Characterization of individual spores of two biological insecticides, Bacillus thuringiensis and Lysinibacillus sphaericus, in response to glutaraldehyde using single-cell optical approaches.

Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) are the most widely used microbial insecticides. Both encounter unfavorable environmental factors and pesticides in the field. Here, the responses of Bt and Ls spores to glutaraldehyde were characterized using Raman spectroscopy and differential interference contrast imaging at the single-cell level. Bt spores were more sensitive to glutaraldehyde than Ls spores under prolonged exposure: <1.0% of Bt spores were viable after 10 min of 0.5% (v/v) glutaraldehyde treatment, compared to ~ 20% of Ls spores. The Raman spectra of glutaraldehyde-treated Bt and Ls spores were almost identical to those of untreated spores; however, the germination process of individual spores was significantly altered. The time to onset of germination, the period of rapid Ca2+-2,6-pyridinedicarboxylic acid (CaDPA) release, and the period of cortex hydrolysis of treated Bt spores were significantly longer than those of untreated spores, with dodecylamine germination being particularly affected. Similarly, the germination of treated Ls spores was significantly prolonged, although the prolongation was less than that of Bt spores. Although the interiors of Bt and Ls spores were undamaged and CaDPA did not leak, proteins and structures involved in spore germination could be severely damaged, resulting in slower and significantly prolonged germination. This study provides insights into the impact of glutaraldehyde on bacterial spores at the single cell level and the variability in spore response to glutaraldehyde across species and populations.

Comparing resistance of bacterial spores and fungal conidia to pulsed light and UVC radiation at a wavelength of 254 nm.

Pulsed light (PL) inactivates microorganisms by UV-rich, high-irradiance and short time pulses (250 µs) of white light with wavelengths from 200 nm to 1100 nm. PL is applied for disinfection of food packaging material and food-contact equipment. Spores of seven Bacillus ssp. strains and one Geobacillus stearothermophilus strain and conidia of filamentous fungi (One strain of Aspergillus brasiliensis, A. carbonarius and Penicillium rubens) were submitted to PL (fluence from 0.23 J/cm2 to 4.0 J/cm2) and UVC (at λ = 254 nm; fluence from 0.01 J/cm2 to 3.0 J/cm2). One PL flash at 3 J/cm2 allowed at least 3 log-reduction of all tested microorganisms. The emetic B. cereus strain F4810/72 was the most resistant of the tested spore-forming bacteria. The PL fluence to 3 log-reduction (F3 PL) of its spores suspended in water was 2.9 J/cm2 and F3 UVC was 0.21 J/cm2, higher than F3 PL and F3 UVC of spores of B. pumilus SAFR-032 2.0 J/cm2 and 0.15 J/cm2, respectively), yet reported as a highly UV-resistant spore-forming bacterium. PL and UVC sensitivity of bacterial spores was correlated. Aspergillus spp. conidia suspended in water were poorly sensitive to PL. In contrast, PL inactivated Aspergillus spp. conidia spread on a dry surface more efficiently than UVC. The F2 PL of A. brasiliensis DSM1988 was 0.39 J/cm2 and F2 UVC was 0.83 J/cm2. The resistance of spore-forming bacteria to PL could be reasonably predicted from the knowledge of their UVC resistance. In contrast, the sensitivity of fungal conidia to PL must be specifically explored.

Use of semi-permeable bag materials to facilitate on-site treatment of biological agent-contaminated waste.

Clean up following the wide-area release of a persistent biological agent has the potential to generate significant waste. Waste containing residual levels of biological contaminants may require off-site shipment under the U.S. Department of Transportation's (US DOT) solid waste regulations for Category A infectious agents, which has packaging and size limitations that do not accommodate large quantities. Treating the waste on-site to inactivate the bio-contaminants could alleviate the need for Category A shipping and open the possibility for categorizing the waste as conventional solid waste with similar shipping requirements as municipal garbage. To collect and package waste for on-site treatment, a semi-permeable nonwoven-based fabric was developed. The fabric was designed to contain residual bio-contaminants while providing sufficient permeability for penetration by a gaseous decontamination agent. The nonwoven fabric was tested in two bench-scale experiments. First, decontamination efficacy and gas permeability were evaluated by placing test coupons inoculated with spores of a Bacillus anthracis surrogate inside the nonwoven material. After chlorine dioxide fumigation, the coupons were analyzed for spore viability and results showed a ≥6 Log reduction on all test materials except glass. Second, filters cut from the nonwoven material were tested in parallel with commercially available cellulose acetate filters having a known pore size (0.45 µm) and results demonstrate that the two materials have similar permeability characteristics. Overall, results suggest that the nonwoven material could be used to package waste at the point of generation and then moved to a nearby staging area where it could be fumigated to inactivate bio-contaminants.

Multifactorial resistance of Bacillus subtilis spores to low-pressure plasma sterilization.

Common sterilization techniques for labile and sensitive materials have far-reaching applications in medical, pharmaceutical, and industrial fields. Heat inactivation, chemical treatment, and radiation are established methods to inactivate microorganisms, but pose a threat to humans and the environment and can damage susceptible materials or products. Recent studies have demonstrated that cold low-pressure plasma (LPP) treatment is an efficient alternative to common sterilization methods, as LPP's levels of radicals, ions, (V)UV-radiation, and exposure to an electromagnetic field can be modulated using different process gases, such as oxygen, nitrogen, argon, or synthetic (ambient) air. To further investigate the effects of LPP, spores of the Gram-positive model organism Bacillus subtilis were tested for their LPP susceptibility including wild-type spores and isogenic spores lacking DNA-repair mechanisms such as non-homologous end-joining (NHEJ) or abasic endonucleases, and protective proteins like α/ß-type small acid-soluble spore proteins (SASP), coat proteins, and catalase. These studies aimed to learn how spores resist LPP damage by examining the roles of key spore proteins and DNA-repair mechanisms. As expected, LPP treatment decreased spore survival, and survival after potential DNA damage generated by LPP involved efficient DNA repair following spore germination, spore DNA protection by α/ß-type SASP, and catalase breakdown of hydrogen peroxide that can generate oxygen radicals. Depending on the LPP composition and treatment time, LPP treatment offers another method to efficiently inactivate spore-forming bacteria.IMPORTANCESurface-associated contamination by endospore-forming bacteria poses a major challenge in sterilization, since the omnipresence of these highly resistant spores throughout nature makes contamination unavoidable, especially in unprocessed foods. Common bactericidal agents such as heat, UV and γ radiation, and toxic chemicals such as strong oxidizers: (i) are often not sufficient to completely inactivate spores; (ii) can pose risks to the applicant; or (iii) can cause unintended damage to the materials to be sterilized. Cold low-pressure plasma (LPP) has been proposed as an additional method for spore eradication. However, efficient use of LPP in decontamination requires understanding of spores' mechanisms of resistance to and protection against LPP.

Selective purification and rapid quantitative detection of spores using a "stepped" magnetic flow device.

A study on the inactivation and germination mechanism of spores is very important in the application of spores, as such high-purity spores are the basis of related research. However, spores and vegetative cells of bacteria often coexist, and it is difficult to separate them. In this study, a magnetic flow device for the purification of spores in the culture medium system was developed based on a "stepped" structure with a magnetic force that could absorb vegetative cells with magnetic nanoparticles. The operation process was as follows: first, vancomycin functionalized nanoparticles were used to prepare Van-Fe3O4 NPs, which were then combined with vegetative cells to form a magnetic conjugate. Subsequently, the magnetic conjugate (vegetative cells) flowed through the "stepped" magnetic flow device and was adsorbed. Meanwhile, the spores moved through the channel and were collected. The achieved purity of the collected spores was more than 95%. Further, the number of the obtained spores was quickly quantified using Raman spectroscopy. The entire purification and quantitative process can be completed within 30 min and the limit of detection was 5 CFU mL-1. This study showed outstanding spore purification ability and provided a new method for purification and rapid quantitative detection of spores.

The design, synthesis, and inhibition of Clostridioides difficile spore germination by acyclic and bicyclic tertiary amide analogs of cholate.

Clostridioides difficile infection (CDI) is a major identifiable cause of antibiotic-associated diarrhea. In our previous study (J. Med. Chem., 2018, 61, 6759-6778), we have identified N-phenyl-cholan-24-amide as a potent inhibitor of spore germination. The most potent compounds in our previous work are N-arylamides. We were interested in the role that the conformation of the amide plays in activity. Previous research has shown that secondary N-arylamides exist exclusively in the coplanar trans conformation while tertiary N-methyl-N-arylamides exist in a non-planar, cis conformation. The N-methyl-N-phenyl-cholan-24-amide was 17-fold less active compared to the parent compounds suggesting the importance of the orientation of the phenyl ring. To lock the phenyl ring into a trans conformation, cyclic tertiary amides were prepared. Indoline and quinoline cholan-24-amides were both inhibitors of spore germination; however, the indoline analogs were most potent. Isoindoline and isoquinoline amides were inactive. We found that the simple indoline derivative gave an IC50 value of 1 µM, while the 5'-fluoro-substituted compound (5d) possessed an IC50 of 400 nM. To our knowledge, 5d is the most potent known spore germination inhibitor described to date. Taken together, our results indicate that the trans, coplanar conformation of the phenyl ring is required for potent inhibition.

Correlating Antibiotic-Induced Dysbiosis to Clostridioides difficile Spore Germination and Host Susceptibility to Infection Using an Ex Vivo Assay.

Antibiotic-induced microbiota disruption and its persistence create conditions for dysbiosis and colonization by opportunistic pathogens, such as those causing Clostridioides difficile (C. difficile) infection (CDI), which is the most severe hospital-acquired intestinal infection. Given the wide differences in microbiota across hosts and in their recovery after antibiotic treatments, there is a need for assays to assess the influence of dysbiosis and its recovery dynamics on the susceptibility of the host to CDI. Germination of C. difficile spores is a key virulence trait for the onset of CDI, which is influenced by the level of primary vs secondary bile acids in the intestinal milieu that is regulated by the microbiota composition. Herein, the germination of C. difficile spores in fecal supernatant from mice that are subject to varying degrees of antibiotic treatment is utilized as an ex vivo assay to predict intestinal dysbiosis in the host based on their susceptibility to CDI, as determined by in vivo CDI metrics in the same mouse model. Quantification of spore germination down to lower detection limits than the colony-forming assay is achieved by using impedance cytometry to count single vegetative bacteria that are identified based on their characteristic electrical physiology for distinction vs aggregated spores and cell debris in the media. As a result, germination can be quantified at earlier time points and with fewer spores for correlation to CDI outcomes. This sets the groundwork for a point-of-care tool to gauge the susceptibility of human microbiota to CDI after antibiotic treatments.

Cell behaviors underlying Myxococcus xanthus aggregate dispersal.

IMPORTANCE: Understanding the processes behind bacterial biofilm formation, maintenance, and dispersal is essential for addressing their effects on health and ecology. Within these multicellular communities, various cues can trigger differentiation into distinct cell types, allowing cells to adapt to their specific local environment. The soil bacterium Myxococcus xanthus forms biofilms in response to starvation, marked by cells aggregating into mounds. Some aggregates persist as spore-filled fruiting bodies, while others disperse after initial formation for unknown reasons. Here, we use a combination of cell tracking analysis and computational simulations to identify behaviors at the cellular level that contribute to aggregate dispersal. Our results suggest that cells in aggregates actively determine whether to disperse or persist and undergo a transition to sporulation based on a self-produced cue related to the aggregate size. Identifying these cues is an important step in understanding and potentially manipulating bacterial cell-fate decisions.

[Identification of Clinical Isolates of the Bacillus cereus Group and Their Characterization by Mass Spectrometry and Electron Microscopy].

Bacillus cereus is a spore-forming bacterium found in the environment mainly in soil. Bacillus spores are known to be extremely resistant not only to environmental factors, but also to various sanitation regimes. This leads to spore contamination of toxin-producing strains in hospital and food equipment and, therefore, poses a great threat to human health. Two clinical isolates identified as B. cereus and B. cytotoxicus were used in the present work. It was shown that their calcium ion content was significantly lower than that of the reference strains. According to electron microscopy, one of the SRCC 19/16 isolates has an enlarged exosporium, and the SRCC 1208 isolate has large electron-dense inclusions of an unclear nature during sporulation. We can assume that these contain a biologically active component with a cytotoxic effect and possibly play a role in pathogenesis. Comparative chemical, biochemical, physiological, and ultrastructural analysis of spores of clinical isolates and reference strains of B. cereus was performed. The results we obtained deepen our understanding of the properties of spores that contribute to the increased pathogenicity of B. cereus group species.

Proteomics-based analysis of the stress response of Bacillus cereus spores under ultrasound and electrolyzed water treatment.

Ultrasound is a green nonthermal technology with promising applications in microbial inactivation. Electrolyzed water has been investigated and found to have a synergistic inactivation effect of ultrasound on spores. This study used a data-independent-acquisition method to analyze the stress response of Bacillus cereus spores following ultrasound combined with electrolyzed water treatment. We identified 197 differentially expressed proteins under ultrasound combined with an electrolyzed water treatment for which the ratio in the metabolic pathway was the highest. Spores downregulated key proteins in energy metabolic and transportation pathways, in particular in phosphotransferase systems and ATP synthase under ultrasound, electrolyzed water, and combined stress. The results of this study revealed that the key proteins in intracellular metabolism decreased after ultrasound treatment, and the expression of small acid-soluble spore protein and cell wall biosynthesis protein increased. Meanwhile, DNA integration, recombination, and inversion protein and small acid-soluble spore protein were upregulated after electrolyzed water treatment. In general, the spores exhibited stress resistance under external stress. The inactivation of spores by further stress was reduced, which we called "cross-protection."

Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop.

Clostridioides difficile infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation co-germinants in the small intestine. While bile acid germinants are essential for C. difficile spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, are essential for inducing germination, while another proposes that either co-germinant class can induce germination. The former model is based on the finding that spores defective in releasing large stores of internal Ca2+ in the form of calcium dipicolinic acid (CaDPA) cannot germinate when germination is induced with bile acid germinant and amino acid co-germinant alone. However, since the reduced optical density of CaDPA-less spores makes it difficult to accurately measure their germination, we developed a novel automated, time-lapse microscopy-based germination assay to analyze CaDPA mutant germination at the single-spore level. Using this assay, we found that CaDPA mutant spores germinate in the presence of amino acid co-germinant and bile acid germinant. Higher levels of amino acid co-germinants are nevertheless required to induce CaDPA mutant spores to germinate relative to WT spores because CaDPA released by WT spores during germination can function in a feedforward loop to potentiate the germination of other spores within the population. Collectively, these data indicate that Ca2+ is not essential for inducing C. difficile spore germination because amino acid and Ca2+ co-germinant signals are sensed by parallel signaling pathways. IMPORTANCE Clostridioides difficile spore germination is essential for this major nosocomial pathogen to initiate infection. C. difficile spores germinate in response to sensing bile acid germinant signals alongside co-germinant signals. There are two classes of co-germinant signals: Ca2+ and amino acids. Prior work suggested that Ca2+ is essential for C. difficile spore germination based on bulk population analyses of germinating CaDPA mutant spores. Since these assays rely on optical density to measure spore germination and the optical density of CaDPA mutant spores is reduced relative to WT spores, this bulk assay is limited in its capacity to analyze germination. To overcome this limitation, we developed an automated image analysis pipeline to monitor C. difficile spore germination using time-lapse microscopy. With this analysis pipeline, we demonstrate that, although Ca2+ is dispensable for inducing C. difficile spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores.

Spore germination receptors - a new paradigm.

David Rudner and his team (Gao et al.) predict a pentameric structure for the GerA alanine-responsive germination receptor of Bacillus subtilis and demonstrate that it behaves as a nutrient-gated ion channel, finally establishing a function for this novel family of receptors and focussing research on early ion movements in germination.

Microbial Protocols for Spacecraft: 3. Spore Monolayer Preparation Methods for Ultraviolet Irradiation Exposures.

Developing robust microbial survival models for interplanetary and planetary spacecraft requires precise inactivation kinetics for vehicle bioburdens. To generate such data, reliable protocols are required for preparing, testing, and assaying microbial cells or spores on simulated spacecraft materials. New data are presented on the utility of the liquid droplet protocol for applying Bacillus subtilis spores to aluminum coupons. Results indicate that low-density spore monolayers should be created between 2 and 5 × 106 spores per cm2 on individual coupons to prevent the formation of aggregates or multilayers of spores. Such aggregation or multilayers will interfere with the precision of characterizing the effects of UV irradiation on spore survival. Optimum spore monolayers are defined as spore monolayers without overlapping or clustered cells and in which all spores will receive UV photons during assays. The best spore monolayers were created with sterile deionized water (SDIW) on uncoated aluminum coupons, or with SDIW + Triton X-100 (at 0.5 × of the critical micellar concentration) on either uncoated Al-coupons or on Chemfilm Class 1A-coated coupons. The Triton X-100 surfactant improved the uniformity of the monolayers without affecting the sensitivity of the spores to UV irradiation. Furthermore, spore layers created at either 2 × 107 or 2 × 108 spores/cm2 created multi-stacking effects that clearly reduced the precision of the UV irradiation assays. A set of standardized protocols is suggested for spacecraft processing and planetary protection communities to permit directly comparing results from divergent labs.

The effect of cold storage and cooking on the viability of Clostridioides difficile spores in consumer foods.

The increased detection of clinical cases of Clostridioides difficile coupled with the persistence of clostridial spores at various stages along the food chain suggest that this pathogen may be foodborne. This study examined C. difficile (ribotypes 078 and 126) spore viability in chicken breast, beef steak, spinach leaves and cottage cheese during refrigerated (4 °C) and frozen (-20 °C) storage with and without a subsequent sous vide mild cooking (60 °C, 1 h). Spore inactivation at 80 °C in phosphate buffer solution, beef and chicken were also investigated to provide D80°C values and determine if PBS was a suitable model system for real food matrices. There was no decrease in spore concentration after chilled or frozen storage and/or sous vide cooking at 60 °C. Non-log-linear thermal inactivation was observed for both C. difficile ribotypes at 80 °C in phosphate buffer solution (PBS), beef and chicken. The predicted PBS D80°C values of 5.72±[2.90, 8.55] min and 7.50±[6.61, 8.39] min for RT078 and RT126, respectively, were in agreement with the food matrices D80°C values of 5.65 min (95% CI range from 4.29 to 8.89 min) for RT078 and 7.35 min (95% CI range from 6.81 to 7.01 min) for RT126. It was concluded that C. difficile spores survive chilled and frozen storage and mild cooking at 60 °C but may be inactivated at 80 °C. Moreover thermal inactivation in PBS was representative of that observed in real food matrices (beef and chicken).

Effects of sequential treatments using radio frequency energy and ultraviolet light on inactivation of Bacillus cereus spores and quality attributes of buckwheat.

Buckwheat kernels were susceptible to be contaminated by heat-resistant spores. This study aimed to investigate effects of radio frequency (RF) heating, ultraviolet (UV) light and their combination treatment on the inactivation of B. cereus spores and quality attributes of buckwheat kernels. Results showed that Weibull model well fitted the inactivation curves of B. cereus spores under RF heating or UV light, and both of the two techniques had a tailing phenomenon (n < 1) in the decontamination process. But the inactivation levels of spores significantly increased by the combined treatments of RF and UV, regardless of the treatment sequence. Treatment by individual RF heating at 105 °C for 30 min or UV exposure at 5.00 mW/cm2 for 60 min resulted in >2.0 log CFU/g reduction of B. cereus spores. The similar inactivation effect could be achieved with shorter processing times by combined treatments (RF temperature-holding time + UV intensity-irradiation time: 85-10 + 3.50-10, 90-0 + 2.25-10, and 95-5 + 1.00-10). Besides, the colors, antioxidant compounds and antioxidant activities of buckwheat were not significantly deteriorated after these combined treatments, but the enzymatic activities were reduced, which was beneficial for long-term storage. Therefore, the proposed sequential treatment of RF heating and UV light in this study holds great potential to assure the food safety of grains.

Comparative Genomics and Physiological Characterization of Two Aerobic Spore Formers Isolated from Human Ileal Samples.

Spore formers are ubiquitous microorganisms commonly isolated from most environments, including the gastro-intestinal tract (GIT) of insects and animals. Spores ingested as food and water contaminants safely transit the stomach and reach the intestine, where some of them germinate and temporarily colonize that niche. In the lower part of the GIT, they re-sporulate and leave the body as spores, therefore passing through their entire life cycle in the animal body. In the intestine, both un-germinated spores and germination-derived cells interact with intestinal and immune cells and have health-beneficial effects, which include the production of useful compounds, protection against pathogenic microorganisms, contribution to the development of an efficient immune system and modulation of the gut microbial composition. We report a genomic and physiological characterization of SF106 and SF174, two aerobic spore former strains previously isolated from ileal biopsies of healthy human volunteers. SF106 and SF174 belong respectively to the B. subtilis and Alkalihalobacillus clausii (formerly Bacillus clausii) species, are unable to produce toxins or other metabolites with cytotoxic activity against cultured human cells, efficiently bind mucin and human epithelial cells in vitro and produce molecules with antimicrobial and antibiofilm activities.

Water-Content-Dependent Morphologies and Mechanical Properties of Bacillus subtilis Spores' Cortex Peptidoglycan.

Peptidoglycan (PG), bacterial spores' major structural component in their cortex layers, was recently found to regulate the spore's water content and deform in response to relative humidity (RH) changes. Here, we report that the cortex PG dominates the Bacillus subtilis spores' water-content-dependent morphological and mechanical properties. When exposed to an environment having RH varied between 10% and 90%, the spores and their cortex PG reversibly expand and contract by 30.7% and 43.2% in volume, which indicates that the cortex PG contributes to 67.3% of a spore's volume change. The spores' and cortex PG's significant volumetric changes also lead to changes in their Young's moduli from 5.7 and 9.0 GPa at 10% RH to 0.62 and 1.2 GPa at 90% RH, respectively. Interestingly, these significant changes in the spores' and cortex PG's morphological and mechanical properties are only caused by a minute amount of the cortex PG's water exchange that occupies 28.0% of the cortex PG's volume. The cortex PG's capability in sensing and responding to environmental RH and effectively changing its structures and properties could provide insight into spores' high desiccation resistance and dormancy mechanisms.

Time-Resolved Proteomics of Germinating Spores of Bacillus cereus.

Bacillus cereus is a spore-forming human pathogen that is a burden to the food chain. Dormant spores are highly resistant to harsh environmental conditions, but lose resistance after germination. In this study, we investigate the B. cereus spore proteome upon spore germination and outgrowth so as to obtain new insights into the molecular mechanisms involved. We used mass spectrometry combined with co-expression network analysis and obtained a unique global proteome view of the germination and outgrowth processes of B. cereus spores by monitoring 2211 protein changeovers. We are the first to examine germination and outgrowth models of B. cereus spores experimentally by studying the dynamics of germinant receptors, other proteins involved in spore germination and resistance, and coat and exosporium proteins. Furthermore, through the co-expression analysis of 1175 proteins identified with high quality data, germination proteome data were clustered into eight modules (termed black, blue, brown, green, red, turquoise, grey, and yellow), whose associated functions and expression profiles were investigated. Germination related proteins were clustered into blue and brown modules, the abundances of which decreased after finishing germination. In the brown and blue we identified 124 proteins that could be vital during germination. These proteins will be very interesting to study in future genetic studies regarding their function in spore revival in B. cereus.

An electric alarm clock for spores.

Inactive spores integrate stimuli over time through stored electrochemical potential.

Electrochemical potential enables dormant spores to integrate environmental signals.

The dormant state of bacterial spores is generally thought to be devoid of biological activity. We show that despite continued dormancy, spores can integrate environmental signals over time through a preexisting electrochemical potential. Specifically, we studied thousands of individual Bacillus subtilis spores that remain dormant when exposed to transient nutrient pulses. Guided by a mathematical model of bacterial electrophysiology, we modulated the decision to exit dormancy by genetically and chemically targeting potassium ion flux. We confirmed that short nutrient pulses result in step-like changes in the electrochemical potential of persistent spores. During dormancy, spores thus gradually release their stored electrochemical potential to integrate extracellular information over time. These findings reveal a decision-making mechanism that operates in physiologically inactive cells.