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Peter Setlow - One of the best experts on this subject based on the ideXlab platform.
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Spore germination.
Current opinion in microbiology, 2020Co-Authors: Peter SetlowAbstract:The germination of dormant Spores of Bacillus species is the first crucial step in the return of Spores to vegetative growth, and is induced by nutrients and a variety of non-nutrient agents. Nutrient germinants bind to receptors in the spore's inner membrane and this interaction triggers the release of the spore core's huge depot of dipicolinic acid and cations, and replacement of these components by water. These latter events trigger the hydrolysis of the spore's peptidoglycan cortex by either of two redundant enzymes in B. subtilis, and completion of cortex hydrolysis and subsequent germ cell wall expansion allows full spore core hydration and resumption of spore metabolism and macromolecular synthesis.
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A Quasi-chemical Model for Bacterial Spore Germination Kinetics by High Pressure
Food Engineering Reviews, 2017Co-Authors: Christopher J. Doona, Florence E. Feeherry, Kenneth Kustin, Haiqing Chen, Runze Huang, X. Philip Ye, Peter SetlowAbstract:High pressure processing (HPP) is an emerging non-thermal technology that is growing exponentially in use worldwide for the pasteurization of commercial foodstuffs. At combinations of elevated pressures and temperatures, HPP inactivates bacterial Spores, but HPP has not yet been implemented commercially for food sterilization. Studies of the mechanisms of bacterial spore inactivation by HPP using primarily Spores of Bacillus species have shown that spore germination precedes inactivation, with the release of dipicolinic acid from the spore core as the rate-determining step. Investigations probing spore resistance to and germination by HPP using Bacillus subtilis , a number of selected B. subtilis mutants, Bacillus amyloliquefaciens , and Clostridium difficile Spores have compiled a wealth of detailed mechanistic information, while also accumulating abundant germination kinetics data that has not previously been analyzed by predictive models. Presently, we devise a “quasi-chemical” model for bacterial spore germination dynamics by HPP. This quasi-chemical germination model (QCGM) hypothesizes a three-step mechanism and derives a set of ordinary differential equations to model the observed germination dynamics. The results with this model are viewed in the context of historical studies of spore activation, germination, and inactivation, with an eye toward potentially integrating differential equation models for germination and inactivation into a single, comprehensive model for spore dynamics by HPP. With the increasing use of high hydrostatic pressure to investigate mechanisms of bacterial spore resistance and physiology, the QCGM results help promote the efficient control of bacterial Spores, whether for the inactivation of Clostridium botulinum Spores in low-acid foods or aerosolized Bacillus anthracis Spores on textiles used in protective clothing, tents, or shelters.
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Spore Resistance Properties
Microbiology spectrum, 2014Co-Authors: Peter SetlowAbstract:Spores of various Bacillus and Clostridium species are among the most resistant life forms known. Since the Spores of some species are causative agents of much food spoilage, food poisoning, and human disease, and the Spores of Bacillus anthracis are a major bioweapon, there is much interest in the mechanisms of spore resistance and how these Spores can be killed. This article will discuss the factors involved in spore resistance to agents such as wet and dry heat, desiccation, UV and γ-radiation, enzymes that hydrolyze bacterial cell walls, and a variety of toxic chemicals, including genotoxic agents, oxidizing agents, aldehydes, acid, and alkali. These resistance factors include the outer layers of the spore, such as the thick proteinaceous coat that detoxifies reactive chemicals; the relatively impermeable inner spore membrane that restricts access of toxic chemicals to the spore core containing the spore's DNA and most enzymes; the low water content and high level of dipicolinic acid in the spore core that protect core macromolecules from the effects of heat and desiccation; the saturation of spore DNA with a novel group of proteins that protect the DNA against heat, genotoxic chemicals, and radiation; and the repair of radiation damage to DNA when Spores germinate and return to life. Despite their extreme resistance, Spores can be killed, including by damage to DNA, crucial spore proteins, the spore's inner membrane, and one or more components of the spore germination apparatus.
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germination of Spores of bacillus species what we know and do not know
Journal of Bacteriology, 2014Co-Authors: Peter SetlowAbstract:Spores of Bacillus species can remain in their dormant and resistant states for years, but exposure to agents such as specific nutrients can cause Spores' return to life within minutes in the process of germination. This process requires a number of spore-specific proteins, most of which are in or associated with the inner spore membrane (IM). These proteins include the (i) germinant receptors (GRs) that respond to nutrient germinants, (ii) GerD protein, which is essential for GR-dependent germination, (iii) SpoVA proteins that form a channel in Spores' IM through which the spore core's huge depot of dipicolinic acid is released during germination, and (iv) cortex-lytic enzymes (CLEs) that degrade the large peptidoglycan cortex layer, allowing the spore core to take up much water and swell, thus completing spore germination. While much has been learned about nutrient germination, major questions remain unanswered, including the following. (i) How do nutrient germinants penetrate through Spores' outer layers to access GRs in the IM? (ii) What happens during the highly variable and often long lag period between the exposure of Spores to nutrient germinants and the commitment of Spores to germinate? (iii) What do GRs and GerD do, and how do these proteins interact? (iv) What is the structure of the SpoVA channel in Spores' IM, and how is this channel gated? (v) What is the precise state of the spore IM, which has a number of novel properties even though its lipid composition is very similar to that of growing cells? (vi) How is CLE activity regulated such that these enzymes act only when germination has been initiated? (vii) And finally, how does the germination of Spores of clostridia compare with that of Spores of bacilli?
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summer meeting 2013 when the sleepers wake the germination of Spores of bacillus species
Journal of Applied Microbiology, 2013Co-Authors: Peter SetlowAbstract:Summary Spores of Bacillus species can remain dormant and resistant for years, but can rapidly ‘come back to life’ in germination triggered by agents, such as specific nutrients, and non-nutrients, such as CaDPA, dodecylamine and hydrostatic pressure. Major events in germination include release of spore core monovalent cations and CaDPA, hydrolysis of the spore cortex peptidoglycan (PG) and expansion of the spore core. This leads to a well-hydrated spore protoplast in which metabolism and macromolecular synthesis begin. Proteins essential for germination include the GerP proteins that facilitate germinant access to Spores' inner layers, germinant receptors (GRs) that recognize and respond to nutrient germinants, GerD important in rapid GR-dependent germination, SpoVA proteins important in CaDPA release and cortex-lytic enzymes that degrade cortex PG. Rates of germination of individuals in spore populations are heterogeneous, and methods have been developed recently to simultaneously analyse the germination of multiple individual Spores. Spore germination heterogeneity is due primarily to large variations in GR levels among individual Spores, with Spores that germinate extremely slowly and termed superdormant having very low GR levels. These and other aspects of spore germination will be discussed in this review, and major unanswered questions will also be discussed.
Ralf Moeller - One of the best experts on this subject based on the ideXlab platform.
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Investigating the detrimental effects of low pressure plasma sterilization on the survival of bacillus subtilis Spores using live cell microscopy
Journal of Visualized Experiments, 2017Co-Authors: F.m. Fuchs, Marcel Fiebrandt, Katharina Stapelmann, Marina Raguse, Kazimierz Madela, Michael Laue, Peter Awakowicz, Ralf MoellerAbstract:© 2017 Journal of Visualized Experiments. Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use Spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of Spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of Spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual Spores. Our observations reveal that the fraction of germinating and outgrowing Spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few Spores and developed in all outgrowing cells with a slight elevation in LPP-treated Spores. Moreover, some of the vegetative bacteria derived from LPP-treated Spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual Spores could be exemplary for the study of other aspects of spore germination and outgrowth.
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Understanding of the importance of the spore coat structure and pigmentation in the Bacillus subtilis spore resistance to low-pressure plasma sterilization
Journal of Physics D: Applied Physics, 2016Co-Authors: Marina Raguse, Marcel Fiebrandt, Katharina Stapelmann, Adam Driks, Peter Eaton, Benjamin Denis, Patrick Eichenberger, Peter Awakowicz, Ralf MoellerAbstract:© 2016 IOP Publishing Ltd. Low-pressure plasmas have been evaluated for their potential in biomedical and defense purposes. The sterilizing effect of plasma can be attributed to several active agents, including (V)UV radiation, charged particles, radical species, neutral and excited atoms and molecules, and the electric field. Spores of Bacillus subtilis were used as a bioindicator and a genetic model system to study the sporicidal effects of low-pressure plasma decontamination. Wild-type Spores, Spores lacking the major protective coat layers (inner, outer, and crust), pigmentation-deficient Spores or spore impaired in encasement (a late step in coat assembly) were systematically tested for their resistance to low-pressure argon, hydrogen, and oxygen plasmas with and without admixtures. We demonstrate that low-pressure plasma discharges of argon and oxygen discharges cause significant physical damage to spore surface structures as visualized by atomic force microscopy. Spore resistance to low-pressure plasma was primarily dependent on the presence of the inner, and outer spore coat layers as well as spore encasement, with minor or less importance of the crust and spore pigmentation, whereas spore inactivation itself was strongly influenced by the gas composition and operational settings.
Barbara Setlow - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the germination of Bacillus megaterium Spores lacking enzymes that degrade the spore cortex
Journal of Applied Microbiology, 2009Co-Authors: Barbara Setlow, Lixin Peng, Charles A. Loshon, Yong-qing Li, Graham Christie, Peter SetlowAbstract:Aims: To determine roles of cortex lytic enzymes (CLEs) in Bacillus megaterium spore germination. Methods and Results: Genes for B. megaterium CLEs CwlJ and SleB were inactivated and effects of loss of one or both on germination were assessed. Loss of CwlJ or SleB did not prevent completion of germination with agents that activate the spore’s germinant receptors, but loss of CwlJ slowed the release of dipicolinic acid (DPA). Loss of both CLEs also did not prevent release of DPA and glutamate during germination with KBr. However, cwlJ sleB Spores had decreased viability, and could not complete germination. Loss of CwlJ eliminated spore germination with Ca2+ chelated to DPA (Ca-DPA), but loss of CwlJ and SleB did not affect DPA release in dodecylamine germination. Conclusions: CwlJ and SleB play redundant roles in cortex degradation during B. megaterium spore germination, and CwlJ accelerates DPA release and is essential for Ca-DPA germination. The roles of these CLEs are similar in germination of B. megaterium and Bacillus subtilis Spores. Significance and Impact of the Study: These results indicate that redundant roles of CwlJ and SleB in cortex degradation during germination are similar in Spores of Bacillus species; consequently, inhibition of these enzymes will prevent germination of Bacillus Spores.
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germination of Spores of bacillus subtilis with dodecylamine
Journal of Applied Microbiology, 2003Co-Authors: Barbara Setlow, Anne E Cowan, Peter SetlowAbstract:Aims: To determine the properties of Bacillus subtilis Spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine-induced spore germination. Methods and Results: Spores of B. subtilis prepared in liquid medium were germinated efficiently by dodecylamine, while Spores prepared on solid medium germinated more poorly with this agent. Dodecylamine germination of Spores was accompanied by release of almost all spore dipicolinic acid (DPA), degradation of the spore's peptidoglycan cortex, release of the spore's pool of free adenine nucleotides and the killing of the Spores. The dodecylamine-germinated Spores did not initiate metabolism, did not degrade their pool of small, acid-soluble spore proteins efficiently and had a significantly lower level of core water than did Spores germinated by nutrients. As measured by DPA release, dodecylamine readily induced germination of B. subtilis Spores that: (a) were decoated, (b) lacked all the receptors for nutrient germinants, (c) lacked both the lytic enzymes either of which is essential for cortex degradation, or (d) had a cortex that could not be attacked by the spore's cortex-lytic enzymes. The DNA in dodecylamine-germinated wild-type Spores was readily stained, while the DNA in dodecylamine-germinated Spores of strains that were incapable of spore cortex degradation was not. These latter germinated Spores also did not release their pool of free adenine nucleotides. Conclusions: These results indicate that: (a) the spore preparation method is very important in determining the rate of spore germination with dodecylamine, (b) wild-type Spores germinated by dodecylamine progress only part way through the germination process, (c) dodecylamine may trigger spore germination by a novel mechanism involving the activation of neither the spore's nutrient germinant receptors nor the cortex-lytic enzymes, and (d) dodecylamine may trigger spore germination by directly or indirectly activating release of DPA from the spore core, through the opening of channels for DPA in the spore's inner membrane. Significance and Impact of the Study: These results provide new insight into the mechanism of spore germination with the cationic surfactant dodecylamine, and also into the mechanism of spore germination in general. New knowledge of mechanisms to stimulate spore germination may have applied utility, as germinated Spores are much more sensitive to processing treatments than are dormant Spores.
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mechanisms of killing Spores of bacillus subtilis by acid alkali and ethanol
Journal of Applied Microbiology, 2002Co-Authors: Barbara Setlow, Anne E Cowan, Charles A. Loshon, P C Genest, C Setlow, Peter SetlowAbstract:Aims: To determine the mechanisms of killing of Bacillus subtilis Spores by ethanol or strong acid or alkali. Methods and Results: Killing of B. subtilis Spores by ethanol or strong acid or alkali was not through DNA damage and the spore coats did not protect Spores against these agents. Spores treated with ethanol or acid released their dipicolinic acid (DPA) in parallel with spore killing and the core wet density of ethanol- or acid-killed Spores fell to a value close to that for untreated Spores lacking DPA. The core regions of Spores killed by these two agents were stained by nucleic acid stains that do not penetrate into the core of untreated Spores and acid-killed Spores appeared to have ruptured. Spores killed by these two agents also did not germinate in nutrient and non-nutrient germinants and were not recovered by lysozyme treatment. Spores killed by alkali did not lose their DPA, did not exhibit a decrease in their core wet density and their cores were not stained by nucleic acid stains. Alkali-killed Spores released their DPA upon initiation of spore germination, but did not initiate metabolism and degraded their cortex very poorly. However, Spores apparently killed by alkali were recovered by lysozyme treatment. Conclusions: The data suggest that spore killing by ethanol and strong acid involves the disruption of a spore permeability barrier, while spore killing by strong alkali is due to the inactivation of spore cortex lytic enzymes. Significance and Impact of the Study: The results provide further information on the mechanisms of spore killing by various chemicals.
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Properties of Spores of Bacillus subtilis Blocked at an Intermediate Stage in Spore Germination
Journal of Bacteriology, 2001Co-Authors: Barbara Setlow, E. Melly, Peter SetlowAbstract:Germination of mutant Spores of Bacillus subtilis unable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water. However, compared to wild-type germinated Spores in which the cortex has been degraded, the germinated mutant Spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation. We propose that the germinated Spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.
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characterization of Spores of bacillus subtilis which lack dipicolinic acid
Journal of Bacteriology, 2000Co-Authors: Madan Paidhungat, Adam Driks, Barbara Setlow, Peter SetlowAbstract:Spores of Bacillus subtilis with a mutation in spoVF cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the Spores of a strain lacking the three major germinant receptors (termed Δger3), as well as spoVF, can be isolated, although they spontaneously germinate much more readily than Δger3 Spores. The Δger3 spoVF Spores lack DPA and have higher levels of core water than Δger3 Spores, although sporulation with DPA restores close to normal levels of DPA and core water to Δger3 spoVF Spores. The DPA-less Spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of Spores with DPA. The Δger3 spoVF Spores also contain minimal levels of the processed active form (termed P41) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P41 formed in Δger3 spoVF Spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-γ. Analysis of the resistance of wild-type, Δger3, and Δger3 spoVF Spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type Spores are more resistant than Δger3 Spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.
Adam Driks - One of the best experts on this subject based on the ideXlab platform.
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Understanding of the importance of the spore coat structure and pigmentation in the Bacillus subtilis spore resistance to low-pressure plasma sterilization
Journal of Physics D: Applied Physics, 2016Co-Authors: Marina Raguse, Marcel Fiebrandt, Katharina Stapelmann, Adam Driks, Peter Eaton, Benjamin Denis, Patrick Eichenberger, Peter Awakowicz, Ralf MoellerAbstract:© 2016 IOP Publishing Ltd. Low-pressure plasmas have been evaluated for their potential in biomedical and defense purposes. The sterilizing effect of plasma can be attributed to several active agents, including (V)UV radiation, charged particles, radical species, neutral and excited atoms and molecules, and the electric field. Spores of Bacillus subtilis were used as a bioindicator and a genetic model system to study the sporicidal effects of low-pressure plasma decontamination. Wild-type Spores, Spores lacking the major protective coat layers (inner, outer, and crust), pigmentation-deficient Spores or spore impaired in encasement (a late step in coat assembly) were systematically tested for their resistance to low-pressure argon, hydrogen, and oxygen plasmas with and without admixtures. We demonstrate that low-pressure plasma discharges of argon and oxygen discharges cause significant physical damage to spore surface structures as visualized by atomic force microscopy. Spore resistance to low-pressure plasma was primarily dependent on the presence of the inner, and outer spore coat layers as well as spore encasement, with minor or less importance of the crust and spore pigmentation, whereas spore inactivation itself was strongly influenced by the gas composition and operational settings.
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characterization of Spores of bacillus subtilis which lack dipicolinic acid
Journal of Bacteriology, 2000Co-Authors: Madan Paidhungat, Adam Driks, Barbara Setlow, Peter SetlowAbstract:Spores of Bacillus subtilis with a mutation in spoVF cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the Spores of a strain lacking the three major germinant receptors (termed Δger3), as well as spoVF, can be isolated, although they spontaneously germinate much more readily than Δger3 Spores. The Δger3 spoVF Spores lack DPA and have higher levels of core water than Δger3 Spores, although sporulation with DPA restores close to normal levels of DPA and core water to Δger3 spoVF Spores. The DPA-less Spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of Spores with DPA. The Δger3 spoVF Spores also contain minimal levels of the processed active form (termed P41) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P41 formed in Δger3 spoVF Spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-γ. Analysis of the resistance of wild-type, Δger3, and Δger3 spoVF Spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type Spores are more resistant than Δger3 Spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.
Gordon Dougan - One of the best experts on this subject based on the ideXlab platform.
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use of purified clostridium difficile Spores to facilitate evaluation of health care disinfection regimens
Applied and Environmental Microbiology, 2010Co-Authors: Trevor D Lawley, Simon Clare, David Goulding, Laura J Deakin, Claire Raisen, Cordelia Brandt, Jon Lovell, Fiona J Cooke, Taane G Clark, Gordon DouganAbstract:Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile Spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor Spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile Spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure Spores and blocking spore-mediated transmission. Complete inactivation of 106 pure C. difficile Spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile Spores (17 to 29 Spores/cm2) and block host transmission. Thus, pure C. difficile Spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.
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proteomic and genomic characterization of highly infectious clostridium difficile 630 Spores
Journal of Bacteriology, 2009Co-Authors: Trevor D Lawley, Nicholas J Croucher, Lu Yu, Simon Clare, Mohammed Sebaihia, David Goulding, Derek Pickard, Julian Parkhill, Jyoti S Choudhary, Gordon DouganAbstract:Clostridium difficile is a gram-positive, spore-forming, anaerobic bacterium that can asymptomatically colonize the intestinal tracts of humans and other mammals (3, 30, 39). Antibiotic treatment can result in C. difficile overgrowth and can lead to clinical disease, ranging from diarrhea to life-threatening pseudomembranous colitis, particularly in immunocompromised hosts (2, 4, 7). In recent years, C. difficile has emerged as the major cause of nosocomial antibiotic-induced diarrhea, and it is frequently associated with outbreaks (21, 22). A contributing factor is that C. difficile can be highly infectious and difficult to contain, especially when susceptible patients are present in the same hospital setting (13). Person-to-person transmission of C. difficile is associated with the excretion of highly resistant Spores in the feces of infected patients, creating an environmental reservoir that can confound many infection control measures (29, 44). Bacterial Spores, which are metabolically dormant cells that are formed following asymmetric cell division, normally have thick concentric external layers, the spore coat and cortex, that protect the internal cytoplasm (15, 42). Upon germination, Spores lose their protective external layers and resume vegetative growth (24, 27, 36). Bacillus Spores and the Spores of most Clostridium species germinate in response to amino acids, carbohydrates, or potassium ions (24, 36). In contrast, C. difficile Spores show an increased level of germination in response to cholate derivatives found in bile (40, 41). Thus, Spores are well adapted for survival and dispersal under a wide range of environmental conditions but will germinate in the presence of specific molecular signals (24, 36). While the Spores of a number of Bacillus species, such as Bacillus subtilis and Bacillus anthracis, and those of other Clostridium species, such as Clostridium perfringens (15, 20), have been well characterized, research on C. difficile Spores has been relatively limited. A greater understanding of C. difficile spore biology could be exploited to rationalize disinfection regimes, molecular diagnostics, and the development of targeted treatments such as vaccines. Here we describe a novel method to isolate highly purified C. difficile Spores that maintain their resistance and infectious characteristics, thus providing a unique opportunity to study C. difficile Spores in the absence of vegetative cells. A thorough proteomic and genomic analysis of the spore provides novel insight into the unique composition and predictive biological properties of C. difficile Spores that should underpin future research into this high-profile but poorly understood pathogen.
