Peptidoglycan analysis reveals that synergistic deacetylase activity in vegetative Clostridium difficile impacts the host response.

Clostridium difficile is an anaerobic and spore-forming bacterium responsible for 15-25% of postantibiotic diarrhea and 95% of pseudomembranous colitis. Peptidoglycan is a crucial element of the bacterial cell wall that is exposed to the host, making it an important target for the innate immune system. The C. difficile peptidoglycan is largely N-deacetylated on its glucosamine (93% of muropeptides) through the activity of enzymes known as N-deacetylases, and this N-deacetylation modulates host-pathogen interactions, such as resistance to the bacteriolytic activity of lysozyme, virulence, and host innate immune responses. C. difficile genome analysis showed that 12 genes potentially encode N-deacetylases; however, which of these N-deacetylases are involved in peptidoglycan N-deacetylation remains unknown. Here, we report the enzymes responsible for peptidoglycan N-deacetylation and their respective regulation. Through peptidoglycan analysis of several mutants, we found that the N-deacetylases PdaV and PgdA act in synergy. Together they are responsible for the high level of peptidoglycan N-deacetylation in C. difficile and the consequent resistance to lysozyme. We also characterized a third enzyme, PgdB, as a glucosamine N-deacetylase. However, its impact on N-deacetylation and lysozyme resistance is limited, and its physiological role remains to be dissected. Finally, given the influence of peptidoglycan N-deacetylation on host defense against pathogens, we investigated the virulence and colonization ability of the mutants. Unlike what has been shown in other pathogenic bacteria, a lack of N-deacetylation in C. difficile is not linked to a decrease in virulence.

Molecular features of lipoprotein CD0873: A potential vaccine against the human pathogen Clostridioides difficile.

Clostridioides difficile is the primary cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease resulting in a significant fatality rate. Colonization of the gut is critical for C. difficile pathogenesis. The bacterial molecules essential for efficient colonization therefore offer great potential as vaccine candidates. Here we present findings demonstrating that the C. difficile immunogenic lipoprotein CD0873 plays a critical role in pathogen success in vivo We found that in a dixenic colonization model, a CD0873-positive strain of C. difficile significantly outcompeted a CD0873-negative strain. Immunization of mice with recombinant CD0873 prevented long-term gut colonization and was correlated with a strong secretory IgA immune response. We further present high-resolution crystal structures of CD0873, at 1.35-2.50 Å resolutions, offering a first view of the ligand-binding pocket of CD0873 and provide evidence that this lipoprotein adhesin is part of a tyrosine import system, an amino acid key in C. difficile infection. These findings suggest that CD0873 could serve as an effective component in a vaccine against C. difficile.

N-Deacetylases required for muramic-δ-lactam production are involved in Clostridium difficile sporulation, germination, and heat resistance.

Spores are produced by many organisms as a survival mechanism activated in response to several environmental stresses. Bacterial spores are multilayered structures, one of which is a peptidoglycan layer called the cortex, containing muramic-δ-lactams that are synthesized by at least two bacterial enzymes, the muramoyl-l-alanine amidase CwlD and the N-deacetylase PdaA. This study focused on the spore cortex of Clostridium difficile, a Gram-positive, toxin-producing anaerobic bacterial pathogen that can colonize the human intestinal tract and is a leading cause of antibiotic-associated diarrhea. Using ultra-HPLC coupled with high-resolution MS, here we found that the spore cortex of the C. difficile 630Δerm strain differs from that of Bacillus subtilis Among these differences, the muramic-δ-lactams represented only 24% in C. difficile, compared with 50% in B. subtilis CD630_14300 and CD630_27190 were identified as genes encoding the C. difficile N-deacetylases PdaA1 and PdaA2, required for muramic-δ-lactam synthesis. In a pdaA1 mutant, only 0.4% of all muropeptides carried a muramic-δ-lactam modification, and muramic-δ-lactams were absent in the cortex of a pdaA1-pdaA2 double mutant. Of note, the pdaA1 mutant exhibited decreased sporulation, altered germination, decreased heat resistance, and delayed virulence in a hamster infection model. These results suggest a much greater role for muramic-δ-lactams in C. difficile than in other bacteria, including B. subtilis In summary, the spore cortex of C. difficile contains lower levels of muramic-δ-lactams than that of B. subtilis, and PdaA1 is the major N-deacetylase for muramic-δ-lactam biosynthesis in C. difficile, contributing to sporulation, heat resistance, and virulence.

The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance in Clostridium difficile.

Clostridium difficile is the leading cause of antibiotic-associated diarrhea in adults. During infection, C. difficile must detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC of C. difficile is an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion of prkC affects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A ΔprkC mutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a ΔprkC mutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkC mutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority of C. difficile proteins associated with the cell wall were less abundant in the ΔprkC mutant than the wild-type strain. Finally, in a hamster model of infection, the ΔprkC mutant had a colonization delay that did not significantly affect overall virulence.

The alternative sigma factor σB plays a crucial role in adaptive strategies of Clostridium difficile during gut infection.

Clostridium difficile is a major cause of diarrhoea associated with antibiotherapy. Exposed to stresses in the gut, C. difficile can survive by inducing protection, detoxification and repair systems. In several firmicutes, most of these systems are controlled by the general stress response involving σB . In this work, we studied the role of σB in the physiopathology of C. difficile. We showed that the survival of the sigB mutant during the stationary phase was reduced. Using a transcriptome analysis, we showed that σB controls the expression of ∼25% of genes including genes involved in sporulation, metabolism, cell surface biogenesis and the management of stresses. By contrast, σB does not control toxin gene expression. In agreement with the up-regulation of sporulation genes, the sporulation efficiency is higher in the sigB mutant than in the wild-type strain. sigB inactivation also led to increased sensitivity to acidification, cationic antimicrobial peptides, nitric oxide and ROS. In addition, we showed for the first time that σB also plays a crucial role in oxygen tolerance in this strict anaerobe. Finally, we demonstrated that the fitness of colonisation by the sigB mutant is greatly affected in a dixenic mouse model of colonisation when compared to the wild-type strain.

Virulence factors of Clostridium difficile and their role during infection.

Clostridium difficile is the prominent etiological agent of healthcare-associated diarrhea. The disease symptoms range from mild diarrhea to life-threatening pseudomembranous colitis. The main risk factor for developing an infection after contamination by the resistant spores is the disruption of the gut microbiota, allowing the spores to germinate. The colonization of the gut is likely to be governed by the bacterial resistance to the host response and the bacterial adhesion to the mucosa. To date, several putative adhesins have been identified, most of them displaying MSCRAMM function, and studies of adhesin mutants have clearly underlined the multi-factorial feature of C. difficile adhesion to the host. Flagella have also been involved in the colonisation process, but their role depends on the tested strains. The clinical signs are mainly due to two large glucosylating toxins, TcdA and TcdB, which are essential for the disease manifestations. The importance of each toxin differs according to strains and experimental conditions, but TcdB seems to be the prominent one, as showed by mutant studies and the natural occurrence of pathogenic strains that do not produce TcdA. The role of the ADP ribosylating binary toxin expressed by some strains, including epidemic lineages, is not clearly established, although it has been related to higher morbidity and mortality. Production of low level of the glucosylating toxins and of the binary toxin seems to promote adhesion to host cells. Expression of the tcdA and tcdB genes is under the control of the second messenger c-di-GMP. This is also the case for other virulence factors, in particular for flagellar, pili type IV and some adhesin genes. Indeed, several studies using knock-out mutants suggest that C. difficile may undergo a switch between the adhesion phenotype and the motility phenotype during the course of infection, regulated by the c-di-GMP intracellular level. In vivo, this could result in biofilm formation that, associated with persistence of spores, could promote the occurrence of relapses observed in at least 20% of patients.

Biofilms of Clostridium species.

The biofilm is a microbial community embedded in a synthesized matrix and is the main bacterial way of life. A biofilm adheres on surfaces or is found on interfaces. It protects bacteria from the environment, toxic molecules and may have a role in virulence. Clostridium species are spread throughout both environments and hosts, but their biofilms have not been extensively described in comparison with other bacterial species. In this review we describe all biofilms formed by Clostridium species during both industrial processes and in mammals where biofilms may be formed either during infections or associated to microbiota in the gut. We have specifically focussed on Clostridium difficile and Clostridium perfringens biofilms, which have been studied in vitro. Regulatory processes including sporulation and germination highlight how these Clostridium species live in biofilms. Furthermore, biofilms may have a role in the survival and spreading of Clostridium species.

The state of play of rodent models for the study of Clostridioides difficile infection.

Clostridioides difficile is the most common cause of nosocomial antibiotic-associated diarrhoea and is responsible for a spectrum of diseases characterized by high levels of recurrence and morbidity. In some cases, complications can lead to death. Currently, several types of animal models have been developed to study various aspects of C. difficile infection (CDI), such as colonization, virulence, transmission and recurrence. These models have also been used to test the role of environmental conditions, such as diet, age and microbiome that modulate infection outcome, and to evaluate several therapeutic strategies. Different rodent models have been used successfully, such as the hamster model and the gnotobiotic and conventional mouse models. These models can be applied to study either the initial CDI infectious process or recurrences. The applications of existing rodent models and their advantages and disadvantages are discussed here.

Adaptive strategies and pathogenesis of Clostridium difficile from in vivo transcriptomics.

Clostridium difficile is currently the major cause of nosocomial intestinal diseases associated with antibiotic therapy in adults. In order to improve our knowledge of C. difficile-host interactions, we analyzed the genome-wide temporal expression of C. difficile 630 genes during the first 38 h of mouse colonization to identify genes whose expression is modulated in vivo, suggesting that they may play a role in facilitating the colonization process. In the ceca of the C. difficile-monoassociated mice, 549 genes of the C. difficile genome were differentially expressed compared to their expression during in vitro growth, and they were distributed in several functional categories. Overall, our results emphasize the roles of genes involved in host adaptation. Colonization results in a metabolic shift, with genes responsible for the fermentation as well as several other metabolic pathways being regulated inversely to those involved in carbon metabolism. In addition, several genes involved in stress responses, such as ferrous iron uptake or the response to oxidative stress, were regulated in vivo. Interestingly, many genes encoding conserved hypothetical proteins (CHP) were highly and specifically upregulated in vivo. Moreover, genes for all stages of sporulation were quickly induced in vivo, highlighting the observation that sporulation is central to the persistence of C. difficile in the gut and to its ability to spread in the environment. Finally, we inactivated two genes that were differentially expressed in vivo and evaluated the relative colonization fitness of the wild-type and mutant strains in coinfection experiments. We identified a CHP as a putative colonization factor, supporting the suggestion that the in vivo transcriptomic approach can unravel new C. difficile virulence genes.

Influence of environmental conditions on the expression and the maturation process of the Clostridium difficile surface associated protease Cwp84.

Expression of the Clostridium difficile protease gene, cwp84, was moderately up-regulated by decreasing pH due to glucose metabolism. Purification under different pH conditions influenced the proteolytic process of Cwp84. Given this, acidic pH could favor the appearance of different forms of Cwp84 that may have different roles during the infection.

Polysaccharide II Surface Anchoring, the Achilles' Heel of Clostridioides difficile.

Cell wall glycopolymers (CWPGs) in Gram-positive bacteria have been reported to be involved in several bacterial processes. These polymers, pillars for proteins and S-layer, are essential for the bacterial surface setup, could be essential for growth, and, in pathogens, participate most often in virulence. CWGPs are covalently anchored to peptidoglycan by proteins that belong to the LytR-CpsA-PSr (LCP) family. This anchoring, important for growth, was reported as essential for some bacteria such as Bacillus subtilis, but the reason why CWGP anchoring is essential remains unknown. We studied LcpA and LcpB of Clostridioides difficile and showed that they have a redundant activity. To delete both lcp genes, we set up the first conditional-lethal mutant method in C. difficile and showed that polysaccharide II (PSII) anchoring at the bacterial surface is essential for C. difficile survival. In the conditional-lethal mutant, C. difficile morphology was impaired, suggesting that peptidoglycan synthesis was affected. Because Lcp proteins are transferring CWPGs from the C55-undecaprenyl phosphate (also needed in the peptidoglycan synthesis process), we assumed that there was competition between PSII and peptidoglycan synthesis pathways. We confirmed that UDP-MurNAc-pentapeptide precursor was accumulated, showing that peptidoglycan synthesis was blocked. Our results provide an explanation for the essentiality of PSII anchoring in C. difficile and suggest that the essentiality of the anchoring of CWPGs in other bacteria can also be explained by the blocking of peptidoglycan synthesis. To conclude, our results suggest that Lcps are potential new targets to combat C. difficile infection. IMPORTANCE Cell wall glycopolymers (CWGPs) in Gram-positive bacteria have been reported to be involved in several bacterial processes. CWGP anchoring to peptidoglycan is important for growth and virulence. We set up the first conditional-lethal mutant method in Clostridioides difficile to study LcpA and LcpB involved in the anchoring of CWPGs to peptidoglycan. This study offers new tools to reveal the role of essential genes in C. difficile. LcpA and LcpB activity was shown to be essential, suggesting that they are potential new targets to combat C. difficile infection. In this study, we also showed that there is competition between the polysaccharide II synthesis pathway and peptidoglycan synthesis that probably exists in other Gram-positive bacteria. A better understanding of these mechanisms allows us to define the Lcp proteins as a therapeutic target for potential design of novel antibiotics against pathogenic Gram-positive bacteria.

Impact of Subinhibitory Concentrations of Metronidazole on Morphology, Motility, Biofilm Formation and Colonization of Clostridioides difficile.

Clostridioides difficile infection (CDI) is the primary cause of health-care-associated infectious diarrhea. Treatment requires mostly specific antibiotics such as metronidazole (MTZ), vancomycin or fidaxomicin. However, approximately 20% of treated patients experience recurrences. Treatment with MTZ is complicated by reduced susceptibility to this molecule, which could result in high failure and recurrence rates. However, the mechanism remains unclear. In this study, we investigated the impact of subinhibitory concentrations of MTZ on morphology, motility, biofilm formation, bacterial adherence to the intestinal Caco-2/TC7 differentiated monolayers, and colonization in monoxenic and conventional mouse models of two C. difficile strains (VPI 10463 and CD17-146), showing different susceptibility profiles to MTZ. Our results revealed that in addition to the inhibition of motility and the downregulation of flagellar genes for both strains, sub-inhibitory concentrations of MTZ induced various in vitro phenotypes for the strain CD17-146 exhibiting a reduced susceptibility to this antibiotic: elongated morphology, enhanced biofilm production and increased adherence to Caco-2/TC7 cells. Weak doses of MTZ induced higher level of colonization in the conventional mouse model and a trend to thicker 3-D structures entrapping bacteria in monoxenic mouse model. Thus, sub-inhibitory concentrations of MTZ can have a wide range of physiological effects on bacteria, which may contribute to their persistence after treatment.

Inhibition of In Vitro Clostridioides difficile Biofilm Formation by the Probiotic Yeast Saccharomyces boulardii CNCM I-745 through Modification of the Extracellular Matrix Composition.

Clostridioides difficile is responsible for post-antibiotic diarrhea and most of the pseudomembranous colitis cases. Multiple recurrences, one of the major challenges faced in C. difficile infection (CDI) management, can be considered as chronic infections, and the role of biofilm formation in CDI recurrences is now widely considered. Therefore, we explored if the probiotic yeast Saccharomyces boulardii CNCM I-745 could impact the in vitro formation of C. difficile biofilm. Biomass staining and viable bacterial cell quantification showed that live S. boulardii exerts an antagonistic effect on the biofilm formation for the three C. difficile strains tested. Confocal laser scanning microscopy observation revealed a weakening and an average thickness reduction of the biofilm structure when C. difficile is co-incubated with S. boulardii, compared to the single-species bacterial biofilm structure. These effects, that were not detected with another genetically close yeast, S. cerevisiae, seemed to require direct contact between the probiotic yeast and the bacterium. Quantification of the extrapolymeric matrix components, as well as results obtained after DNase treatment, revealed a significant decrease of eDNA, an essential structural component of the C. difficile biofilm matrix, in the dual-species biofilm. This modification could explain the reduced cohesion and robustness of C. difficile biofilms formed in the presence of S. boulardii CNCM I-745 and be involved in S. boulardii clinical preventive effect against CDI recurrences.

Localization of the Clostridium difficile cysteine protease Cwp84 and insights into its maturation process.

Clostridium difficile is a nosocomial pathogen involved in antibiotic-associated diarrhea. C. difficile expresses a cysteine protease, Cwp84, which has been shown to degrade some proteins of the extracellular matrix and play a role in the maturation of the precursor of the S-layer proteins. We sought to analyze the localization and the maturation process of this protease. Two identifiable forms of the protease were found to be associated in the bacteria: a form of ∼80 kDa and a cleaved one of 47 kDa, identified as the mature protease. They were found mainly in the bacterial cell surface fractions and weakly in the extracellular fraction. The 80-kDa protein was noncovalently associated with the S-layer proteins, while the 47-kDa form was found to be tightly associated with the underlying cell wall. Our data supported that the anchoring of the Cwp84 47-kDa form is presumably due to a reassociation of the secreted protein. Moreover, we showed that the complete maturation of the recombinant protein Cwp84(30-803) is a sequential process beginning at the C-terminal end, followed by one or more cleavages at the N-terminal end. The processing sites of recombinant Cwp84 are likely to be residues Ser-92 and Lys-518. No proteolytic activity was detected with the mature recombinant protease Cwp84(92-518) (47 kDa). In contrast, a fragment including the propeptide (Cwp84(30-518)) displayed proteolytic activity on azocasein and fibronectin. These results showed that Cwp84 is processed essentially at the bacterial cell surface and that its different forms may display different proteolytic activities.

Characterization of Non-Toxigenic Clostridioides difficile Strains Isolated from Preterm Neonates and In Vivo Study of Their Protective Effect.

In a previous monocentric study in preterm neonates (PN), we described a high Clostridioides difficile colonization rate (74%) with two uncommon non-toxigenic strains (NTCD) belonging to PCR-ribotype (RT) (CE)847 and (CE)032. To determine the extent of carriage of both NTCD in other spatio-temporal settings, strains isolated in PN stools from two multicenter cohorts were characterized by PCR-ribotyping, MLVA and MLST. We also evaluated the protective role of two NTCD from these RT against C. difficile infection in a hamster caecitis model. Animals were administered either each NTCD alone (n = 7), or followed by a 027 strain (n = 9). A control group received only the 027 strain (n = 8). Clinical activity and colonization by C. difficile in stools were monitored daily until death or sacrifice at D20. We isolated 18 RT(CE)032 (ST-83) strains and 2 RT(CE)847 (ST-26) strains among 247 PN from both cohorts. Within each RT, strains were genetically related. The survival rate was significantly increased when animals received a RT(CE)847 or (CE)032 strain before the 027 strain (4/9 deaths, p = 0.029; 1/9 death, p = 0.0004, respectively). We describe two predominant uncommon NTCD strains, in a PN population from different healthcare facilities. Both NTCD provide a potential protection against C. difficile infection.

Effects of subinhibitory concentrations of antibiotics on colonization factor expression by moxifloxacin-susceptible and moxifloxacin-resistant Clostridium difficile strains.

Recent outbreaks of Clostridium difficile infection have been related to the emergence of the NAP1/027 epidemic strain. This strain demonstrates increased virulence and resistance to the C-8-methoxyfluoroquinolones gatifloxacin and moxifloxacin. These antibiotics have been implicated as major C. difficile infection-inducing agents. We investigated by real-time reverse transcription-PCR the impact of subinhibitory concentrations of ampicillin, clindamycin, ofloxacin, and moxifloxacin on the expression of genes encoding three colonization factors, the protease Cwp84, the high-molecular-weight S-layer protein, and the fibronectin-binding protein Fbp68. We have previously shown in six non-NAP1/027 moxifloxacin-susceptible strains that the presence of ampicillin or clindamycin induced an upregulation of these genes, whereas the presence of fluoroquinolones did not. The objective of this study was to analyze the expression of these genes under the same conditions in four NAP1/027 strains, one moxifloxacin susceptible and three moxifloxacin resistant. Two in vitro-selected moxifloxacin-resistant mutants were also analyzed. Moxifloxacin resistance was associated with the Thr82-->Ile substitution in GyrA in all but one of the moxifloxacin-resistant strains. The expression of cwp84 and slpA was strongly increased after culture with ampicillin or clindamycin in NAP1/027 strains. Interestingly, after culture with fluoroquinolones, the expression of cwp84 and slpA was only increased in four moxifloxacin-resistant strains, including the NAP1/027 strains and one of the in vitro-selected mutants. The overexpression of cwp84 was correlated with increased production of the protease Cwp84. The historical NAP1/027 moxifloxacin-susceptible strain and its mutant appear to be differently regulated by fluoroquinolones. Overall, fluoroquinolones appear to favor the expression of some colonization factor-encoding genes in resistant C. difficile strains. The fluoroquinolone resistance of the NAP1/027 epidemic strains could be considered an ecological advantage. This could also increase their colonization fitness and promote the infection.

2,4-Disubstituted 5-Nitroimidazoles Potent against Clostridium difficile.

Metronidazole is one of the first-line treatments for non-severe Clostridium difficile infections (CDI). However, resistance limits its use in cases of severe and complicated CDI. Structure-activity relationships previously described for the 5-nitroimidazole series have shown that functionalization at the 2- and 4-positions can impart better activity against parasites and anaerobic bacteria than metronidazole. Herein we report the synthesis of new 2,4-disubstituted 5-nitroimidazole compounds that show potent antibacterial activity against C. difficile. We used a vicarious nucleophilic substitution of hydrogen (VNS) reaction to introduce a phenylmethylsulfone at the 4-position and a unimolecular radical nucleophilic substitution (SRN 1) reaction to introduce an ethylenic function at the 2-position of the 5-nitroimidazole scaffold.

Antibiotics involved in Clostridium difficile-associated disease increase colonization factor gene expression.

Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. Antibiotics are presumed to disturb the normal intestinal microbiota, leading to depletion of the barrier effect and colonization by pathogenic bacteria. This first step of infection includes adherence to epithelial cells. We investigated the impact of various environmental conditions in vitro on the expression of genes encoding known, or putative, colonization factors: three adhesins, P47 (one of the two S-layer proteins), Cwp66 and Fbp68, and a protease, Cwp84. The conditions studied included hyperosmolarity, iron depletion and exposure to several antibiotics (ampicillin, clindamycin, ofloxacin, moxifloxacin and kanamycin). The analysis was performed on three toxigenic and three non-toxigenic C. difficile isolates using real-time PCR. To complete this work, the impact of ampicillin and clindamycin on the adherence of C. difficile to Caco-2/TC7 cells was analysed. Overall, for the six strains of C. difficile studied, exposure to subinhibitory concentrations (1/2 MIC) of clindamycin and ampicillin led to the increased expression of genes encoding colonization factors. This was correlated with the increased adherence of C. difficile to cultured cells under the same conditions. The levels of gene regulation observed among the six strains studied were highly variable, cwp84 being the most upregulated. In contrast, the expression of these genes was weakly, or not significantly, modified in the presence of ofloxacin, moxifloxacin or kanamycin. These results suggest that, in addition to the disruption of the normal intestinal microbiota and its barrier effect, the high propensity of antibiotics such as ampicillin and clindamycin to induce C. difficile infection could also be explained by their direct role in enhancing colonization by C. difficile.

Targeting Clostridium difficile Surface Components to Develop Immunotherapeutic Strategies Against Clostridium difficile Infection.

New therapies are needed to prevent and treat Clostridium difficile infection and to limit the rise in antibiotic resistance. Besides toxins, several surface components have been characterized as colonization factors and have been shown as immunogenic. This review will focus on passive and active immunization strategies targeting C. difficile surface components to combat C. difficile. Concerning passive immunization, the first strategies used antisera raised against the entire bacterium to prevent infection in the hamster model. Then, surface components such as the flagellin and the S-layer proteins were used for immunization and the passive transfer of antibodies was protective in animal models. Passive immunotherapy with polyvalent immunoglobulins was used in humans and bovine immunoglobulin concentrates were evaluated in clinical trials. Concerning active immunization, vaccine assays targeting surface components were tested mainly in animal models, mouse models of colonization and hamster models of infection. Bacterial extracts, spore proteins and surface components of vegetative cells such as cell wall proteins, flagellar proteins, and polysaccharides were used as vaccine targets. Vaccine assays were performed by parenteral and mucosal routes of immunization. Both gave promising results and pave the way to development of new vaccines.

Inhibitory Effect of Ursodeoxycholic Acid on Clostridium difficile Germination Is Insufficient to Prevent Colitis: A Study in Hamsters and Humans.

Introduction: Bile acids (BA) influence germination and growth of Clostridium difficile. Ursodeoxycholic acid (UDCA), a BA minor in human, used for cholestatic liver diseases, inhibits germination and growth of C. difficile in vitro, but was never tested in vivo with an infectious challenge versus control. We hypothesized that UDCA could prevent CDI. We evaluated the effects of UDCA on C. difficile in vitro and in hamsters, with pharmacokinetics study and with an infectious challenge. Then, we studied CDI incidence in UDCA-treated patients. Methods: We evaluated germination and growth of C. difficile, with 0.01, 0.05, and 0.1% UDCA. We analyzed fecal BA of hamsters receiving antibiotics and UDCA (50 mg/kg/day), antibiotics, or UDCA alone. Then, we challenged with spores of C. difficile at D6 hamsters treated with UDCA (50 mg/kg/day) from D1 to D13, versus control. In human, we analyzed the database of a cohort on CDI in acute flares of inflammatory bowel disease (IBD). As PSC-IBD patients were under UDCA treatment, we compared PSC-IBD patients to IBD patients without PSC. Results: In vitro, UDCA inhibited germination and growth of C. difficile at 0.05 and 0.1%, competing with 0.1% TCA (with 0.1%: 0.05% ± 0.05% colony forming unit versus 100% ± 0%, P < 0.0001). In hamsters, UDCA reached high levels only when administered with antibiotics (43.5% UDCA at D5). Without antibiotics, UDCA was in small amount in feces (max. 4.28%), probably because of UDCA transformation into LCA by gut microbiota. During infectious challenge, mortality was similar in animals treated or not with UDCA (62.5%, n = 5/8, P = 0.78). UDCA percentage was high, similar and with the same kinetics in dead and surviving hamsters. However, dead hamsters had a higher ratio of primary over secondary BA compared to surviving hamsters. 9% (n = 41/404) of IBD patients without PSC had a CDI, versus 25% (n = 4/12) of PSC-IBD patients treated with UDCA. Conclusion: We confirmed the inhibitory effect of UDCA on growth and germination of C. difficile in vitro, with 0.05 or 0.1% UDCA. However, in our hamster model, UDCA was inefficient to prevent CDI, despite high levels of UDCA in feces. Patients with PSC-IBD treated with UDCA did not have less CDI than IBD patients.