Review 2: "Combination of Mitomycin C and low-dose Metronidazole Synergistically against Clostridioides Difficile Infection and Recurrence Prevention"

RR\ID Evidence Scale rating by reviewer:

• Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review: Gong et al sought to add mitomycin C to metronidazole for the treatment of Clostridioides difficile. I applaud the authors for this work as there are limited options to treat this terrible disease that significantly reduces the quality of life of patients. Although I find the claims/conclusions from this manuscript to be reasonable from their results, I have concerns with the methodology. The minimum bactericidal concentration (MBC) assay is unclear whether this is to evaluate biofilm or purely MBC. It reads as a minimum biofilm eradication assay (MBEC) but is not represented by the results. Minimum inhibitory concentration (MIC) assay utilizes E-test when it is recommended to utilize agar dilution methods (CLSI M11). Although other studies have utilized E-test, I recommend against it for this as the study evaluates metronidazole and it has been found that resistance (via nimB as mentioned) is under-recognized without the addition of heme to the agar. (Nature communications 2023 - https://doi.org/10.1038/s41467-023-39429-x). Additionally, I found the mice model grouping unclear in its current format. Lastly, the incubation periods differ between each assay as well as CFU. Understandably, these may differ from assay to assay, especially biofilm versus not. However, I question the 18 hours of incubation for the checkerboard (usually 24 hours) and the 24 hours for the MIC testing (usually 48h for C. difficile/anaerobes). Overall, the foundational research question is of clinical importance and applaud the in vitro, in vivo, and ex vivo methodological robustness.