Feuerstadt, P., Theriault, N. & Tillotson, G. The burden of CDI in the United States: a multifactorial challenge. BMC Infect. Dis. 23, 132 (2023).
European Centre for Disease Control and Prevention. Clostridioides difficile infections – Annual Epidemiological Report for 2018−2020; 2024. Available from: https://www.ecdc.europa.eu/en/publications-data/clostridioides-difficile-infections-annual-epidemiological-report-2018-2020.
Guh, A.Y. et al. Trends in U.S. Burden of Clostridioides difficile Infection and Outcomes. N. Engl. J. Med. 382, 1320–1330 (2020).
Czepiel, J. et al. Clostridium difficile infection: review. Eur. J. Clin. Microbiol. Infect. Dis. 38, 1211–1221 (2019).
Feuerstadt, P. et al. Healthcare resource utilization and direct medical costs associated with index and recurrent Clostridioides difficile infection: a real-world data analysis. J. Med. Econ. 23, 603–609 (2020).
Bartlett, J. G. & Gerding, D. N. Clinical Recognition and Diagnosis of Clostridium difficile Infection. Clin. Infect. Dis. 46, S12–S18 (2008).
Zhu, D., Sorg, J. A. & Sun, X. Clostridioides difficile Biology: Sporulation, Germination, and Corresponding Therapies for C. difficile Infection. Front. Cell. Infect. Microbiol. 8, 29 (2018).
Baktash, A. et al. Mechanistic Insights in the Success of Fecal Microbiota Transplants for the Treatment of Clostridium difficile Infections. Front. Microbiol. 9, 1242 (2018).
McDonald, L. C. et al. Clinical Practice Guidelines for Clostridium difficile Infection in Adults and Children: 2017 Update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA). Clin. Infect. Dis. 66, 987–994 (2018).
Johnson, S. et al. Clinical Practice Guideline by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA): 2021 Focused Update Guidelines on Management of Clostridioides difficile Infection in Adults. Clin. Infect. Dis. 73, e1029–e1044 (2021).
Al-Jashaami, L. S. & DuPont, H. L. Management of Clostridium difficile Infection. Gastroenterol. Hepatol. 12, 609–616 (2016).
Tsigrelis, C. Recurrent Clostridioides difficile infection: Recognition, management, prevention. Clevel. Clin. J. Med. 87, 347–359 (2020).
Wang, R. Clostridioides difficile infection: microbe-microbe interactions and live biotherapeutics. Front. Microbiol. 14, 1182612 (2023).
Gonzales-Luna, A. J. et al. Reduced Susceptibility to Metronidazole Is Associated With Initial Clinical Failure in Clostridioides difficile Infection. Open Forum Infect. Dis. 8, ofab365 (2021).
van Prehn, J. et al. European Society of Clinical Microbiology and Infectious Diseases: 2021 update on the treatment guidance document for Clostridioides difficile infection in adults. Clin. Microbiol. Infect. 27, S1–S21 (2021).
Freeman, J. et al. Antimicrobial susceptibility testing of Clostridioides difficile: a dual-site study of three different media and three therapeutic antimicrobials. Clin. Microbiol. Infect. 6, 1011–1017 (2025).
Marchandin, H. et al. In vivo emergence of a still uncommon resistance to fidaxomicin in the urgent antimicrobial resistance threat Clostridioides difficile. J. Antimicrobial Chemother. 78, 1992–1999 (2023).
Smits, W. K., Garey, K. W., Riley, T. V. & Johnson, S. Clostridioides difficile is a bacterial priority pathogen. Anaerobe 93, 102965 (2025).
Eubank, T. A. et al. Reduced Vancomycin Susceptibility in Clostridioides difficile Is Associated With Lower Rates of Initial Cure and Sustained Clinical Response. Clin. Infect. Dis. 79, 15–21 (2024).
Fishbein, S. et al. Randomized Controlled Trial of Oral Vancomycin Treatment in Clostridioides difficile-Colonized Patients. mSphere 6, e00936–00920 (2021).
Benech, N. et al. Update on microbiota-derived therapies for recurrent Clostridioides difficile infections. Clin. Microbiol. Infect. 30, 462–468 (2024).
Pribyl, A. L., Hugenholtz, P. & Cooper, M. A. A decade of advances in human gut microbiome-derived biotherapeutics. Nat. Microbiol. 10, 301–312 (2025).
Wang, Y., Hunt, A., Danziger, L. & Drwiega, E. N. A Comparison of Currently Available and Investigational Fecal Microbiota Transplant Products for Recurrent Clostridioides difficile Infection. Antibiotics 13, 436 (2024).
McMillan, A. S. & Theriot, C. M. Bile acids impact the microbiota, host, and C. difficile dynamics providing insight into mechanisms of efficacy of FMTs and microbiota-focused therapeutics. Gut Microbes 16, 2393766 (2024).
Birch, C. R. et al. Cost-effectiveness of faecal microbiota transplantation compared with vancomycin monotherapy for early Clostridioides difficile infection: economic evaluation alongside a randomized controlled trial. J. Hospital Infect. 155, 145–149 (2025).
Baunwall, S. M. D. et al. Faecal microbiota transplantation for first or second Clostridioides difficile infection (EarlyFMT): a randomised, double-blind, placebo-controlled trial. Lancet Gastroenterol. Hepatol. 7, 1083–1091 (2022).
OpenBiome. FMT Update & Future Directions. [Press Release]; 2024. Available from https://openbiome.org/feature/fmt-update-future-directions/.
Davidson, J. OpenBiome No Longer Distributing FMT For Recurrent C. difficile Infections. [Gastroenterology & Endoscopy News]; 2024. Available from https://www.gastroendonews.com/PRN/Article/12-24/OpenBiome-No-Longer-Distributing-FMT-For-Recurrent-C-difficile-Infections/75606.
Jain, N., Umar, T. P., Fahner, A.-F. & Gibietis, V. Advancing therapeutics for recurrent clostridioides difficile infections: an overview of vowst’s FDA approval and implications. Gut Microbes 15, 2232137 (2023).
Mullard, A. FDA approves second microbiome-based C. difficile therapy. Nat. Rev. Drug Discov. 22, 436 (2023).
Shirley, D.-A., Tornel, W., Warren, C. A. & Moonah, S. Clostridioides difficile Infection in Children: Recent Updates on Epidemiology, Diagnosis, Therapy. Pediatrics 152, e2023062307 (2023).
Carlson, T. J., Gonzales-Luna, A. J. & Garey, K. W. Fulminant Clostridioides difficile Infection: A Review of Treatment Options for a Life-Threatening Infection. Semin. Respiratory Crit. Care Med. 43, 028–038 (2022).
van Groesen, E. et al. Semisynthetic guanidino lipoglycopeptides with potent in vitro and in vivo antibacterial activity. Sci. Transl. Med. 16, eabo4736 (2024).
Theriot, C. M. et al. Cefoperazone-treated mice as an experimental platform to assess differential virulence of Clostridium difficile strains. Gut Microbes 2, 326–334 (2011).
Blake, S. et al. Salicylanilide Analog Minimizes Relapse of Clostridioides difficile Infection in Mice. J. Medicinal Chem. 63, 6898–6908 (2020).
Seekatz, A. M. et al. Fecal Microbiota Transplantation Eliminates Clostridium difficile in a Murine Model of Relapsing Disease. Infect. Immun. 83, 3838–3846 (2015).
Hou, K. et al. Microbiota in health and diseases. Signal Transduct. Target. Ther. 7, 135 (2022).
Seekatz, A. M. & Young, V. B. Clostridium difficile and the microbiota. J. Clin. Investig. 124, 4182–4189 (2014).
Theriot, C. M., Bowman, A. A. & Young, V. B. Antibiotic-Induced Alterations of the Gut Microbiota Alter Secondary Bile Acid Production and Allow for Clostridium difficile Spore Germination and Outgrowth in the Large Intestine. mSphere 1, e00045–00015 (2016).
McMillan, A. S. et al. Metagenomic, metabolomic, and lipidomic shifts associated with fecal microbiota transplantation for recurrent Clostridioides difficile infection. mSphere 9, e00706–e00724 (2024).
Buffie, C. G. et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 517, 205–208 (2015).
Girinathan, B. P. et al. In vivo commensal control of Clostridioides difficile virulence. Cell Host Microbe 29, 1693–1708.e1697 (2021).
Kang, J. D. et al. Bile Acid 7α-Dehydroxylating Gut Bacteria Secrete Antibiotics that Inhibit Clostridium difficile: Role of Secondary Bile Acids. Cell Chem. Biol. 26, 27–34.e24 (2019).
Fishbein, S. R. S. et al. Commensal-pathogen dynamics structure disease outcomes during Clostridioides difficile colonization. Cell Host Microbe 33, 30–41.e36 (2025).
Aguirre, A. M. et al. Bile acid-independent protection against Clostridioides difficile infection. PLOS Pathog. 17, e1010015 (2021).
Walsh, C. T. et al. Bacterial resistance to vancomycin: Five genes and one missing hydrogen bond tell the story. Chem. Biol. 3, 21–28 (1996).
van Groesen, E. et al. Vancomyxins: Vancomycin-Polymyxin Nonapeptide Conjugates That Retain Anti-Gram-Positive Activity with Enhanced Potency against Gram-Negative Strains. ACS Infect. Dis. 7, 2746–2754 (2021).
Li, Q. et al. Outer-membrane-acting peptides and lipid II-targeting antibiotics cooperatively kill Gram-negative pathogens. Commun. Biol. 4, 31 (2021).
Antonoplis, A. et al. Vancomycin-Arginine Conjugate Inhibits Growth of Carbapenem-Resistant E. coli and Targets Cell-Wall Synthesis. ACS Chem. Biol. 14, 2065–2070 (2019).
Sarkar, P. et al. Vancomycin Derivative Inactivates Carbapenem-resistant Acinetobacter baumannii and Induces Autophagy. ACS Chem. Biol. 15, 884–889 (2020).
Bian, X. et al. Single Amine or Guanidine Modification on Norvancomycin and Vancomycin to Overcome Multidrug-Resistance through Augmented Lipid II Binding and Increased Membrane Activity. J. Medicinal Chem. 67, 20639–20663 (2024).
Babakhani, F. et al. Fidaxomicin Inhibits Spore Production in Clostridium difficile. Clin. Infect. Dis. 55, S162–S169 (2012).
Yamaguchi, T. et al. The gut microbiome diversity of Clostridioides difficile-inoculated mice treated with vancomycin and fidaxomicin. J. Infect. Chemother. 26, 483–491 (2020).
Louie, T. J. et al. OPT-80 Eliminates Clostridium difficile and Is Sparing of Bacteroides Species during Treatment of C. difficile Infection. Antimicrobial Agents Chemother. 53, 261–263 (2009).
Louie, T. J. et al. Fidaxomicin versus Vancomycin for Clostridium difficile Infection. N. Engl. J. Med. 364, 422–431 (2011).
van Eijk, E. et al. Complete genome sequence of the Clostridium difficile laboratory strain 630Δerm reveals differences from strain 630, including translocation of the mobile element CTn5. BMC Genomics 16, 31 (2015).
Hussain, H. A., Roberts, A. P. & Mullany, P. Generation of an erythromycin-sensitive derivative of Clostridium difficile strain 630 (630Δerm) and demonstration that the conjugative transposon Tn916ΔE enters the genome of this strain at multiple sites. J. Med. Microbiol. 54, 137–141 (2005).
Kassam, Z., Lee, C. H. & Hunt, R. H. Review of the Emerging Treatment of Clostridium difficile Infection with Fecal Microbiota Transplantation and Insights into Future Challenges. Clin. Lab. Med. 34, 787–798 (2014).
Rohlke, F. & Stollman, N. Fecal microbiota transplantation in relapsing Clostridium difficile infection. Therapeutic Adv. Gastroenterol. 5, 403–420 (2012).
Blair, H. A. SER-109 (VOWST™): A Review in the Prevention of Recurrent Clostridioides difficile Infection. Drugs 84, 329–336 (2024).
DeFilipp, Z. et al. Drug-Resistant E. coli Bacteremia Transmitted by Fecal Microbiota Transplant. N. Engl. J. Med. 381, 2043–2050 (2019).
Tan, X. & Johnson, S. Fecal microbiota transplantation (FMT) for C. difficile infection, just say ‘No’. Anaerobe 60, 102092 (2019).
Peery, A. F. et al. AGA Clinical Practice Guideline on Fecal Microbiota–Based Therapies for Select Gastrointestinal Diseases. Gastroenterology 166, 409–434 (2024).
Peixoto, R. S. et al. Harnessing the microbiome to prevent global biodiversity loss. Nat. Microbiol. 7, 1726–1735 (2022).
Khosravi, A. & Mazmanian, S. K. Disruption of the gut microbiome as a risk factor for microbial infections. Curr. Opin. Microbiol. 16, 221–227 (2013).
Durack, J. & Lynch, S. V. The gut microbiome: Relationships with disease and opportunities for therapy. J. Exp. Med. 216, 20–40 (2019).
Afzaal, M. et al. Human gut microbiota in health and disease: Unveiling the relationship. Front. Microbiol. 13, 999001 (2022).
Carlson, T. J. & Gonzales-Luna, A. J. Antibiotic Treatment Pipeline for Clostridioides difficile Infection (CDI): A Wide Array of Narrow-Spectrum Agents. Curr. Infect. Dis. Rep. 22, 20 (2020).
Quan, M. et al. Fighting against Clostridioides difficile infection: Current medications. Int. J. Antimicrobial Agents 64, 107198 (2024).
World Health Organization. 2023 Antibacterial agents in clinical and preclinical development: an overview and analysis. Available from https://www.who.int/publications/i/item/9789240094000 (2024).
Okhuysen, P. C. et al. A Randomized, Double-Blind, Phase 3 Safety and Efficacy Study of Ridinilazole Versus Vancomycin for Treatment of Clostridioides difficile Infection: Clinical Outcomes With Microbiome and Metabolome Correlates of Response. Clin. Infect. Dis. 78, 1462–1472 (2024).
Taylor, N. P. Summit makes case for changes to antibiotic R&D after sharing data from failed phase 3 trial. 2022. Available from https://www.fiercebiotech.com/biotech/summit-makes-case-changes-antibiotic-rd-after-sharing-data-failed-phase-3-trial.
DEINOVE – Update on the receivership proceedings. [Press release]; 2023. Available from https://firstwordpharma.com/story/5697000.
Lomeli, B. K. et al. Multiple-Ascending-Dose Phase 1 Clinical Study of the Safety, Tolerability, and Pharmacokinetics of CRS3123, a Narrow-Spectrum Agent with Minimal Disruption of Normal Gut Microbiota. Antimicrobial Agents Chemother. 64, e01395–01319 (2019).
Crestone Inc. Crestone Announces Positive Data from Phase 2 Clinical Trial of CRS3123 for C. Difficile Infections (CDI). [Press release]; 2024. Available from https://crestonepharma.com/positive-data-phase-2-clinical-trial-crs3123/.
Eubank, T. A. et al. P-1109. A phase 2, randomized, double-blind study of ibezapolstat compared with vancomycin for the treatment of Clostridioides difficile infection: clinical and microbiome evaluation. Open Forum Infect. Dis. 12, ofae631.1297 (2025).
MGB Biopharma. MGB Biopharma Announces Successful Outcome from Phase II Clinical Study with MGB-BP-3 – a Potential New Gold Standard, First-Line Treatment for Clostridium difficile Infection (CDI). [Press release]; 2020. Available from https://www.mgb-biopharma.com/mgb-biopharma-announces-successful-outcome-from-phase-ii-clinical-study-with-mgb-bp-3-a-potential-new-gold-standard-first-line-treatment-for-clostridium-difficile-infection-cdi/.
Critchley, I. A. et al. Spectrum of activity and mode of action of REP3123, a new antibiotic to treat Clostridium difficile infections. J. Antimicrobial Chemother. 63, 954–963 (2009).
Murray, B. et al. In vitro activity of the novel antibacterial agent ibezapolstat (ACX-362E) against Clostridioides difficile. J. Antimicrobial Chemother. 75, 2149–2155 (2020).
Citron, D. M. et al. Comparative in vitro activity of REP3123 against Clostridium difficile and other anaerobic intestinal bacteria. J. Antimicrobial Chemother. 63, 972–976 (2009).
Garey, K. W. et al. Efficacy, Safety, Pharmacokinetics, and Microbiome Changes of Ibezapolstat in Adults with Clostridioides difficile Infection: A Phase 2a Multicenter Clinical Trial. Clin. Infect. Dis. 75, 1164–1170 (2022).
McPherson, J. K. et al. The microbiome-restorative potential of ibezapolstat for the treatment of Clostridioides difficile infection is predicted through variant PolC-type DNA polymerase III in Lachnospiraceae and Oscillospiraceae. Antimicrobial Agents Chemother. 69, e01679–01624 (2025).
Vacca, M. et al. The Controversial Role of Human Gut Lachnospiraceae. Microorganisms 8, 573 (2020).
Reeves, A. E., Koenigsknecht, M. J., Bergin, I. L. & Young, V. B. Suppression of Clostridium difficile in the Gastrointestinal Tracts of Germfree Mice Inoculated with a Murine Isolate from the Family Lachnospiraceae. Infect. Immun. 80, 3786–3794 (2012).
Tejada, J. N. et al. Prevention and cure of murine C. difficile infection by a Lachnospiraceae strain. Gut Microbes 16, 2392872 (2024).
Zaplana, T., Miele, S. & Tolonen, A. C. Lachnospiraceae are emerging industrial biocatalysts and biotherapeutics. Front. Bioeng. Biotechnol. 11, 1324396 (2024).
Feuerstadt, P. et al. SER-109, an Oral Microbiome Therapy for Recurrent Clostridioides difficile Infection. N. Engl. J. Med. 386, 220–229 (2022).
Knetsch, C. W. et al. Comparative analysis of an expanded Clostridium difficile reference strain collection reveals genetic diversity and evolution through six lineages. Infect., Genet. Evolution 12, 1577–1585 (2012).
Baktash, A. et al. Comparison of Whole-Genome Sequence-Based Methods and PCR Ribotyping for Subtyping of Clostridioides difficile. J. Clin. Microbiol. 60, e01737–01721 (2022).
Winston, J. A., Thanissery, R., Montgomery, S. A. & Theriot, C. M. Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291. J. Vis. Exp. 118, e54850 (2016).
Perez, J., Springthorpe, V. S. & Sattar, S. A. Clospore: A Liquid Medium for Producing High Titers of Semi-purified Spores of Clostridium difficile. J. AOAC Int. 94, 618–626 (2011).
Warren, C. A. et al. Amixicile, a Novel Inhibitor of Pyruvate:Ferredoxin Oxidoreductase, Shows Efficacy against Clostridium difficile in a Mouse Infection Model. Antimicrobial Agents Chemother. 56, 4103–4111 (2012).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Bolyen, E. et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37, 852–857 (2019).
Callahan, B. J. et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583 (2016).
Bokulich, N. A. et al. Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier plugin. Microbiome 6, 90 (2018).
Robeson, M. S. et al. RESCRIPt: Reproducible sequence taxonomy reference database management. PLoS Computational Biol. 17, e1009581 (2021).
Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590–D596 (2012).
R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing. https://www.R-project.org/ (2021).
McMurdie, P. J. & Holmes, S. phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data. PLoS ONE 8, e61217 (2013).
Lahti, L. & Shetty, S. microbiome R package. https://doi.org/10.18129/B9.bioc.microbiome
Barnett, D. J. M., Arts, I. C. W. & Penders, J. microViz: an R package for microbiome data visualization and statistics. J. Open Source Softw. 6, 3201 (2021).
Oksanen, J. et al. vegan: Community Ecology Package. R package version 2.6-4 https://CRAN.R-project.org/package=vegan (2022).
Simpson, G. L. & Oksanen, J. ggvegan: ‘ggplot2’ Plots for the ‘vegan’ Package. R package version 0.1.999 https://gavinsimpson.r-universe.dev/ggvegan/ggvegan.pdf (2023).
Wickham, H. ggplot2: Elegant Graphics for Data Analysis. https://doi.org/10.1007/978-0-387-98141-3 (Springer-Verlag, 2009).
Ducarmon, Q. R. et al. Clostridioides difficile infection with isolates of cryptic clade C-II: a genomic analysis of polymerase chain reaction ribotype 151. Clin. Microbiol. Infect. 29, 538.e531–538.e536 (2023).