Basic Study
Copyright ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Pathophysiol. Aug 24, 2023; 14(4): 71-85
Published online Aug 24, 2023. doi: 10.4291/wjgp.v14.i4.71
Novel, non-colonizing, single-strain live biotherapeutic product ADS024 protects against Clostridioides difficile infection challenge in vivo
Christopher K Murphy, Michelle M O’Donnell, James W Hegarty, Sarah Schulz, Colin Hill, R Paul Ross, Mary C Rea, Ronald Farquhar, Laurent Chesnel
Christopher K Murphy, Laurent Chesnel, Research and Development, Adiso Therapeutics Inc., Concord, MA 01742, United States
Michelle M O’Donnell, Sarah Schulz, Colin Hill, R Paul Ross, APC Microbiome Ireland, University College Cork, Cork T12 K8AF, Ireland
James W Hegarty, Mary C Rea, Teagasc Food Research Centre, Moorepark Fermoy, Cork P61 C996, Ireland
Ronald Farquhar, Executive Leadership Team, Adiso Therapeutics Inc., Concord, MA 01742, United States
Author contributions: Murphy CK, O’Donnell MM, Hill C, Ross RP, Rea MC, Farquhar R, and Chesnel L designed the experiments; O’Donnell MM, Hegarty JW, and Schulz S conducted the research; data was acquired and interpreted by O’Donnell MM, Hegarty JW, Schulz S, and Chesnel L; Chesnel L drafted the manuscript; all authors reviewed the manuscript and approved the final version.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of NeoSome Life Sciences (IACUC Protocol No. NLS17-002).
Conflict-of-interest statement: Hill C, Ross RP, and Rea MC received consulting fees from Adiso Therapeutics, Inc. Murphy CK, Farquhar R, and Chesnel L are employees of Adiso Therapeutics, Inc. O’Donnell MM, Hegarty JW, and Schulz S are supported by an Adiso Therapeutics, Inc., research grant to UCC/APC.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Laurent Chesnel, PhD, Research and Development, Adiso Therapeutics, Inc., 530 Virginia Road, Suite 300, Concord, MA 01742, United States.
Received: June 1, 2023
Peer-review started: June 1, 2023
First decision: July 19, 2023
Revised: August 2, 2023
Accepted: August 7, 2023
Article in press: August 7, 2023
Published online: August 24, 2023

The Centers for Disease Control and Prevention estimate that Clostridioides difficile (C. difficile) causes half a million infections (CDI) annually and is a major cause of total infectious disease death in the United States, causing inflammation of the colon and potentially deadly diarrhea. We recently reported the isolation of ADS024, a Bacillus velezensis (B. velezensis) strain, which demonstrated direct in vitro bactericidal activity against C. difficile, with minimal collateral impact on other members of the gut microbiota. In this study, we hypothesized that in vitro activities of ADS024 will translate in vivo to protect against CDI challenge in mouse models.


To investigate the in vivo efficacy of B. velezensis ADS024 in protecting against CDI challenge in mouse models.


To mimic disruption of the gut microbiota, the mice were exposed to vancomycin prior to dosing with ADS024. For the mouse single-dose study, the recovery of ADS024 was assessed via microbiological analysis of intestinal and fecal samples at 4 h, 8 h, and 24 h after a single oral dose of 5 × 108 colony-forming units (CFU)/mouse of freshly grown ADS024. The single-dose study in miniature swine included groups that had been pre-dosed with vancomycin and that had been exposed to a dose range of ADS024, and a group that was not pre-dosed with vancomycin and received a single dose of ADS024. The ADS024 colonies [assessed by quantitative polymerase chain reaction (qPCR) using ADS024-specific primers] were counted on agar plates. For the 28-d miniature swine study, qPCR was used to measure ADS024 levels from fecal samples after oral administration of ADS024 capsules containing 5 × 109 CFU for 28 consecutive days, followed by MiSeq compositional sequencing and bioinformatic analyses to measure the impact of ADS024 on microbiota. Two studies were performed to determine the efficacy of ADS024 in a mouse model of CDI: Study 1 to determine the effects of fresh ADS024 culture and ADS024 spore preparations on the clinical manifestations of CDI in mice, and Study 2 to compare the efficacy of single daily doses vs dosing 3 times per day with fresh ADS024. C. difficile challenge was performed 24 h after the start of ADS024 exposure. To model the human distal colon, an anerobic fecal fermentation system was used. MiSeq compositional sequencing and bioinformatic analyses were performed to measure microbiota diversity changes following ADS024 treatment. To assess the potential of ADS024 to be a source of antibiotic resistance, its susceptibility to 18 different antibiotics was tested.


In a mouse model of CDI challenge, single daily doses of ADS024 were as efficacious as multiple daily doses in protecting against subsequent challenge by C. difficile pathogen-induced disease. ADS024 showed no evidence of colonization based on the observation that the ADS024 colonies were not recovered 24 h after single doses in mice or 72 h after single doses in miniature swine. In a 28-d repeat-dose study in miniature swine, ADS024 was not detected in fecal samples using plating and qPCR methods. Phylogenetic analysis performed in the human distal colon model showed that ADS024 had a selective impact on the healthy human colonic microbiota, similarly to the in vivo studies performed in miniature swine. Safety assessments indicated that ADS024 was susceptible to all the antibiotics tested, while in silico testing revealed a low potential for off-target activity or virulence and antibiotic-resistance mechanisms.


Our findings, demonstrating in vivo efficacy of ADS024 in protecting against CDI challenge in mouse models, support the use of ADS024 in preventing recurrent CDI following standard antibiotic treatment.

Keywords: Gut, Single-strain live biotherapeutic product, ADS024, Clostridioides difficile, Clostridioides difficile challenge model, Microbiota

Core Tip:Clostridioides difficile (C. difficile), a Gram positive pathogen associated with life-threatening gastrointestinal disease, colonizes millions of healthy people worldwide, causing disease in individuals with disrupted gut microbiomes. Here, we demonstrate in vivo efficacy of recently isolated Bacillus velezensis ADS024, without colonization, in protecting against C. difficile infection (CDI) challenge in mouse models. We also show that this novel strain has minimal effects on the gut microbiome in a human distal colon model and miniature swine. This study supports further investigation of ADS024 as a single-strain, live biotherapeutic product candidate for preventing recurrent CDI following successful standard-of-care antibiotic therapy.