Contribution of Inhibitory Metabolites and Competition for Nutrients to Colonization Resistance against Clostridioides difficile by Commensal Clostridium

Round 1

Reviewer 1 Report

This review is very important to understand mechanisms of action C, difficile and other Clostridium spp. of the gut microbiota. I was impressed by so many biochemical data and mechanisms described. This paper gives many questions to answer in the future, especially important for treatment of CDI and other enteric infections. For many years we use to used the term "colonization resistance" and try to understand how it works. These 2 authors explained many details in the mechanism of colonization resistance and also showed the way for future science in this field.

I recommend to publish this paper in Microorganisms.

Sincerely

Gayane Martirosian, M.D., Ph.D. Prof.

Department of Medical Microbiology

Medical University of Silesia in Katowice, Poland

e-mail: gmartir@sum.edu.pl

Comments for author File: Comments.docx

Author Response

We thank the reviewer for his/her positive comments and are glad the reviewer is satisfied with our work.

Reviewer 2 Report

The manuscript by Reed and Theriot is an insightful review by experts at the intersection between bile salts and C. difficile infection. There a thorough, yet engaging description of bile salt analysis and the biology and pathogenesis of C. difficile. There are a few issues that need to be addressed.

 

Major Criticisms:

There is no abstract

Please include one to two conceptual figures

 

Minor Criticisms

Line 50: “hydroxysteroid” is misspelled

Line 64: “shocked” is imprecise

Line 131: “Clostridium rarely encode bshs,” This should be "BSHs". Genes themselves are not encoded, it is proteins that are encoded by the genes.

Line 132: There is evidence for BSH activity, and a bsh gene in Clostridium hiranonis (PMID:17152920). Protein ID (QEK20610).

Line 225: The 3alpha-HSDH in VPI 12708 is baiA, which is specific for CoA and commits the bile acid to DCA formation. As used in the sentence, it is implied that this enzyme and organism is involved in epimerization to iso-derivatives with organisms like C. innocuum. However, the initial oxidation by BaiA is also reduced intracellularly by BaiA with a 3a-OH product produced. Mention of E. lenta or R. gnavus would be better here, as they are mentioned very soon after in the text.

Line 352: please correct “model of CDI, it the mice challenged”

Line 357 and thereafter: “of a commensal Clostridia” should either be “commensal clostridia” or “commensal Clostridium”. Please correct subsequent examples of this (as well as the title).

 

 

Author Response

The authors thank the reviewers for providing their time and expertise, author comments to the reviews are in bold.

Reviewer 2

Major Criticisms:

There is no abstract

Authors: An abstract has been written and included.

Please include one to two conceptual figures.

Authors: Two conceptual figures have been included. See Figures 1 and 2.

Minor Criticisms

Line 50: “hydroxysteroid” is misspelled

Authors: The misspelling has been corrected, thank you for the notation.

Line 64: “shocked” is imprecise

Authors: While shocked was the term used by the authors of the study referenced, the term has been changed to “exposed to” in the text.

Line 131: “Clostridium rarely encode bshs,” This should be "BSHs". Genes themselves are not encoded, it is proteins that are encoded by the genes.

Authors: The error has been corrected, thank you for the notation.

Line 132: There is evidence for BSH activity, and a bsh gene in Clostridium hiranonis (PMID:17152920). Protein ID (QEK20610).

Authors: The information that C. hiranonis has a bsh has been included in the manuscript. See below:

“…although C. hiranonis and the pathogen Clostridium perfringens both encode BSHs and have demonstrated BSH activity [21,44]. C. hiranonis is the only bacterium to date known to have the capability for both 7α-dehydroxylation and deconjugation[44].”

Line 225: The 3alpha-HSDH in VPI 12708 is baiA, which is specific for CoA and commits the bile acid to DCA formation. As used in the sentence, it is implied that this enzyme and organism is involved in epimerization to iso-derivatives with organisms like C. innocuum. However, the initial oxidation by BaiA is also reduced intracellularly by BaiA with a 3a-OH product produced. Mention of E. lenta or R. gnavus would be better here, as they are mentioned very soon after in the text.

Authors: As suggested, the mention of VPI 12708 has been changed to R. gnavus to remove the implication that the 3alpha-HSDH in VPI 12708 is involved in epimerization.

Line 352: please correct “model of CDI, it the mice challenged”

Authors: The error has been corrected, thank you for the notation. The line now reads “…model of CDI, the mice challenged”

Line 357 and thereafter: “of a commensal Clostridia” should either be “commensal clostridia” or “commensal Clostridium”. Please correct subsequent examples of this (as well as the title).

Authors: All examples of this were found and corrected.

Reviewer 3 Report

The authors did a nice job of summarizing the current understanding of the role of bile acid and other metabolites produced primarily by commensal Clostridia in inhibiting the growth of Clostridioides difficile. I have no major suggestions for revisions. Minor corrections/suggestions are listed below:

Line 94-95. This sentence should be re-written to be more accessible to the general reader. Specifically, you should indicate that LCA and CDCA inhibit rounding of human fibroblasts. (As written its unclear whether host cells or C. difficilecells are rounding.)

Lines 104-107: This sentence should be re-stated in light of your own work that demonstrated that C. difficile can germinate in the presence of small intestinal contents from non-antibiotic treated mice ex vivo (Koenigsknect et al, DOI: 10.1128/IAI.02768-14)

Lines 145-163: You should also comment upon how deconjugation affects solubility and bioavailability of bile acids.

Line 233/239: phrase “as a toxicity reducer” is awkward, use “to reduce toxicity”

Line 246: “bile acids produced by commensal Clostridia” should be re-written, perhaps “bile acids modified by commensal Clostridia” as it is modification rather than de novo bile acid synthesis that is occuring.

Lines 258-270 should also cite your work on modulation of innate immune response (Winston et al, DOI: 10.1128/IAI.00045-20)

Line 329 – Section header “Competition of nutrients” should be “Competition for nutrients”

Line 336 – “The RT027 strain gained a point mutation” is awkward phrasing, probably better to restate that “In the RT027 strain, a point mutation occurred”

Line 352 – “it the mice” should just be “the mice”

 

Author Response

The authors thank the reviewers for providing their time and expertise, author comments to the reviews are in bold.

Reviewer 3

Minor criticisms:

Line 94-95. This sentence should be re-written to be more accessible to the general reader. Specifically, you should indicate that LCA and CDCA inhibit rounding of human fibroblasts. (As written its unclear whether host cells or C. difficile cells are rounding.)

Authors: The sentence was split into two to make it less confusing to read, and the rounding of the fibroblast cells (as opposed to C. difficile cells) was clarified. See below for revised version.

“LCA and CDCA are able to bind to TcdB with high efficiency and they are able to inhibit cell rounding, a sign of cell death, in human fibroblast cells[31]. DCA binds to TcdB with lower efficiency than LCA and CDCA, and does not inhibit cell rounding in human fibroblasts [31].”

Lines 104-107: This sentence should be re-stated in light of your own work that demonstrated that C. difficile can germinate in the presence of small intestinal contents from non-antibiotic treated mice ex vivo (Koenigsknect et al, DOI: 10.1128/IAI.02768-14)

Authors: The sentence was expanded to clarify that while cecal contents from non-antibiotic treated mice inhibit germination of C. difficile spores, small intestinal contents do not. See below for revised version.

 “Cecal extracts from mice made susceptible to CDI stimulate C. difficile spore germination, while cecal extracts from mice resistant to CDI inhibit spore germination, indicating that antibiotic induced changes in bile acid levels in vivo are sufficient to induce germination and outgrowth of C. difficile spores [36,37]. However, C. difficile spores are able germinate in the small intestine prior to antibiotics, indicating that the bile acids present in the small intestine do not protect against CDI[37].”

Lines 145-163: You should also comment upon how deconjugation affects solubility and bioavailability of bile acids.

Authors: Information about the effect of deconjugation on solubility and bioavailability of bile acids and how this may affect toxicity was added, see below.

“…an increase in toxicity for at least some members of the gut microbiota [53,54]. Conjugated bile acids are more soluble than deconjugated bile acids, so the increased toxicity observed may be offset by the decreased bioavailability that occurs when micelles form[55,56].”

Line 233/239: phrase “as a toxicity reducer” is awkward, use “to reduce toxicity”

Authors: The phrasing has been changed as suggested in both instances.

Line 246: “bile acids produced by commensal Clostridia” should be re-written, perhaps “bile acids modified by commensal Clostridia” as it is modification rather than de novo bile acid synthesis that is occuring.

Authors: The phrasing has been changed as suggested to increase clarity.

Lines 258-270 should also cite your work on modulation of innate immune response (Winston et al, DOI: 10.1128/IAI.00045-20)

Authors: The relevant work has been cited, see below.

“The levels of bile acids can also affect FXR receptor expression, as giving mice exogenous UCDA increases the expression of TGR5 and FXR, causing alterations to the bile acid metabolome[43].”

Line 329 – Section header “Competition of nutrients” should be “Competition for nutrients”

Authors: The phrasing has been corrected, thank you for the notation

Line 336 – “The RT027 strain gained a point mutation” is awkward phrasing, probably better to restate that “In the RT027 strain, a point mutation occurred”

Authors: The phrasing has been corrected, thank you for the notation

Line 352 – “it the mice” should just be “the mice”

Authors: The error has been corrected, thank you for the notation. The line now reads “…model of CDI, the mice challenged”