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Clostridium difficile spores: a major threat to the hospital environment

Jonathan Barra-Carrasco1 & Daniel Paredes-Sabja*,1,2

ABSTRACT: Clostridium difficile is a Gram-positive, anaerobic spore former and is an important nosocomial and community-acquired pathogenic bacterium. C. difficile infections (CDI) are a leading cause of infections worldwide with elevated rates of morbidity. Despite the fact that two major virulence factors, the enterotoxin TcdA and the cytotoxin TcdB, are essential in the development of CDI, C. difficile spores are the main vehicle of infection, and persistence and transmission of CDI and are thought to play an essential role in episodes of CDI recurrence and horizontal transmission. Recent research has unmasked several properties of C. difficile’s unique strategy to form highly transmissible spores and to persist in the colonic environment. Therefore, the aim of this article is to summarize recent advances in the biological properties of C. difficile spores, which might be clinically relevant to improve the management of CDI in hospital environments. The obligate anaerobic pathogen, Clostridium difficile, is a Gram-positive, spore-forming bacteria that can be present as both toxigenic and nontoxigenic strains [1] . C. difficile infections (CDI) are a major cause of nosocomial infections and antibiotic-associated diarrhea [1] . The severity of CDI can vary from mild self-limiting diarrhea to pseudomembranous colitis and toxic megacolon [2] . During the last decade, CDI epidemiology has experienced significant changes (i.e., emergence of hypervirulent strains) that have led to an increase in CDI cases, resulting in colectomy and death, with mortality rates reaching up to 15% in several severe outbreaks [3–5] . The emergence of PCR ribotype 027 in the early 2000s significantly increased CDI incidence [6,7] , and has also been associated with more severe outcomes [8,9] . A recent study demonstrated that ribotype 078, also found in food animals, leads to severe CDI [10] . In part, these changes could be attributed to the remarkable degree of chromosomal plasticity of C. difficile genomes such as deletion events and high propensity to horizontal gene transfer [11–14] . For example, He et al. provided strong evidence that the acquisition of fluoroquinolone resistance is associated with the emergence of C. difficile ribotype 027 [15] . Higher toxin production due to an in-frame shift mutation in the anti-sigma factor, tcdC, thought to be a negative regulator of toxin production [16] , was initially thought to be associated with higher toxin production in vitro, yet the role of TcdC remains controversial since another study failed to observe an association with tcdC genotype and toxin production [17] . Acquisition of novel genetic regions that lead to diversification of the pan genome and consequent participation in virulence and/or host adaptation may also have some impact on CDI severity [18] . C. difficile produces two major virulence factors, TcdA and TcdB, which belong to the family of large clostridial toxins [19] . Both toxins harbor a receptor-binding domain, a transmembrane

KEYWORDS 

• Clostridium difficile • exosporium • nosocomial diarrhea • spores • sporulation

Laboratorio de Mecanismos de Patogénesis Bacteriana, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago, Chile 2 Department of Biomedical Sciences, Oregon State University, Corvallis, OR, USA *Author for correspondence: [email protected] 1

10.2217/FMB.14.2 © 2014 Future Medicine Ltd

Future Microbiol. (2014) 9(4), 475–486

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Review  Barra-Carrasco & Paredes-Sabja domain and a glucosyltransferase domain, enabling them to bind to the cell surface and translocate to the cytosol to glucosylate and inactivate host GTPases, including Rac, Rho and Cdc42. These latter events lead to the alteration of the actin cytoskeleton, apoptosis and disruption of the epithelial barrier and most importantly, are essential for the development of clinical symptoms [20] . An additional virulence factor in its own right is the binary toxin, also called the C. difficile transferase (CDT). CDT binds to lipolysis-stimulated lipoprotein receptor [21] , translocates to the cytosol and ADP-ribosylates actin in an irreversible manner, leading to the disruption or rearrangement of the actin cytoskeleton [20] . In Caco-2 cells, CDT induces the formation of long protrusions to which C. difficile vegetative cells can bind, increasing adherence to epithelial surfaces [22] . However, the actual implication of CDT in the pathogenesis of CDI remains elusive. In addition to the aforementioned virulence factor and because it is a strict anaerobe, C. difficile spores are the vehicle by which C. difficile infects a susceptible host [23] , persists in the colonic environment (presumably through attachment to the colonic surfaces, as demonstrated in vitro [24]) and survives attacks of the host’s innate immune system [25] . Indeed, contamination of the hospital environment with C. difficile spores appears to account for approximately 38% of the cases of CDI in hospitalized patients undergoing antibiotic administration [26] . A recent study demonstrated that the intestinal cecal extracts from antibiotic-treated mice were able to trigger germination of C. difficile, mainly because the cecal flora from antibiotictreated mice was less able to modify the germinant taurocholate relative to the cecal flora from untreated mice [27] , suggesting that antibioticaltered gut microbiota has reduced ability to metabolize the main germinants of C. difficile spores (i.e., cholate and derivatives such as taurocholate) [28] . This might lead to increased concentration of germinants in the colonic lumen enhancing the germination rate of C. difficile spores, and establishment of CDI symptoms. Additionally, during the infection cycle, C. difficile initiates an efficient sporulation cycle as demonstrated by the elevated spore counts in stools of patients with CDI [29] . Patients with CDI become super shedders of C. difficile spores [29] and contribute to horizontal transmission through the contamination of surfaces with

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C. difficile spores that may persist for years in the hospital environment. C. difficile spores may also persist in the patients’ colonic tract causing recurring episodes of CDI. Events of CDI recurrence can be classified as: relapse episodes, caused by the initial strain that produced the first episode and accounts for 50–85% of all relapse episodes; or as reinfection, caused by different strains that may account for approximately 25–50% of all relapse episodes of CDI [30] . Despite the significant relevance of the spore morphotype in CDI, studies on C. difficile spores account for only approximately 4.6% of all C. difficile hits in PubMed (until 15 September 2013). Consequently, the aim of this article is to summarize what is known about C. difficile spore resistance, transmission, and the molecular basis of sporulation and germination and highlight their role in the persistence and transmission of CDI. C. difficile spore formation during CDI In general, bacterial sporulation is initiated when environmental conditions are less favorable for bacterial life leading to the rapid formation of highly resistant and dormant spores that are able to return to life once environmental conditions are supportive of bacterial growth. In vitro sporulation of Bacillus subtilis and Clostridium perfringens typically requires 8–12 h for the formation of a mature and dormant spore [31] . In this context, C. difficile is able to persist in the host’s colonic tract by producing highly resistant dormant spores that are impermeable to all known anti-CDI therapy. A milestone in our understanding of the pathogenesis of CDI was a recent study by Deakin et al. [32] , which demonstrated that, through the construction of C. difficile strains with a mutation in the master regulation of sporulation, spo0A, sporulation is essential for the persistence and transmission of C. difficile in a mouse model. Indeed, the mice luminal colonic environment triggers sporulation as early as 4 h after infection as demonstrated by transcriptomic analysis, which detected the expression of genes involved in the early stages of sporulation [33] . A rapid increase of spores in stools, reaching 56% of spores relative to vegetative cells, was observed after 14 h of infection [33] , which is much higher than the approximate 10% of spores that appear after 3–10 days in trypticase soy broth or agar plates [34,35] . This suggests that spore formation is an adaptive strategy in order to persist in the colonic environment as

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Clostridium difficile spores: a major threat to the hospital environment  demonstrated with strains unable to form spores [32] . The latter suggests that the host’s colonic environment has molecules sensed by specific histidine kinases able to trigger efficient sporulation of C. difficile through phosphorylation of the master regulator of sporulation, Spo0A [32,36] . Recent progress on the regulatory process of sporulation has been published and suggests that the network that regulates the spatial and temporal expression of genes during sporulation of C. difficile differs from the paradigm of B. subtlis sporulation [37–39] . Considering the rapid rate with which C. difficile sporulates in the gut of a murine model [33,40] , it is tempting to hypothesize that this might also be the case in the human gut; however, these results should be interpreted with caution primarily because the murine gut will most definitely provide different signals than the human gut. Nevertheless, spore formation in the gut may constitute a novel target to combat CDI. Although direct evidence is lacking, there is a general acceptance that C. difficile sporulates in the colonic gut of patients. In this context, an observational study demonstrated that 1–4 weeks after treatment, patients continued to shed spores to the environment [29] , highlighting the need for further work to identify these molecules and their cognate histidine kinases to allow the development of chemical analogs that inhibit in vivo sporulation. To date, the only molecule known to inhibit sporulation of C. difficile is the RNA polymerase inhibitor fidaxomicin; however, the precise mechanism of how it affects sporulation is unclear [41,42] . This concept has been exploited in the inhibition of yeast sporulation by several cationic amphiphilic drugs [43] . C. difficile spores: transmission vector Unlike other enteric diseases caused by nonspore forming enteropathogens, C. difficile, similarly to other clostridial pathogens, employs a unique and highly sophisticated strategy to persist in the host’s colonic environment by transforming into a dormant and highly resistant spore morphotype [1] . As mentioned above, C. difficile spores have unique requirements that allow them to return to life primarily in the colonic environment, but not in presence of epithelial cells or inside professional phagocytic cells [24,25] ; thus, C. difficile spores can survive inside the host and/or spread through the hospital environment, converting C. difficile spores into the perfect transmission vehicle of CDI. Indeed,

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environmental contamination with C. difficile spores is a major contributor to the spread of CDI across patients [29,44,45] . This transmission cycle is believed to be initiated when patients with CDI shed spores through their stools, contaminating their skin, clothing and close-contact bedding surfaces [29,46,47] . Indeed, approximately 50% of the patients that recover from a CDI episode may become asymptomatic shedders of C. difficile spores for up to 1–4 weeks after treatment [29] and thus, a source of transmission [45] . A recent study demonstrated that patients with recent CDI contribute to the transmission of spores during outpatient visits [48] . The outpatient setting may be an underappreciated source of community-associated CDI [26,48] . Thus standard infection-control measures, including placement of infected patients under contact precaution, should be continued. In addition to the intrinsic contamination produced by super shedder patients, horizontal transmission of C. difficile spores may easily occur between hospitalized patients since healthcare workers may also act as transmission vectors [29,46,47] . This has recently been reinforced and is suggested to account for approximately 38% of CDIs [26] . The use of gloves by healthcare workers should be carefully supervised since C. difficile spores are also transmitted to gloved hands after contact with the skin of a patient with CDI [44] . Thus, gloves should be provided as protection to healthcare workers and must be immediately disposed after use. In addition to surfaces in proximity to CDI patients, C. difficile spores have also been found in high horizontal surfaces and air vents [49,50] . A study has demonstrated that C. difficile spores can be transmitted through the air, reaching counts of 53–476 CFU/m3 [51] . Aerial dissemination of C. difficile spores could be attributed to patients’ bedding, activity that has been shown to produce aerial dissemination of C. difficile spores to the air [52–54] . Further studies are required to properly evaluate if surface contamination and aerial dissemination of C. difficile spores could be a contributory factor of environmental transmission of C. difficile spores to hospitalized patients, in which case routine surface disinfection with sporicidal agents and negatively pressurized isolation of rooms harboring CDI patients could help prevent the spread of CDI in clinical settings [51] . In summary, eradication of C. difficile spores from the hospital environment is essential to eradicate or reduce the rates of infection.

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Review  Barra-Carrasco & Paredes-Sabja Resistance of bacterial spores to disinfectants C. difficile spores, as other bacterial spores, are naturally resistant to environmental stress that would otherwise inactivate vegetative cells. In hospital settings, C. difficile spores are exposed to a wide array of disinfectants commonly used to disinfect contaminated surfaces [55] and, if not treated, they can attach to surfaces and remain viable, suffering only 1 decimal reduction in spore viability after 1 year [56] . Although the precise levels of resistance of C. difficile spores to various environmental stressors are unclear, their intrinsic resistance properties seem to be sufficient to successfully contribute to their persistence in hospital environments [55] . Several studies have characterized the survival of C. difficile spores to various disinfectant chemicals typically used in disinfection routines [55–57] . Strikingly, commonly used ethanol-based disinfectants have no effect on the reduction of C. difficile spore viability [56] . Indeed, incubation of C. difficile spores in 50% ethanol at 4°C for 14 months causes no significant reduction in spore viability [56] . Similarly, quaternary ammonium compounds are cationic surfactants typically utilized in disinfectant protocols; however, their sporicidal activity against C. difficile spores is negligible [55] . Alternative disinfectants with sporicidal activity are sodium hypochlorite, which at concentrations of 5000 ppm can achieve 7 decimal reductions in spore viability within 6 min of incubation at room temperature [56] . During outbreak conditions, bleach (5000 ppm) has been advocated as an effective disinfectant of C. difficile spores primarily because there are no alternatives as efficient as bleach. However, several safety concerns with 5000 ppm of bleach requires that it is used as a two-step process (i.e., must be wiped off using water) to avoid skin irritation, corrosion of metallic surfaces and damage of sensitive equipment. Acidified nitrite and glutaraldehyde have been shown to achieve approximately 4 decimal reductions in C. difficile spore viability within 30 min of incubation [58] . However, these compounds have been shown to be harmful to healthcare workers, as nitrite compounds by themselves are carcinogenic, while glutaraldehyde has been shown to cause asthma and dermatitis in healthcare workers [59] . Alternatively, peracetyl ions and vapor hydrogen peroxide have been shown to achieve approximately 4 and 7 decimal reductions in 15 and 30 min, respectively [58,60] . These latter two disinfectants have the advantage of

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being harmless and environmentally safe [61] . Far-UV produces an approximate 4.4 decimal reduction in the viability of C. difficile spores after 5 s of exposition [62] . Recently, accelerated hydrogen peroxide was demonstrated to be an alternative cleaning agent, reducing the presence of C. difficile from 48 to 28% of positive culture [63] . These observations highlight the need to develop new protocols that incorporate novel disinfectant compounds. C. difficile spore structure Bacterial spores are structurally different than their vegetative cell counterparts, conferring them with extreme resistance properties, which enable them to survive adverse conditions [64,65] . Several studies have published transmission electron micrographs of C. difficile spores [24,66,67] demonstrating that the basic ultrastructural layers (Figure 1) are similar to those of other endospore-forming species such as B. subtilis and Bacillus anthracis [64] . The C. difficile spore core, albeit dehydrated (i.e., has a water content of 77% of the spore core wet weight) [68] , has twofold higher water content than the spore core of C. perfringens spores (i.e., water content of 32% of the spore core wet weight) [69] . Although not clearly shown in the transmission electron micrographs (TEMs) (Figure 1) , the spore core is surrounded by an inner membrane, which in B. subtilis spores, has been demonstrated to be largely immobile [70] . This might also be the case in C. difficile spores judging by the resistance to ethanol treatment [68] , which in B. subtilis spores is associated with the spore inner membrane low permeability [71] . It is likely that the low permeability of the spore inner membrane of C. difficile spores protects their core against DNA-damaging chemicals typically used in clinical settings, such as quaternary ammonium compounds, formaldehyde, hydrogen peroxide and ethanol [56,57] . Surrounding the spore’s inner membrane is the germ cell wall, composed of a peptidoglycan cortex with a similar structure to that of growing cells (Figure 1) . The germ cell wall layer is surrounded by a layer of spore-specific cortex, with unique modifications in the glycan residues that provide protection against cellular cortex hydrolases (Figure 1) [72,73] . An outer membrane that surrounds the spore cortex is essential for spore formation yet it plays no role in spore resistance and is not visible in TEMs (Figure 1) . This layer is surrounded by a proteinaceous spore coat composed of approximately 70 proteins in the Bacillus

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Clostridium difficile spores: a major threat to the hospital environment 

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Figure 1. Ultrastructural morphotype of Clostridium difficile spores of various strains. Transmission electron micrographs of spores of C. difficile historical strain 630, endemic strain R20291 that belongs to ribotype 027 and Pitt177 that belongs to ribotype 027. Co: Coats; Ex: Exosporium structure; PG: Peptidoglycan cortex; SC: Spore core. Reproduced with permission from [66] © Elsevier (2013).

species; in the Bacillus and Clostridium species it provides protection against reactive chemicals and lytic enzymes and antimicrobial enzymes such as lysozyme. The spore coat is clearly visible in the TEMs of C. difficile spores (Figure 1) and presents similar laminations as in several Bacillus species (Figure 1) . In several species, including C. difficile, the spore coat is surrounded by an outermost layer, called the exosporium (Figure 1) . The C. difficile exosporium layer appears as an electrodense layer closely surrounding the spore coat and unlike the loosely fitting B. anthracis and Bacillus cereus spores. The exosporium layer exhibits significant differences between C. difficile strains, for example, spores of strain 630 have a thick exosporium layer that lacks the typical hair-like nap (Figure 1) observed in spores of the B. cereus group [24,34,64] , while spores of the epidemic strain R20291 have a scruffy hair-like nap (Figure 1) [66,68] . Recent studies have shown that C. difficile’s exosporium plays an important role in adherence to epithelium cells, interacts with the phagosome’s membrane and with inert surfaces, and contributes to spore surface hydrophobicity and spore dormancy [24,25,66,67] . Conserved spore resistance factors in C. difficile As aforementioned, C. difficile spores are resistant to various chemicals, heat, radiation and enzymes. Recent work has identified the first

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two factors with roles in C. difficile spore resistance: the exosporium morphogenetic factor CdeC, which is involved in spore resistance to ethanol, heat and lysozyme resistance [68] , and CotA, which is involved in loss of heat resistance in a subset of the population [74] . Both of these factors confer their role on resistance by altering the assembly process of the outer layers of C. difficile spores. In addition to these factors, there are a lack of molecular studies that characterize the role of the classical factors in C. difficile spore resistance to chemicals, heat, radiation and lysozyme, which have been shown to play a role in resistant B. subtilis and C. perfringens spores. These include a group of small acid soluble spore proteins (SASPs), germination protease, spore maturation protein (SpmAB) and the penicillinbinding-protein DacB. All sequenced C. difficile genomes encode homologs of these classical spore resistance factors. Recent transcriptional studies on sporulation of C. difficile have shown that all are expressed during spore formation [37,39] , suggesting that they may play a role in C. difficile spore resistance. One major factor involved in the chemical resistance of bacterial spores is a group of SASPs), which have a molecular weight of approximately 6–9 kDa [65] , and are exclusively found in the spore core of B. subtilis and C. perfringens spores  [65,75] . They are synthesized during late sporulation in the developing spore and become

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Review  Barra-Carrasco & Paredes-Sabja degraded during early germination, providing the nascent cell with amino acids during the initial stages of spore outgrowth [76,77] . The α/βtype SASPs saturate the spore DNA through nonspecific binding and provide protection against chemicals that target the spore DNA. In vitro work with B. subtilis and C. perfringens spores has demonstrated that α/β-type SASPs provide shielding protection against hydrogen peroxide and UV irradiation [78–80] , commonly used in clinical settings as alternative surface disinfectants [62,81,82] . These α/β-type SASPs also protect B. subtilis and C. perfringens spores against nitrous acid and formaldehyde [78,83] ; however, they do not play a role against alkylating agents [84] . Finally, these proteins also provide protection to B. subtilis and C. perfringens spores against UV radiation, which is a commonly used surface disinfectant method in clinical settings. Furthermore. C. difficile’s genome encodes several α/β-type SASPs homologs, suggesting that these proteins could play an important role in the resistance of C. difficile spores against common hospital decontamination procedures. In B. subtilis, these molecules are degraded during spore germination by a germination protease that becomes activated during the early minutes of germination [85] ; therefore, triggering germination of C. difficile spores would contribute to the degradation of these proteins and increase C. difficile spores’ susceptibility to routinely used disinfectants [40] . Another major spore resistance factor located in the core of dormant spores is the large depot (∼25% of core dry weight) of pyridine-2–6-dicarboxylic acid (dipicolinic acid; DPA), which is mainly chelated in a 1:1 ratio with divalent cations, mainly Ca 2+ (Ca-DPA). Bacillus and Clostridium spores that lack DPA, have a high spore core water content and lose most of their resistance properties to chemicals, UV and heat [86,87] . C. difficile spores also posses DPA and, although the precise amount of DPA is unknown, it is released during germination [88] , which is consistent with germinated C. difficile spores losing their spore resistance properties and increased susceptibility to commonly used disinfectants and antimicrobial compounds [82] . The aforementioned factors are the main components involved in the resistance of bacterial spores. Other auxiliary factors that contribute to bacterial spore resistance act during the spore formation process and include the spore maturation proteins (i.e., SpmA and SpmB), shown to be involved

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in the resistance of B. subtilis and C. perfringens spores to UV irradiation and various chemicals [69,89] . Similarly, the spore cortex possesses a lower degree of crosslinking of the glycan chains than the cortex layer of vegetative cells, which provides elasticity and contributes to maintenance of heat resistance properties of B. subtilis and C. perfringens spores. This process is regulated during spore formation by several penicillin-binding proteins, the most essential being DacB [69,89,90] . C. difficile genomes encodes orthologs of SpmAB and DacB, which have recently been demonstrated to be expressed during sporulation [37,39] , suggesting that similar mechanisms might also play a role in C. difficile spore resistance. Despite the enormous resistance of bacterial spores to various stressors, all of the aforementioned resistance properties are lost during spore germination. Thus, strategies able to modulate the germination machinery will promote the loss of spore resistance factors and render spores with an increased susceptibility to routinely used disinfectant regimes [40] . Resistance of C. difficile spores to antibiotics C. difficile spores are intrinsically resistant to all known antibiotic therapies mainly because they are metabolically dormant and also due to the spore’s inner membrane impermeability properties, blocking the entrance of antibiotics to the spore core [91–94] . Recently, Allen et al. demonstrated in vitro that vancomycin and fidaxomixin do not inhibit progression of germination of C. difficile spores, but rather inhibit outgrowth [95] . However, fidaxomicin at subinhibitory concentrations, unlike vancomycin and metronidazole, inhibits spore formation in vitro [42] . The high rates of recurrence of CDI in patients treated with metronidazole and vancomycin are not only because they contribute with alterations to the colonic microbiota [96] , but also presumably because spore production is not inhibited during treatment with these antibiotics during treatment of CDI [91,92] . By contrast, clinical evidence has demonstrated that fidaxomicin has some effect on recurrence [97,98] , presumably by inhibition of spore formation. Reactivation of C. difficile spores: germinants & germination Dormant bacterial spores are able to return to life within minutes in the presence of nutrients, termed germinants. These nutrient germinants are species specific and are sensed by specific

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Clostridium difficile spores: a major threat to the hospital environment  germinant receptors that localize to the spore inner membrane [72] . Unlike other Bacillus and Clostridium species, C. difficile lacks the canonical GerA germinant receptors [72] and their spores mainly germinate in the presence of cholate derivatives and several amino acids [99–101] , which are sensed, at least in part, by one of the three serine proteases encoded by C. difficile, CspC. This protease is catalytically dead and acts as a germinant receptor, leading to the release of the spore’s deposit of Ca-DPA [88] . CspBA is expressed as a fusion gene that is processed to incorporate CspB during C. difficile spore assembly [102] . CspC seems to be required for CspB-mediated proteolytic activation of proSleC into mature SleC [102] . All three components of the germination machinery (i.e., CspC, CspB and SleC) are essential for germination of C. difficile spores [88,102] . The spore cortex also contributes to bacterial spore resistance, by acting as a compressing force jacket, yet the CspA portion lacks the catalytic triad and, during its incorporation into the spore core, it contributes to the spores’ low core water content and to the low permeability of the spore’s inner membrane [70] . Although the spore’s inner membrane properties have not been demonstrated in C. difficile spores, it is expected that degradation of the spore cortex allows spore core expansion, loss of the permeability barrier of the spore’s inner membrane and core rehydration with the consequent loss of all of the spore-resistant factors. Recent evidence suggests that the exosporium layer of C. difficile spores is a repressor of germination and might control reactivation of C. difficile spores where those spores with an intact exosporium are more dormant than those lacking the exosporium layer [66] . Two-step strategies, where a first step involves activation and germination of C. difficile spores followed by a second step where germinated spores are inactivated by commonly used in vitro or in vivo stressors, should aid in increasing the susceptibility of C. difficile spores to the host innate immune system. These strategies require an in depth understanding of the reactivation kinetics of C. difficile spores. Recent evidence indicates that taurocholate is sufficient to trigger germination by itself and l-glycine acts as a co-germinant of taurocholate increasing the germination response [99] . C. difficile spores are also able to increase their germination ability when they are in contact with epithelial cells [103] . There is also strain to strain variability in the response to

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taurocholate, yet the inhibitor chenodeoxycholate does not inhibit germination in all strains [101] , suggesting that there might be differences in the levels of CspC among clinical C. difficile isolates. The germination efficiency of C. difficile spores is also variable, in fact, approximately 2–5% of 630 and R20291 spores form colonies [66,68] , indicating that in contrast to the low superdormant population observed in B. subtilis spores (less than ∼1% of the spores) [104] , a higher fraction of the C. difficile spore population might remain superdormant and might be responsible for recurrent CDI. Indeed, dormant C. difficile spores are more resistant than germinated spores to attacks by professional phagocytic cells [25] . These studies suggest that reactivation of C. difficile spores with taurocholate and amino acids requires further research to develop wide spectrum germination strategies that aim to activate C. difficile spores with different germination phenotypes, rendering the entire spore population sensitive to the host’s gut innate immunity. Novel strategies to kill C. difficile spores Several attempts to develop novel strategies to eradicate C. difficile spores include a two-step process [82,105] . The rationale behind this is that most factors involved in spore resistance (i.e., α/β-type SASP, low spore core water content, Ca-DPA and the spore cortex) are lost during germination. However, germination of C. difficile spores in the presence of oxygen progresses to a lesser extent than in the absence of oxygen, at least with strains ATCC 43593 and 43598. Indeed, treating C. difficile spores with ethanol produced a 1.6 decimal reduction in viability of C. difficile spores germinated under anaerobic but not aerobic conditions [82] , suggesting that oxygen exposure might prevent C. difficile spores from fully progressing to outgrowth [82] . This lack of germinability of C. difficile spores under aerobic conditions can be modulated by the presence of osmotic solutes enhancing C. difficile spore killing with antimicrobials that attack the spore’s inner membrane such as nisin [105] . However, extensive germination studies have been performed by Sorg’s laboratory with C. difficile strain UK1 where complete progression of germination has been observed under aerobic conditions [99] , which suggests that this might be strain dependent. In addition to activation of C. difficile spores, strategies that exploit inhibition of germination

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Review  Barra-Carrasco & Paredes-Sabja have also been shown to successfully reduce the virulence of CDI in a mouse model [40] , where an antigermination chemical analog of taurocholate is able to inhibit germination [106] and attenuate virulence of CDI [40] . This highlights the potential use of inhibitors of spore germination by germinant analogs as a prophylactic strategy to prevent CDI. Clearly, further research efforts are needed to trigger or block C. difficile spore germination either on inert surfaces or in vivo to develop more efficient strategies that may fully eliminate C. difficile spores from the hospital environment and the host. Conclusion Similar to other bacterial spores, C. difficile spores are highly resistant to various stressors used in healthcare settings and therefore are capable of persisting in the hospital environment. During CDI, C. difficile initiates a sporulation cycle, which has been demonstrated to be efficient and rapid in a murine model, contributing to the persistence of C. difficile spores in the patient’s colonic environment and to horizontal transmission of CDI by the dissemination of C. difficile spores. Spores present in the hospital environment may persist for extended periods of time (i.e., years) and act as a reservoir of C. difficile for hospital-acquired CDI. Bleach-free routine disinfectants are unable to kill dormant C. difficile spores. Molecular studies have been conducted and have identified some factors involved in spore resistance, yet the classical spore-resistance factors identified in the well-studied B. subtilis and C. perfringens spores, are also encoded in all sequenced genomes of C. difficile strains, suggesting that the mechanism of resistance to chemicals, heat, radiation and enzymes might be similar and most importantly, that these are lost during germination. C. difficile spores return to active

growth upon sensing taurocholate and several amino acids, which are detected in part by a unique taurocholate receptor, the inactive serine protease CspC, which strikingly contrasts with the classical germinant receptors that belong to the GerA family of germinant receptors. CspC seems to subsequently activate the CspB-SleC machinery for the complete degradation of the spore’s cortex, rendering spores susceptible to disinfectant regimens. However, the presence of superdormant C. difficile spores and the high germination variability observed within strains has hampered the development of efficient two-step spore inactivation strategies. Future perspective C. difficile spores play a crucial role in pathogenesis of CDI, as strains unable to form spores are unable to persist in the host and transmit CDI between hosts. Although orthologs of spore resistance factors are present in C. difficile genomes of sequenced strains, much work will be required to fully understand how these factors contribute to C. difficile spore survival in the hospital environment. Future work should also be oriented towards the modulation of C. difficile spore germination in the presence of oxygen, knowledge that will allow the development of two-step inactivation strategies (i.e., germination and inactivation) to eradicate C. difficile spores from contaminated surfaces. Similarly, in vivo modulation of C. difficile spore germination could either inhibit the return of C. difficile spores to active growth or trigger their germination to be subsequently inactivated by anti-C. difficile therapy. Financial & competing interests disclosure This work was supported by grants from the Fondo Nacional de Ciencia y Tecnología de Chile (FONDECYT REGULAR Grant 1110569), from the Research Office of

EXECUTIVE SUMMARY ●●

Clostridium difficile initiates an efficient cycle of spore formation in vivo facilitating their persistence and dissemination.

●●

C. difficile spores have similar structural and resistance properties as other bacterial spores.

●●

I n contrast to other bacterial spores, C. difficile spores lack the canonical GerA, and use the inactive serine protease, CspC, as a germinant receptor for taurocholate-triggered germination.

●●

ompletion of germination upon hydrolysis of the spore peptidoglycan cortex occurs through the essential CspB-SleC C machinery.

●●

. difficile spore superdormancy and the great variability observed between strains may hamper the development of C spore inactivation strategies that are based on a initial germination stage.

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Clostridium difficile spores: a major threat to the hospital environment  Universidad Andres Bello (DI-275–13/R) and from the Fondo de Fomento al Desarrollo Científico y Tecnológico (FONDEF IdeA Grant CA13I10077) to D Paredes-Sabja. The authors have no other relevant affiliations or financial involvementwith any organization or entity with a

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