The chaperone ClpC participates in sporulation, motility, biofilm, and toxin production of Clostridioides difficile.

OBJECTIVES: Clostridioides difficile is a nosocomial pathogen that is associated with the use of antibiotics. One of the most worrying aspects of C. difficile infection is its ability to resist antimicrobial therapies, owing to spore formation. In several bacterial pathogens, proteases of the Clp family participate in phenotypes associated with persistence and virulence. This suggests that these proteins could be involved in virulence-related traits. In this study, we analysed the role of ClpC chaperone-protease of C. difficile in virulence-related traits by comparing the phenotypes of wild-type and mutant strains lacking the clpC gene (ΔclpC). METHODS: We performed biofilm, motility, spore formation, and cytotoxicity assays. RESULTS: Our results show significant differences between the wild-type and ΔclpC strains in all analysed parameters. CONCLUSIONS: Based on these findings, we conclude that clpC plays a role in the virulence properties of C. difficile.

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.

Role of the Spore Coat Proteins CotA and CotB, and the Spore Surface Protein CDIF630_02480, on the Surface Distribution of Exosporium Proteins in Clostridioides difficile 630 Spores.

Clostridioides difficile is Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea. During disease, C. difficile forms metabolically dormant spores that persist in the host and contribute to recurrence of the disease. The outermost surface of C. difficile spores, termed the exosporium, plays an essential role in interactions with host surfaces and the immune system. The main exosporium proteins identified to date include three orthologues of the BclA family of collagen-like proteins, and three cysteine-rich proteins. However, how the underlying spore coat influences exosporium assembly remains unclear. In this work, we explore the contribution of spore coat proteins cotA and cotB, and the spore surface protein, CDIF630_02480, to the exosporium ultrastructure, formation of the polar appendage and the surface accessibility of exosporium proteins. Transmission electron micrographs of spores of insertional inactivation mutants demonstrate that while cotB contributes to the formation of thick-exosporium spores, cotA and CDIF630_02480 contribute to maintain proper thickness of the spore coat and exosporium layers, respectively. The effect of the absence of cotA, cotB and CDIF630_02480 on the surface accessibility of the exosporium proteins CdeA, CdeC, CdeM, BclA2 and BclA3 to antibodies was affected by the presence of the spore appendage, suggesting that different mechanisms of assembly of the exosporium layer might be implicated in each spore phenotype. Collectively, this work contributes to our understanding of the associations between spore coat and exosporium proteins, and how these associations affect the assembly of the spore outer layers. These results have implications for the development of anti-infecting agents targeting C. difficile spores.

Intra-species diversity of Clostridium perfringens: A diverse genetic repertoire reveals its pathogenic potential.

Clostridium perfringens is the causative agent of many enterotoxic diseases in humans and animals, and it is present in diverse environments (soil, food, sewage, and water). Multilocus Sequence Typing (MLST) and Whole Genome Sequencing (WGS) have provided a general approach about genetic diversity of C. perfringens; however, those studies are limited to specific locations and often include a reduced number of genomes. In this study, 372 C. perfringens genomes from multiple locations and sources were used to assess the genetic diversity and phylogenetic relatedness of this pathogen. In silico MLST was used for typing the isolates, and the resulting sequence types (ST) were assigned to clonal complexes (CC) based on allelic profiles that differ from its founder by up to double-locus variants. A pangenome analysis was conducted, and a core genome-based phylogenetic tree was created to define phylogenetic groups. Additionally, key virulence factors, toxinotypes, and antibiotic resistance genes were identified using ABRicate against Virulence Factor Database (VFDB), TOXiper, and Resfinder, respectively. The majority of the C. perfringens genomes found in publicly available databases were derived from food (n = 85) and bird (n = 85) isolates. A total of 195 STs, some of them shared between sources such as food and human, horses and dogs, and environment and birds, were grouped in 25 CC and distributed along five phylogenetic groups. Fifty-three percent of the genomes were allocated to toxinotype A, followed by F (32%) and G (7%). The most frequently found virulence factors based on > 70% coverage and 99.95% identity were plc (100%), nanH (99%), ccp (99%), and colA (98%), which encode an alpha-toxin, a sialidase, an alpha-clostripain, and a collagenase, respectively, while tetA (39.5%) and tetB (36.2%), which mediate tetracycline resistance determinants, were the most common antibiotic resistance genes detected. The analyses conducted here showed a better view of the presence of this pathogen across several host species. They also confirm that the genetic diversity of C. perfringens is based on a large number of virulence factors that vary among phylogroups, and antibiotic resistance markers, especially to tetracyclines, aminoglycosides, and macrolides. Those characteristics highlight the importance of C. perfringens as a one of the most common causes of foodborne illness.

Assembly of the exosporium layer in Clostridioides difficile spores.

Clostridioides difficile is a Gram-positive, spore-forming obligate anaerobe and a major threat to the healthcare system world-wide. Because of its strict anaerobic requirements, the infectious and transmissible morphotype is the dormant spore. During infection, C. difficile produces spores that can persist in the host and are responsible for disease recurrence and transmission, especially between hospitalized patients. Although the C. difficile spore surface mediates critical interactions with host surfaces, this outermost layer, known as the exosporium, is poorly conserved when compared to members of the Bacillus genus. Notably, the exosporium has been shown to be important for the persistence of C. difficile in the host. In this review, the ultrastructural properties, composition, and morphogenesis of the exosporium will be discussed.

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.

FastMLST: A Multi-core Tool for Multilocus Sequence Typing of Draft Genome Assemblies.

Multilocus Sequence Typing (MLST) is a precise microbial typing approach at the intra-species level for epidemiologic and evolutionary purposes. It operates by assigning a sequence type (ST) identifier to each specimen, based on a combination of alleles of multiple housekeeping genes included in a defined scheme. The use of MLST has multiplied due to the availability of large numbers of genomic sequences and epidemiologic data in public repositories. However, data processing speed has become problematic due to the massive size of modern datasets. Here, we present FastMLST, a tool that is designed to perform PubMLST searches using BLASTn and a divide-and-conquer approach that processes each genome assembly in parallel. The output offered by FastMLST includes a table with the ST, allelic profile, and clonal complex or clade (when available), detected for a query, as well as a multi-FASTA file or a series of FASTA files with the concatenated or single allele sequences detected, respectively. FastMLST was validated with 91 different species, with a wide range of guanine-cytosine content (%GC), genome sizes, and fragmentation levels, and a speed test was performed on 3 datasets with varying genome sizes. Compared with other tools such as mlst, CGE/MLST, MLSTar, and PubMLST, FastMLST takes advantage of multiple processors to simultaneously type up to 28 000 genomes in less than 10 minutes, reducing processing times by at least 3-fold with 100% concordance to PubMLST, if contaminated genomes are excluded from the analysis. The source code, installation instructions, and documentation of FastMLST are available at https://github.com/EnzoAndree/FastMLST.

Updating changes in human gut microbial communities associated with Clostridioides difficile infection.

Clostridioides difficile is the causative agent of antibiotic-associated diarrhea, a worldwide public health problem. Different factors can promote the progression of C. difficile infection (CDI), mainly altered intestinal microbiota composition. Microbial species belonging to different domains (i.e., bacteria, archaea, eukaryotes, and even viruses) are synergistically and antagonistically associated with CDI. This review was aimed at updating changes regarding CDI-related human microbiota composition using recent data and an integral approach that included the different microorganism domains. The three domains of life contribute to intestinal microbiota homeostasis at different levels in which relationships among microorganisms could explain the wide range of clinical manifestations. A holistic understanding of intestinal ecosystem functioning will facilitate identifying new predictive factors for infection and developing better treatment and new diagnostic tools, thereby reducing this disease's morbidity and mortality.

Landscapes and bacterial signatures of mucosa-associated intestinal microbiota in Chilean and Spanish patients with inflammatory bowel disease.

Inflammatory bowel diseases (IBDs), which include ulcerative colitis (UC) and Crohn's disease (CD), cause chronic inflammation of the gut, affecting millions of people worldwide. IBDs have been frequently associated with an alteration of the gut microbiota, termed dysbiosis, which is generally characterized by an increase in abundance of Proteobacteria such as Escherichia coli, and a decrease in abundance of Firmicutes such as Faecalibacterium prausnitzii (an indicator of a healthy colonic microbiota). The mechanisms behind the development of IBDs and dysbiosis are incompletely understood. Using samples from colonic biopsies, we studied the mucosa-associated intestinal microbiota in Chilean and Spanish patients with IBD. In agreement with previous studies, microbiome comparison between IBD patients and non-IBD controls indicated that dysbiosis in these patients is characterized by an increase of pro-inflammatory bacteria (mostly Proteobacteria) and a decrease of commensal beneficial bacteria (mostly Firmicutes). Notably, bacteria typically residing on the mucosa of healthy individuals were mostly obligate anaerobes, whereas in the inflamed mucosa an increase of facultative anaerobe and aerobic bacteria was observed. We also identify potential co-occurring and mutually exclusive interactions between bacteria associated with the healthy and inflamed mucosa, which appear to be determined by the oxygen availability and the type of respiration. Finally, we identified a panel of bacterial biomarkers that allow the discrimination between eubiosis from dysbiosis with a high diagnostic performance (96% accurately), which could be used for the development of non-invasive diagnostic methods. Thus, this study is a step forward towards understanding the landscapes and alterations of mucosa-associated intestinal microbiota in patients with IBDs.

Evolution and Epidemic Spread of SARS-CoV-2 in Colombia: A Year into the Pandemic.

Current efforts to understand the epidemiology, transmission dynamics and emergence of novel SARS-CoV-2 variants worldwide has enabled the scientific community to generate critical information aimed at implementing disease surveillance and control measures, as well as to reduce the social, economic and health impact of the pandemic. Herein, we applied an epidemic model coupled with genomic analysis to assess the SARS-CoV-2 transmission dynamics in Colombia. This epidemic model allowed to identify the geographical distribution, Rt dynamics and predict the course of the pandemic considering current implementation of countermeasures. The analysis of the incidence rate per 100,000 inhabitants carried out across different regions of Colombia allowed visualizing the changes in the geographic distribution of cases. The cumulative incidence during the timeframe March 2020 to March 2021 revealed that Bogotá (8063.0), Quindío (5482.71), Amazonas (5055.68), Antioquia (4922.35) and Tolima (4724.41) were the departments with the highest incidence rate. The highest median Rt during the first period evaluated was 2.13 and 1.09 in the second period; with this model, we identified improving opportunities in health decision making related to controlling the pandemic, diagnostic testing capacity, case registration and reporting, among others. Genomic analysis revealed 52 circulating SARS-CoV-2 lineages in Colombia detected from 774 genomes sequenced throughout the first year of the pandemic. The genomes grouped into four main clusters and exhibited 19 polymorphisms. Our results provide essential information on the spread of the pandemic countrywide despite implementation of early containment measures. In addition, we aim to provide deeper phylogenetic insights to better understand the evolution of SARS-CoV-2 in light of the latent emergence of novel variants and how these may potentially influence transmissibility and infectivity.

Clostridioides difficile spores stimulate inflammatory cytokine responses and induce cytotoxicity in macrophages.

Clostridioides difficile is a gram-positive, spore-forming anaerobic bacterium, and the leading cause of antibiotic-associated diarrhea worldwide. During C. difficile infection, spores germinate in the presence of bile acids into vegetative cells that subsequently colonize the large intestine and produce toxins. In this study, we demonstrated that C. difficile spores can universally adhere to, and be phagocytosed by, murine macrophages. Only spores from toxigenic strains were able to significantly stimulate the production of inflammatory cytokines by macrophages and subsequently induce significant cytotoxicity. Spores from the isogenic TcdA and TcdB double mutant induced significantly lower inflammatory cytokines and cytotoxicity in macrophages, and these activities were restored by pre-exposure of the spores to either toxins. These findings suggest that during sporulation, spores might be coated with C. difficile toxins from the environment, which could affect C. difficile pathogenesis in vivo.

Major genetic discontinuity and novel toxigenic species in Clostridioides difficile taxonomy.

Clostridioides difficile infection (CDI) remains an urgent global One Health threat. The genetic heterogeneity seen across C. difficile underscores its wide ecological versatility and has driven the significant changes in CDI epidemiology seen in the last 20 years. We analysed an international collection of over 12,000 C. difficile genomes spanning the eight currently defined phylogenetic clades. Through whole-genome average nucleotide identity, and pangenomic and Bayesian analyses, we identified major taxonomic incoherence with clear species boundaries for each of the recently described cryptic clades CI-III. The emergence of these three novel genomospecies predates clades C1-5 by millions of years, rewriting the global population structure of C. difficile specifically and taxonomy of the Peptostreptococcaceae in general. These genomospecies all show unique and highly divergent toxin gene architecture, advancing our understanding of the evolution of C. difficile and close relatives. Beyond the taxonomic ramifications, this work may impact the diagnosis of CDI.

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.

Visualization of fidaxomicin association with the exosporium layer of Clostridioides difficile spores.

BACKGROUND: Fidaxomicin has novel pharmacologic effects on C. difficile spore formation including outgrowth inhibition and persistent spore attachment. However, the mechanism of fidaxomicin attachment on spores has not undergone rigorous microscopic studies. MATERIALS & METHODS: Fidaxomicin attachment to C. difficile spores of three distinct ribotypes and C. difficile mutant spores with inactivation of exosporium or spore-coat protein-coding genes were visualized using confocal microscopy with a fidaxomicin-bodipy compound (green fluorescence). The pharmacologic effect of the fidaxomicin-bodipy compound was determined. Confocal microscopy experiments included direct effect on C. difficile wild-type and mutant spores, effect of exosporium removal, and direct attachment to a comparator spore forming organism, Bacillus subtilis. RESULTS: The fidaxomicin-bodipy compound MIC was 1 mg/L compared to 0.06 mg/L for unlabeled fidaxomicin, a 16-fold increase. Using confocal microscopy, the intracellular localization of fidaxomicin into vegetative C. difficile cells was observed consistent with its RNA polymerase mechanism of action and inhibited spore outgrowth. The fidaxomicin-bodipy compound was visualized outside of the core of C. difficile spores with no co-localization with the membrane staining dye FM4-64. Exosporium removal reduced fidaxomicin-bodipy association with C. difficile spores. Reduced fidaxomicin-bodipy was observed in C. difficile mutant spores for the spore surface proteins CdeC and CotE. CONCLUSION: This study visualized a direct attachment of fidaxomicin to C. difficile spores that was diminished with mutants of specific exosporium and spore coat proteins. These data provide advanced insight regarding the anti-spore properties of fidaxomicin.

Comprehensive genome analyses of Sellimonas intestinalis, a potential biomarker of homeostasis gut recovery.

Sellimonas intestinalis is a Gram-positive and anaerobic bacterial species previously considered as uncultivable. Although little is known about this Lachnospiraceae family member, its increased abundance has been reported in patients who have recovered from intestinal homeostasis after dysbiosis events. In this context, the aim of the present study was to take advantage of a massive in vitro culture protocol that allowed the recovery of extremely oxygen-sensitive species from faecal samples, which led to isolation of S. intestinalis. Whole genome analyses of 11 S. intestinalis genomes revealed that this species has a highly conserved genome with 99.7 % 16S rRNA gene sequence similarity, average nucleotide polymorphism results >95, and 50.1 % of its coding potential being part of the core genome. Despite this, the variable portion of its genome was informative enough to reveal the existence of three lineages (lineage-I including isolates from Chile and France, lineage-II from South Korea and Finland, and lineage-III from China and one isolate from the USA) and evidence of some recombination signals. The identification of a cluster of orthologous groups revealed a high number of genes involved in metabolism, including amino acid and carbohydrate transport as well as energy production and conversion, which matches with the metabolic profile previously reported for microbiota from healthy individuals. Additionally, virulence factors and antimicrobial resistance genes were found (mainly in lineage-III), which could favour their survival during antibiotic-induced dysbiosis. These findings provide the basis of knowledge about the potential of S. intestinalis as a bioindicator of intestinal homeostasis recovery and contribute to advancing the characterization of gut microbiota members with beneficial potential.

Nasal Immunization with the C-Terminal Domain of Bcla3 Induced Specific IgG Production and Attenuated Disease Symptoms in Mice Infected with Clostridioides difficile Spores.

Clostridioides difficile is a Gram-positive, spore-forming bacterium that causes a severe intestinal infection. Spores of this pathogen enter in the human body through the oral route, interact with intestinal epithelial cells and persist in the gut. Once germinated, the vegetative cells colonize the intestine and produce toxins that enhance an immune response that perpetuate the disease. Therefore, spores are major players of the infection and ideal targets for new therapies. In this context, spore surface proteins of C. difficile, are potential antigens for the development of vaccines targeting C. difficile spores. Here, we report that the C-terminal domain of the spore surface protein BclA3, BclA3CTD, was identified as an antigenic epitope, over-produced in Escherichia coli and tested as an immunogen in mice. To increase antigen stability and efficiency, BclA3CTD was also exposed on the surface of B. subtilis spores, a mucosal vaccine delivery system. In the experimental conditions used in this study, free BclA3CTD induced antibody production in mice and attenuated some C. difficile infection symptoms after a challenge with the pathogen, while the spore-displayed antigen resulted less effective. Although dose regimen and immunization routes need to be optimized, our results suggest BclA3CTD as a potentially effective antigen to develop a new vaccination strategy targeting C. difficile spores.

Characterization of Exosporium Layer Variability of Clostridioides difficile Spores in the Epidemically Relevant Strain R20291.

Clostridioides difficile is a Gram-positive anaerobic intestinal pathogenic bacterium and the causative agent of antibiotic-associated diarrhea. C. difficile spore is a dormant state which acts as a vehicle of transmission and infection. In C. difficile spores, the outermost exosporium layer is the first barrier of interaction with the host and should carry spore ligands involved in spore-host interactions. C. difficile forms two types of spores (i.e., thin and thick exosporium layers). In this communication, we contribute to understand several biological aspects of these two exosporium morphotypes. By transmission electron microscopy, we demonstrate that both exosporium morphotypes appear simultaneously during sporulation and that spore-coat laminations are formed under anaerobic conditions. Nycodenz density-gradient allows enrichment of spores with a thick-exosporium layer morphotype and presence of polar appendage. Using translational fluorescent fusions with exosporium proteins BclA3, CdeA, CdeC, and CdeM as well as with several spore coat proteins, we observed that expression intensity and distribution of SNAP-translational fusions in R20291 strain is highly heterogeneous. Electron micrographs demonstrate that multicopy expression of CdeC, but not CdeM, SNAP translational fusion, increases the abundance of the thick exosporium morphotype. Collectively, these results raise further questions on how these distinctive exosporium morphotypes are made during spore formation.

Origin, genomic diversity and microevolution of the Clostridium difficile B1/NAP1/RT027/ST01 strain in Costa Rica, Chile, Honduras and Mexico.

Clostridium difficile B1/NAP1/RT027/ST01 has been responsible for outbreaks of antibiotic-associated diarrhoea in clinical settings worldwide and is associated with severe disease presentations and increased mortality rates. Two fluoroquinolone-resistant (FQR) lineages of the epidemic B1/NAP1/RT027/ST01 strain emerged in the USA in the early 1990s and disseminated trans continentally (FQR1 and FQR2). However, it is unclear when and from where they entered Latin America (LA) and whether isolates from LA exhibit unique genomic features when compared to B1/NAP1/RT027/ST01 isolates from other regions of the world. To answer the first issue we compared whole-genome sequences (WGS) of 25 clinical isolates typed as NAP1, RT027 or ST01 in Costa Rica (n=16), Chile (n=5), Honduras (n=3) and Mexico (n=1) to WGS of 129 global isolates from the same genotype using Bayesian phylogenomics. The second question was addressed through a detailed analysis of the number and type of mutations of the LA isolates and their mobile resistome. All but two B1/NAP1/RT027/ST01 isolates from LA belong to the FQR2 lineage (n=23, 92 %), confirming its widespread distribution. As indicated by analysis of a dataset composed of 154 WGS, the B1/NAP1/RT027/ST01 strain was introduced into the four LA countries analysed between 1998 and 2005 from North America (twice) and Europe (at least four times). These events occurred soon after the emergence of the FQR lineages and more than one decade before the first report of the detection of the B1/NAP1/RT027/ST01 in LA. A total of 552 SNPs were identified across all genomes examined (3.8-4.3 Mb) in pairwise comparisons to the R20291 reference genome. Moreover, pairwise SNP distances were among the smallest distances determined in this species so far (0 to 55). Despite this high level of genomic conservation, 39 unique SNPs (7 %) in genes that play roles in the infection process (i.e. slpA) or antibiotic resistance (i.e. rpoB, fusA) distinguished the LA isolates. In addition, isolates from Chile, Honduras and Mexico had twice as many antibiotic resistance genes (ARGs, n=4) than related isolates from other regions. Their unique set of ARGs includes a cfr-like gene and tetM, which were found as part of putative mobile genetic elements whose sequences resemble undescribed integrative and conjugative elements. These results show multiple, independent introductions of B1/NAP1/RT027/ST01 isolates from the FQR1 and FQR2 lineages from different geographical sources into LA and a rather rapid accumulation of distinct mutations and acquired ARG by the LA isolates.

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.