Blocking toxin function and modulating the gut microbiota: caffeic acid phenethyl ester as a potential treatment for Clostridioides difficile infection

Reviewer #1 (Public review):

Summary:

In this manuscript, Guo and colleagues used a cell rounding assay to screen a library of compounds for inhibition of TcdB, an important toxin produced by Clostridioides difficile. Caffeic acid and derivatives were identified as promising leads, and caffeic acid phenethyl ester (CAPE) was further investigated.

Strengths:

Considering the high morbidity rate associated with C. difficile infections (CDI), this manuscript presents valuable research in the investigation of novel therapeutics to combat this pressing issue. Given the rising antibiotic resistance in CDI, the significance of this work is particularly noteworthy. The authors employed a robust set of methods and confirmatory tests, which strengthen the validity of the findings. The explanations provided are clear, and the scientific rationale behind the results is well-articulated. The manuscript is extremely well written and organized. There is a clear flow in the description of the experiments performed. Also, the authors have investigated the effects of CAPE on TcdB in careful detail, and reported compelling evidence that this is a meaningful and potentially useful metabolite for further studies.

Weaknesses:

The authors have made some changes in the revised version. However, many of the changes were superficial, and some concerns still need to be addressed. Important details are still missing from the description of some experiments. Authors should carefully revise the manuscript to ascertain that all details that could affect interpretation of their results are presented clearly. For instance, authors still need to include details of how the metabolomics analyses were performed. Just stating that samples were "frozen for metabolomics analyses" is not enough. Was this mass-spec or NMR-based metabolomics. Assuming it was mass-spec, what kind? How was metabolite identity assigned, etc? These are important details, which need to be included. Even in cases where additional information was included, the authors did not discuss how the specific way in which certain experiments were performed could affect interpretation of their results. One example is the potential for compound carryover in their experiments. Another important one is the fact that CAPE affects bacterial growth and sporulation. Therefore, it is critical that authors acknowledge that they cannot discard the possibility that other factors besides compound interactions with the toxin are involved in their phenotypes. As stated previously, authors should also be careful when drawing conclusions from the analysis of microbiota composition data, and changes to the manuscript should be made to reflect this. Ascribing causality to correlational relationships is a recurring issue in the microbiome field. Again, I suggest authors carefully revise the manuscript and tone down some statements about the impact of CAPE treatment on the gut microbiota.

Reviewer #2 (Public review):

I appreciate the author's responses to my original review. This is a comprehensive analysis of CAPE on C. difficile activity. It seems like this compound affects all aspects of C. difficile, which could make it effective during infection but also make it difficult to understand the mechanism. Even considering the authors responses, I think it is critical for the authors to work on the conclusions regarding the infection model. There is some protection from disease by CAPE but some parameters are not substantially changed. For instance, weight loss is not significantly different in the C. difficile only group versus the C. difficile + CAPE group. Histology analysis still shows a substantial amount of pathology in the C. difficile + CAPE group. This should be discussed more thoroughly using precise language.

Reviewer #3 (Public review):

Summary:

The study is well written, and the results are solid and well demonstrated. It shows a field that can be explored for the treatment of CDI

Strengths:

Results are really good, and the CAPE shows a good and promising alternative for treating CDI.

Weaknesses:

Some references are too old or missing.

Comments on revisions:

I have read your study after comments made by all referees, and I noticed that all questions and suggestions addressed to the authors were answered and well explained. Some of the minor and major issues related to the article were also solved. I am satisfied with all the effort given by the authors to improve their manuscript.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this manuscript, Guo and colleagues used a cell rounding assay to screen a library of compounds for inhibition of TcdB, an important toxin produced by Clostridioides difficile. Caffeic acid and derivatives were identified as promising leads, and caffeic acid phenethyl ester (CAPE) was further investigated.

Strengths:

Considering the high morbidity rate associated with C. difficile infections (CDI), this manuscript presents valuable research in the investigation of novel therapeutics to combat this pressing issue. Given the rising antibiotic resistance in CDI, the significance of this work is particularly noteworthy. The authors employed a robust set of methods and confirmatory tests, which strengthened the validity of the findings. The explanations provided are clear, and the scientific rationale behind the results is well-articulated. The manuscript is extremely well-written and organized. There is a clear flow in the description of the experiments performed. Also, the authors have investigated the effects of CAPE on TcdB in careful detail and reported compelling evidence that this is a meaningful and potentially useful metabolite for further studies.

Weaknesses:

This is really a manuscript about CAPE, not caffeic acid, and the title should reflect that. Also, a few details are missing from the description of the experiments. The authors should carefully revise the manuscript to ascertain that all details that could affect the interpretation of their results are presented clearly. Just as an example, the authors state in the results section that TcdB was incubated with compounds and then added to cells. Was there a wash step in between? Could compound carryover affect how the cells reacted independently from TcdB? This is just an example of how the authors should be careful with descriptions of their experimental procedures. Lastly, authors should be careful when drawing conclusions from the analysis of microbiota composition data. Ascribing causality to correlational relationships is a recurring issue in the microbiome field. Therefore, I suggest authors carefully revise the manuscript and tone down some statements about the impact of CAPE treatment on the gut microbiota.

Thanks for your constructive suggestion. We have carefully revised the manuscript, including the description of title, results and methods sections.

Reviewer #2 (Public review):

Summary:

This work is towards the development of nonantibiotic treatment for C. difficile. The authors screened a chemical library for activity against the C. difficile toxin TcdB, and found a group of compounds with antitoxin activity. Caffeic acid derivatives were highly represented within this group of antitoxin compounds, and the remaining portion of this work involves defining the mechanism of action of caffeic acid phenethyl ester (CAPE) and testing CAPE in mouse C. difficile infection model. The authors conclude CAPE attenuates C. difficile disease by limiting toxin activity and increasing microbial diversity during C. difficile infection.

Strengths/ Weaknesses:

The strategy employed by the authors is sound although not necessarily novel. A compound that can target multiple steps in the pathogenies of C. difficile would be an exciting finding. However, the data presented does not convincingly demonstrate that CAPE attenuates C. difficile disease and the mechanism of action of CAPE is not convincingly defined. The following points highlight the rationale for my evaluation.

(1) The toxin exposure in tissue culture seems brief (Figure 1). Do longer incubation times between the toxin and cells still show CAPE prevents toxin activity?

Thanks for your comments. The cytotoxicity assay was employed to directly assess the protective capacity of CAPE against cell death induced by TcdB. Our observations at 1 and 12 h post-TcdB exposure revealed that CAPE effectively mitigated the toxic effects of the TcdB at both time points, demonstrating its potent protective role. Please see Figure S1.

(2) The conclusion that CAPE has antitoxin activity during infection would be strengthened if the mouse was pretreated with CAPE before toxin injections (Figure 1D).

Thanks for your constructive comments. According to your suggestion, we administered TcdB 2 h after pretreatment with CAPE. The outcomes demonstrated that CAPE pretreatment significantly enhanced the survival rate of the intoxicated mice, confirming that CAPE retains its antitoxin efficacy during the infection process. Please see Figure S2.

(3) CAPE does not bind to TcdB with high affinity as shown by SPR (Figure 4). A higher affinity may be necessary to inhibit TcdB during infection. The GTD binds with millimolar affinity and does not show saturable binding. Is the GTD the binding site for CAPE? Auto processing is also affected by CAPE indicating CAPE is binding non-GTD sites on TcdB.

Thanks for your comments. Our findings indicate that the GTD domain is a critical binding site for CAPE. CAPE exerts its protective effects at multiple stages of TcdB-mediated cell death, including inhibiting TcdB's self-cleavage and blocking the activity of GTD, thereby preventing the glycosylation modification of Rac1 by TcdB.

(4) In the infection model, CAPE does not statistically significantly attenuate weight loss during C. difficile infection (Figure 6). I recognize that weight loss is an indirect measure of C. difficile disease but histopathology also does not show substantial disease alleviation (see below).

Thanks for your comments. Our comparative analysis revealed a notable distinction in the body weight of mice on the third day post-infection (Figure 6B). Similarly, the dry/wet stool ratio exhibited a comparable pattern, suggesting that treatment with phenethyl caffeic acid ameliorated Clostridium difficile-induced diarrhea to a significant degree (Figure 6C).

(5) In the infection model (Figure 6), the histopathology analysis shows substantial improvement in edema but limited improvement in cellular infiltration and epithelial damage. Histopathology is probably the most critical parameter in this model and a compound with disease-modifying effects should provide substantial improvements.

Thanks for your comments. Edema, inflammatory factor infiltration, and epithelial damage served as key evaluation metrics. Statistical analysis revealed that the pathological scores of mice treated with CAPE were markedly reduced compared to those in the model group (Figure 6F).

(6) The reduction in C. difficile colonization is interesting. It is unclear if this is due to antitoxin activity and/or due to CAPE modifying the gut microbiota and metabolites (Figure 6). To interpret these data, a control is needed that has CAPE treatment without C. difficile infection or infection with an atoxicogenic strain.

The observed reduction in C. difficile fecal colonization following drug treatment may be attributed to the CAPE's antitoxin properties or its capacity to modify the intestinal microbiota and metabolites. These two mechanisms likely work in tandem to combat CDI. CDI is primarily triggered by the toxins A (TcdA) and B (TcdB) secreted by the bacterium. Certain therapies, including monoclonal antibodies like bezlotoxumab, target CDI by neutralizing these toxins, thereby mitigating gut damage and subsequent C. difficile colonization(1,2). The establishment of C. difficile in the gut is intricately linked to the equilibrium of the intestinal microbiota. Although antibiotic treatments can inhibit C. difficile growth, they may also disrupt the microbial balance, potentially facilitating the overgrowth of other pathogens. Consequently, interventions such as fecal microbiota transplantation (FMT) are designed to reestablish gut flora balance and consequently decrease C. difficile colonization(3,4). Moreover, the administration of probiotics and prebiotics is considered to reduce C. difficile colonization by modifying the gut environment(5,6).

(7) Similar to the CAPE data, the melatonin data does not display potent antitoxin activity and the mouse model experiment shows marginal improvement in the histopathological analysis (Figure 9). Using 100 µg/ml of melatonin (~ 400 micromolar) to inactivate TcdB in cell culture seems high. Can that level be achieved in the gut?

The uptake and dissemination of melatonin within the body varies with the dose administered. For instance, in rats, the bioavailability of melatonin following administration was found to be 53.5%, whereas in dogs, bioavailability was nearly complete (100%) at a dose of 10 mg/kg, yet it decreased to 16.9% at a lower dose of 1 mg/kg(7). This data suggests that the absorption of melatonin differs across various animal species and is influenced by the dose administered. Moreover, it underscores the higher potential bioavailability of melatonin, implying that a dose of 200 mg/kg should be adequate to achieve the desired concentration in the body post-administration.

(8) The following parameters should be considered and would aid in the interpretation of this work. Does CAPE directly affect the growth of C. difficile? Does CAPE affect the secretion of TcdB from C. difficile? Does CAPE alter the sporulation and germination of C. diffcile?

We incorporated CAPE into the MIC assay for detecting C. difficile, as well as for assessing the sporulation capacity of C. difficile and evaluating the secretion level of TcdB. The findings revealed that CAPE markedly repressed tcdB transcription at a concentration of 16 μg/mL and effectively suppressed the growth and sporulation of C. difficile BAA-1870 at a concentration of 32 μg/mL. Please see Figure S3.

References:

(1) Skinner AM, et al. Efficacy of bezlotoxumab to prevent recurrent Clostridioides difficile infection (CDI) in patients with multiple prior recurrent CDI. Anaerobe. 2023 Dec; 84: 102788.

(2) Wilcox MH, et al. Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection. N Engl J Med. 2017 Jan 26;376(4):305-317.

(3) Khoruts A, Sadowsky MJ. Understanding the mechanisms of faecal microbiota transplantation. Nat Rev Gastroenterol Hepatol. 2016 Sep;13(9):508-16.

(4) Khoruts A, Staley C, Sadowsky MJ. Faecal microbiota transplantation for Clostridioides difficile: mechanisms and pharmacology. Nat Rev Gastroenterol Hepatol. 2021 Jan;18(1):67-80.

(5) Mills JP, Rao K, Young VB. Probiotics for prevention of Clostridium difficile infection. Curr Opin Gastroenterol. 2018 Jan;34(1):3-10.

(6) Lau CS, Chamberlain RS. Probiotics are effective at preventing Clostridium difficile-associated diarrhea: a systematic review and meta-analysis. Int J Gen Med. 2016 Feb 22; 9:27-37.

(7) Yeleswaram K, et al. Pharmacokinetics and oral bioavailability of exogenous melatonin in preclinical animal models and clinical implications. J Pineal Res. 1997 Jan;22(1):45-51.

Reviewer #3 (Public review):

Summary:

The study is well written, and the results are solid and well demonstrated. It shows a field that can be explored for the treatment of CDI.

Strengths:

The results are really good, and the CAPE shows a good and promising alternative for treating CDI. The methodology and results are well presented, with tables and figures that corroborate them. It is solid work and very promising.

Weaknesses:

Some references are too old or missing.

Thanks for your constructive suggestion. We have included and refreshed several references to enhance the manuscript.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

While the manuscript convincingly demonstrates that CAPE affects the TcdB toxin and reduces its toxicity in vitro, it would be beneficial to include data on the effect of CAPE on the growth of C. difficile. This would help ensure that the observed in vivo effects are not merely due to reduced bacterial growth but rather due to the specific action of CAPE on the toxin.

Thanks for your constructive suggestion. We have augmented our findings with the impact of CAPE on the bacteria themselves, revealing that CAPE not only hampers the growth of the bacterial cells but also suppresses their capacity to produce spores. Please see Figure S3.

(1) Line 41, line 115 - authors should clarify what they mean when mentioning Bacteroides within parentheses.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(2) Line 71 - Is C. difficile really found "in the environment"?

Thanks for your comments. C. difficile is prevalent across various natural settings, including soil and water ecosystems. A study has identified highly diverse strains of this bacterium within environmental samples(1). Moreover, the significant presence of C. difficile in soil and lawn specimens collected near Australian hospitals indicates that the organism is indeed a common inhabitant in the environment(2).

(3) Lines 128-130 - Was there a wash step here? What could be the impact of compound carryover in this experiment?

Thanks for your comments. Following pre-incubation of TcdB with CAPE, remove the compounds that have not bound to TcdB through centrifugation. The persistence of the compound in the culture post-washing could result in an inflated assessment of its efficacy, particularly if it continues to engage with TcdB or the cells beyond the initial 1-hour pre-incubation window. The carryover of the compound might also give rise to misleading positive results, where the compound seems to confer protection or inhibition against TcdB-mediated cell rounding, whereas such effects are actually due to the lingering activity of the compound. This carryover could skew the determination of the compound's minimum effective concentration, as the effective concentration interacting with the cells might be inadvertently elevated. Furthermore, if the compounds possess cytotoxic properties or impact cell viability, carryover could generate artifacts in cell morphology that are unrelated to the direct interaction between TcdB and the compounds.

(4) Lines 133-134 - I suggest authors mention how many caffeic acid derivatives there were in the entire library so that the suggested "enrichment" of them in the group of bioactive compounds can be better judged.

Thanks for your comments. The natural compound library contained eight caffeic acid derivatives, of which methyl caffeic acid and ferulic acid displayed no efficacy. This information has been incorporated into the manuscript.

(5) Line 135 - I recommend the authors add the molarity of the compound solutions used.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(6) Line 247 - I think the term "CAPE mice" is confusing. Please use a full description.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(7) Line 248 - I also think the terms "model mice" and "model group" are confusing. Maybe call them "control mice"?

Thanks for your comments. The terms "model mice" and "model group" are indeed synonymous, and we have subsequently clarified that control mice refer to those that have not been infected with C. difficile.

(8) Line 273 - "most abundant species at the genus level" is incorrect. I think what you mean is "most abundant TAXA".

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(9) Line 278 - Please include your p-value cut-off together with the LDA score.

Thanks for your comments. We have revised the above description to “LDA score > 3.5, p < 0.05”.

(10) Line 292 - Details on how metabolomics was performed should be included here.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(11) Line 299 - 1.5 is a fairly low cut-off. The authors should at a minimum also include the p-value cut-off used.

Response: Thanks for your comments. We have revised the above description to “fold change > 1.5, p < 0.05”.

(12) Line 307 - Purine "degradation" would be better here.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(13) Line 328 onward - The melatonin experiment is a weird one. Although I fully understand the rationale behind testing the effect of melatonin in the mouse model, the idea that just because melatonin levels changed in the gut it would act as a direct inhibitor of TcdB was very far-fetched, even though it ended up working. Authors should explain this in the manuscript.

Thanks for your comments. Furthermore, beyond our murine studies, we have confirmed that melatonin significantly diminishes TcdB-induced cytotoxicity at the cellular level (Figure 9A). Additionally, it has been documented that melatonin, acting as an antimicrobial adjuvant and anti-inflammatory agent, can decrease the recurrence of CDI(3). Consequently, we contend that the aforementioned statement is substantiated.

(14) Lines 429-435 - There are seemingly contradictory pieces of information here. The authors state that adenosine is released from cells upon inflammation and that CAPE treatment caused an increase in adenosine levels. Later in this section, the authors state that adenosine prevents TcdA-mediated damage and inflammation. This should be clarified and better discussed.

Thanks for your comments. Adenosine modulates immune responses and inflammatory cascades by interacting with its receptors, including its capacity to suppress the secretion of specific pro-inflammatory mediators. We have updated this depiction in the manuscript.

(15) Lines 513-514 - How was this phenotype quantified?

Thanks for your comments. Initially, we introduced TcdB at a final concentration of 0.2 ng/mL along with various concentrations of compounds into 1 mL of medium for a 1-h pre-incubation period. Subsequently, unbound compounds were removed through centrifugation, and the resulting mixture was then applied to the cells.

(16) Figure 3 - panels are labeled incorrectly.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(17) Figure 5C - it is unclear what the different colors and labels represent.

Thanks for your comments. In the depicted graph, blue denotes the total binding energy, red signifies the electrostatic interactions, green corresponds to the van der Waals forces, and orange indicates solvation or hydration effects. The horizontal axis represents the mutation of the amino acid residue at the respective position to alanine. As illustrated in Figure 5C, the mutations W520A and GTD exhibit the highest binding energies.

References:

(1) Janezic S, et al. Highly Divergent Clostridium difficile Strains Isolated from the Environment. PLoS One. 2016 Nov 23;11(11): e0167101.

(2) Perumalsamy S, Putsathit P, Riley TV. High prevalence of Clostridium difficile in soil, mulch and lawn samples from the grounds of Western Australian hospitals. Anaerobe. 2019 Dec; 60:102065.

(3) Sutton SS, et al. Melatonin as an Antimicrobial Adjuvant and Anti-Inflammatory for the Management of Recurrent Clostridioides difficile Infection. Antibiotics (Basel). 2022 Oct 25;11(11):1472.

Reviewer #2 (Recommendations for the authors):

Minor comments and questions.

(1) Which form of TcdB is being used in these experiments?

Thanks for your comments. The TcdB proteins used in this study are TcdB1 subtypes.

(2) Why are THP-1 cells being used in these assays?

Thanks for your comments. For the purposes of this study, we employed a diverse array of cell lines, including Vero, HeLa, THP-1, Caco-2, and HEK293T. Each cell line was selected to serve a specific experimental objective. The inclusion of the THP-1 cell line was necessitated by the need to incorporate a macrophage cell line to ensure the comprehensive nature of our experiments, allowing for the testing of both epithelial cells and macrophages. C. difficile is a kind of intestinal pathogenic bacteria, and immune clearance plays a vital role in the process of pathogen infection, so THP-1 cells are used as important immune cells.

(3) Please improve the quality of the microscopy images in Figure 1.

Thanks for your comments. We have improved the quality of the microscopy images in Figure 1.

(4) Does the flow cytometry experiment in Figure 2B show internalization? Surface-bound toxins would provide the same histogram.

Thanks for your comments. Figure 2B was employed to assess the internalization of TcdB, and the findings indicate that CAPE does not influence the internalization process of TcdB.

(5) The sensogram in Figure 4A does not look typical and should be clarified.

Thanks for your comments. Typically, small molecules and proteins engage in a rapid binding and dissociation dynamic. However, as depicted in Figure 4A, the interaction between CAPE and TcdB demonstrates a gradual progression towards equilibrium. This behavior can be primarily explained by the swift occupation of the protein's primary binding sites by the small molecule in the initial stages. Subsequently, CAPE binds to secondary or lower affinity sites, extending the time needed to reach equilibrium. Additionally, the likelihood of CAPE binding to multiple sites on TcdB requires time for the exploration and occupation of these diverse locations before equilibrium is attained, we have incorporated an analysis of this potential scenario into the manuscript.

Reviewer #3 (Recommendations for the authors):

These are my suggestions for the text:

(1) Line 29: high recurrent rates.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(2) Line 32: Where is the caffeic acid identified? I think a line should be included.

Thanks for your comments. Caffeic acid was identified from natural compounds library and we have completed the corresponding modifications according to the suggestions.

(3) Line 39: C. difficile is not italic.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(4) Line 41: Bacteroides spp.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(5) Line 56: This number of casualties 56.000 is still happening or it was in the past?

Thanks for your comments. The mortality rates reported in the manuscript reflect a downturn in the incidence and fatality of CDI around 2017(1), as the infection gained broader recognition. Nonetheless, a recent study reveals that the mortality rate for CDI cases in Germany can soar to 45.7% within a year, with the overall economic burden amounting to approximately 1.6 billion euros. This underscores the ongoing significance of CDI as a global public health challenge(2).

(6) Line 104: Where did the idea of testing caffeic acid come from? Any previous study of the authors? Any studies with the inhibition of other pathogens?

Thanks for your comments. Initially, we conducted a screen of a compound library comprising 2,076 compounds and identified several potent inhibitors, which, upon structural analysis, were revealed to be caffeic acid derivatives. Prior to our investigation, no studies had explored the potential of CAPE in this context.

(7) Line 115: Bacteroides spp.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

Results section

(8) Did the authors try the caffeic acid with the TcdA or binary toxin? I know this is not the purpose of the study, but TcdA toxin has a high identity structure with TcdB and generates inflammation in the gut via neutrophils. Negative strains for the major toxins and positive for the binary toxin also cause severe cases of CDI.

Thanks for your comments. Although we acknowledge the significance of TcdA and binary toxins in CDI, we did not investigate the impact of CAPE on these toxins. Our focus was exclusively on the effect of CAPE against TcdB, as it is the primary virulence factor in C. difficile pathogenesis. Since TcdA and TcdB are highly similar in structure, we will analyze the neutralization effect of CAPE on TcdA in later studies.

(9) Does caffeic acid have any effect on C. difficle? Or does it only gain the toxins? That would be ideal.

Thanks for your comments. We have included additional related assays in our study. Beyond directly neutralizing TcdB, CAPE also demonstrates the capacity to inhibit the growth and spore formation of C. difficile.

(10) Line 230: C. difficile BAA-1870 is a clinical strain? There are no details about it in the paper.

Thanks for your comments. C. difficile BAA-1870 (RT027/ST1), a highly virulent isolate frequently employed in research(3-6), was kindly donated by Professor Aiwu Wu. We have meticulously noted the PCR ribotype in our manuscript.

(11) Line 236: Did the mice fully recover from CDI after the administration of the CAPE? Was one dose enough?

Thanks for your comments. CAPE was administered orally at 24 h intervals, commencing with the initial dose on Day 0. By the time a significant difference was observed on Day 3, the treatment had been administered a total of three times.

Methodology

(12) Most of the methods do not have a reference.

Thanks for your comments. We have added several references to the methods.

Discussion section

(13) The first two paragraphs of the discussion should be summarized. Those details were already explained in the introduction.

Thanks for your comments. The discussion section and the introduction address slightly different focal points; therefore, we aim to retain the first two paragraphs to maintain continuity and context.

(14) Line 382: Bezolotoxumab was approved by the FDA in 2016. It is not recent.

Thanks for your comments. We have revised the above description.

(15) Line 410: "Despite the high 410 cure rate and increasing popularity of FMT, its safety remains controversial. Although this is true, recently (2022) the FDA approved the Rebyota, which was later cited by the authors.

Thanks for your comments. We have revised the above description.

(16) Lines 415-416: "the abundance of Bacteroides, a critical gut microbiota component that is required for C. difficile resistance". There is only one reference cited by the authors. I suppose that if it is true, more studies should be mentioned. Why are probiotics with Bacteroides spp. not available in the market?

Thanks for your comments. We have supplemented additional references. The scarcity of probiotic products containing Bacteroides spp. on the market is primarily attributable to the stringent requirements of their survival conditions. As most Bacteroides spp. are anaerobic, they thrive in oxygen-deprived environments. This unique survival trait poses challenges in maintaining their viability during product preservation and distribution, which in turn escalates production costs and complexity. Furthermore, despite the significant role of Bacteroides in gut health, research into its potential probiotic benefits and safety is comparatively underexplored.

References:

(1) Guh AY, et al. Emerging Infections Program Clostridioides difficile Infection Working Group. Trends in U.S. Burden of Clostridioides difficile Infection and Outcomes. N Engl J Med. 2020 Apr 2;382(14):1320-1330.

(2) Schley K, et al. Costs and Outcomes of Clostridioides difficile Infections in Germany: A Retrospective Health Claims Data Analysis. Infect Dis Ther. 2024 Nov 20.

(3) Saito R, et al. Hypervirulent clade 2, ribotype 019/sequence type 67 Clostridioides difficile strain from Japan. Gut Pathog. 2019 Nov 4; 11:54.

(4) Pellissery AJ, Vinayamohan PG, Venkitanarayanan K. In vitro antivirulence activity of baicalin against Clostridioides difficile. J Med Microbiol. 2020 Apr;69(4):631-639.

(5) Shao X, et al. Chemical Space Exploration around Thieno[3,2-d]pyrimidin-4(3H)-one Scaffold Led to a Novel Class of Highly Active Clostridium difficile Inhibitors. J Med Chem. 2019 Nov 14;62(21):9772-9791.

(6) Mooyottu S, Flock G, Venkitanarayanan K. Carvacrol reduces Clostridium difficile sporulation and spore outgrowth in vitro. J Med Microbiol. 2017 Aug;66(8):1229-1234.