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Measurements of the pH within dormant and germinated bacterial spores

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Abstract

The pH within the core or central region of dormant spores of Bacillus cereus and B. megaterium is 6.3-6.4 irrespective of the external pH. However, the spore's internal pH rises to 7.3-7.5 upon germination. The low internal pH of the dormant spore may be a contributing factor to its metabolic dormancy.
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... (30) Transmission can also occur through contact with contaminated surfaces or via direct inoculation into the bowel. (39) There is also evidence for airborne transmission of C. difficile spores during periods of activity and with bed making (40,41) . ...
... The inner layer is a permeability barrier inner membrane followed by germ cell wall surrounded by thick spore cortex which is in turn enclosed in spore coat and external exosporium. (37)(38)(39)(40) In response, external stimuli including bile acids, amino acids, nutrients and electrolytes by CSP proteases various germination pathways are activated including bile acid-amino acid, alanine racemose dependent D-amino acid, or bile salt-divalent pathways. (23,41,42) Each of these pathways leads to activation of the gene, Slec which initiates degradation of cortex resulting in full core rehydration and release of Ca-DPA and initiation of germination. ...
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Introduction: Clostridioides difficile is a motile, gram-negative, toxin-producing bacteria responsible for the majority of the nosocomial as well as community-acquired infection in the US and worldwide. A high rate of morbidity, mortality, recurrences, healthcare cost coupled with frequent epidemic outbreaks with drug-resistant strains has classified C. difficile as a pathogen with an urgent threat to human health. Current guidelines regarding disinfectant use are either limited or absent or are not effective in reducing the environmental burden of C. difficile. The aims for this study was first to determine the in vitro efficacy of select hospital disinfectants in eradicating the spores of C. difficile both in planktonic form as well as in biofilm and secondly, to determine the effect of community disinfectants on the eradication and propagation of C. difficile. Methods Following standardized protocol, low, medium and high category spores were produced and exposed to the original concentration of disinfectants at both label determined and standardized time in presence or absence of organic matter. Minimum disinfectant concentration and minimum organic matter concentration for with bactericidal effect were also determined. The efficacy of hospital disinfectants on the strictly anaerobic mono organism, mixed multi organism and strictly aerobic multi organism 72 hours and 120 hours C. difficile biofilm was quantified using both microbiological techniques as well as microscopically. The effect of community household disinfectants on C. difficile spores was determined using time-kill experiments as well as time-kill curves. Germination assay, Ca-DPA assay, spore cortex fragmentation assay, the effect on the dormant vs. activated spore, qPCR and microscopy was done to characterize the role of community disinfectants on C. difficile propagation. Results The effect of 7 hospital disinfectant and 7 community disinfectant were tested against16 C. difficile spores of 6 different ribotypes. All tested hospital disinfectants were effective in eradicating almost 100% C. difficile spores at manufacturer suggested contact time in absence of organic matter and at all spore concentration. Using the general linear model, sporicidal activity of the disinfectants was affected by a shorter contact time (0.52±0.10 log decrease; p<0.0001), and presence of organic matter (low organic substance: 0.77±0.11 log decrease; p<0.0001; high organic substance: 2.02±0.12 log decrease; p<0.0001). C. difficile vegetative cells and spores were recovered from biofilms regardless of type or duration of biofilm formation. No disinfectant was able to completely eliminate C. difficile from the biofilms. Overall, Clorox, OPA, and Virex were most effective at killing C. difficile spores regardless of biofilm age, ribotype, or wash conditions (p= 0.001, each). Clorox and OPA were also effective at killing total vegetative cell growth (P=0.001, each) but Virex was found to be ineffective against the total vegetative cell growth (p=0.77). Clorox and Virex were most effective in reducing biomass followed by Nixall, OPA, and Vital oxide. None of the community disinfectants except Clorox was found to be effective against C. difficile spores. Increased spore germination was observed for Lysol hydrogen peroxide and Lysol. Effect on active vs dormant spores, germination assay, Ca-DPA assay, spore cortex fragmentation assay, qPCR and microscopy ass indicate the potential for Lysol hydrogen peroxide to act as a germinant for C. difficile spores. Conclusion: None of the disinfectants could consistently reduce C. difficile spore count under all conditions. Furthermore, this is the first study to look at the efficacy of hospital disinfectants on C. difficile spores encased in a biofilm. This study reported for the very first time about the germinant potential of certain household disinfectants. Thus, the findings for this study may help to inform the infection control guidelines and aid to fulfill the CDCs goal of reducing C. difficile infection by 30% by 2020.
... The inner membrane of the spore serves as a chemical protection barrier for the spore core ( Setlow, 2010 ). It has extremely low permeability to some small molecules and even water ( Setlow & Setlow, 1980 ). More, it is reported that the extremely low permeability of the inner membrane is related to the fluidity of lipids on the spore inner membrane, but not associated with the composition of the inner membrane ( Swerdlow et al., 1981 ). ...
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Bacterial spores, one of the main microorganisms, can cause food spoilage and food-borne diseases. However, due to their high resistance to stress, the inactivation of spores has been a pressing issue in the food industry. “Sprout first and inactivate later” is the traditional idea of inactivating spores. However, different germination factors cause different germination effects, which in turn affect the inactivating results. Heat sterilization is a simple and effective sterilization method, but it will destroy the quality and nutrition of food. As an emerging non-thermal sterilization technology, cold plasma (CP) has shown great potential in food sterilization. But the CP inactivating effect and mechanism to spores still need more research and attention. Therefore, the spore structure, formation, and germination were reviewed. The CP sterilization mechanism and its application in spore inactivation were summarized. This review work provides a reference for spore control in food science and industry.
... The fluorescence intensity of DiIC 12 and FM5-95 stained spores dropped dramatically upon the start of the rapid decline in spore brightness, and the drop was completed around the 'time to germination' (Figs. 3, 4). In a word, just as with the dynamics of SpoVAEa-SGFP2, the change of the IM during germination is also highly correlated with the rapid Ca 2+ -DPA release and cortex hydrolysis, which lead to the increase of spore core pH, and water content 26,27 . The FM5-95 stained spore also had a fluorescent spot, whereas DiIC 12 spores had relative uniform staining (Figs. 3C, 4C). ...
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The SpoVA proteins make up a channel in the inner membrane (IM) of Bacillus subtilis spores. This channel responds to signals from activated germinant receptors (GRs), and allows release of Ca²⁺-DPA from the spore core during germination. In the current work, we studied the location and dynamics of SpoVAEa in dormant spores. Notably, the SpoVAEa-SGFP2 proteins were present in a single spot in spores, similar to the IM complex formed by all GRs termed the germinosome. However, while the GRs’ spot remains in one location, the SpoVAEa-SGFP2 spot in the IM moved randomly with high frequency. It seems possible that this movement may be a means of communicating germination signals from the germinosome to the IM SpoVA channel, thus stimulating CaDPA release in germination. The dynamics of the SpoVAEa-SGFP2 and its surrounding IM region as stained by fluorescent dyes were also tracked during spore germination, as the dormant spore IM appeared to have an immobile germination related functional microdomain. This microdomain disappeared around the time of appearance of a germinated spore, and the loss of fluorescence of the IM with fluorescent dyes, as well as the appearance of peak SpoVAEa-SGFP2 fluorescent intensity occurred in parallel. These observed events were highly related to spores’ rapid phase darkening, which is considered as due to rapid Ca²⁺DPA release. We also tested the response of SpoVAEa and the IM to thermal treatments at 40–80 °C. Heat treatment triggered an increase of green autofluorescence, which is speculated to be due to coat protein denaturation, and 80 °C treatments induce the appearance of phase-grey-like spores. These spores presumably have a similar intracellular physical state as the phase grey spores detected in the germination but lack the functional proteins for further germination events.
... Beyond persisters and antibiotic survival, a drop in the cellular pH is more commonly associated with metabolic perturbations and dormancy. For instance, the pH of spores of Bacillus megaterium and Bacillus cereus is 1 to 1.3 pH units lower than the pH of growing cells, but rises rapidly upon germination [101][102][103] . In yeast, cytoplasmic pH was identified as a second messenger for glucose levels where low intracellular glucose induces acidification 104 . ...
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... The fluorescence intensity of DiIC 12 and FM5-95 stained spores dramatically dropped upon the start of the spore's brightness rapid decline, and the drop was completed around the 'time to germination' (Fig. 3, 4). In a word, just as with the dynamics of SpoVAEa-SGFP2, the change of the IM during germination is also highly correlated with the Ca 2+ -DPA rapid release, which leads to the increase of spore core pH, and water content [25,26]. The FM5-95 stained spore also had a fluorescent spot, whereas DiIC 12 spores had relative uniform staining (Fig. 3C, 4C). ...
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The SpoVA proteins make up a channel in the inner membrane (IM) of B. subtilis spore. This channel responds to signals from activated germinant receptors (GRs), and allows release of Ca ²⁺ -DPA from the spore core during germination. In the current work, we studied the location and dynamics of SpoVAEa in dormant spores. Notably, the SpoVAEa-SGFP2 proteins were present in a single spot in spores, similar to the complex formed by all GRs. However, while the GRs’ spot remains in one location, the SpoVAEa-SGFP2 spot in the IM moved randomly with high frequency. The dynamics of the SpoVAEa-SGFP2 and its surrounding IM region as stained by fluorescent dyes were also tracked during spore germination, as the dormant spore IM appeared to have an immobile germination related functional microdomain. This microdomain disappeared around the time of appearance of a germinated spore, the loss of fluorescence of the IM by fluorescent dyes, as well as the appearance of SpoVAEa-SGFP2 peak fluorescent intensity occurred in parallel. These observed events were highly related to the rapid phase darkening, which is considered as the Ca ²⁺ DPA rapid release. We also tested the response of SpoVAEa and the IM to thermal treatments at 40-80°C. Heat treatment triggered an increase of green autofluorescence, which is speculated to be due to coat protein denaturation, and 80°C treatments induce the appearance of phase-grey-like spores. These spores presumably have a similar intracellular physical state as the phase grey spores detected in the germination but lack the functional proteins for further germination events.
... The question of how much metabolic activity occurs within ''quiescent'' spores has received attention in some species [58,59]. Our results strongly suggest that starvation-induced M. xanthus spores themselves release a social germination factor prior to the onset of morphological transformation. ...
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Many microbes produce stress-resistant spores to survive unfavorable conditions [1-4] and enhance dispersal [1, 5]. Cooperative behavior is integral to the process of spore formation in some species [3, 6], but the degree to which germination of spore populations involves social interactions remains little explored. Myxococcus xanthus is a predatory soil bacterium that upon starvation forms spore-filled multicellular fruiting bodies that often harbor substantial diversity of endemic origin [7, 8]. Here we demonstrate that germination of M. xanthus spores formed during fruiting-body development is a social process involving at least two functionally distinct social molecules. Using pairs of natural isolates each derived from a single fruiting body that emerged on soil, we first show that spore germination exhibits positive density dependence due to a secreted "public-good" germination factor. Further, we find that a germination defect of one strain under saline stress in pure culture is complemented by addition of another strain that germinates well in saline environments and mediates cheating by the defective strain. Glycine betaine, an osmo-protectant utilized in all domains of life, is found to mediate saline-specific density dependence and cheating. Density dependence in non-saline conditions is mediated by a distinct factor, revealing socially complex spore germination involving multiple social molecules.
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