed-bed reactor with TiO2-coated glass rings, under artificial UV-A radiation. Calculations of the radiation effectively absorbed inside the reactor were carried out by Monte Carlo simulations. The photocatalytic inactivation was assessed by analyzing the viability of the microorganisms retained by the coated glass rings inside the reactor at different irradiation periods. The initial concentration of the spores was reduced by almost 55% at the end of the experiment (12 h). Complementary assays were carried out employing Bacillus subtilis vegetative cells, obtaining a reduction of more than 96% under the same conditions. Two efficiency parameters were computed to assess the reactor performance: the photonic efficiency and the quantum efficiency of inactivation. Results of the efficiency parameters allow an objective comparison of the reactor performance under different experimental conditions and configurations.

Effect of UV-C light or hydrogen peroxide wipes on the inactivation of methicillin-resistant Staphylococcus aureus, Clostridium difficile spores and norovirus surrogate.

AIMS: The current study aimed to assess the potential of a new high dose ultraviolet (UV) disinfection device to inactivate methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile and a norovirus surrogate on handheld mobile devices, and to compare the efficacy of the UV-C device to hydrogen peroxide disinfection wipes. METHODS AND RESULTS: Suspensions of MRSA, C. difficile spores and a surrogate for norovirus (MS2) were inoculated onto glass or plastic coupons, with or without organic contamination and were exposed to continuous UV-C light for 15-60 s (165-646 mJ cm-2 ) in a self-contained UV-C chamber or treated with hydrogen peroxide wipes. Increasing the UV-C dose from 310 to 650 mJ cm-2 did not result in greater levels of inactivation. UV-C light inactivated all three micro-organisms, in the absence of organic contamination, by >2·9 log. Treatment of MRSA, C. difficile spores or MS2, in the presence of organic contamination, with UV-C light (310-646 mJ cm-2 ) resulted in 2·3-3·7 log reductions. Treatment of MRSA with UV-C light provided levels of inactivation comparable to treatment with hydrogen peroxide wipes used following the manufacturer's instructions. CONCLUSIONS: UV-C light and hydrogen peroxide wipes had strong antimicrobial activity against MRSA, C. difficile spores and a norovirus surrogate, in the presence or absence of organic contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: Chemical disinfection wipes are widely used in healthcare facilities, but they are not recommended for use on handheld mobile devices which may harbour pathogenic micro-organisms. The powerful bactericidal, sporicidal and virucidal activity of this high dose UV-C light device, shows that this technology is a promising alternative to chemical disinfectants, particularly for control of MRSA.

Modelling of ultraviolet light inactivation kinetics of methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, Clostridium difficile spores and murine norovirus on fomite surfaces.

AIMS: Quantitative data on the doses needed to inactivate micro-organisms on fomites are not available for ultraviolet applications. The goal of this study was to determine the doses of UV light needed to reduce bacteria and murine norovirus (MNV) on hard surface fomites through experimentation and to identify appropriate models for predicting targeted levels of reduction. METHODS AND RESULTS: Stainless steel and Formica laminate coupons were selected as they are common surfaces found in healthcare settings. Test organisms included methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Clostridium difficile and MNV. The fomites were inoculated with 105 -107 bacteria or virus and exposed to a range of UV doses. The order of resistance to UV irradiation was virus, bacterial spore and vegetative cell. The best fitting inactivation curves suggested nonlinear responses to increasing doses after a 3-4 log reduction in the test organisms. The average UV doses required for a 3 log reduction in the C. difficile, MRSA and VRE were 16 000, 6164 and 11 228 (mJ-s cm-2 ) for stainless steel, respectively, and 16 000, 11 727 and 12 441 (mJ-s cm-2 ) for Formica laminate, respectively. CONCLUSIONS: Higher UV light doses are required to inactivate bacteria and viruses on hard surfaces than in suspension. Greater doses are needed to inactivate bacterial spores and MNV compared to vegetative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantitative data and models on UV light doses needed to inactivate bacteria and MNV on hard surfaces are now available. The generalizable results of this study can be used to estimate required UV dosages to achieve targeted levels of inactivation based on estimated levels of contamination or to support quantitative microbial risk assessments.

Inactivation of Bacillus subtilis spores at various germination and outgrowth stages using intense pulsed light.

It is important to inactivate spore-forming bacteria in foods because their spores are highly resistant to various stresses. Although thermal treatment is an effective inactivation method, the associated high temperatures can cause changes in food quality. Intense pulsed light (IPL) is a nonthermal technique that can effectively improve food safety. This study evaluated the inactivation effects of IPL at various fluences on Bacillus subtilis spores. IPL treatment at a total fluence of 7.40 J/cm2 resulted in a 7 log reduction, indicating the potential of IPL to effectively inactivate bacterial spores. The sensitivity of B. subtilis spores to IPL during germination and outgrowth was also measured. The resistance to the IPL increased temporarily until 1 h after the start of incubation, and then gradually decreased for longer incubation periods. This temporary increase in resistance at the early stage of incubation was attributed to the leakage of dipicolinic acid from the spores. The results also showed that the inactivation efficiency increases after 1 h pre-incubation because the numbers of vegetative cells increased with the incubation time.

Inactivation of Bacillus and Clostridium Spores in Coconut Water by Ultraviolet Light.

Bacterial spores are generally more resistant than vegetative bacteria to ultraviolet (UV) inactivation. The UV sensitivity of these spores must be known for implementing UV disinfection of low acid liquid foods. UV inactivation kinetics of bacterial spores in coconut water (CW) and distilled sterile water was studied. Populations of Bacillus cereus and Clostridium sporogenes dormant spores were reduced by more than 5.5 log10 at the UV-C photon fluence of 1142 µE·m-2 and 1919 µE·m-2 respectively. C. sporogenes spores showed higher UV-C resistance than B. cereus, with the photon fluence 300 µE·m-2 required for one log inactivation (D10) and 194 µE·m-2, respectively. No significant difference was observed in D10 values of spores suspended in the two fluid types (p > 0.05). The inactivation kinetics of microorganisms were described by log linear models with low root mean square error and high coefficient of determination (R2 > 0.98). This study clearly demonstrated that high levels of inactivation of bacterial spores can be achieved in CW. The baseline data generated from this study will be used to conduct spore inactivation studies in continuous flow UV systems. Further proliferation of the technology will include conducting extensive pilot studies.

Preservation of red pepper flakes using microwave-combined cold plasma treatment.

BACKGROUND: Red pepper flakes are often contaminated with various microorganisms; however, any technologies aiming to decontaminate the flakes should also maintain their quality properties. This study investigated the effect of microwave-combined cold plasma treatment (MCPT) at different microwave power densities on microbial inactivation and preservation of red pepper flakes. Red pepper flake samples inoculated with spores of Bacillus cereus or Aspergillus flavus and without inoculation were subjected to MCPT at 900 W for 20 min at either low microwave power density (LMCPT, 0.17 W m-2 ) or high microwave power density (HMCPT, 0.25 W m-2 ). RESULTS: The numbers of B. cereus and A. flavus spores on red pepper flakes after LMCPT and HMCPT were initially reduced by 0.7 ± 0.1 and 1.4 ± 0.3 log spores cm-2 and by 1.5 ± 0.3 and 1.5 ± 0.2 log spores cm-2 respectively and remained constant for 150 days at 25 °C. Immediately after HMCPT, the concentrations of capsaicin and ascorbic acid in the flakes were significantly lower than in untreated samples; however, no difference in concentration was detected during storage. Neither LMCPT nor HMCPT affected the antioxidant activity or color of the flakes during storage. LMCPT also did not affect the sensory properties and the concentrations of capsaicin and dihydrocapsaicin of the flakes, indicating its suitability in preserving their quality properties. CONCLUSION: MCPT may provide an effective non-thermal treatment for food preservation which can improve the microbial safety and stability of red pepper flakes while maintaining intact their qualitative properties. © 2018 Society of Chemical Industry.

Fatty Acid Oxidation Is Required for Myxococcus xanthus Development.

Myxococcus xanthus cells produce lipid bodies containing triacylglycerides during fruiting body development. Fatty acid ß-oxidation is the most energy-efficient pathway for lipid body catabolism. In this study, we used mutants in fadJ (MXAN_5371 and MXAN_6987) and fadI (MXAN_5372) homologs to examine whether ß-oxidation serves an essential developmental function. These mutants contained more lipid bodies than the wild-type strain DK1622 and 2-fold more flavin adenine dinucleotide (FAD), consistent with the reduced consumption of fatty acids by ß-oxidation. The ß-oxidation pathway mutants exhibited differences in fruiting body morphogenesis and produced spores with thinner coats and a greater susceptibility to thermal stress and UV radiation. The MXAN_5372/5371 operon is upregulated in sporulating cells, and its expression could not be detected in csgA, fruA, or mrpC mutants. Lipid bodies were found to persist in mature spores of DK1622 and wild strain DK851, suggesting that the roles of lipid bodies and ß-oxidation may extend to spore germination.IMPORTANCE Lipid bodies act as a reserve of triacylglycerides for use when other sources of carbon and energy become scarce. ß-Oxidation is essential for the efficient metabolism of fatty acids associated with triacylglycerides. Indeed, the disruption of genes in this pathway has been associated with severe disorders in animals and plants. Myxococcus xanthus, a model organism for the study of development, is ideal for investigating the complex effects of altered lipid metabolism on cell physiology. Here, we show that ß-oxidation is used to consume fatty acids associated with lipid bodies and that the disruption of the ß-oxidation pathway is detrimental to multicellular morphogenesis and spore formation.

Avirulent Bacillus anthracis Strain with Molecular Assay Targets as Surrogate for Irradiation-Inactivated Virulent Spores.

The revelation in May 2015 of the shipment of γ irradiation-inactivated wild-type Bacillus anthracis spore preparations containing a small number of live spores raised concern about the safety and security of these materials. The finding also raised doubts about the validity of the protocols and procedures used to prepare them. Such inactivated reference materials were used as positive controls in assays to detect suspected B. anthracis in samples because live agent cannot be shipped for use in field settings, in improvement of currently deployed detection methods or development of new methods, or for quality assurance and training activities. Hence, risk-mitigated B. anthracis strains are needed to fulfill these requirements. We constructed a genetically inactivated or attenuated strain containing relevant molecular assay targets and tested to compare assay performance using this strain to the historical data obtained using irradiation-inactivated virulent spores.

A Standard Method To Inactivate Bacillus anthracis Spores to Sterility via Gamma Irradiation.

In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 × 1010 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D10) values and irradiation doses required to ensure sterility (DSAL) to the point at which the probability of detecting a viable spore is 10-6 Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques.IMPORTANCE The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.