PONE-D-21-19140

Gut microbiome affects the metabolism of metronidazole in mice through regulation of hepatic cytochromes P450 expression

PLOS ONE

Dear Dr. Jourová,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The reviewers pointed to information missing in the Materials and Methods section (e.g., Western blots should be presented following the PLOSone reporting requirements, etc.), potentially inappropriate statistical analyses, and an overinterpretation of the data (e.g., lack of consideration of other drug metabolizing enzymes).  I also concur with the suggestions from both reviewers regarding the presentation of the data. 

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript has used germ free (GF) and specific pathogen free (SPF) to demonstrate that the absence of gut microbiota appeared to result in a temporal trend of increase (although not statistically significant) in the plasma metronidazole 2 hours after oral administration, but not at other time points. The hydroxylated metabolite determined was not changed. In addition, this study demonstrated that oral metronidazole the mRNA expression of selected P450s (CYP1-3) and xenobiotic-sensing transcription factors to a different extent between SPF and GF mice, although common patterns were also observed such as an up-regulation of Cyp2b10 by metronidazole in both mouse groups, over the time course. In contrast, the protein and enzyme activities were not readily altered significantly, although certain trend of increase or decrease was observed.

Major points:

1. Figure 2 – Figure 5: it is strongly recommended that the SPF and GF data should be plotted in the same figure, so one can evaluate the basal differences between SPF and GF. If this results in too many bars in one figure, the plots can be split per time point. This is important because it is highly possible that the tendency in the change in the metronidazole pharmacokinetics is not due to the differential response patterns of the P450s to metronidazole treatment between SPF and GF, but simply due to the basal differences in these enzymes between SPF and GF.

2. Please show the western blot result as the actual immuno-blots (currently Table 1), so that one can evaluate the specificity of the bands, sample size, and biological variations. Please include the standard deviations or standard errors in Table 1.

3. Method section: The RT-qPCR normalization method claimed that the data were expressed as ddCq and normalized to the Hprt house-keeping gene. However, It looks like Figure 3 and Figure 4 actually artificially assigned both SPF control and GF control to 1. This might not be the best way to express the data because the SPF control may be different from the GF control. Please consider re-plotting the data so it matches the Method section.

4. Method section: statistical method: one-way ANOVA was performed. However, this study design has two factors, namely enterotype (SPF vs. GF) and treatment (control vs. metronidazole over a time course). Thus two-way ANOVA is a more appropriate method.

5. From the plasma metronidazole result (Figure 2A), I think the 2h time point is the most critical for evaluation regarding mRNA expression, protein levels, and enzyme activities. Please especially take a look into this time point to connect the Figure 2A result with the other figures.

6. Figure 2A and 2B: because the hydroxylated metabolite did not decrease to a similar extent as the apparent increase of the parent compound, it is possible that this may be a disposition issue. Please consider measuring hepatic metronidazole and its metabolite (and maybe even urine). Also one needs to consider transporters and Ugts in addition to the P450s.

7. Introduction section says CYP2A6 in humans is the most predominant enzyme for metronidazole metabolism. However the mouse data on the Cyp2a isoforms do not seem to explain the trend of increase in plasma metronidazole at 2 hours; in fact there was a clear trend of decrease in CYP2A enzyme activities to a greater extent in SPF mice than in GF mice (opposite to the expectations) at 2h. The mRNA increase in Cyp2a was only observed after 6h and is not likely responsible for the increase in plasma metronidazole at 2h. Would other P450 isoforms be responsible for the mouse metabolism of this drug? Please discuss based on your findings.

Minor points:

1. Method section: Western blot: please include the catalog numbers of the antibodies.

2. Method section: please define the age, sex, and sample size of the mice.

3. Abstract: please be more specific in describing the key findings on mRNA, protein, and enzyme activities.

4. Title: it seems that the study was mainly determining the effect of the absence of gut microbiome on the hepatic P450s, not necessarily the effect of the gut microbiome on the hepatic P450s.

5. Typo: “could not be determinate.” → “ could not be determined.”

Reviewer #2: Manuscript ID: PONE-D-21-19140

Title: Gut microbiome affects the metabolism of metronidazole in mice through regulation of hepatic cytochromes P450 expression

The manuscript described the effect of gut microbiome on the fate of metronidazole by profiling the metronidazole’s pharmacokinetics changes (Cmax and metabolic ratio). And the selected P450 enzymes related changes (mRNA expression, protein expression and enzyme activity) were compared in both SPF and germ-free (GF) mice after administration metronidazole within 24 hours. And the results provided the possibility of the selected hepatic P450 enzymes altered metronidazole metabolism.

1. There were some wording issues e.g. CYP (line171) and one cited literature [36] information about metronidazole has not been found within the mentioned literature content.

2. The structure and enzymatic information that mentioned in the introduction section was not clearly marked in the Figure 1.

3. Some antibiotics mechanism that mentioned in the manuscript (Introduction section) has been summarized in the related review [1] and should be mentioned in the introduction.

4. A lot of details of Section ‘cytochrome P450 enzyme activity assays’ were missing. Such as the particle size of column; the UV/ fluorescence detection parameters, and the gradient ranges.

5. The metabolic ratios were mentioned with only the values (line 234-238). Did the metabolic ratio calculate individually? What is the mean and SD for the metabolic ratio?

6. Could the Figure 3-5 changed into a box plot with individual values plus mean and SD to facilitate the data trend observation?

7. Literature has reported that co-dosing metronidazole with neomycin, vancomycin, and ampicillin for 3 weeks could obtain pseudo germ-free mice [2]. Could the short-term treatment with metronidazole in SPF mice regulate similar metabolic pathway? If so, did the comparison between SPF mice and GF mice after administration metronidazole describe the difference between pseudo germ-free mice versus germ-free mice metabolic pathway?

8. The pharmacokinetic data showed in Figure 2 have a clear overlap in Cmax value. Especially, the SD of the GF mice Cmax approached the SPF mice Cmax mean value. Was there a possibility that there is an outlier plasma concentration within the limited sample numbers? Could the individual Cmax values be attached as supplementary data?

9. Besides the described results, were there any RNA-seq screening data and/or proteomics data results support the selected metabolic enzyme regulation? Or does there any other metabolic enzyme also significantly involved into the metronidazole metabolism?

Reference

[1] Leitsch D. A review on metronidazole: an old warhorse in antimicrobial chemotherapy. Parasitology. 2019 Aug;146(9):1167-78.

[2] Liang W, Zhao L, Zhang J, Fang X, Zhong Q, Liao Z, Wang J, Guo Y, Liang H, Wang L. Colonization Potential to Reconstitute a Microbe Community in Pseudo Germ-Free Mice After Fecal Microbe Transplant From Equol Producer. Frontiers in Microbiology. 2020 Jun 5;11:1221.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Reviewer comments plos one P450 metronidazole.docx

Attachment

Submitted filename: Reviewer response.docx

PONE-D-21-19140R1Gut microbiome affects the metabolism of metronidazole in mice through regulation of hepatic cytochromes P450 expressionPLOS ONE

Dear Dr. Jourová,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.  Some major concerns must be addressed.

Please submit your revised manuscript within 60 days. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Gianfranco D. Alpini

Academic Editor

PLOS ONE

Journal Requirements:

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: (No Response)

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: (No Response)

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: (No Response)

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: (No Response)

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

Reviewer #3: In the revised version of the manuscript, the authors have responded some comments and added some new data. However, there are still several concerns should be addressed before it can be considered for publication.

1. Better and individual animal western blot bands are requested.

2. Figures should be arranged in order with results.

3. Some results and Figures (order) are not match.

4. Missing X-axis in Figure 4 C and G.

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Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Gut microbiome affects the metabolism of metronidazole in mice through regulation of hepatic cytochromes P450 expression

PONE-D-21-19140R2

Dear Dr. Jourová,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Gianfranco D. Alpini

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: The authors addressed all of my previous concerns and I have no more comments or issues regarding this manuscript.

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No