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:
General comments:
In its current form, the manuscript is potentially informative. The conclusion that the combination of metronidazole and mitomycin C is synergistic in the mouse model, leading to improved survival of C. difficle infection, is well-supported with appropriate experiments. The suggestion that the combination prevents recurrent episodes is not correct and should be changed. Accordingly, the title of the manuscript should be changed.
Metronidazole is currently considered as an inferior antibiotic for C. difficile infections. Mitomycin has been tested for various species of multidrug resistant bacteria, but is not used clinically for the treatment of infections.Therefore, the clinical relevance is unclear. It also remains unclear whether this effect of metronidazole with mitomycin is replicable with other combinations of anti-CDI antibiotics involving mitomycin or with C. difficile strains other than RT 027.
Other comments:
C. difficile can result in severe infections and frequently relapses (20%). A key strength of this study is the use of a mouse model to evaluate various combinations of mitomycin and metronidazole on survival outcomes of the hypervirulent RT 027 strain. Figures 4B and 5B present the most notable findings, demonstrating that the combination therapy significantly improves survival when mice are challenged with the RT 027 reference strain. While survival is a definitive outcome measure, it does not directly correspond to the definition of "recurrence of CDI," which is defined by an extended recovery of spores (not a clinical relapse) from fecal samples following the challenge infection. A more appropriate term to describe this phenomenon would be the "persistence” or “prolonged excretion” of C. difficile.
An important remaining question is whether the observed combination effect is specific to metronidazole or also extends to fidaxomicin and vancomycin. Vancomycin was tested on only one RT 027 strain, yielding an FIC of 0.5 (fig 1B), and the data were very similar to the metronidazole data for 2 of 3 tested strains. I also encourage the authors to expand their investigation by testing additional C. difficile strains in the mouse model, including well-characterized metronidazole-resistant strains.
Various in vitro assays were conducted prior to the mouse model experiments, but some tests are not appropriate, such as E-tests on BHIS media (performed without heme). Is it correct that the medium used in the checkerboard assay is also without heme? Instead, additional whole-genome sequencing (WGS) data is needed for the strains used, particularly to identify genetic determinants of metronidazole resistance (and mitomycin?) Furthermore, more information on the effect of mitomycin on phage production of the tested strain is necessary to understand the synergy of the combination better.
The authors mention that metronidazole resistance can result from the overexpression of nimB genes (and mutations in the promoter of nimB, ref 44) but have not investigated this in the strains studied. Similarly, other mechanisms for metronidazole resistance have been described that are difficult to detect without appropriate tests, such as plasmid mediated metronidazole resistance (seePMID: 33876817) .These phenotypic techniques required to assess heme-dependent metronidazole resistance differ from those employed in this study (see also ref 44 and PMID: 33876817). As a result, the authors’ conclusions may not fully apply to "metronidazole-susceptible tested" strains .
Technical comments:
The challenged mice in the study were sacrificed on day 7 post-infection to analyze the morphology and quantify the cecal and colonic tissues (Figures 4D and 5D). Would it not have been possible to include histological analysis of the tissues, with grading of the severity of inflammation?
Some comments on the figures. Figure 1C shows a RT 078 strain with higher FC than the other strains; what could be a reason? Metronidazole resistant? The Y-axis of figure 3B is probably incorrect, please elucidate. Figure 4; please define “mock” (also in the MM). Figure 5E; seems that the combination of mityomycin with metronidazole leads to a lower serum BUN than vancomycine in the mouse model?
Discussion:
The discussion section of the manuscript is brief and requires greater balance. For example, metronidazole is no longer recommended for the treatment of C. difficile infections in the US and Europe, according to the IDSA and ESCMID guidelines. The hypothesis that the combination therapy minimizes disruption of the microbiota composition is unlikely, as it assumes that mitomycin mitigates the microbiome damage caused by metronidazole. However, the primary message of the paper is that the combination is synergistic in killing bacteria, such as C. difficile. Both metronidazole and mitomycin have a broad antibacterial spectrum. Additional data on the effects of mitomycin on gut microbiota composition are needed to test this hypothesis. Lastly, the potential clinical application of the proposed combination therapy remains highly uncertain and is not well addressed in the manuscript.
Conclusion:
In conclusion, while the experiments support the key finding that mitomycin C exhibits synergistic effects in the mouse model for RT 027, there remains significant uncertainty regarding whether this synergy applies universally to all C. difficile strains.
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