Heterogeneity in practices to reduce the risk of transmission of Clostridioides difficile in healthcare settings: a survey of ESCMID Study Group for Clostridioides difficile (ESGCD) members.

Clostridioides difficile is a leading cause of healthcare-associated infections. The main objective was to assess the current landscape of CDI infection prevention and control (IPC) practices. An anonymous survey of IPC practices for CDI was conducted between July 25 and October 31, 2022. Precautions for symptomatic patients were applicable for 75.9% and were discontinued 48 h minimum after the resolution of diarrhea for 40.7% of respondents. Daily cleaning of CDI patients' rooms was reported by 23 (42.6%). There was unexpected heterogeneity in IPC practices regarding the hospital management of CDI.

Culture-independent detection of low-abundant Clostridioides difficile in environmental DNA via PCR.

Clostridioides difficile represents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examined C. difficile strains originated from clinical specimen and were isolated under selective conditions employing antibiotics. This suggests a significant bias among analyzed C. difficile strains, which impedes a holistic view on this pathogen. In order to support extensive isolation of C. difficile strains from environmental samples, we designed a detection PCR that targets the hpdBCA-operon and thereby identifies low abundances of C. difficile in environmental samples. This operon encodes the 4-hydroxyphenylacetate decarboxylase, which catalyzes the production of the antimicrobial compound para-cresol. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific for C. difficile and successfully detected C. difficile despite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of the hpdBCA detection PCR sequence for initial phylogenetic classification, which allows assessment of C. difficile diversity in environmental samples via amplicon sequencing. Our findings furthermore showed that C. difficile strains isolated under antibiotic treatment from environmental samples were originally dominated by other strains according to PCR amplicon results. This provided evidence for selective cultivation of under-represented but antibiotic-resistant isolates. Thereby, we revealed a substantial bias in C. difficile isolation and research.IMPORTANCEClostridioides difficile is a main cause of diarrheic infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence has focused on bacterial isolation from clinical specimens under antibiotic treatment, which implies a substantial bias in isolated strains. Comprehensive studies, however, require an unbiased strain collection, which is accomplished by isolation of C. difficile from diverse environmental samples and avoidance of antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from our C. difficile-specific detection PCR, which rapidly verifies C. difficile presence in environmental samples and further allows estimation of the C. difficile diversity by using next-generation sequencing.

Antibacterial activity of ibezapolstat against antimicrobial-resistant clinical strains of Clostridioides difficile.

Antimicrobial resistance is emerging in clinical strains of Clostridioides difficile. Ibezapolstat (IBZ) is a DNA polymerase IIIC inhibitor that has completed phase II clinical trials. IBZ has potent in vitro activity against wild-type, susceptible strains but its effect on C. difficile strains with reduced susceptibility to metronidazole (MTZ), vancomycin (VAN), or fidaxomicin (FDX) has not been tested. The primary objective of this study was to test the antibacterial properties of IBZ against multidrug-resistant C. difficile strains. The in vitro activity, bactericidal, and time-kill activity of IBZ versus comparators were evaluated against 100 clinical strains of which 59 had reduced susceptibility to other C. difficile antibiotics. Morphologic changes against a multidrug resistance strain were visualized by light and scanning electron microscopy. The overall IBZ MIC50/90 values (µg/mL) for evaluated C. difficile strains were 4/8, compared with 2/4 for VAN, 0.5/1 for FDX, and 0.25/4 for MTZ. IBZ MIC50/90 values did not differ based on non-susceptibility to antibiotic class or number of classes to which strains were non-susceptible. IBZ bactericidal activity was similar to the minimum inhibitory concentration (MIC) and maintained in wild-type and non-susceptible strains. Time-kill assays against two laboratory wild-type and two clinical non-susceptible strains demonstrated sustained IBZ activity despite reduced killing by comparator antibiotics for IBZ and VAN non-susceptible strains. Microscopy visualized increased cell lengthening and cellular damage in multidrug-resistant strains exposed to IBZ sub-MIC concentrations. This study demonstrated the potent antibacterial activity of IBZ against a large collection of C. difficile strains including multidrug-resistant strains. This study highlights the therapeutic potential of IBZ against multidrug-resistant strains of C. difficile.

Characterizing the flavodoxin landscape in Clostridioides difficile.

Clostridioides difficile infections have become a major challenge in medical facilities. The bacterium is capable of spore formation allowing the survival of antibiotic treatment. Therefore, research on the physiology of C. difficile is important for the development of alternative treatment strategies. In this study, we investigated eight putative flavodoxins of C. difficile 630. Flavodoxins are small electron transfer proteins of specifically low potential. The unusually high number of flavodoxins in C. difficile suggests that they are expressed under different conditions. We determined high transcription levels for several flavodoxins during the exponential growth phase, especially for floX. Since flavodoxins are capable of replacing ferredoxins under iron deficiency conditions in other bacteria, we also examined their expression in C. difficile under low iron and no iron levels. In particular, the amount of fldX increased with decreasing iron concentration and thus could possibly replace ferredoxins. Moreover, we demonstrated that fldX is increasingly expressed under different oxidative stress conditions and thus may play an important role in the oxidative stress response. While increased fldX expression was detectable at both RNA and protein level, CD2825 showed increased expression only at mRNA level under H2O2 stress with sufficient iron availability and may indicate hydroxyl radical-dependent transcription. Although the exact function of the individual flavodoxins in C. difficile needs to be further investigated, the present study shows that flavodoxins could play an important role in several physiological processes and under infection-relevant conditions. IMPORTANCE: The gram-positive, anaerobic, and spore-forming bacterium Clostridioides difficile has become a vast problem in human health care facilities. The antibiotic-associated infection with this intestinal pathogen causes serious and recurrent inflammation of the intestinal epithelium, in many cases with a severe course. To come up with novel targeted therapies against C. difficile infections, a more detailed knowledge on the pathogen's physiology is mandatory. Eight putative flavodoxins, an extraordinarily high copy number of this type of small electron transfer proteins, are annotated for C. difficile. Flavodoxins are known to be essential electron carriers in other bacteria, for instance, during infection-relevant conditions such as iron limitation and oxidative stress. This work is a first and comprehensive overview on characteristics and expression profiles of the putative flavodoxins in the pathogen C. difficile.

Rapid Isothermal Detection of Pathogenic Clostridioides difficile Using Recombinase Polymerase Amplification.

Nosocomial-associated diarrhea due to Clostridioides difficile infection (CDI) is diagnosed after sample precultivation by the detection of the toxins in enzyme immunoassays or via toxin gene nucleic acid amplification. Rapid and direct diagnosis is important for targeted treatment to prevent severe cases and recurrence. We developed two singleplex and a one-pot duplex fluorescent 15 min isothermal recombinase polymerase amplification (RPA) assays targeting the toxin genes A and B (tcdA and tcdB). Furthermore, we adapted the singleplex RPA to a 3D-printed microreactor device. Analytical sensitivity was determined using a DNA standard and DNA extracts of 20 C. difficile strains with different toxinotypes. Nineteen clostridial and gastrointestinal bacteria strains were used to determine analytical specificity. Adaptation of singleplex assays to duplex assays in a 50 µL volume required optimized primer and probe concentrations. A volume reduction by one-fourth (12.4 µL) was established for the 3D-printed microreactor. Mixing of RPA was confirmed as essential for optimal analytical sensitivity. Detection limits (LOD) ranging from 119 to 1411 DNA molecules detected were similar in the duplex tube format and in the singleplex 3D-printed microreactor format. The duplex RPA allows the simultaneous detection of both toxins important for the timely and reliable diagnosis of CDI. The 3D-printed reaction chamber can be developed into a microfluidic lab-on-a-chip system use at the point of care.

Comparative Genomics of Clostridioides difficile.

Clostridioides difficile, a Gram-positive spore-forming anaerobic bacterium, has rapidly emerged as the leading cause of nosocomial diarrhoea in hospitals. The availability of large numbers of genome sequences, mainly due to the use of next-generation sequencing methods, has undoubtedly shown their immense advantages in the determination of C. difficile population structure. The implementation of fine-scale comparative genomic approaches has paved the way for global transmission and recurrence studies, as well as more targeted studies, such as the PaLoc or CRISPR/Cas systems. In this chapter, we provide an overview of recent and significant findings on C. difficile using comparative genomic studies with implications for epidemiology, infection control and understanding of the evolution of C. difficile.

Antibiotic Resistances of Clostridioides difficile.

The rapid evolution of antibiotic resistance in Clostridioides difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are a matter of concern for public health. Antibiotic resistance plays an important role in driving C. difficile epidemiology. Emergence of new types is often associated with the emergence of new resistances, and most of the epidemic C. difficile clinical isolates is currently resistant to multiple antibiotics. In particular, it is to worth to note the recent identification of strains with reduced susceptibility to the first-line antibiotics for CDI treatment and/or for relapsing infections. Antibiotic resistance in C. difficile has a multifactorial nature. Acquisition of genetic elements and alterations of the antibiotic target sites, as well as other factors, such as variations in the metabolic pathways or biofilm production, contribute to the survival of this pathogen in the presence of antibiotics. Different transfer mechanisms facilitate the spread of mobile elements among C. difficile strains and between C. difficile and other species. Furthermore, data indicate that both genetic elements and alterations in the antibiotic targets can be maintained in C. difficile regardless of the burden imposed on fitness, and therefore resistances may persist in C. difficile population in absence of antibiotic selective pressure.

Membrane Vesicles of Clostridioides difficile and Other Clostridial Species.

Membrane vesicles are secreted by growing bacterial cells and are important components of the bacterial secretome, with a role in delivering effector molecules that ultimately enable bacterial survival. Membrane vesicles of Clostridioides difficile likely contribute to pathogenicity and is a new area of research on which there is currently very limited information. This chapter summarizes the current knowledge on membrane vesicle formation, content, methods of characterization and functions in Clostridia and model Gram-positive species.

Exogenous butyrate inhibits butyrogenic metabolism and alters virulence phenotypes in Clostridioides difficile.

The gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile, but the molecular basis of this colonization resistance is incompletely understood. A prominent class of gut microbiome-produced metabolites important for colonization resistance against C. difficile is short-chain fatty acids (SCFAs). In particular, one SCFA (butyrate) decreases the fitness of C. difficile in vitro and is correlated with C. difficile-inhospitable gut environments, both in mice and in humans. Here, we demonstrate that butyrate-dependent growth inhibition in C. difficile occurs under conditions where C. difficile also produces butyrate as a metabolic end product. Furthermore, we show that exogenous butyrate is internalized into C. difficile cells and is incorporated into intracellular CoA pools where it is metabolized in a reverse (energetically unfavorable) direction to crotonyl-CoA and (S)-3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA. This internalization of butyrate and reverse metabolic flow of a butyrogenic pathway(s) in C. difficile coincides with alterations in toxin release and sporulation. Together, this work highlights butyrate as a marker of a C. difficile-inhospitable environment to which C. difficile responds by releasing its diarrheagenic toxins and producing environmentally resistant spores necessary for transmission between hosts. These findings provide foundational data for understanding the molecular and genetic basis of how C. difficile growth is inhibited by butyrate and how butyrate alters C. difficile virulence in the face of a highly competitive and dynamic gut environment.IMPORTANCEThe gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile, but the molecular basis of this colonization resistance is incompletely understood, which hinders the development of novel therapeutic interventions for C. difficile infection (CDI). We investigated how C. difficile responds to butyrate, an end-product of gut microbiome community metabolism which inhibits C. difficile growth. We show that exogenously produced butyrate is internalized into C. difficile, which inhibits C. difficile growth by interfering with its own butyrate production. This growth inhibition coincides with increased toxin release from C. difficile cells and the production of environmentally resistant spores necessary for transmission between hosts. Future work to disentangle the molecular mechanisms underlying these growth and virulence phenotypes will likely lead to new strategies to restrict C. difficile growth in the gut and minimize its pathogenesis during CDI.

Host-defense piscidin peptides as antibiotic adjuvants against Clostridioides difficile.

The spore-forming intestinal pathogen Clostridioides difficile causes multidrug resistant infection with a high rate of recurrence after treatment. Piscidins 1 (p1) and 3 (p3), cationic host defense peptides with micromolar cytotoxicity against C. difficile, sensitize C. difficile to clinically relevant antibiotics tested at sublethal concentrations. Both peptides bind to Cu2+ using an amino terminal copper and nickel binding motif. Here, we investigate the two peptides in the apo and holo states as antibiotic adjuvants against an epidemic strain of C. difficile. We find that the presence of the peptides leads to lower doses of metronidazole, vancomycin, and fidaxomicin to kill C. difficile. The activity of metronidazole, which targets DNA, is enhanced by a factor of 32 when combined with p3, previously shown to bind and condense DNA. Conversely, the activity of vancomycin, which acts at bacterial cell walls, is enhanced 64-fold when combined with membrane-active p1-Cu2+. As shown through microscopy monitoring the permeabilization of membranes of C. difficile cells and vesicle mimics of their membranes, the adjuvant effect of p1 and p3 in the apo and holo states is consistent with a mechanism of action where the peptides enable greater antibiotic penetration through the cell membrane to increase their bioavailability. The variations in effects obtained with the different forms of the peptides reveal that while all piscidins generally sensitize C. difficile to antibiotics, co-treatments can be optimized in accordance with the underlying mechanism of action of the peptides and antibiotics. Overall, this study highlights the potential of antimicrobial peptides as antibiotic adjuvants to increase the lethality of currently approved antibiotic dosages, reducing the risk of incomplete treatments and ensuing drug resistance.

Elucidation of bezlotoxumab binding specificity to toxin B in Clostridioides difficile.

C. difficile or Clostridioides difficile infection (CDI) is currently one of the major causes of epidemics worldwide. Toxin B from Clostridioides difficile toxin B (TcdB) infection is the main target protein inhibiting CDI recurrence. Clinical research suggested that bezlotoxumab's (Bez) efficiency is significantly reduced in neutralizing the B2 strain compared to the B1 strain. The monoclonal antibody (mAb) functions by binding to the epitope 1 and 2 regions in the combined repetitive oligopeptide (CROP) domain. Some binding residues are distinctively different between B1 and B2 strains. In this work, we aimed to elucidate and compare insights into the interaction of toxins B1 and B2 in complex with Bez by using all-atom molecular dynamics (MD) simulations and binding free energy calculations. The predicted ΔGbinding values suggested that the antibody (Ab) could bind to toxin B1 significantly better than B2, supported by higher salt bridge and hydrogen bonding (H-bonding) interactions, as well as the number of contact residues between the two focused proteins. The toxin B1 residues important for binding with Bez were E1878, T1901, E1902, F1905, N1941, V1946, N2031, T2032, E2033, V2076, V2077, and E2092. The lower susceptibility of Bez towards toxin B2 was primarily due to a change of residue E2033 from glutamate to alanine (A2033) and the loss of E1878 and E1902 contributions, as determined by the intermolecular interaction changes from the dynamic residue interaction network (dRIN) analysis. The obtained data strengthen our understanding of Bez/toxin B binding.

Diagnostic prediction models to identify patients at risk for healthcare-facility-onset Clostridioides difficile: A systematic review of methodology and reporting.

OBJECTIVE: To systematically review the methodology, performance, and generalizability of diagnostic models for predicting the risk of healthcare-facility-onset (HO) Clostridioides difficile infection (CDI) in adult hospital inpatients (aged ≥18 years). BACKGROUND: CDI is the most common cause of healthcare-associated diarrhea. Prediction models that identify inpatients at risk of HO-CDI have been published; however, the quality and utility of these models remain uncertain. METHODS: Two independent reviewers evaluated articles describing the development and/or validation of multivariable HO-CDI diagnostic models in an inpatient setting. All publication dates, languages, and study designs were considered. Model details (eg, sample size and source, outcome, and performance) were extracted from the selected studies based on the CHARMS checklist. The risk of bias was further assessed using PROBAST. RESULTS: Of the 3,030 records evaluated, 11 were eligible for final analysis, which described 12 diagnostic models. Most studies clearly identified the predictors and outcomes but did not report how missing data were handled. The most frequent predictors across all models were advanced age, receipt of high-risk antibiotics, history of hospitalization, and history of CDI. All studies reported the area under the receiver operating characteristic curve (AUROC) as a measure of discriminatory ability. However, only 3 studies reported the model calibration results, and only 2 studies were externally validated. All of the studies had a high risk of bias. CONCLUSION: The studies varied in their ability to predict the risk of HO-CDI. Future models will benefit from the validation on a prospective external cohort to maximize external validity.

Abnormalities in Clostridioides and related metabolites before ACTH treatment may be associated with its efficacy in patients with infantile epileptic spasm syndrome.

OBJECTIVE: Adrenocorticotropic hormone (ACTH) is the first-line treatment of infantile epileptic spasm syndrome (IESS). Its reported effectiveness varies, and our current understanding regarding the role of gut microbiota composition in IESS treatment response is limited. This study assessed the microbiome-metabolome association to understand the role and mechanism of gut microbiota composition in IESS treatment outcomes. METHODS: Children with IESS undergoing ACTH treatment were enrolled. Pre-treatment stool and serum samples were collected for 16S rRNA gene sequencing and liquid chromatography-tandem mass spectrometry, respectively. The children were divided into "responsive" and "non-responsive" groups, and gut microbiota and serum metabolome differences were analyzed. RESULTS: Of the 30 patients with IESS, 14 responded to ACTH and 16 did not. The "non-responsive" group had larger maleficent Clostridioides and Peptoclostridium_phage_p630P populations (linear discriminant analysis >2; false discovery rate q < 0.05). Ten metabolites were upregulated (e.g., xanthurenic acid) and 15 were downregulated (e.g., vanillylmandelic acid) (p < 0.05). Association analysis of the gut microbiome and serum metabolome revealed that Clostridioides and Peptoclostridium_phage_p630P2 were positively correlated with linoleic and xanthurenic acids, while Clostridioides was negatively correlated with vanillylmandelic acid (p < 0.05). A classifier using differential gut bacteria and metabolites achieved an area under the receiver operating characteristic curve of 0.906 to distinguish responders from non-responders. CONCLUSION: This study found significant differences in pre-treatment gut microbiota and serum metabolome between children with IESS who responded to ACTH and those who did not. Additional exploration may provide valuable information for treatment selection and potential interventions. Our results suggest that varying ACTH responses in patients with IESS may be associated with increased gut Clostridioides bacteria and kynurenine pathway alteration, but additional experiments are needed to verify this association.

Design, Synthesis, and Evaluation of Cephamycin-Based Antisporulation Agents targeting Clostridioides difficile.

With the aim of discovering small molecule inhibitors of the sporulation process in Clostridioides difficile, we prepared a series of C-7 α-(4-substituted-1H-1,2,3-triazol-1-yl)acetamide analogues of cefotetan, a known inhibitor of the C. difficile sporulation-specific protein target CdSpoVD. These analogues were evaluated using both in vitro binding assays with CdSpoVD and antisporulation assays against C. difficile. Further design concepts were aided utilizing the predicted docking scores (DS) using both AlphaFold (AF) models, and a crystal structure of the CdSpoVD protein (PDB 7RCZ). Despite being 1 order of magnitude more potent as a sporulation inhibitor than cefotetan, in vivo studies on compound 6a in a murine-model of C. difficile infection demonstrated comparable spore shedding capabilities as cefotetan. Importantly, compound 6a had no concerning broad spectrum antibacterial activities, toxicity, or hemolytic activity and thus has potential for further drug development.

The Role of the Gut Microbiome in Cancer: A Review, With Special Focus on Colorectal Neoplasia and Clostridioides difficile.

The gut microbiome has coevolved with humans to aid in physiologic functions and prevent disease. An increasing prevalence of gut dysbiosis in modern society exists and has strong linkages to multiple disease processes common in the developed world. Mechanisms for microbiome-human interactions that impact host homeostasis include bacterial metabolite/toxin production, biofilm formation with mucous layer infiltration, and host immune system modulation. Most of this crosstalk occurs at the epithelial layer of the gut, and as such the role of these interactions in the induction of colorectal cancer-a highly prevalent disease globally and one undergoing significant epidemiologic shifts-is under increasing scrutiny. Although multiple individual gut bacteria have been hypothesized as possible driver organisms in the oncogenic process, no bacterium has been definitively identified as a causal agent of colorectal cancer, suggesting that host lifestyle factors, microbiome community interactions, and the mucosal and/or systemic immune response may play a critical role in the process. Recent evidence has emerged implicating the ubiquitous human pathogen Clostridioides difficile as a possible promoter of colorectal cancer through chronic toxin-mediated cellular changes. Although much remains to be defined regarding the natural history of infections caused by this pathogen and its potential for oncogenesis, it provides a strong model for the role of both individual bacteria and of the gut microbial community as a whole in the development of colorectal cancer.

Biochemical Characterizations of the Putative Amidase Endolysin Ecd18980 Catalytic Domain from Clostridioides difficile.

Clostridioides difficile is the major causative pathogen of pseudomembranous colitis, and novel antimicrobial agents are required for treatment. Phage-derived endolysins exhibiting species-specific lytic activity have potential as novel antimicrobial agents. We surveyed the genome of C. difficile strain 630 and identified a gene encoding an endolysin, Ecd18980, which has an amidase_3 domain at the N-terminus but unknown C-terminal domain. The genes encoding Ecd18980 and its catalytic domain (Ecd18980CD) were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. These purified proteins showed lytic activity against C. difficile. Ecd18980CD showed higher lytic activity than the wild-type enzyme and near-specific lytic activity against C. difficile. This species specificity is thought to depend on substrate cleavage activity rather than binding. We also characterized the biochemical properties of Ecd18980CD, including optimal pH, salt concentration, and thermal stability.

Clostridioides difficile utilizes siderophores as an iron source and FhuDBGC contributes to ferrichrome uptake.

IMPORTANCE: This study is the first example of C. difficile growing with siderophores as the sole iron source and describes the characterization of the ferric hydroxamate uptake ABC transporter (FhuDBGC). This transporter shows specificity to the siderophore ferrichrome. While not required for pathogenesis, this transporter highlights the redundancy in iron acquisition mechanisms that C. difficile uses to compete for iron during an infection.

A sporulation signature protease is required for assembly of the spore surface layers, germination and host colonization in Clostridioides difficile.

A genomic signature for endosporulation includes a gene coding for a protease, YabG, which in the model organism Bacillus subtilis is involved in assembly of the spore coat. We show that in the human pathogen Clostridioidesm difficile, YabG is critical for the assembly of the coat and exosporium layers of spores. YabG is produced during sporulation under the control of the mother cell-specific regulators σE and σK and associates with the spore surface layers. YabG shows an N-terminal SH3-like domain and a C-terminal domain that resembles single domain response regulators, such as CheY, yet is atypical in that the conserved phosphoryl-acceptor residue is absent. Instead, the CheY-like domain carries residues required for activity, including Cys207 and His161, the homologues of which form a catalytic diad in the B. subtilis protein, and also Asp162. The substitution of any of these residues by Ala, eliminates an auto-proteolytic activity as well as interdomain processing of CspBA, a reaction that releases the CspB protease, required for proper spore germination. An in-frame deletion of yabG or an allele coding for an inactive protein, yabGC207A, both cause misassemby of the coat and exosporium and the formation of spores that are more permeable to lysozyme and impaired in germination and host colonization. Furthermore, we show that YabG is required for the expression of at least two σK-dependent genes, cotA, coding for a coat protein, and cdeM, coding for a key determinant of exosporium assembly. Thus, YabG also impinges upon the genetic program of the mother cell possibly by eliminating a transcriptional repressor. Although this activity has not been described for the B. subtilis protein and most of the YabG substrates vary among sporeformers, the general role of the protease in the assembly of the spore surface is likely to be conserved across evolutionary distance.

A phase 3 study evaluating the lot consistency, immunogenicity, safety, and tolerability of a Clostridioides difficile vaccine in healthy adults 65 to 85 years of age.

BACKGROUND: A toxoid-based Clostridioides difficile vaccine is currently in development. Here, we report lot-to-lot consistency, immunogenicity, safety, and tolerability of 3 C difficile vaccine doses in healthy older adults. METHODS: This phase 3, placebo-controlled study randomized (1:1:1:1) healthy adults 65 to 85 years of age to 1 of 3 C difficile vaccine lots or placebo. Participants received C difficile vaccine (200 µg total toxoid) or placebo (Months 0, 1, 6). The primary immunogenicity objective was lot-to-lot consistency (2-sided 95 % CIs within 0.5 and 2 for comparisons of geometric mean concentration [GMC] ratios) for toxins A- and B-specific neutralizing antibody levels 1 month after Dose 3. Safety outcomes included local reactions and systemic events ≤7 days after vaccination, adverse events (AEs), and serious AEs (SAEs). RESULTS: Of 1317 enrolled participants, 1218 completed the study. C difficile vaccine immunogenicity was consistent across lots, with neutralizing antibody responses 1 month after Dose 3 for both toxin A (GMC [95 % CI]: lot 1, 878.8 [786.3, 982.2]; lot 2, 873.0 [779.2, 978.1]; lot 3, 872.9 [782.6, 973.5]) and toxin B (lot 1, 5823.9 [5041.0, 6728.4]; lot 2, 5462.8 [4733.4, 6304.7]; lot 3, 5426.0 [4724.4, 6231.8]). Two-sided 95 % CIs for GMC ratios were within 0.5 and 2 for toxins A and B, indicating lot-to-lot consistency was achieved. C difficile vaccine was well tolerated, with similar rates of local reactions and systemic events among vaccine lots. AE and SAE rates were similar across C difficile vaccine (36.5 % and 4.5 %, respectively) and placebo (35.3 % and 6 %). CONCLUSIONS: Three doses (Months 0,1,6) of toxoid-based C difficile vaccine induced robust neutralizing antibody responses and were well tolerated in healthy participants 65 to 85 years of age. Lot-to-lot consistency was excellent, indicating the manufacturing process for this C difficile vaccine formulation was well controlled. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03579459.