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. 2022 Jun 3;12(1):9283.
doi: 10.1038/s41598-022-13248-4.

Identification of ADS024, a newly characterized strain of Bacillus velezensis with direct Clostridiodes difficile killing and toxin degradation bio-activities

Michelle M O'Donnell  1 , James W Hegarty  2 , Brian Healy  1 , Sarah Schulz  1 , Calum J Walsh  1   2 , Colin Hill  1 , R Paul Ross  1 , Mary C Rea  2 , Ronald Farquhar  3 , Laurent Chesnel  4
Affiliations

Identification of ADS024, a newly characterized strain of Bacillus velezensis with direct Clostridiodes difficile killing and toxin degradation bio-activities

Michelle M O'Donnell et al. Sci Rep. .

Abstract

Clostridioides difficile infection (CDI) remains a significant health threat worldwide. C. difficile is an opportunistic, toxigenic pathogen that takes advantage of a disrupted gut microbiome to grow and produce signs and symptoms ranging from diarrhea to pseudomembranous colitis. Antibiotics used to treat C. difficile infection are usually broad spectrum and can further disrupt the commensal gut microbiota, leaving patients susceptible to recurrent C. difficile infection. There is a growing need for therapeutic options that can continue to inhibit the outgrowth of C. difficile after antibiotic treatment is completed. Treatments that degrade C. difficile toxins while having minimal collateral impact on gut bacteria are also needed to prevent recurrence. Therapeutic bacteria capable of producing a range of antimicrobial compounds, proteases, and other bioactive metabolites represent a potentially powerful tool for preventing CDI recurrence following resolution of symptoms. Here, we describe the identification and initial characterization of ADS024 (formerly ART24), a novel therapeutic bacterium that can kill C. difficile in vitro with limited impact on other commensal bacteria. In addition to directly killing C. difficile, ADS024 also produces proteases capable of degrading C. difficile toxins, the drivers of symptoms associated with most cases of CDI. ADS024 is in clinical development for the prevention of CDI recurrence as a single-strain live biotherapeutic product, and this initial data set supports further studies aimed at evaluating ADS024 in future human clinical trials.

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Conflict of interest statement

The authors of this manuscript have several competing interests. PR, CH, and MCR received consulting fees from Adiso Therapeutics, Inc. LC and RF are employees of Adiso Therapeutics, Inc. MMOD, BH, JH, SS, CJW are supported by an Adiso Therapeutics, Inc. research grant to UCC/APC. All authors declare that they have no non-financial interests.

Figures

Figure 1
Figure 1
A culture-based screen of fecal samples identified ADS024. (a) Representative isolates from a single healthy human donor (ADS024) demonstrating zones of inhibition in a deferred antagonism assay with C. difficile. (b) Random amplified polymorphic DNA analysis demonstrating that 4 of the 5 isolates belong to the same bacterial strain.
Figure 2
Figure 2
Results of ANI calculation using FastANI for the 187 publicly available genomes of B. velezensis, B. amyloliquefaciens and B. siamensis. Pairwise ANIs were calculated using the tool FastANI (Jain 2018), version 1.1, which computes a very fast and relatively accurate approximation of ANI. FastANI is based on all homologous genome regions between two genomes. Only genome pairs with FastANI exceeding the threshold for species delineation are shown (94%, the hypothetical same-species range).
Figure 3
Figure 3
Bacillus-like bacteria can be isolated from fermented foods. (a) Representative agar plates of cultures of natural miso, organic miso, doenjang paste, and tojang. B. amyloliquefaciens/B.velezensis were detected only in organic miso and doenjang paste. (b) A subset of 13 representative colony PCR amplicons is shown in (b). Colony PCR from 13 representative isolates of B. amyloliquefaciens/B.velezensis from fermented foods were compared to ADS024 genomic DNA. Five isolates were selected for whole genome sequencing and confirmed to be B. velezensis. (c) Supernatants from food-derived B. amyloliquefaciens operational group members were compared to supernatant from human-derived ADS024 in the well diffusion assay against C. difficile. Only human-derived ADS024 (red circles) displayed clear zones of inhibition after 24 h and 48 h.
Figure 4
Figure 4
CFS and IPA extracts from ADS024 can inhibit C. difficile in a well diffusion assay. Shown are the inhibition zone diameters across 42 C. difficile isolates. Diamonds indicate a fixed zone diameter, and rectangles describe a range from two independent experiments. Red indicates CFS; grey indicates IPA extract.
Figure 5
Figure 5
ADS024 can kill C. difficile in liquid co-culture. ADS024 was cultured with C. difficile at the ratios shown, and C. difficile viability was assayed at time zero and 24 h later. Shown is one representative experiment.
Figure 6
Figure 6
ADS024 CFS, and lyophilate degrade C. difficile toxin A and toxin B. Lane 1 shows a Novex Hi Mark pre-stained protein ladder. Toxin A (left) and toxin B (right) were incubated with reconstituted lyophilized ADS024 (lanes 2–7), or CFS (lane 11) in the amounts shown, and toxin proteolytic cleavage was detected by western blot to toxin A and toxin B, respectively. Reconstituted lyophilate was tested once using duplicate samples. Lane 12 and lane 13 use a CFS of Lot 2 and Lot 3, respectively. Images were captured with a Licor Odyssey gel imager. See full uncropped image of Lanes 1–13 in Supplementary Fig. 1 in the supplemental information.
Figure 7
Figure 7
The killing capability of ADS024 is limited to Clostridium and Bacillus species. The killing activity of ADS024 was evaluated across a panel of gut commensals in a single well diffusion assay using ADS024 CFS. Shown are the zone diameters for each bacterium tested. In cases where the zone diameter was < 10 mm, the zone diameter was noted as “ < 10 mm.” The bore well size was 7 mm, therefore a “10 mm zone” indicates 2–3 mm in actual size. In cases where no zone was visible, the result is listed as “no zone.”
Figure 8
Figure 8
ADS024 CFS and IPA extracts can inhibit translation and disrupt B. firmus cell membranes. (a) B. firmus was incubated with ADS024 CFS for 30 min and 24 h, and bacterial cytological profiling was used to visualize morphological changes. B. firmus cell membranes are shown in red, and B. firmus DNA is shown in blue. SYTOX Green fluorescence indicates permeabilization of the B. firmus cell membrane. (b) Similar experiment as in (a) except that ADS024 IPA extracts were used. (c) Similar experiment as in (a) except that E. coli was used in place of B. firmus. (d) Similar experiment as in (c) in which E. coli was exposed to ADS024 IPA extracts. White scale bar for all images is 1 micron. Experiments were conducted in duplicate.
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