Redistribution of the Novel Clostridioides difficile Spore Adherence Receptor E-Cadherin by TcdA and TcdB Increases Spore Binding to Adherens Junctions.

Clostridioides difficile causes antibiotic-associated diseases in humans, ranging from mild diarrhea to severe pseudomembranous colitis and death. A major clinical challenge is the prevention of disease recurrence, which affects nearly ~20 to 30% of the patients with a primary C. difficile infection (CDI). During CDI, C. difficile forms metabolically dormant spores that are essential for recurrence of CDI (R-CDI). In prior studies, we have shown that C. difficile spores interact with intestinal epithelial cells (IECs), which contribute to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence that C. difficile spores interact with E-cadherin, contributing to spore adherence and internalization into IECs. C. difficile toxins TcdA and TcdB lead to adherens junctions opening and increase spore adherence to IECs. Confocal micrographs demonstrate that C. difficile spores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA and TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and immunogold labeling, we observed that E-cadherin binds to C. difficile spores, specifically to the hairlike projections of the spore, reducing spore adherence to IECs. Overall, these results expand our knowledge of how C. difficile spores bind to IECs by providing evidence that E-cadherin acts as a spore adherence receptor to IECs and by revealing how toxin-mediated damage affects spore interactions with IECs.

Different presentations of the Buccal Bifurcation Cyst: A case series.

The buccal bifurcation cyst (BBC) is an uncommon odontogenic inflammatory cyst affecting the vestibular aspects of the first or second mandibular molar of pediatric patients. Its etiopathogenesis is not fully understood, but it is hypothesized that food and detritus impacting buccal periodontal pockets in titled tooth would be responsible for inflammation of the pericoronal tissues, leading to proliferation of epithelial rests and subsequent cystic formation. The true prevalence of the BBC is not known, but it is estimated to be less than 1% of all the inflammatory cysts. Most cases are unilateral but bilateral cases may account for up to 30% of all BBCs, which can generate confusion to unfamiliar clinicians. Maxillary cases are extremely uncommon, and to our knowledge, there are no cases published in the English literature. In this case series, we present five BBC cases; two unilateral, two bilateral, and one affecting the maxilla. We included clinical, imaging, and histopathological information to highlight the different presentations that this cyst might have, with the final aim to aid clinicians in its diagnosis and ultimately, its treatment.

[Skin test (Skin Prick Test) in food allergy]. / Pruebas cutáneas (Skin Prick Test) en alergia alimentaria.

Skin tests, also known as prick tests, in food allergies are restricted to IgE-mediated food allergies, with results that barely indicate sensitization to a certain food and do not necessarily suggest food allergy. The clinical context of the patient is the decisive point before performing any type of food allergy skin test; Contextualizing and relating the patient's clinical picture with the immunological mechanism of IgE-mediated allergies will allow a better selection of the allergy test for each case. Positive tests should be interpreted more carefully, and consider that during early childhood it is common that several patients may have negative specific serum IgE tests in the blood for food allergens compared to those performed on the skin, which are positive. Skin testing can be carried out with standardized strata of foods, but they are not always available for all foods and some are very unstable. Skin tests can even be performed with raw foods, using the prick to prick technique to perform the puncture with fresh foods (especially fruits or vegetables) or other products that are not commercially available. The skin prick test is a test where, after having placed the allergenic extract in drops or with fresh food itself, the puncture must be carried out with a standardized lancet.

Las pruebas cutáneas, también conocidas como prick test, en alergia alimentaria están restrictas a las alergias a alimentos mediadas por IgE, con resultados que apenas indican sensibilización a cierto alimento y no necesariamente sugieren alergia alimentaria. El contexto clínico del paciente es el punto decisivo antes de realizar cualquier tipo de prueba cutánea de alergia a alimentos; contextualizar y relacionar el cuadro clínico del paciente con el mecanismo inmunológico de las alergias mediadas por IgE permitirá una mejor selección de la prueba de alergia para cada caso. Las pruebas positivas deben interpretarse con más cuidado, y considerar que durante la infancia precoz es común que varios pacientes pueden tener pruebas IgE séricas especificas negativas en sangre para alérgenos alimentarios comparadas con las realizadas en piel, que resultan positivas. La prueba cutánea puede llevarse a cabo con estratos estandarizados de alimentos, pero no siempre se encuentran disponibles para todos los alimentos y algunos son muy inestables. Incluso pueden realizarse pruebas cutáneas con alimentos in natura, mediante la técnica prick to prick para efectuar la punción con alimentos frescos (especialmente con frutas o vegetales) u otros productos que no estén disponibles comercialmente. El skin prick test es una prueba donde después de haber colocado el extracto alergénico en gotas o con el propio alimento fresco debe llevarse a cabo la punción con una lanceta estandarizada.

Using a ligate intestinal loop mouse model to investigate Clostridioides difficile adherence to the intestinal mucosa in aged mice.

Interaction of Clostridioides difficile spores with the intestinal mucosa contributes to the persistence and recurrence of the infection. Advanced age is one of the main risk factors for C. difficile infection and recurrence of the disease. However, interaction of C. difficile spores with the intestinal mucosa during aging has not been evaluated. In the present work, using intestinal ligated loop technique in a mouse model, we analyzed C. difficile spore adherence and internalization to the ileum and colonic mucosa during aging. Additionally, we provide visual documentation of the critical steps of the procedure. Consequently, our data suggest that spore internalization in the ileum and colonic mucosa is higher in elderly mice rather than adults or young mice. Also, our data suggest that spore adherence to the ileum and colonic mucosa decreases with aging.

Entry of spores into intestinal epithelial cells contributes to recurrence of Clostridioides difficile infection.

Clostridioides difficile spores produced during infection are important for the recurrence of the disease. Here, we show that C. difficile spores gain entry into the intestinal mucosa via pathways dependent on host fibronectin-α5ß1 and vitronectin-αvß1. The exosporium protein BclA3, on the spore surface, is required for both entry pathways. Deletion of the bclA3 gene in C. difficile, or pharmacological inhibition of endocytosis using nystatin, leads to reduced entry into the intestinal mucosa and reduced recurrence of the disease in a mouse model. Our findings indicate that C. difficile spore entry into the intestinal barrier can contribute to spore persistence and infection recurrence, and suggest potential avenues for new therapies.

Value of bronchial reversibility to salbutamol, exhaled nitric oxide and responsiveness to methacholine to corroborate the diagnosis of asthma in children

INTRODUCTION AND OBJECTIVES: Functional and inflammatory measures have been recommended to corroborate asthma diagnosis in schoolchildren, but the evidence in this regard is conflicting. We aimed to determine, in real-life clinical situation, the value of spirometry, spirometric bronchial reversibility to salbutamol (BDR), bronchial responsiveness to methacholine (MCT) and fractional exhaled nitric oxide (FENO), to corroborate the diagnosis of asthma in children on regular inhaled corticosteroids (ICS) referred from primary care. METHODS: One hundred and seventy-seven schoolchildren with mild-moderate persistent asthma, on treatment with regular ICS, participated in the study. Abnormal tests were defined as FENO ≥ 27 ppb, BDR (FEV1 ≥ 12%) and methacholine PC20 ≤ 4 mg/mL. RESULTS: The proportions of positive BDR, FENO and MCT, were 16.4%, 33.3%, and 87.0%, respectively. MCT was associated with FENO (p < 0.03) and BDR (p = 0.001); FENO was associated with BDR (p = 0.045), family history of asthma (p = 0.003) and use of asthma medication in the first two years of life (p = 0.004). BDR was significantly related with passive tobacco exposure (p = 0.003). CONCLUSIONS: Spirometry, BDR and BDR had a poor performance for corroborating diagnosis in our asthmatic children on ICS treatment; on the contrary, MCT was positive in most of them, which agrees with previous reports. Although asthma tests are useful to corroborate asthma when positive, clinical diagnosis remains the best current approach for asthma diagnosis, at least while better objective and feasible measurements at the daily practice are available. At present, these tests may have a better role for assessing the management and progression of the condition

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Plasmablastic lymphoma as a manifestation of the human immunodeficiency virus: Case report.

Plasmablastic lymphoma is a rare subtype of non-Hodgkin's lymphoma, which generally presents an aggressive clinical course and low survival rates. It is strongly associated with HIV infection and the most common site of involvement of the territory of the head and neck is Waldeyer's lymphatic ring, followed by the gastrointestinal tract, lymph nodes and skin. The morphological characteristics of PBL in the oral cavity / jaw in the context of HIV infection show diffuse sheets of large immunoblastic cells with abundant cytoplasm, vesicular chromatin and prominent nucleus, a small located in the center with plasma cells differentiation. The main goal of this article is to review the literature of the plasmablastic lymphoma and report a case. Key words:Plasmablastic lymphoma, PBL, HIV, AIDS, Non Hodgkin Lynphoma.

Hemorragia digestiva alta como complicação de esofagite eosinofílica / Upper gastrointestinal bleeding as a complication of eosinophilic esophagitis

A hemorragia digestiva alta (HDA) é uma condição médica comum, que permanece com uma taxa de mortalidade aproximadamente de 10%. Doenças alérgicas habitualmente não configuram risco para HDA. Entretanto, o aumento recente de doenças alérgicas que afetam cronicamente o trato digestório poderia mudar esse cenário. Este artigo relata um caso de HDA após hematêmese provocada por impactação alimentar. Realizada endoscopia digestiva alta (EDA) e diagnosticada esofagite eosinofílica (EoE), que após tratamento adequado, apresentou melhora dos sintomas. A EoE é uma doença inflamatória crônica esofágica emergente, com aumento do número de casos diagnosticados ao redor do mundo. Atualmente, considera-se a causa mais prevalente de disfagia e impactação alimentar em crianças e adultos jovens. Os sintomas de EoE não são específicos para cada faixa etária, e podem variar desde sintomas mais leves, como sintomas de doença do refluxo gastroesofágico, até disfagia e impactação alimentar. Existe atraso no diagnóstico e tratamento, propiciando um aumento de complicações, cujo risco mais temido seria rotura do esôfago. Revisando a literatura até o presente relato, constatamos que a EoE nunca foi descrita como uma causa de HDA. Além da apresentação incomum da HDA levando ao diagnóstico de EoE, esse caso ressalta a importância do atendimento multidisciplinar e cooperação entre especialidades. Portanto, há necessidade de diagnóstico mais precoce e preciso, buscando ampliar o conhecimento para não negligenciar características específicas da disfagia, e evitar complicações com o tratamento adequado.

Upper gastrointestinal bleeding (UGIB) is a common medical condition whose mortality rate remains at about 10%. Allergic diseases are no usual risk for UGIB. However, the recent increase in allergic diseases that chronically affect the gastrointestinal tract could change this scenario. This article reports a case of UGIB after hematemesis caused by food impaction. Upper gastrointestinal endoscopy was performed and eosinophilic esophagitis (EoE) was diagnosed. EoE is an emerging chronic esophageal inflammatory disease with an increasing number of diagnosed cases around the world. Currently, it is considered the most prevalent cause of dysphagia and food impaction in children and young adults. EoE symptoms are not specific to each age group and may range from mild symptoms such as those of gastroesophageal reflux disease to dysphagia and food impaction. There is a delay in diagnosis and treatment that leads to increased complications, including esophageal rupture, the most feared risk. Our literature review showed that EoE had never been described as a cause of UGIB. In addition to the unusual presentation of UGIB leading to the diagnosis of EoE, this case highlights the importance of multidisciplinary care and cooperation between specialties. Therefore, there is a need for earlier and more accurate diagnosis, which would lead to expanded knowledge that could be used to not disregard specific characteristics of dysphagia and avoid complications with appropriate treatment.

Value of bronchial reversibility to salbutamol, exhaled nitric oxide and responsiveness to methacholine to corroborate the diagnosis of asthma in children.

INTRODUCTION AND OBJECTIVES: Functional and inflammatory measures have been recommended to corroborate asthma diagnosis in schoolchildren, but the evidence in this regard is conflicting. We aimed to determine, in real-life clinical situation, the value of spirometry, spirometric bronchial reversibility to salbutamol (BDR), bronchial responsiveness to methacholine (MCT) and fractional exhaled nitric oxide (FENO), to corroborate the diagnosis of asthma in children on regular inhaled corticosteroids (ICS) referred from primary care. METHODS: One hundred and seventy-seven schoolchildren with mild-moderate persistent asthma, on treatment with regular ICS, participated in the study. Abnormal tests were defined as FENO ≥ 27 ppb, BDR (FEV1 ≥ 12%) and methacholine PC20 ≤ 4 mg/mL. RESULTS: The proportions of positive BDR, FENO and MCT, were 16.4%, 33.3%, and 87.0%, respectively. MCT was associated with FENO (p < 0.03) and BDR (p = 0.001); FENO was associated with BDR (p = 0.045), family history of asthma (p = 0.003) and use of asthma medication in the first two years of life (p = 0.004). BDR was significantly related with passive tobacco exposure (p = 0.003). CONCLUSIONS: Spirometry, BDR and BDR had a poor performance for corroborating diagnosis in our asthmatic children on ICS treatment; on the contrary, MCT was positive in most of them, which agrees with previous reports. Although asthma tests are useful to corroborate asthma when positive, clinical diagnosis remains the best current approach for asthma diagnosis, at least while better objective and feasible measurements at the daily practice are available. At present, these tests may have a better role for assessing the management and progression of the condition.

Induction of a Specific Humoral Immune Response by Nasal Delivery of Bcla2ctd of Clostridioides difficile.

Clostridioides difficile, formerly known as Clostridium difficile, is a spore-forming bacterium considered as the most common cause of nosocomial infections in developed countries. The spore of C. difficile is involved in the transmission of the pathogen and in its first interaction with the host; therefore, a therapeutic approach able to control C. difficile spores would improve the clearance of the infection. The C-terminal (CTD) end of BclA2, a spore surface protein of C. difficile responsible of the interaction with the host intestinal cells, was selected as a putative mucosal antigen. The BclA2 fragment, BclA2CTD, was purified and used to nasally immunize mice both as a free protein and after adsorption to the spore of Bacillus subtilis, a well-established mucosal delivery vehicle. While the adsorption to spores increased the in vitro stability of BclA2CTD, in vivo both free and spore-adsorbed BclA2CTD were able to induce a similar, specific humoral immune response in a murine model. Although in the experimental conditions utilized the immune response was not protective, the induction of specific IgG indicates that free or spore-bound BclA2CTD could act as a putative mucosal antigen targeting C. difficile spores.

Effect of antibiotic to induce Clostridioides difficile-susceptibility and infectious strain in a mouse model of Clostridioides difficile infection and recurrence.

The anaerobic bacterium Clostridioides difficile is the leading cause of antibiotic-associated diarrhea that can culminate in life-threating colitis. During the C. difficile infection (CDI), C. difficile produces toxins that generate the clinical symptoms of the disease, and produce spores, which persist in the host during antibiotic treatment and can cause recurrent CDI (R-CDI). In this work, we aimed to compare three antibiotic regimens in the susceptibility of mice to CDI and R-CDI (i.e., antibiotic cocktail followed by clindamycin, 5 days of cefoperazone and 10 days of cefoperazone) with three different C. difficile isolates (i.e., strains 630; R20291, and VPI 10463). We observed that the severity of the clinical symptoms of CDI and R-CDI was dependent on the antibiotic treatment used to induce C. difficile-susceptibility, and that the three strains generated a different onset to diarrhea and weight loss in mice that were administrated with the same antibiotic treatment and which differed in comparison to the effect previously reported by other research groups. Our results suggest that, in our experimental conditions, in those animals treated with antibiotic cocktail followed by clindamycin, infection with strain R20291 had the highest diarrhea manifestation in comparison to strains 630 and VPI 10463. In animals treated with cefoperazone for 5 days, infection with strains R20291 or 630 had the highest diarrhea manifestation in comparison to VPI 10463, while in animals treated with cefoperazone for 10 days, infection with strain R20291 or VPI 10463, but not 630, had the highest diarrhea manifestation.

Indomethacin increases severity of Clostridium difficile infection in mouse model.

AIM: To evaluate the effect on the nonsteroidal anti-inflammatory drug indomethacin on Clostridium difficile infection (CDI) severity. MATERIALS & METHODS: Indomethacin was administered in two different mouse models of antibiotic-associated CDI in two different facilities, using a low and high dose of indomethacin. RESULTS: Indomethacin administration caused weight loss, increased the signs of severe infection and worsened histopathological damage, leading to 100% mortality during CDI. Indomethacin-treated, antibiotic-exposed mice infected with C. difficile had enhanced intestinal inflammation with increased expression of KC, IL-1ß and IL-22 compared with infected mice unexposed to indomethacin. CONCLUSION: These results demonstrate a negative impact of nonsteroidal anti-inflammatory drugs on antibiotic-associated CDI in mice and suggest that targeting the synthesis or signaling of prostaglandins might be an approach to ameliorating the severity of CDI.

Clostridium difficile exosporium cysteine-rich proteins are essential for the morphogenesis of the exosporium layer, spore resistance, and affect C. difficile pathogenesis.

Clostridium difficile is a Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea that can culminate in fatal colitis. During the infection, C. difficile produces metabolically dormant spores, which persist in the host and can cause recurrence of the infection. The surface of C. difficile spores seems to be the key in spore-host interactions and persistence. The proteome of the outermost exosporium layer of C. difficile spores has been determined, identifying two cysteine-rich exosporium proteins, CdeC and CdeM. In this work, we explore the contribution of both cysteine-rich proteins in exosporium integrity, spore biology and pathogenesis. Using targeted mutagenesis coupled with transmission electron microscopy we demonstrate that both cysteine rich proteins, CdeC and CdeM, are morphogenetic factors of the exosporium layer of C. difficile spores. Notably, cdeC, but not cdeM spores, exhibited defective spore coat, and were more sensitive to ethanol, heat and phagocytic cells. In a healthy colonic mucosa (mouse ileal loop assay), cdeC and cdeM spore adherence was lower than that of wild-type spores; while in a mouse model of recurrence of the disease, cdeC mutant exhibited an increased infection and persistence during recurrence. In a competitive infection mouse model, cdeC mutant had increased fitness over wild-type. Through complementation analysis with FLAG fusion of known exosporium and coat proteins, we demonstrate that CdeC and CdeM are required for the recruitment of several exosporium proteins to the surface of C. difficile spores. CdeC appears to be conserved exclusively in related Peptostreptococcaeace family members, while CdeM is unique to C. difficile. Our results sheds light on how CdeC and CdeM affect the biology of C. difficile spores and the assembly of the exosporium layer and, demonstrate that CdeC affect C. difficile pathogenesis.

Census of Cnidaria (Medusozoa) and Ctenophora from South American marine waters.

We have compiled available records in the literature for medusozoan cnidarians and ctenophores of South America. New records of species are also included. Each entry (i.e., identified species or still as yet not determined species referred to as "sp." in the literature) includes a synonymy list for South America, taxonomical remarks, notes on habit, and information on geographical occurrence. We have listed 800 unique determined species, in 958 morphotype entries: 5 cubozoans, 905 hydrozoans, 25 scyphozoans, 3 staurozoans, and 20 ctenophores. Concerning nomenclatural and taxonomical decisions, two authors of this census (Miranda, T.P. & Marques, A.C.) propose Podocoryna quitus as a nomen novum for the junior homonym Hydractinia reticulata (Fraser, 1938a); Euphysa monotentaculata Zamponi, 1983b as a new junior synonym of Euphysa aurata Forbes, 1848; and Plumularia spiralis Milstein, 1976 as a new junior synonym of Plumularia setacea (Linnaeus, 1758). Finally, we also reassign Plumularia oligopyxis Kirchenpauer, 1876 as Kirchenpaueria oligopyxis (Kirchenpauer, 1876) and Sertularella margaritacea Allman, 1885 as Symplectoscyphus margaritaceus (Allman, 1885).

Characterization of the Adherence of Clostridium difficile Spores: The Integrity of the Outermost Layer Affects Adherence Properties of Spores of the Epidemic Strain R20291 to Components of the Intestinal Mucosa.

Clostridium difficile is the causative agent of the most frequently reported nosocomial diarrhea worldwide. The high incidence of recurrent infection is the main clinical challenge of C. difficile infections (CDI). Formation of C. difficile spores of the epidemic strain R20291 has been shown to be essential for recurrent infection and transmission of the disease in a mouse model. However, the underlying mechanisms of how these spores persist in the colonic environment remains unclear. In this work, we characterized the adherence properties of epidemic R20291 spores to components of the intestinal mucosa, and we assessed the role of the exosporium integrity in the adherence properties by using cdeC mutant spores with a defective exosporium layer. Our results showed that spores and vegetative cells of the epidemic R20291 strain adhered at high levels to monolayers of Caco-2 cells and mucin. Transmission electron micrographs of Caco-2 cells demonstrated that the hair-like projections on the surface of R20291 spores are in close proximity with the plasma membrane and microvilli of undifferentiated and differentiated monolayers of Caco-2 cells. Competitive-binding assay in differentiated Caco-2 cells suggests that spore-adherence is mediated by specific binding sites. By using spores of a cdeC mutant we demonstrated that the integrity of the exosporium layer determines the affinity of adherence of C. difficile spores to Caco-2 cells and mucin. Binding of fibronectin and vitronectin to the spore surface was concentration-dependent, and depending on the concentration, spore-adherence to Caco-2 cells was enhanced. In the presence of an aberrantly-assembled exosporium (cdeC spores), binding of fibronectin, but not vitronectin, was increased. Notably, independent of the exosporium integrity, only a fraction of the spores had fibronectin and vitronectin molecules binding to their surface. Collectively, these results demonstrate that the integrity of the exosporium layer of strain R20291 contributes to selective spore adherence to components of the intestinal mucosa.

Ultrastructural Variability of the Exosporium Layer of Clostridium difficile Spores.

The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the transmission, infectious, and persistent morphotype, and the outermost exosporium layer is likely to play a major role in spore-host interactions during the first contact of C. difficile spores with the host and for spore persistence during recurrent episodes of infection. Although some studies on the biology of the exosporium have been conducted (J. Barra-Carrasco et al., J Bacteriol 195:3863-3875, 2013, http://dx.doi.org/10.1128/JB.00369-13; J. Phetcharaburanin et al., Mol Microbiol 92:1025-1038, 2014, http://dx.doi.org/10.1111/mmi.12611), there is a lack of information on the ultrastructural variability and stability of this layer. In this work, using transmission electron micrographs, we analyzed the variability of the spore's outermost layers in various strains and found distinctive variability in the ultrastructural morphotype of the exosporium within and between strains. Through transmission electron micrographs, we observed that although this layer was stable during spore purification, it was partially lost after 6 months of storage at room temperature. These observations were confirmed by indirect immunofluorescence microscopy, where a significant decrease in the levels of two exosporium markers, the N-terminal domain of BclA1 and CdeC, was observed. It is also noteworthy that the presence of the exosporium marker CdeC on spores obtained from C. difficile biofilms depended on the biofilm culture conditions and the strain used. Collectively, these results provide information on the heterogeneity and stability of the exosporium surface of C. difficile spores. These findings have direct implications and should be considered in the development of novel methods to diagnose and/or remove C. difficile spores by using exosporium proteins as targets.

Outcome of relapsing Clostridium difficile infections do not correlate with virulence-, spore- and vegetative cell-associated phenotypes.

One of the main clinical challenges of Clostridium difficile infections (CDI) is the high rate of relapse episodes. The main determinants involved in relapse of CDI include the presence of antibiotic-resistant C. difficile spores in the colonic environment and a permanent state of dysbiosis of the microbiota caused by antibiotic therapy. A possible scenario is that phenotypes related to the persistence of C. difficile spores might contribute to relapsing infections. In this study, 8 C. difficile isolates recovered from 4 cases with relapsing infection, and 9 isolates recovered from single infection cases were analyzed for PCR ribotyping and the presence of tcdA, tcdB and cdtAB genes. Factors associated to spore persistence, sporulation, spore adherence and biofilm formation and sporulation during biofilm formation were characterized. We also evaluated motility and cytotoxicity. However, we observed no significant difference in the analyzed phenotypes among the different clinical outcomes, most likely due to the high variability observed among strains within clinical backgrounds in each phenotype and the small sample size. It is noteworthy that C. difficile spores adhered to similar extents to undifferentiated and differentiated Caco-2 cells. By contrast, spores of all clinical isolates tested had increased germination efficiency in presence of taurocholate, while decreased sporulation rate during biofilm development in the presence of glucose. In conclusion, these results show that, at least in this cohort of patients, the described phenotypes are not detrimental in the clinical outcome of the disease.

Protein composition of the outermost exosporium-like layer of Clostridium difficile 630 spores.

Clostridium difficile spores are considered the morphotype of infection, transmission and persistence of C. difficile infections. There is a lack of information on the composition of the outermost exosporium layer of C. difficile spores. Using recently developed exosporium removal methods combined with MS/MS, we have established a gel-free approach to analyze the proteome of the exosporium of C. difficile spores of strain 630. A total of 184 proteins were found in the exosporium layer of C. difficile spores. We identified 7 characterized spore coat and/or exosporium proteins; 6 proteins likely to be involved in spore resistance; 6 proteins possibly involved in pathogenicity; 13 uncharacterized proteins; and 146 cytosolic proteins that might have been encased into the exosporium during assembly, similarly as reported for Bacillus anthracis and Bacillus cereus spores. We demonstrate through Flag-fusions that CotA and CotB are mainly located in the spore coat, while the exosporium collagen-like glycoproteins (i.e. BclA1, BclA2 and BclA3), the exosporium morphogenetic proteins CdeC and CdeM, and the uncharacterized exosporium proteins CdeA and CdeB are mainly located in the exosporium layer of C. difficile 630 spores. This study offers novel candidates of C. difficile exosporium proteins as suitable targets for detection, removal and spore-based therapies. BIOLOGICAL SIGNIFICANCE: This study offers a novel strategy to identify proteins of the exosporium layer of C. difficile spores and complements previous proteomic studies on the entire C. difficile spores and spore coat since it defines the proteome of the outermost layer of C. difficile spores, the exosporium. This study suggests that C. difficile spores have several proteins involved in protection against environmental stress as well as putative virulence factors that might play a role during infection. Spore exosporium structural proteins were also identified providing the ground basis for further functional studies of these proteins. Overall this work provides new protein target for the diagnosis and/or therapeutics that may contribute to combat C. difficile infections.