Difficile R20291 Research Articles

Identification and preclinical evaluation of MMV676558 as a promising therapeutic candidate against Clostridioides difficile

Clostridioides difficile, a gram-positive, toxin-producing, spore-forming anaerobe, is a major cause of antibiotic-associated diarrhoea. The bacterium's intrinsic drug resistance limits current treatment options to fidaxomicin and vancomycin for initial episodes, with anti-toxin B monoclonal antibody or faecal microbiota transplantation recommended for complicated or recurrent cases. This underscores the urgent need for novel therapeutics. In this study, we screened the MMV Pathogen Box at a 10 µM concentration against C. difficile R20291. Primary hits were evaluated for minimum inhibitory concentrations (MIC), killing kinetics, and biofilm inhibition. Bacterial cytological profiling (BCP) and transmission electron microscopy (TEM) were employed to study the mode of action. MMV676558 was further tested in a mouse model to assess survival, histopathology, and gut microbiota effects. We identified nineteen hits that inhibited over 50 % of C. difficile growth. MIC assays revealed three hits with MICs below 16 µg/mL: MMV676558, MMV688755, and MMV690027. These hits were effective against various C. difficile ribotypes. Killing kinetics were comparable or superior to vancomycin and fidaxomicin, and biofilm assays showed inhibitory effects. BCP and TEM analyses suggested membrane function disruption as the mode of action. Furthermore, MMV676558 demonstrated a protective effect in mice, with favourable histopathology and gut microbiota profiles. Given the urgent threat posed by C. difficile antibiotic resistance, discovering new treatments is a top priority. Our study identified three promising hits from the MMV Pathogen Box, with MMV676558 showing significant in vivo potential for further evaluation.

MicroRNA miR-27a-5p reduces intestinal inflammation induced by Clostridioides difficile flagella by regulating the NF-κB signaling pathway.

Clostridioides difficile is a major cause of nosocomial post-antibiotic infections, often resulting in severe inflammation and watery diarrhea. Previous studies have highlighted the role of C. difficile flagellin FliC in activating the TLR5 receptor and triggering NF-κB cell signaling, leading to the release of pro-inflammatory cytokines. However, the microRNAs (miRNAs) mediated regulatory mechanisms underlying the FliC-induced inflammatory response remain unclear. miRNA expression levels were analyzed in Caco-2 intestinal epithelial cells following FliC stimulation, infection with the epidemic C. difficile R20291 strain, or its unflagellated mutant by RT-qPCR. Chemical inhibitors were used to block NF-κB signaling, and their impact on miR-27a-5p expression was assessed. Knockdown and overexpression experiments with miRNA inhibitor and mimic were conducted to elucidate miR-27a-5p's functional role in FliC-induced inflammatory responses. Additionally, a mouse model of C. difficile infection was treated with miR-27a-5p to evaluate its therapeutic potential in vivo. miR-27a-5p showed significant FliC-dependent overexpression in Caco-2 cells. Inhibition of NF-κB signaling suppressed miR-27a-5p overexpression. Knockdown of miR-27a-5p increased NF-κB activation and TNF-α and IL-8 cytokine production, while its overexpression had the opposite effect. Moreover, miR-27a-5p was overexpressed in the caeca of C. difficile-infected mice, correlating with intestinal IL-8 levels. Treatment of infected mice with miR-27a-5p mimic reduced disease severity and intestinal inflammation. miR-27a-5p plays a crucial role in regulating C. difficile-induced inflammation, suggesting its potential as a therapeutic target for controlling severe infection. These findings offer valuable insights into potential therapeutic strategies for managing C. difficile infection and associated inflammatory complications.

Porcine Monocyte DNA Traps Formed during Infection with Pathogenic Clostridioides difficile Strains.

Clostridioides (Clostridium) difficile is an enteric pathogen of several mammalian species including man, frequently involving nosocomial resurgence, following oral administration of broad-spectrum antibiotics, but also with human-to-human infection occurring, and neonatal pigs with zoonotic transmission. To date, the immune response to C. difficile has mostly focused on neutrophils and cytokine/chemokines, particularly in human infection. The neonatal pig is now recognized as a valuable model for human infection. We show that porcine monocytes respond to C. difficile differently compared with many other bacterial infections. Infection of porcine monocytes with human C. difficile strains CD630 (Ribotype 078) or R20291 (Ribotype 027) for 3 or 24 h post-infection (pi) resulted in a lack of oxidative burst or nitrite ion production when compared to uninfected controls (p > 0.05). The survival dynamics of both CD630 and R20291 in monocytes were similar with intracellular bacterial numbers being similar at 3 h pi and 24 h pi (p > 0.05). However, we show that porcine monocytes entrap C. difficile via extracellular DNA traps. This process began as early as 3 h pi, and at 24 h pi the nuclei appeared to be depleted of DNA, although extracellular DNA was associated with the cell membrane. Our preliminary study also suggests that entrapment of C. difficile by extracellular DNA may occur via a process of monocyte etosis.

One size does not fit all - Trehalose metabolism by Clostridioides difficile is variable across the five phylogenetic lineages.

Clostridioides difficile, the leading cause of antibiotic-associated diarrhoea worldwide, is a genetically diverse species which can metabolise a number of nutrient sources upon colonising a dysbiotic gut environment. Trehalose, a disaccharide sugar consisting of two glucose molecules bonded by an α 1,1-glycosidic bond, has been hypothesised to be involved in the emergence of C. difficile hypervirulence due to its increased utilisation by the RT027 and RT078 strains. Here, growth in trehalose as the sole carbon source was shown to be non-uniform across representative C. difficile strains, even though the genes for its metabolism were induced. Growth in trehalose reduced the expression of genes associated with toxin production and sporulation in the C. difficile R20291 (RT027) and M120 (RT078) strains in vitro, suggesting an inhibitory effect on virulence factors. Interestingly, the R20291 TreR transcriptional regulatory protein appeared to possess an activator function as its DNA-binding ability was increased in the presence of its effector, trehalose-6-phosphate. Using RNA-sequencing analysis, we report the identification of a putative trehalose metabolism pathway which is induced during growth in trehalose: this has not been previously described within the C. difficile species. These data demonstrate the metabolic diversity exhibited by C. difficile which warrants further investigation to elucidate the molecular basis of trehalose metabolism within this important gut pathogen.

The microbial-derived bile acid lithocholate and its epimers inhibit Clostridioides difficile growth and pathogenicity while sparing members of the gut microbiota.

Clostridioides difficile is a Gram-positive, spore-forming anaerobe that causes clinical diseases ranging from diarrhea and pseudomembranous colitis to toxic megacolon and death. C. difficile infection (CDI) is associated with antibiotic usage, which disrupts the indigenous gut microbiota and causes the loss of microbial-derived secondary bile acids that normally provide protection against C. difficile colonization. Previous work has shown that the secondary bile acid lithocholate (LCA) and its epimer isolithocholate (iLCA) have potent inhibitory activity against clinically relevant C. difficile strains. To further characterize the mechanisms by which LCA and its epimers iLCA and isoallolithocholate (iaLCA) inhibit C. difficile, we tested their minimum inhibitory concentration against C. difficile R20291 and a commensal gut microbiota panel. We also performed a series of experiments to determine the mechanism of action by which LCA and its epimers inhibit C. difficile through bacterial killing and effects on toxin expression and activity. Additionally, we tested the cytotoxicity of these bile acids through Caco-2 cell apoptosis and viability assays to gauge their effects on the host. Here, we show that the epimers iLCA and iaLCA strongly inhibit C. difficile growth in vitro while sparing most commensal Gram-negative gut microbes. We also show that iLCA and iaLCA have bactericidal activity against C. difficile, and these epimers cause significant bacterial membrane damage at subinhibitory concentrations. Finally, we observe that iLCA and iaLCA decrease the expression of the large cytotoxin tcdA, while LCA significantly reduces toxin activity. Although iLCA and iaLCA are both epimers of LCA, they have distinct mechanisms for inhibiting C. difficile. LCA epimers, iLCA and iaLCA, represent promising compounds that target C. difficile with minimal effects on members of the gut microbiota that are important for colonization resistance. IMPORTANCE In the search for a novel therapeutic that targets Clostridioides difficile, bile acids have become a viable solution. Epimers of bile acids are particularly attractive as they may provide protection against C. difficile while leaving the indigenous gut microbiota largely unaltered. This study shows that LCA epimers isolithocholate (iLCA) and LCA epimers isoallolithocholate (iaLCA) specifically are potent inhibitors of C. difficile, affecting key virulence factors including growth, toxin expression, and activity. As we move toward the use of bile acids as therapeutics, further work will be required to determine how best to deliver these bile acids to a target site within the host intestinal tract.

Transcriptome analysis of the Clostridioides difficile response to a specific lactobacilli probiotic formulation: explanations for its mechanisms of action.

Clostridioides difficile infections (CDI) are a major cause of morbidity and mortality in hospitalized patients. A probiotic formulation (Bio-K+) comprised of Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacti. rhamnosus CLR2 strains have been shown to reduce the incidence of CDI and antibiotic-associated diarrhea (AAD). This research aims to therefore elucidate the mechanism of action of the three probiotic strained against C. difficile R20291, independently of the acidification of the environment. . Antitoxin activity was evaluated using ELISA method and the expression of C. difficile genes was evaluated using transcriptomic analysis in co-culture assays conducted in a bioreactor allowing precise control of the pH. The fermentation results demonstrated a decrease for toxin A and many genes directly related to C. difficile virulence were underexpressed in the co-cultures. The lactobacilli tested could have a role in the motility, the quorum sensing (QS), the survival of the spores, and the germination potential of the spores, which are essential elements for the virulence of C. difficile. .

Flagellum and toxin phase variation impacts intestinal colonization and disease development in a mouse model of Clostridioides difficile infection

ABSTRACT Clostridioides difficile is a major nosocomial pathogen that can cause severe, toxin-mediated diarrhea and pseudomembranous colitis. Recent work has shown that C. difficile exhibits heterogeneity in swimming motility and toxin production in vitro through phase variation by site-specific DNA recombination. The recombinase RecV reversibly inverts the flagellar switch sequence upstream of the flgB operon, leading to the ON/OFF expression of flagellum and toxin genes.How this phenomenon impacts C. difficile virulence in vivo remains unknown. We identified mutations in the right inverted repeat that reduced or prevented flagellar switch inversion by RecV. We introduced these mutations into C. difficile R20291 to create strains with the flagellar switch “locked” in either the ON or OFF orientation. These mutants exhibited a loss of flagellum and toxin phase variation during growth in vitro, yielding precisely modified mutants suitable for assessing virulence in vivo. In a hamster model of acute C. difficile infection, the phase-locked ON mutant caused greater toxin accumulation than the phase-locked OFF mutant but did not differ significantly in the ability to cause acute disease symptoms. In contrast, in a mouse model, preventing flagellum and toxin phase variation affected the ability of C. difficile to colonize the intestinal tract and to elicit weight loss, which is attributable to differences in toxin production during infection. These results show that the ability of C. difficile to phase vary flagella and toxins influences colonization and disease development and suggest that the phenotypic variants generated by flagellar switch inversion have distinct capacities for causing disease.

FliW and CsrA Govern Flagellin (FliC) Synthesis and Play Pleiotropic Roles in Virulence and Physiology of Clostridioides difficile R20291.

Clostridioides difficile flagellin FliC is associated with toxin gene expression, bacterial colonization, and virulence, and is also involved in pleiotropic gene regulation during in vivo infection. However, how fliC expression is regulated in C. difficile remains unclear. In Bacillus subtilis, flagellin homeostasis and motility are coregulated by flagellar assembly factor (FliW), flagellin Hag (FliC homolog), and Carbon storage regulator A (CsrA), which is referred to as partner-switching mechanism “FliW-CsrA-Hag.” In this study, we characterized FliW and CsrA functions by deleting or overexpressing fliW, csrA, and fliW-csrA in C. difficile R20291. We showed that fliW deletion, csrA overexpression in R20291, and csrA complementation in R20291ΔWA (fliW-csrA codeletion mutant) dramatically decreased FliC production, but not fliC gene transcription. Suppression of fliC translation by csrA overexpression can be relieved mostly when fliW was coexpressed, and no significant difference in FliC production was detected when only fliW was complemented in R20291ΔWA. Further, loss of fliW led to increased biofilm formation, cell adhesion, toxin production, and pathogenicity in a mouse model of C. difficile infection (CDI), while fliW-csrA codeletion decreased toxin production and mortality in vivo. Our data suggest that CsrA negatively modulates fliC expression and FliW indirectly affects fliC expression through inhibition of CsrA post-transcriptional regulation. In light of “FliW-CsrA-Hag” switch coregulation mechanism reported in B. subtilis, our data also suggest that “FliW-CsrA-fliC/FliC” can regulate many facets of C. difficile R20291 pathogenicity. These findings further aid us in understanding the virulence regulation in C. difficile.

What's a SNP between friends: The lineage of Clostridioides difficile R20291 can effect research outcomes

Clostridioides difficile R20291 is the most studied PCR-Ribotype 027 isolate. The two predominant lineages of this hypervirulent strain, however, exhibit substantive phenotypic differences and possess genomes that differ by a small number of nucleotide changes. It is important that the source of R20291 is taken into account in research outcomes.

The Acid-Dependent and Independent Effects of Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacticaseibacillus rhamnosus CLR2 on Clostridioides difficile R20291.

Clostridioides difficile infections (CDI) result from antibiotic use and cause severe diarrhea which is life threatening and costly. A specific probiotic containing Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacticaseibacillus rhamnosus CLR2 has demonstrated a strong inhibitory effect on the growth of several nosocomial C. difficile strains by production of antimicrobial metabolites during fermentation. Though there are several lactobacilli shown to inhibit C. difficile growth by processes relying on acidification, this probiotic has demonstrated potency for CDI prevention among hospitalized patients. Here, we describe the acid-dependent and independent mechanisms by which these strains impair the cytotoxicity of a hypervirulent strain, C. difficile R20291 (CD). These bacteria were co-cultured in a series of experiments under anaerobic conditions in glucose-rich and no-sugar medium to inhibit or stimulate CD toxin production, respectively. In glucose-rich medium, there was low CD toxin production, but sufficient amounts to cause cytotoxic damage to human fibroblast cells. In co-culture, there was acidification by the lactobacilli resulting in growth inhibition as well as ≥99% reduced toxin A and B production and no observable cytotoxicity. In the absence of glucose, CD produced much more toxin. In co-culture, the lactobacilli did not acidify the medium and CD growth was unaffected; yet, the amount of detected toxin A and B was decreased by 20% and 41%, respectively. Despite the high concentration of toxin, cells exposed to the supernatant from the co-culture were able to survive. These results suggest that in addition to known acid-dependent effects, the combination of L. acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2 can interfere with CD pathogenesis without acidification: (1) reduced toxin A and B production and (2) toxin neutralization. This might explain the strain specificity of this probiotic in potently preventing C. difficile-associated diarrhea in antibiotic-treated patients compared with other probiotic formulae.

Evaluation of functionality of type II toxin-antitoxin systems of Clostridioides difficile R20291

Clostridioides difficile is a nosocomial, Gram-positive, strictly anaerobic, spore-forming pathogen capable of colonizing and proliferating in the human intestine. In bacteria, it has been shown that the Toxin-Antitoxin systems mediate the cellular response to external stress by initiating processes such as biofilm formation and programmed cell death. This work aims to evaluate the functionality of four type II TA modules of Clostridioides difficile R20291. We performed bioinformatic analysis to search for putative TA systems using the TADB platform. Then we performed a heterologous expression assay to evaluate the functionality of these systems. Our results showed that the MazEF and RelBE systems were functional, suggesting that their corresponding toxins possess an endoribonuclease activity. In conclusion, MazEF and RelBE systems of C. difficile R20291 are functional in a heterologous expression system.

The enteric pathogen Clostridium difficile chemotaxes towards mucin glycans during the early colonization phase of infection

BackgroundInfection with the pathogen Clostridium difficile is a major cause of antibiotic‐induced diarrhea and colitis. In the United States, C. difficile infects millions of patients each year and results in healthcare associated costs of over 1 billion dollars. Despite the significant burden of C. difficile infection, the exact mechanisms of C. difficile host colonization remain unknown. It is well known that antibiotic use sets the stage for infection and that toxin production induces symptoms. However, how C. difficile identifies and colonizes its ecological niche are unclear. Similar to other pathogens, flagellum‐mediated motility and the ability to interact with the intestinal mucus layer are likely critical factors for C. difficile colonization. We hypothesized that C. difficile would recognize mucin glycans present on human MUC2 and would chemotax towards these oligosaccharides. We also speculated that MUC2 would influence C. difficile gene expression and promote colonization.Methods & ResultsTo address these gaps in knowledge we fluorescently tagged C. difficile R20291 with CFDA‐SE and measured chemotaxis using a standard capillary assay. We observed that C. difficile was able to chemotax towards purified human MUC2 from a variety of sources, including human goblet cell lines HT29‐MTX, LS174T, T84 as well as human stool. We also found that C. difficile could recognize oligosaccharides present on MUC2 glycans, such as fucose, galactose, mannose, GalNac, GluNAc, and neurominic acid. The greatest accumulation of C. difficile was observed with mannose. To confirm these findings, we live‐imaged C. difficile chemotaxis in IBIDI chemotaxis slides and found that the presence of MUC2 or mannose increased the velocity and distance traveled by C. difficile compared to buffer medium alone. Examination of C. difficile genomes from several ribotypes revealed that C. difficile lacks the glycosyl hydrolases (GH) required for O‐linked mucin glycan degradation, although it did contain GH family 38, which cleaves mannose residues. To assess whether C. difficile could use mucin oligosaccharides as a primary carbon source, we grew C. difficile in a fully defined minimal medium (CDMM) in the absence of glucose supplemented with individual glycan oligosaccharides or MUC2. Consistent with our GH analysis, C. difficile was unable to grow on intact MUC2 alone. However, C. difficile was able to utilize all mucin oligosaccharides for growth. Further analysis of the C. difficile genome confirmed the transporters required for uptake of these sugars are present in all species. Finally, RNAseq demonstrated shifts in gene expression in C. difficile R20291 in response to human MUC2.ConclusionsThese data indicate that C. difficile recognizes and utilizes mucin‐derived oligosaccharides. We also demonstrate that C. difficile does not possess the enzymatic capacity to degrade these glycans on its own. Therefore, it must require the presence of other mucin‐degrading microbes for growth, pointing to the role of the microbial community in supporting C. difficile colonization.

2568. Mechanisms of a Specific Probiotic Comprised of Lactobacillus acidophilus CL1285, L. casei LBC80R and L. rhamnosus CLR2 that Interferes with Clostridioides difficile 20291 Toxin Production

Background Clostridioides difficile infections (CDI) result from antibiotic use and cause severe diarrhea (C. difficile-associated diarrhea, CDAD) which is life-threatening and costly. A specific probiotic containing Lactobacillus acidophilus CL1285, L. casei LBC80R and L. rhamnosus CLR2 (Bio-K+) has demonstrated benefits in preventing CDI and has a strong inhibitory effect on the growth of several nosocomial C. difficile strains in vitro. Many Lactobacilli can inhibit CD growth though lactic acidification. Here, we have investigated novel acid-independent mechanisms by which these strains impair C. difficile virulence.MethodsThe hypervirulent strain C. difficile R20291 was co-cultured anaerobically with Bio-K+ probiotic strains in various media and glucose concentrations (5 g/L, 3 g/L, 0 g/L), for 24 hours at 37°C. Parameters such as Log CFU, pH, Toxin A and B, cell cytotoxicity were measured. Statistical comparisons using ANOVA one-way was performed in order to determine whether the groups were significantly different.ResultsAt 5 g/L glucose, no C. difficile toxin was produced and co-culture with these lactobacilli resulted in potent acidification and growth inhibition. At 3 g/L glucose, C. difficile toxin production occurred and acidification by the lactobacilli resulted in growth inhibition as well as >99% reduced Toxin A and B production. In the absence of glucose and a starting pH of 7.0, TY broth, the lactobacilli did not acidify the medium and C. difficile growth was normal yet Toxin A and B production was partially reduced at, 20% and 41% lower. Toxin B from the supernatant of C. difficile grown in TY was cytotoxic to human fibroblast cells, but this was less cytotoxic when co-cultured with the Lactobacilli.ConclusionThese results suggest that the combination of L. acidophilus CL1285, L. casei LBC80R and L. rhamnosus CLR2 interferes with C. difficile pathogenesis through: 1) inhibition of C. difficile growth (via lactic acid secretion), 2) reduced toxin A/B synthesis and (3) toxin neutralization. These results might explain the strain specificity of Bio-K+ probiotic bacteria in potentially preventing C. difficile-associated diarrhea in antibiotic treated patients. Disclosures All authors: No reported disclosures.

A novel conjugal donor strain for improved DNA transfer into Clostridium spp.

Clostridium encompasses species which are relevant to human and animal disease as well as species which have industrial potential, for instance, as producers of chemicals and fuels or as tumour delivery vehicles. Genetic manipulation of these target organisms is critical for advances in these fields. DNA transfer efficiencies, however, vary between species. Low efficiencies can impede the progress of research efforts.A novel conjugal donor strain of Escherichia coli has been created which exhibits a greater than 10-fold increases in conjugation efficiency compared to the traditionally used CA434 strain in the three species tested; C. autoethanogenum DSM 10061, C. sporogenes NCIMB 10696 and C. difficile R20291. The novel strain, designated ‘sExpress’, does not methylate DNA at Dcm sites (CCWGG) which allows circumvention of cytosine-specific Type IV restriction systems.A robust protocol for conjugation is presented which routinely produces in the order of 105 transconjugants per millilitre of donor cells for C. autoethanogenum, 106 for C. sporogenes and 102 for C. difficile R20291. The novel strain created is predicted to be a superior conjugal donor in a wide range of species which possess Type IV restriction systems.

Time-resolved transcriptome analysis of Clostridium difficile R20291 response to cysteine

The incidence of Clostridium difficile infection has been steadily rising over the past decade. The increase in the rate of incidence is associated with the specific NAP1/BI/027 strains which are “hypervirulent” and have led to several large outbreaks since their emergence. However, the relation between these outbreaks and virulence regulation mechanisms remains unclear. It has been reported that the major virulence factor TcdA and TcdB in C. difficile could be repressed by cysteine. Here, we investigated the functional and virulence-associated regulation of C. difficile R20291 response to cysteine by using a time-resolved genome-wide transcriptome analysis. Dramatic changes of gene expression in C. difficile revealed functional processes related to transport, metabolism, and regulators in the presence of cysteine during different phases of growth. Flagellar and ribosomal genes were significantly down-regulated in long-term response to cysteine. Many NAP1/BI/027- specific genes were also modulated by cysteine. In addition, cdsB inactivation in C. difficile R20291 could remove the repression of toxin synthesis but could not remove the repression of butyrate production in the presence of cysteine. This suggests that toxin synthesis and butyrate production might have different regulatory controls in response to cysteine. Altogether, our research provides important insights into the regulatory mechanisms of C. difficile response to cysteine.

Identification of Clostridium difficile Immunoreactive Spore Proteins of the Epidemic Strain R20291.

Clostridium difficile infections are the leading cause of diarrhea associated with the use of antibiotics. During infection, C. difficile initiates a sporulation cycle leading to the persistence of C. difficile spores in the host and disease dissemination. The development of vaccine and passive immunization therapies against C. difficile has focused on toxins A and B. In this study, an immunoproteome-based approach to identify immunogenic proteins located on the outer layers of C. difficile spores as potential candidates for the development of immunotherapy and/or diagnostic methods against this devastating infection is used. To identify potential immunogenic proteins on the surface of C. difficile R20291, spore coat/exosporium extracts are separated by 2D electrophoresis (2-DE) and analyzed for reactivity against C. difficile spore-specific goat sera. Finally, the selected spots are in-gel digested with chymotrypsin, peptides generated are separated by nanoUPLC followed by MS/MS using Quad-TOF-MS, corroborated by Ultimate 3000RS-nano-UHPLC coupled to Q-Exactive-Plus-Orbitrap MS. The analysis identify five immunoreactive proteins: spore coat proteins CotE, CotA, and CotCB; exosporium protein CdeC; and a cytosolic methyltransferase. This data provides a list of spore surface protein candidates as antigens for vaccine development against C. difficile infections.

Pleiotropic roles of Clostridium difficile sin locus

Clostridium difficile is the primary cause of nosocomial diarrhea and pseudomembranous colitis. It produces dormant spores, which serve as an infectious vehicle responsible for transmission of the disease and persistence of the organism in the environment. In Bacillus subtilis, the sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition. In B. subtilis, SinR mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, so their interaction determines whether SinR can inhibit its target gene expression. The C. difficile genome carries two sinR homologs in the operon that we named sinR and sinR’, coding for SinR (112 aa) and SinR’ (105 aa), respectively. In this study, we constructed and characterized sin locus mutants in two different C. difficile strains R20291 and JIR8094, to decipher the locus’s role in C. difficile physiology. Transcriptome analysis of the sinRR’ mutants revealed their pleiotropic roles in controlling several pathways including sporulation, toxin production, and motility in C. difficile. Through various genetic and biochemical experiments, we have shown that SinR can regulate transcription of key regulators in these pathways, which includes sigD, spo0A, and codY. We have found that SinR’ acts as an antagonist to SinR by blocking its repressor activity. Using a hamster model, we have also demonstrated that the sin locus is needed for successful C. difficile infection. This study reveals the sin locus as a central link that connects the gene regulatory networks of sporulation, toxin production, and motility; three key pathways that are important for C. difficile pathogenesis.

Characterization of Chicken IgY Specific to Clostridium difficile R20291 Spores and the Effect of Oral Administration in Mouse Models of Initiation and Recurrent Disease.

Clostridium difficile infection (CDI) are the leading cause of world-wide nosocomial acquired diarrhea. The current main clinical challenge in CDI is the elevated rate of infection recurrence that may reach up to 30% of the patients, which has been associated to the formation of dormant spores during the infection. We sought to characterize the effects of oral administration of specific anti-spore IgY in mouse models of CDI and recurrent CDI. The specificity of anti-spore IgY was evaluated in vitro. In both, initiation mouse model and recurrence mouse model, we evaluated the prophylactic and therapeutic effect of anti-spore IgY, respectively. Our results demonstrate that anti-spore IgY exhibited high specificity and titers against C. difficile spores and reduced spore adherence to intestinal cells in vitro. Administration of anti-spore IgY to C57BL/6 mice prior and during CDI delayed the appearance of the diarrhea by 1.5 day, and spore adherence to the colonic mucosa by 90%. Notably, in the recurrence model, co-administration of anti-spore IgY coupled with vancomycin delayed the appearance of recurrent diarrhea by a median of 2 days. Collectively, these observations suggest that anti-spore IgY antibodies may be used as a novel prophylactic treatment for CDI, or in combination with antibiotics to treat CDI and prevent recurrence of the infection.

High metabolic versatility of different toxigenic and non-toxigenic Clostridioides difficile isolates

Clostridioides difficile (formerly Clostridium difficile) is a major nosocomial pathogen with an increasing number of community-acquired infections causing symptoms from mild diarrhea to life-threatening colitis. The pathogenicity of C. difficile is considered to be mainly associated with the production of genome-encoded toxins A and B. In addition, some strains also encode and express the binary toxin CDT. However; a large number of non-toxigenic C. difficile strains have been isolated from the human gut and the environment.In this study, we characterized the growth behavior, motility and fermentation product formation of 17 different C. difficile isolates comprising five different major genomic clades and five different toxin inventories in relation to the C. difficile model strains 630Δerm and R20291. Within 33 determined fermentation products, we identified two yet undescribed products (5-methylhexanoate and 4-(methylthio)-butanoate) of C. difficile. Our data revealed major differences in the fermentation products obtained after growth in a medium containing casamino acids and glucose as carbon and energy source. While the metabolism of branched chain amino acids remained comparable in all isolates, the aromatic amino acid uptake and metabolism and the central carbon metabolism-associated fermentation pathways varied strongly between the isolates. The patterns obtained followed neither the classification of the clades nor the ribotyping patterns nor the toxin distribution. As the toxin formation is strongly connected to the metabolism, our data allow an improved differentiation of C. difficile strains. The observed metabolic flexibility provides the optimal basis for the adaption in the course of infection and to changing conditions in different environments including the human gut.

Inhibition of spore germination, growth, and toxin activity of clinically relevant C. difficile strains by gut microbiota derived secondary bile acids

The changing epidemiology of Clostridium difficile infection over the past decades presents a significant challenge in the management of C. difficile associated diseases. The gastrointestinal tract microbiota provides colonization resistance against C. difficile, and growing evidence suggests that gut microbial derived secondary bile acids (SBAs) play a role. We hypothesized that the C. difficile life cycle; spore germination and outgrowth, growth, and toxin production, of strains that vary by age and ribotype will differ in their sensitivity to SBAs. C. difficile strains R20291 and CD196 (ribotype 027), M68 and CF5 (017), 630 (012), BI9 (001) and M120 (078) were used to define taurocholate (TCA) mediated spore germination and outgrowth, growth, and toxin activity in the absence and presence of gut microbial derived SBAs (deoxycholate, isodeoxycholate, lithocholate, isolithocholate, ursodeoxycholate, ω-muricholate, and hyodeoxycholate) found in the human and mouse large intestine. C. difficile strains varied in their rates of germination, growth kinetics, and toxin activity without the addition of SBAs. C. difficile M120, a highly divergent strain, had robust germination, growth, but significantly lower toxin activity compared to other strains. Many SBAs were able to inhibit TCA mediated spore germination and outgrowth, growth, and toxin activity in a dose dependent manner, but the level of inhibition and resistance varied across all strains and ribotypes. This study illustrates how clinically relevant C. difficile strains can have different responses when exposed to SBAs present in the gastrointestinal tract.