Difficile Germination Research Articles

Insoluble polysaccharides produced in plant cell cultures protect from Clostridioides difficile colitis

Clostridioides difficile infection (CDI) poses a significant health threat due to high recurrence rates. Antimicrobial agents are commonly used to manage CDI-related diarrhoea; however, by aggravating intestinal dysbiosis, antibiotics enable C. difficile spores germination and production of toxins, the main virulence factors. Therefore, the binding of exotoxins using adsorbents represents an attractive alternative medication for the prevention and treatment of relapses. In this study, we provided evidence that the natural insoluble polysaccharides, named ABR119, extracted by plant cell cultures, effectively trap C. difficile toxins. In our experiments, ABR119 exhibited no cytotoxicity in vitro and was safely administered in vivo. In the animal model of C. difficile-associated colitis, ABR119 (50 mg/kg body weight) significantly reduced the colonic myeloperoxidase activity and severity of inflammation, preventing body weight loss. These effects were not evident when we treated animals with wheat bran polysaccharides. We did not detect bacterial killing effects of ABR119 against C. difficile nor against bacterial species of the normal gut microbiota. Moreover, ABR119 did not interfere in vitro with the antimicrobial activities of most clinically used antibiotics. In summary, ABR119 holds promise for treating and preventing C. difficile colitis by trapping the bacterial toxins, warranting further studies to assess the ABR119 potential in human infections caused by C. difficile.

Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop.

Clostridioides difficile infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation co-germinants in the small intestine. While bile acid germinants are essential for C. difficile spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, are essential for inducing germination, while another proposes that either co-germinant class can induce germination. The former model is based on the finding that spores defective in releasing large stores of internal Ca2+ in the form of calcium dipicolinic acid (CaDPA) cannot germinate when germination is induced with bile acid germinant and amino acid co-germinant alone. However, since the reduced optical density of CaDPA-less spores makes it difficult to accurately measure their germination, we developed a novel automated, time-lapse microscopy-based germination assay to analyze CaDPA mutant germination at the single-spore level. Using this assay, we found that CaDPA mutant spores germinate in the presence of amino acid co-germinant and bile acid germinant. Higher levels of amino acid co-germinants are nevertheless required to induce CaDPA mutant spores to germinate relative to WT spores because CaDPA released by WT spores during germination can function in a feedforward loop to potentiate the germination of other spores within the population. Collectively, these data indicate that Ca2+ is not essential for inducing C. difficile spore germination because amino acid and Ca2+ co-germinant signals are sensed by parallel signaling pathways. IMPORTANCE Clostridioides difficile spore germination is essential for this major nosocomial pathogen to initiate infection. C. difficile spores germinate in response to sensing bile acid germinant signals alongside co-germinant signals. There are two classes of co-germinant signals: Ca2+ and amino acids. Prior work suggested that Ca2+ is essential for C. difficile spore germination based on bulk population analyses of germinating CaDPA mutant spores. Since these assays rely on optical density to measure spore germination and the optical density of CaDPA mutant spores is reduced relative to WT spores, this bulk assay is limited in its capacity to analyze germination. To overcome this limitation, we developed an automated image analysis pipeline to monitor C. difficile spore germination using time-lapse microscopy. With this analysis pipeline, we demonstrate that, although Ca2+ is dispensable for inducing C. difficile spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores.

Longitudinal Bile Acid Composition Changes Following Faecal Microbiota Transplantation for Clostridioides difficile Infection in Children With and Without Underlying Inflammatory Bowel Disease.

Faecal microbiota transplant [FMT] is effective in treating recurrent Clostridioides difficile infection [CDI] and restores gut microbiota composition. This is unlikely to account for its entire mechanism of efficacy, as studies have shown that factors such as bile acids influence the risk of infection by affecting Clostridioides difficile germination. We therefore aimed to investigate longitudinal changes in the gut bile acid composition after FMT performed for recurrent CDI, in children with and without inflammatory bowel disease [IBD]. Eight children received FMT; five had underlying IBD. Primary and secondary faecal bile acids were measured by liquid chromatography-mass spectrometry in recipients [pre-FMT and longitudinally post-FMT for up to 6 months] and donors. Pre-FMT, recipients had higher primary and lower secondary bile acid proportions compared with donors. Post-FMT, there was a gradual increase of secondary and decrease of primary bile acids. Whereas gut bacterial diversity had been shown to be restored in all children shortly after FMT, normalisation of bile acids to donor levels occurred only by 6 months. In children with IBD, although microbiota diversity returned to pre-FMT levels within 6 months, secondary bile acids remained at donor levels. The differences in bile acid profiles compared with gut bacterial diversity post-FMT suggests that interactions between the two may be more complex than previously appreciated and may contribute to FMT efficacy in different ways. This initial finding demonstrates the need to further investigate gut metabolites in larger cohorts, with longitudinal sampling to understand the mechanisms of FMT effectiveness.

730. A Phase 3, Randomized, Double-Blind Study to Evaluate the Efficacy and Safety of Ridinilazole Compared with Vancomycin for the Treatment of Clostridioides difficile Infection

Abstract Background Vancomycin (VAN) therapy for C. difficile infection (CDI) is effective with > 80% clinical response (CR) but is associated with 20–30% recurrence rate (rCDI). Secondary bile acids (2° BAs) inhibit C. difficile germination and help prevent rCDI. VAN depletes the gut microbiome decreasing the conversion of primary bile acids to 2° BAs. Ridinilazole (RDZ) is a highly selective anti-CDI, DNA-binding antibiotic in development for the treatment of CDI and prevention of rCDI. Methods A global, double-blinded, randomized Phase 3 trial assessed a 10-day treatment with RDZ 200 mg BID vs VAN 125 mg QID for CDI. The primary endpoint was sustained clinical response (SCR) defined as CR and no rCDI through 30 days post-end of treatment (EOT). Other endpoints included rCDI, microbiome diversity and composition, and microbiome-derived 2° BAs concentration. rCDI was defined as a new episode of diarrhea with confirmed positive free toxin test (FTT), requiring additional CDI therapy. All participants were monitored for treatment emergent adverse events (TEAE). Results Of the 759 patients (pts) enrolled, 745 were included in the mITT population (RDZ n=370, VAN n=375). RDZ achieved a numerically higher SCR rate than VAN (73.0% vs 70.7%) p=0.4672. RDZ resulted in a significant reduction in rCDI rate (8.1% vs 17.3%, p=0.0002) (Fig 1). In a pre-specified subpopulation, this was most notable in pts not receiving other antibiotics (rCDI 6.7% in RDZ vs 16.5% in VAN, p=0.0005). Microbiome alpha diversity was higher for RDZ vs VAN at EOT and EOT+30d (p< 0.0001 and p≤ 0.0007 respectively, Fig 2) as were relative abundance (p< 0.0001 and p=0.0203 respectively), and concentrations of 2° BAs (Fig 3). Higher microbiome diversity and concentrations of 2° BAs at EOT were associated with both lower rCDI and higher SCR rates. RDZ was well tolerated (pts with ≥ 1 TEAE: RDZ 36.4% vs VAN 35.5%, treatment discontinuation due to TEAE: RDZ 0.8% vs. VAN 2.9%). Conclusion RDZ was effective for sustained clinical response and safe for the treatment of patients with CDI. This was most notable in pts not receiving antibiotics. Compared to VAN, RDZ patients had faster recovery of fecal 2° BA, consistent with the preservation of microbiome diversity, resulting in a significantly lower rate of rCDI. Disclosures Pablo C. Okhuysen, MD, AstraZeneca: Stocks/Bonds|Beam Therapeutics: Stocks/Bonds|Biontech: Stocks/Bonds|Deinove: Grant/Research Support|Ferring: Advisor/Consultant|Glaxo Smith Kleine: Stocks/Bonds|Johnson and Johnson: Stocks/Bonds|Melinta: Grant/Research Support|Merck Sharp & Dohme Corp: Grant/Research Support|Moderna: Stocks/Bonds|Napo Pharmaceuticals: Advisor/Consultant|Napo Pharmaceuticals: Grant/Research Support|Novavax: Stocks/Bonds|Pfizer: Stocks/Bonds|Summit: Advisor/Consultant|Summit: Grant/Research Support Kevin W. Garey, PharmD, MS, Acurx: Grant/Research Support|cidara: Advisor/Consultant|cidara: Grant/Research Support|Paratek: Grant/Research Support|Seres Health: Grant/Research Support|Summit: Grant/Research Support Thomas J. Louie, MD, adiso therapeutics: Advisor/Consultant|adiso therapeutics: Grant/Research Support|crestone: Advisor/Consultant|crestone: Grant/Research Support|Finch: Advisor/Consultant|Finch: Grant/Research Support|Seres Therapeutics: Advisor/Consultant|Seres Therapeutics: Grant/Research Support|Seres Therapeutics: Honoraria|summit plc: Grant/Research Support|vedanta biosciences: Advisor/Consultant|vedanta biosciences: Grant/Research Support Jianling LI, MS, Abbott: Stocks/Bonds|Abbvie: Stocks/Bonds|ALX Oncology: Stocks/Bonds|BioNTech: Stocks/Bonds|Bluebird Bio: Stocks/Bonds|Cytokinetics: Stocks/Bonds|I-Mab: Stocks/Bonds|Johnson & Johnson: Stocks/Bonds|Moderna: Stocks/Bonds|TG Therapeutics: Stocks/Bonds Esther Duperchy, PhD, Summit Plc: Employee Jose G. Montoya, MD, Summit: Honoraria|Summit: Stocks/Bonds Lori Styles, MD, Abbvie: Stocks/Bonds|Summit Therapeutics: employee|Summit Therapeutics: Stocks/Bonds Fong Clow, Sc. D, Summit Therapeutics: Employee Danelle James, MD, Summit Therapeutics: Employee.

489. Bile Acid Concentrations in Healthy Volunteers Receiving Oral Omadacycline or Vancomycin

Abstract Background Microbiome disruption associated with Clostridioides difficile infection (CDI) includes reduced metabolism of primary to secondary bile acids leading to increased likelihood of C. difficile germination and CDI recurrence. Omadacycline has potent in vitro activity against C. difficile but its effect on the microbiome is unknown. The purpose of this study was to assess changes in bile acid concentrations in healthy volunteers given omadacycline compared to vancomycin, the most common antibiotic used to treat CDI. Methods As part of an ongoing healthy volunteer study of adults between 18 and 40 years, subjects received a 10-day course of oral omadacycline or vancomycin. Stool samples were collected and bile acids were extracted and quantified via targeted liquid chromatography-mass spectrometry (LC-MS). For this analysis, samples collected at the end of antibiotic therapy (day 9-10) were analyzed for primary and secondary bile acids. Results were compared and visualized using R (ggplot2). Results Between October 2020 and December 2021, 16 healthy volunteers aged 26 ± 5 years (male: 69%; Caucasian: 31%; mean body mass index: 23.6 ± 3.8 kg/m2) were enrolled. Concentrations of primary bile acids (cholic acid and chenodeoxycholic acid) were higher in patients receiving oral vancomycin than those receiving omadacycline (Table 1). Secondary bile acids were higher in the omadacycline arm compared to vancomycin. Secondary:primary bile ratio was higher for omadacycline (0.43) than vancomycin (0.03). Conclusion Omadacycline preserved bile acid homeostasis in the gut to a higher extent that vancomycin, suggesting reduced microbiome dysbiosis. With potent in vitro C. difficile activity, availability as an oral and IV formulations, and favorable microbiome properties, further development of omadacycline for the treatment of CDI is warranted. Disclosures Kevin W. Garey, PharmD, MS, Acurx Pharmaceuticals: Grant/Research Support|Paratek Pharmaceuticals: Grant/Research Support|Seres Therapeutics: Grant/Research Support|Summit Pharmaceuticals: Grant/Research Support.

Long-Term Lactulose Administration Improves Dysbiosis Induced by Antibiotic and C. difficile in the PathoGutTM SHIME Model.

Clostridioides difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and an important nosocomial infection with different severity degrees. Disruption of the gut microbiota by broad-spectrum antibiotics creates a proper environment for C. difficile colonization, proliferation, and clinical disease onset. Restoration of the gut microbial ecosystem through prebiotic interventions can constitute an effective complementary treatment of CDI. Using an adapted simulator of the human gut microbial ecosystem, the PathoGutTM SHIME, the effect of different long-term and repeated dose lactulose treatments was tested on C. difficile germination and growth in antibiotic-induced dysbiotic gut microbiota environments. The results showed that lactulose reduced the growth of viable C. difficile cells following clindamycin treatment, shifted the antibiotic-induced dysbiotic microbial community, and stimulated the production of health-promoting metabolites (especially butyrate). Recovery of the gut microenvironment by long-term lactulose administration following CDI was also linked to lactate production, decrease in pH and modulation of bile salt metabolism. At a structural level, lactulose showed a significant bifidogenic potential and restored key commensal members of the gut ecosystem such as Lactobacillaceae, Veillonellaceae and Lachnospiraceae. These results support further human intervention studies aiming to validate the in vitro beneficial effects of lactulose on gut microbiome recovery during antibiotic exposure and CDI.

The evaluation of fecal microbiota transplantation vs vancomycin in a Clostridioides difficile infection model.

Vancomycin is the preferred treatment for Clostridioides difficile infection (CDI) but has been associated with a high recurrence rate of CDI in treated patients. Fecal microbiota transplantation (FMT) has emerged as a remarkably successful treatment for recurrent CDI (rCDI). Herein, we present a mouse model of CDI to further define the changes in intestinal inflammation, flora, and metabolites following FMT versus vancomycin treatment and to find the potential therapy to restore colonization resistance. Both FMT and vancomycin treatment could ameliorate CDI-induced clinical features and intestinal tissue damage, with decrease in the levels of inflammatory mediators like IL-1β, IL-6, TNF-α, G-CSF, and MCP-1 in the colon and plasma. Observing the fecal gut microbiome profile revealed that unlike vancomycin, FMT could replenish intestinal microbiota by augmenting the relative abundance of the phylum Bacteroidetes and eliminating the abundance of the phylum Proteobacteria. FMT also reduced the levels of several carbohydrates, such as raffinose and fructose-6-phosphate, and amino acids, including tryptophan and glutamyl-valine, in the gut metabolome, thus suppressing C. difficile germination and growth. Our results suggest that the FMT-induced reconstruction of a specific gut community structure and restoration of metabolites promote the recovery of colonization resistance in mice better than vancomycin, thus offering new insights for the prevention of rCDI. KEY POINTS: • Both FMT and vancomycin ameliorate CDI-induced inflammatory response. • FMT restores a specific community structure and gut metabolites. • Mice treated with FMT may promote the recovery of colonization resistance and has a better outcome.

Gut Dysbiosis and Clostridioides difficile Infection in Neonates and Adults.

In this review, we focus on gut microbiota profiles in infants and adults colonized (CDC) or infected (CDI) with Clostridioides difficile. After a short update on CDI epidemiology and pathology, we present the gut dysbiosis profiles associated with CDI in adults and infants, as well as the role of dysbiosis in C. difficile spores germination and multiplication. Both molecular and culturomic studies agree on a significant decrease of gut microbiota diversity and resilience in CDI, depletion of Firmicutes, Bacteroidetes, and Actinobacteria phyla and a high abundance of Proteobacteria, associated with low butyrogenic and high lactic acid-bacteria levels. In symptomatic cases, microbiota deviations are associated with high levels of inflammatory markers, such as calprotectin. In infants, colonization with Bifidobacteria that trigger a local anti-inflammatory response and abundance of Ruminococcus, together with lack of receptors for clostridial toxins and immunological factors (e.g., C. difficile toxins neutralizing antibodies) might explain the lack of clinical symptoms. Gut dysbiosis amelioration through administration of “biotics” or non-toxigenic C. difficile preparations and fecal microbiota transplantation proved to be very useful for the management of CDI.

Descriptive Study of Gut Microbiota in Infected and Colonized Subjects by Clostridiodes difficile.

Clostridiodes difficile can lead to a range of situations from the absence of symptoms (colonization) to severe diarrhea (infection). Disruption of gut microbiota provides an ideal environment for infection to occur. Comparison of gut microbiota of infected and colonized subjects could provide relevant information on susceptible groups or protectors to the development of infection, since the presence of certain genera could be related to the inhibition of transition from a state of colonization to infection. Through high-throughput sequencing of 16S rDNA gene, we performed alpha and beta diversity and composition studies on 15 infected patients (Group CDI), 15 colonized subjects (Group P), and 15 healthy controls (Group CTLR). A loss of alpha diversity and richness and a different structure have been evidenced in the CDI and P groups with respect to the CTRL group, but without significant differences between the first two. In CDI and P groups, there was a strong decrease in phylum Firmicutes and an expansion of potential pathogens. Likewise, there was a loss of inhibitory genus of C. difficile germination in infected patients that were partially conserved in colonized subjects. Therefore, infected and colonized subjects presented a gut microbiota that was completely different from that of healthy controls, although similar to each other. It is in composition where we found that colonized subjects, especially in minority genera, presented differences with respect to those infected.

Reuterin disrupts Clostridioides difficile metabolism and pathogenicity through reactive oxygen species generation

ABSTRACT Antibiotic resistance is one of the world’s greatest public health challenges and adjunct probiotic therapies are strategies that could lessen this burden. Clostridioides difficile infection (CDI) is a prime example where adjunct probiotic therapies could decrease disease incidence through prevention. Human-derived Lactobacillus reuteri is a probiotic that produces the antimicrobial compound reuterin known to prevent C. difficile colonization of antibiotic-treated fecal microbial communities. However, the mechanism of inhibition is unclear. We show that reuterin inhibits C. difficile outgrowth from spores and vegetative cell growth, however, no effect on C. difficile germination or sporulation was observed. Consistent with published studies, we found that exposure to reuterin stimulated reactive oxygen species (ROS) in C. difficile, resulting in a concentration-dependent reduction in cell viability that was rescued by the antioxidant glutathione. Sublethal concentrations of reuterin enhanced the susceptibility of vegetative C. difficile to vancomycin and metronidazole treatment and reduced toxin synthesis by C. difficile. We also demonstrate that reuterin is protective against C. difficile toxin-mediated cellular damage in the human intestinal enteroid model. Overall, our results indicate that ROS are essential mediators of reuterin activity and show that reuterin production by L. reuteri is compatible as a therapeutic in a clinically relevant model.

A Revised Understanding of Clostridioides difficile Spore Germination.

The dormant resistant spores of Clostridioides difficile are transformed into metabolically active cells through the process of germination. Spore germination in C. difficile is regulated by the detection of bile salt germinants and amino acid cogerminants by pseudoproteases CspC and CspA, respectively. The germinant signal is transduced to the serine protease CspB, which processes the cortex lytic enzyme SleC, leading to degradation of the spore cortex peptidoglycan and subsequent reactivation of the spore. Divergent C. difficile germination models have been proposed to explain interactions between key regulators and transduction of germinant and cogerminant signals. This review summarises advances in understanding C. difficile germination and outlines current models of germination regulation.

Bile salt metabolism is not the only factor contributing to Clostridioides (Clostridium) difficile disease severity in the murine model of disease

ABSTRACT Susceptibility of patients to antibiotic-associated C. difficile disease is intimately associated with specific changes to gut microbiome composition. In particular, loss of microbes that modify bile salt acids (BSA) play a central role; primary bile acids stimulate spore germination whilst secondary bile acids limit C. difficile vegetative growth. To determine the relative contribution of bile salt (BS) metabolism on C. difficile disease severity, we treated mice with three combinations of antibiotics prior to infection. Mice given clindamycin alone became colonized but displayed no tissue pathology while severe disease, exemplified by weight loss and inflammatory tissue damage occurred in animals given a combination of five antibiotics and clindamycin. Animals given only the five antibiotic cocktails showed only transient colonization and no disease. C. difficile colonization was associated with a reduction in bacterial diversity, an inability to amplify bile salt hydrolase (BSH) sequences from fecal DNA and a relative increase in primary bile acids (pBA) in cecal lavages from infected mice. Further, the link between BSA modification and the microbiome was confirmed by the isolation of strains of Lactobacillus murinus that modified primary bile acids in vitro, thus preventing C. difficile germination. Interestingly, BSH activity did not correlate with disease severity which appeared linked to alternations in mucin, which may indirectly lead to increased exposure of the epithelial surface to inflammatory signals. These data confirm the role of microbial metabolic activity in protection of the gut and highlights the need for greater understanding the function of bacterial communities in disease prevention.

2578. Narrow-Spectrum Antibiotic Treatment of Clostridium difficile Infection Improves Preservation of Intestinal Metabolic Profile

BackgroundCommensal gut bacteria are thought to protect against C. difficile infection (CDI) by producing metabolites that inhibit C. difficile germination and growth. Alternatively, the protective effect could reflect nutrient competition or other mechanisms of chemical communication that also involve the host. CDI treatment using a broad-spectrum antibiotic such as vancomycin (VAN) dramatically depletes commensal bacteria. This dysbiosis can persist for several weeks after end-of-therapy (EOT), and is associated with increased recurrence risk. In this study, we investigate the hypothesis that treating CDI with a more selective antibiotic reduces collateral damage to the intestinal microbiota, preserving or restoring a CDI-inhibitory metabolic profile.MethodsStool samples were collected from CDI patients treated with either a narrow- (RDZ) or broad-spectrum antibiotic (VAN) at days 1, 10 (EOT), 25, and 40. Global metabolite profiles were measured by untargeted LC-MS.ResultsUntargeted metabolite analysis showed broad differences in the metabolic activity of intestinal microbiota of RDZ- and VAN-treated subjects (Figure 1). At EOT, 28% of LC-MS features detected in both RDZ and VAN samples were differentially present (FDR corrected P-value <0.05). Over 80% of the differentially present features were elevated in the RDZ group, indicating diminished capacity of microbiota from VAN subjects to generate diverse metabolic products. Pathway analysis found significant differences in purine, taurine, tyrosine, and bile acid metabolites. The VAN group showed a 5-fold decrease in free taurine, a major conjugation substrate of primary bile acids released by bacterial bile salt hydrolases. VAN treatment also decreased fermentation products of aromatic amino acids and amino sugars derived from mucin degradation. Oligosaccharides were the major metabolite class elevated in VAN subjects.ConclusionOur data suggest that RDZ treatment correlates with enhanced preservation of bacteria-derived ligands regulating intestinal immune function and substrates of bacterial metabolism. These metabolic profile differences between a narrow- and broad-spectrum antibiotic may contribute to their varying efficacy in preventing CDI recurrence. Disclosures All authors: No reported disclosures.

In vitro efficacy of sodium selenite in reducing toxin production, spore outgrowth and antibiotic resistance in hypervirulent Clostridium difficile

This study investigated the efficacy of the essential mineral, selenium (sodium selenite), in reducing the toxin production, spore outgrowth and antibiotic resistance of Clostridium difficile in vitro. Two hypervirulent C. difficile isolates were cultured in brain heart infusion broth with and without a sub-minimum inhibitory concentration (sub-MIC) of sodium selenite, and the supernatant and bacterial pellet were harvested for total toxin quantitation and RT-qPCR analysis of toxin-encoding genes, respectively. Additionally, C. difficile isolates were cultured in brain heart infusion broth containing 0.5 or 1× the minimum inhibitory concentration (MIC) of either ciprofloxacin or vancomycin with or without sub-MICs of sodium selenite. Further, the effect of sodium selenite on C. difficile germination and spore outgrowth was also determined by exposing C. difficile spores to a sub-MIC of sodium selenite in a germination medium and measuring the germination and outgrowth by measuring the optical density at 600 nm. Sodium selenite significantly reduced C. difficile toxin synthesis, cytotoxicity and spore outgrowth. Further, the expression of the toxin production genes, tcdA and tcdB, was downregulated in the presence of sodium selenite, while sodium selenite significantly increased the sensitivity of C. difficile to ciprofloxacin , but not vancomycin, as revealed by decreased bacterial growth in samples containing ciprofloxacin+selenium compared to the antibiotic control. Although the sub-MIC of sodium selenite did not inhibit spore germination, it was capable of completely inhibiting spore outgrowth. Our results suggest that sodium selenite could potentially be used to control C. difficile and indicate that future in vivo studies are warranted.

The requirement for co-germinants during Clostridium difficile spore germination is influenced by mutations in yabG and cspA.

Clostridium difficile spore germination is critical for the transmission of disease. C. difficile spores germinate in response to cholic acid derivatives, such as taurocholate (TA), and amino acids, such as glycine or alanine. Although the receptor with which bile acids are recognized (germinant receptor) is known, the amino acid co-germinant receptor has remained elusive. Here, we used EMS mutagenesis to generate mutants with altered requirements for the amino acid co-germinant, similar to the strategy we used previously to identify the bile acid germinant receptor, CspC. Surprisingly, we identified strains that do not require co-germinants, and the mutant spores germinated in response to TA alone. Upon sequencing these mutants, we identified different mutations in yabG. In C. difficile, yabG expression is required for the processing of key germination components to their mature forms (e.g., CspBA to CspB and CspA). A defined yabG mutant exacerbated the EMS mutant phenotype. Building upon this work, we found that small deletions in cspA resulted in spores that germinated in the presence of TA alone without the requirement of a co-germinant. cspA encodes a pseudoprotease that was previously shown to be important for incorporation of the CspC germinant receptor. Herein, our study builds upon the role of CspA during C. difficile spore germination by providing evidence that CspA is important for recognition of co-germinants during C. difficile spore germination. Our work suggests that two pseudoproteases (CspC and CspA) likely function as the C. difficile germinant receptors.

Status of vaccine research and development for Clostridium difficile

Clostridium difficile associated disease is fundamentally associated with dysbiosis of the gut microbiome as a consequence of antibiotic use. This is because this sporulating, obligate anaerobe germinates and proliferates rapidly in the dysbiotic gut, which is an indirect consequence of their use. During its growth, C. difficile produces two toxins, toxin A (TcdA) and toxin B (TcdB), which are responsible for the majority of clinical symptoms associated with the disease. Three parenterally delivered vaccines, based on detoxified or recombinant forms of these toxins, have undergone or are undergoing clinical trials. Each offers the opportunity to generate high titres of toxin neutralising antibodies. Whilst these data suggest these vaccines may reduce primary symptomatic disease, they do not in their current form reduce the capacity of the organism to persist and shed from the vaccinated host. The current progress of vaccine development is considered with advantages and limitations of each highlighted. In addition, several alternative approaches are described that seek to limit C. difficile germination, colonisation and persistence. It may yet prove that the most effective treatments to limit infection, disease and spread of the organism will require a combination of therapeutic approaches. The potential use and efficacy of these vaccines in low and middle income countries will be depend on the development of a cost effective vaccine and greater understanding of the distribution and extent of disease in these countries.

Microbial bile salt hydrolases mediate the efficacy of faecal microbiota transplant in the treatment of recurrent Clostridioides difficile infection

ObjectiveFaecal microbiota transplant (FMT) effectively treats recurrent Clostridioides difficile infection (rCDI), but its mechanisms of action remain poorly defined. Certain bile acids affect C. difficile germination or vegetative growth. We...

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.

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.

Relationship Between Remote History of Cholecystectomy and Risk for Incident Clostridium difficile Infection

Introduction:Clostridium difficile infection (CDI) is the leading cause of healthcare-associated diarrhea and gastroenteritis-associated death. Secondary bile acids inhibit C. difficile germination in vitro and confer resistance to CDI in animals. Cholecystectomy (CCY) alters gut bile acid composition, with increased fecal levels of secondary bile acids. This study tested the hypothesis that remote CCY confers protection against CDI by increasing gut levels of secondary bile acids. Methods: This was a retrospective case control study. The study population included all adult inpatients tested for the C. difficile toxin B gene by stool PCR between 1 January 2010 and 30 June 2017 at our institution, excluding those with a previous positive test for CDI within 90 days. For patients with multiple stool tests for C. difficile, the first test was selected. Remote CCY was defined as a history of CCY at least 6 months prior to the index test. Cases of CDI were defined as a positive stool PCR followed by anti-CDI treatment and were matched 1:1 with non-CDI controls by gender, age, BMI, and recent antibiotics exposure. Additional clinical factors including demographics, baseline comorbidities, medication exposures, and laboratory values were retrieved using automated queries. Conditional logistic regression modeling was used to estimate the relationship between remote CCY and risk for CDI, after adjusting for other factors. Results: The final study population was 4,918, including 2,459 CDI cases and 2,459 controls. Cases and controls did not differ based on gender, age, BMI, or recent antibiotics exposure. Patients with CDI had higher serum creatinine, more baseline comorbidities, and longer hospital stays. They were more likely to have an abnormal white blood cell count, to be in an intensive care unit, and to have recently received proton pump inhibitors. More patients with remote CCY were obese compared to patients without history of CCY (31% vs 25%, p<0.05) and were female (69% vs 50%, p<0.01). Rates of remote CCY did not differ based on CDI status (8.1% vs 7.9%, p=0.83). This result was unchanged after adjusting for additional clinical factors (adjusted OR 1.05, 95% CI 0.85-1.29, see Table) and also unchanged after adjusting for the matching variables. Conclusion: There was no association between remote CCY and risk for CDI in this large retrospective cohort study. Either gut bile acid content is not a major driver of CDI in humans or CCY impacts gut bile acids less than previously believed.168 Figure 1 No Caption available.