Gastroenterology Inhibiting Growth of Clostridioides difficile by Restoring Valerate, Produced by the Intestinal Microbiota --Manuscript Draft-- Manuscript Number: GASTRO-D-18-00449R1 Full Title: Inhibiting Growth of Clostridioides difficile by Restoring Valerate, Produced by the Intestinal Microbiota Article Type: Basic - Alimentary Tract Corresponding Author: Julian Marchesi Cardiff University Cardiff, UNITED KINGDOM Corresponding Author Secondary Information: Corresponding Author's Institution: Cardiff University Corresponding Author's Secondary Institution: First Author: Julie A. K. McDonald, PhD First Author Secondary Information: Order of Authors: Julie A. K. McDonald, PhD Benjamin H. Mullish, MB BChir Alexandros Pechlivanis, PhD Zhigang Liu, PhD Jerusa Brignardello, MSc Dina Kao, MD Elaine Holmes, PhD Jia V. Li, PhD Thomas B Clarke, PhD Mark R. Thursz, MD Julian R. Marchesi, PhD Order of Authors Secondary Information: Abstract: Background & Aims: Faecal microbiota transplantation (FMT) is effective for treating recurrent Clostridioides difficile infection (CDI), but there are concerns regarding its long-term safety. Understanding the mechanisms of the effects of FMT could help us design safer, targeted therapies. We aimed to identify microbial metabolites that are important for C difficile growth. Methods: We used a CDI chemostat model as a tool to study the effects of FMT in vitro. The following analyses were performed: C difficile plate counts, 16S rRNA gene sequencing, 1H-NMR spectroscopy, and UPLC mass spectrometry bile acid profiling. FMT mixtures were prepared using fresh faecal samples provided by donors enrolled in the FMT Programme. Results from chemostat experiments were validated using human stool samples, C difficile batch cultures, and C57BL/6 mice with CDI. Human stool samples were collected from 16 patients with recurrent CDI and 5 healthy donors participating in an FMT trial. Results: In the CDI chemostat model, clindamycin decreased valerate and deoxycholic acid concentrations and increased C difficile total viable counts (TVC) and valerate precursors, taurocholic acid, and succinate concentrations. After we stopped adding Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation clindamycin, levels of bile acids and succinate recovered, whereas levels of valerate and valerate precursors did not. In the CDI chemostat model FMT increased valerate concentrations and decreased C difficile TVC (94% reduction), spore counts (86% reduction), and valerate precursor concentrations—concentrations of bile acids were unchanged. In stool samples from patients with CDI, valerate was depleted before FMT, but restored after FMT. C difficile batch cultures confirmed that valerate decreased vegetative growth, and that taurocholic acid is required for germination but had no effect on vegetative growth. C difficile TVC were decreased by 95% in mice with CDI given glycerol trivalerate compared to phosphate-buffered saline. Conclusions: We identified valerate as a metabolite that is depleted with clindamycin and only recovered with FMT. Valerate is a target for a rationally designed recurrent CDI therapy. Key words: bacteria; stool transplant; gut microbiome; pathogen Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Cover Letter Dr. Julian R. Marchesi Professor of Digestive Health Centre for Digestive and Gut Health Department of Surgery and Cancer Faculty of Medicine Imperial College London Room 1003 Liver Unit, 10th Floor St Mary’s Hospital South Wharf Road London W2 1NY Tel: +44 02033126197 [email protected] 21 June 2018 Dear Professors Peek and Corley, Please consider our revised manuscript entitled "A novel route for inhibiting Clostridioides difficile growth by restoring gut microbiota-produced valerate" by McDonald and colleagues for consideration for publication in Gastroenterology (Category: Basic and Translational, Alimentary Tract, manuscript number GASTRO-D-18-00449R1). We appreciate the positive feedback and interest that the editors and reviewers have taken in our manuscript. We have addressed all the comments and made the requested revisions. The major outstanding concern was the limited clinical perspective without an in vivo mouse interventional study. As such, we performed an interventional study in a C. difficile infection (CDI) mouse model, where C. difficile infected mice were administered valerate (in the form of glycerol trivalerate) or PBS (as a vehicle control). We showed a significant reduction in C. difficile total viable counts in faeces from glycerol trivalerate-treated mice compared to PBS-treated mice (95% reduction after 3 doses, see Figure 6). No adverse effects were noted in glycerol trivalerate-treated mice over the course of the experiment. These results support our other findings and strengthen our manuscript. We have also reworded the text and provided additional clarification as requested by Reviewers 1 and 2. Moreover, we have added a schematic of the key metabolite interactions with C. difficile as requested by Reviewer 2 (see Figure S12). We believe our manuscript has been significantly improved by making these suggested changes. A point-by-point response to the reviewer comments is provided that describes the revisions in more detail. Due to the addition of the mouse experimental data, we have moved text from the main text discussion section to the supplementary discussion section. We have also moved Figure 2 to the supplementary figures to ensure the total number of figures and tables does not exclude 7 in total. The main conclusions of our manuscript remain unchanged. We believe we have exhaustively demonstrated the role of valerate in CDI by presenting results from chemostat experiments, human samples, batch culture experiments, and mouse experiments. We showed that valerate directly inhibits C. difficile growth, and therefore reduction of valerate-producing bacteria in the gut microbiota following antibiotic exposure decreases valerate production and permits C difficile growth. Consequently, restoration of valerate and valerate-producing bacteria in the gut microbiota following FMT decreases C. difficile growth. We believe these findings are important for the development of safer, microorganism- free treatments for CDI/recurrent CDI. Treatment with valerate may be of particular interest to immunocompromised, paediatric, or frail/elderly patients with CDI. Moreover, valerate administration may be beneficial for patients who are expected to undergo further antibiotic treatment for other infections, where antibiotic exposure may render FMT treatment less effective. Therefore, the clear clinical relevance of these findings would be of great interest to Gastroenterology readers. Thank you for your consideration. Should you require any further information, please do not hesitate to contact me. Yours sincerely, Prof. Julian R. Marchesi Professor of Digestive Health at Imperial College London Professor of Human Microbiome Research at Cardiff University Point by Point Response Response to Reviewer Comments EDITORS: This is an interesting study into the pathobiology of C. diff infection in a model system allowing to study the molecular effects of fecal transplantation. The authors show the detrimental effects of Clindamycin, which increased valerate precursors, [taurocholic] acid and succinate, and decreased valerate. After stopping clindamycin the effects on valerate and its precursors persisted, while [TCA] and succinate normalized. FMT counteracted this effect on valerate, and valerate affected C. diff growth. Thus, the authors suggest that valerate is a good target for prevention and treatment of C diff infection and suggest therapeutic studies. While there is general enthusiasm for the study the authors will need to perform to interventional study in mice (a concern also raised by Reviewer 3). In absence of this experiment, this contribution lacks sufficient novelty and clinical perspective for further consideration. The authors will also need to revise the title of the manuscript, as "suppressive metabotype" is too vague. We would like to thank the reviewers and editors for the time and effort put into reviewing the previous version of this manuscript. We appreciate their constructive comments, and we believe this feedback has improved our manuscript. As the editors and Reviewer 3 suggested, we performed an interventional study with valerate (in the form of glycerol trivalerate) in a CDI mouse model. We found a significant decrease in C. difficile total viable counts (TVC) in glycerol trivalerate-treated mice compared to PBS-treated mice (see Figure 6). After only 3 doses, glycerol trivalerate-treated mice had an average of 95% less C. difficile TVC per gram of faeces compared to PBS-treated mice. These results corroborate the other findings presented in this study and support the therapeutic use of valerate to treat CDI. We have discussed these results in more detail in response to Reviewer 3’s comments below. We have also edited the title of the manuscript (as reviewed by Dr. Kristine Novak, Science Editor for Gastroenterology) to remove the term “suppressive metabotype,” and the title now reads: “Inhibiting Growth of Clostridioides difficile by Restoring Valerate, Produced by the Intestinal Microbiota.” REVIEWER 1: This outstanding manuscript is an extension of work by the authors (their refs 12, 14, and 27) and especially the lead author in her investigations of an in vitro, chemostat model of the microbiome.