Effective Colonization by Nontoxigenic Clostridioides difficile REA Strain M3 (NTCD-M3) Spores following Treatment with Either Fidaxomicin or Vancomycin

ABSTRACT Colonization with nontoxigenic Clostridioides difficile strain M3 (NTCD-M3) has been demonstrated in susceptible hamsters and humans when administered after vancomycin treatment. NTCD-M3 has also been shown to decrease risk of recurrent C. difficile infection (CDI) in patients following vancomycin treatment for CDI. As there are no data for NTCD-M3 colonization after fidaxomicin treatment, we studied the efficacy of NTCD-M3 colonization and determined fecal antibiotic levels in a well-studied hamster model of CDI. Ten of 10 hamsters became colonized with NTCD-M3 after 5 days of treatment with fidaxomicin when NTCD-M3 was administered daily for 7 days after treatment discontinuation. The findings were nearly identical to 10 vancomycin-treated hamsters also given NTCD-M3. High fecal levels of OP-1118, the major fidaxomicin metabolite, and vancomycin were noted during treatment with the respective agents and modest levels noted 3 days after treatment discontinuation at the time when most of the hamsters became colonized. These findings support the ongoing development of NTCD-M3 for the prevention of recurrent CDI. IMPORTANCE NTCD-M3 is a novel live biotherapeutic, that has been shown in a Phase 2 clinical trial to prevent recurrence of C. difficile infection (CDI) when administered shortly after antibiotic treatment of the initial CDI episode. Fidaxomicin was not, however, in widespread use at the time this study was conducted. A large multi-center Phase 3 clinical trial is now currently in the planning stage, and it is anticipated that many patients eligible for this study will be treated with fidaxomicin. Since efficacy in the hamster model of CDI has predicted success in patients with CDI, we studied the ability of NTCD-M3 to colonize hamsters after treatment with either fidaxomicin or vancomycin.


NTCD colonization
This is a small hamster study to assess whether NTCD-M3 would colonize these hamsters after either vancomycin or fidaxomicin treatment. All hamsters became colonized with NTCD-M3 with both therapies. This builds upon human data to expand previous findings to now include colonization with fidaxomicin treated arms. Although perhaps not the most novel finding, this does move the development of NTCD-M3 in the right direction and agree with the authors that this supports further development. Figure 2 is an excellent visual summary of results. I don't have any major comments but a few suggestions to improve the clarity of the manuscript.
Statistical analysis (lines 107-12). A description on the analysis plan for NTCD-M3 colonization should be provided here. I'm guessing the authors felt that 100% of hamsters would be colonized and thus, the low number of hamsters used. A power calculation here may be beneficial. Table 1. LC-MS data may not be completely familiar to readers of MIcrobiolog Spectrum. A 1-2 line explanatory in the title may be helpful. This study looked at colonization (Y/N) and no quantitation was performed. This should be mentioned as a limitation including why quantitation was not performed.
Reviewer #2 (Comments for the Author): See attached Word document for detailed suggestions.
Reviewer #3 (Comments for the Author): This is a well written and straight forward study to determine if fidaxomicin inhibits M3 colonization compared to vancomycin. This study is important because fidaxomicin use has been increasing since the first in-human studies of M3, and will likely continue to due so, and, as such, whether fidaxomicin may inhibit M3 colonization is an important question to investigate with the planned phase 3 study.
No major comments.
The minor comment is in the methods it states stool was collected out to day 30, but in the results and figures 1 and 2 stool appears to have been collected out to day 43. Please update the methods.

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Summary
Colonization with a non-toxigenic C. difficile strain following antibiotic treatment has been shown to lower risk of recurrence in humans and hamsters. While colonization of C. difficile strain M3 (NTCD-M3) following vancomycin treatment has been demonstrated, colonization following fidaxomicin has not, despite it being the current clinically-preferred antibiotic. The authors sought to establish that strain NTCD-M3 colonizes similarly in both antibiotic courses (i.e., fidaxomicin in addition to the previously studied vancomycin) using a hamster model of C. difficile colonization that mirrors the authors' phase 2 clinical trial conditions (Gerding et al., JAMA, 2015). They found that hamsters in each group became colonized at similar time periods following the cessation of antibiotics, which tended to associate with gradually decreasing fecal antibiotic concentrations. The authors suggest that this provides a foundation for future clinical trials investigating the treatment efficacy of colonizing with non-toxigenic C. difficile strain NTCD-M3. This is a succinct manuscript bringing novel information to the scientific community regarding possible C. difficile preventative measures. It is well-controlled and generally draws conclusions that can be readily gathered from the data presented.

Specific comments
Major points: 1. None Minor points: 1. L52: if trying to indicate hamsters were housed individually, state "individually housed in polycarbonate cages…" The current reading suggests that several hamsters where group-housed in a couple individual cages. 2. L80: consider adding something along the lines of, "in the absence of study drugmediated microbiota perturbations" to more clearly state the purpose of the control. 3. L130: As a non-clinician, the broader context of the MICs as stated was not readily apparent (i.e., are these normal resistance levels?). Consider adding, "…, which are similar to previously reported MICs for other C. difficile strains" and citing Gargis et al. associate with fecal antibiotic concentrations, particularly the variability in OP-1118 levels? If so, adding a statement to that effect would strengthen the claim at L160 about colonization being associated with gradual decreases in antibiotic concentrations. Otherwise, consider adding a statement indicating the lack of association and that the factors involved in partial colonization were extraneous to this study. 5. L165: "This delay correlated with…" The authors use "correlate" without offering any statistical backing for their statement in the results. Please use an appropriate statistical test or alternative wording as in L159 ("appears to be associated"). 6. Other: If M3 colonization levels were titered (rather than collecting just presence/absence data), this reviewer would very much appreciate those data being included in a supplement. While they are not essential for the manuscript's conclusions, they would be useful to the scientific community.

Response to Reviewers
Reviewer #1: Statistical analysis (lines 107-12). A description on the analysis plan for NTCD-M3 colonization should be provided here. I'm guessing the authors felt that 100% of hamsters would be colonized and thus, the low number of hamsters used. A power calculation here may be beneficial.
Using groups of 10 hamsters allows for the detection of significant declines in colonization of 50% from that of the expected colonization rate (P=0.03) and allows comparison with other data collected historically in our lab. We have now included a brief summary in the statistical methods section. Table 1. LC-MS data may not be completely familiar to readers of MIcrobiolog(y) Spectrum. A 1-2 line explanatory in the title may be helpful.

Response:
We have now included additional explanation of the data in a footnote to the table.
This study looked at colonization (Y/N) and no quantitation was performed. This should be mentioned as a limitation including why quantitation was not performed.
Response: Semi-quantitative cultures were performed (1+ to 4+) but the transition from negative to high levels (3+ to 4+) occurred rapidly and was not captured within our sampling time periods (hamster pellets were collected daily from the cages). Hamsters were typically culture negative one day and 3+ or 4+ the next day. We have included the raw data for semi-quantitative cultures as supplemental files and we have now modified our methodology and results section to include these semi-quantitative culture results.

Reviewer #2:
If trying to indicate hamsters were housed individually, state "individually housed in polycarbonate cages…" The current reading suggests that several hamsters where group-housed in a couple individual cages.

Response:
We have now changed the wording to clarify that hamsters were individually housed rather than group housed.