Adaptation of the infant gut microbiome during the complementary feeding transition

Starin McKeen; Nicole C. Roy; Jane Adair Mullaney; Hannah Eriksen; Amy Lovell; Martin Kussman; Wayne Young; Karl Fraser; Clare R. Wall; Warren C. McNabb

doi:10.1371/journal.pone.0270213

Peer Review History

Original SubmissionAugust 6, 2021
6 Dec 2021 Decision Letter - yinglin xia, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-21-23857Adaptation of the infant gut microbiome during the complementary feeding transitionPLOS ONE Dear Professor McNabb, 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. This is an interesting study. However, this longitudinal and multiple omics study was designed with small sample sizes and the data was analyzed using some inappropriate models or methods. After reading this manuscript I agree with these two reviewers' comments on its limitations of design and methods used. 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We will update your Data Availability statement on your behalf to reflect the information you provide. 7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Additional Editor Comments: 1. This is longitudinal study design. However cross-sectional models were used for almost analyses, Such as the t-test between sampling time points for sequences. This is not correct. 2. Was the data tested for normality before choosing tests or models? 3. Levels 1 to 4 KEGG were analyzed. More information about these levels of KEGG such as what they are about should be described to improve readability. 4. Since this is clinical trial, did the study design was based on power analysis? In other words, the used samples sizes were based on power calculations? How many percentage of power for this study? What model or test was used for the power calculation? 5. Table 1. Taxonomic compositional changes over time at phylum level are not meaningful. The taxa at the lower levels such as genus or species are required to be analyzed. 6. The microbiome data were normalized using the total sum scaling. However, the total sum scaling is the simplest normalization method and was evaluated by several studies as not optimal for microbiome data. [Note: HTML markup is below. Please do not edit.] 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: No Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: No ****** 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: Yes ****** 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: Yes ****** 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: Major Questions/Comments: 1. In Fig. 1, it shows clearly n=40 infants were randomized by sex (as stated in line 101) into goups of n=30 and n=10. Thus this will lead to a higly unbalanced comparison. In addition, the final comparison is based on (valid data of) n=20 versus n=5. Personally I think it's hard to draw reasonal statistical conclusions based on n=5. 2. Correlation cutoffs 0.6/0.5 were used for KEGG pathway/species correlations (as mentioned in line 215/216). I understand different cutoffs for different correlations could be used. But some rationale are expected to be mentioned, i.e., why to use 0.6/0.5 as the cutoffs for KEGG pathway/species correlations? Minor Comments: 1. Extra "is a" at line 73. 2. At line 577, do we mean "Small and insignificant (FDR corrected p-value >0.05) appreant...", i.e., should be 0.05 instead of 0.5 for the "cutoff" for p-values? Reviewer #2: 1. Sample size/power calculation should be performed when design the study. If not. clarify this is a pilot study. 2. For a small sample size, it is hard to determine whether the data follows a normal distribution. Nonparametric method may be considered (e.g. Spearman correlation and Friedman test). Or consider data transformation. 3. Little details were discussed for the two intervention groups. It does not serve the research aims and no comparisons between the intervention were performed. 4. Fig 3AB. What method was used for the confidence bands? 5. Fig 3 CD. T-test is not appropriate. Consider PERMANOVA/Friedman test. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. 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 #1: No Reviewer #2: 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. https://doi.org/10.1371/journal.pone.0270213.r001
Revision 1
17 May 2022 Author Response Thank you for your helpful comments on our manuscript. 1. In line with the journal requirements, we have amended the manuscript to comply with PLOS ONE style from the templates indicated at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Have updated our methods and results sections 3. Have made sure the grant and funding information match 4. Have completed the competing interests 5. Have made the data available as additional supplementary tables https://mfr.osf.io/render?url=https%3A%2F%2Fosf.io%2F3zfre%2Fdownload 6. See 5. 7. Included captions for supporting information files at the end of the manuscript and updated in text citations to match accordingly. Our response to the comments from the reviewers 1. This pilot study was a longitudinal design and multivariate analyses were applied and when ANOVA analysis identified significance, post-hoc testing was done, which were the ‘T tests’ referred to. It is appropriate to apply post-hoc testing if ANOVA finds significance. 2. We applied a permutation ANOVA to the data. Permutation ANOVA is a test that does not require gaussianity. It is therefore robust and resistant to skewed distribution of the data and does not need normality tests prior. 3. We have added more information about the KEGG levels to the manuscript line 140-143. 4. There was no power analysis as this reports the findings from a pilot study which had the purpose of seeing whether this type of study involving infants and a dietary intervention were feasible. There was no primary endpoint and the two groups were either those who included a probiotic or those who added NZ Kumara to the diet of the infants. 5. Taxonomic compositional changes over time at phylum level are meaningful when dealing with an infant undergoing weaning. We know that profound changes to the microbiome occur during the period of introduction to solids. We found that there is a large shift towards taxa of the Firmicutes phylum and away from taxa from the Actinobobacteria phyla when the diet was expanded to include solids. 6. We agree that we could have used alternative normalization strategies for microbiome data. However, infants have a unique situation that they are changing rapidly as they grow and one of the challenges that we hoped to overcome was the ability to measure changes over time that were not confounded by the physical changes in environment space also expanding. The microbiome increases with the infant growth and so we found that for this study, normalization through total sum scaling and then converting to abundance, allowed us to compare the same infant across time. In response to whether we describe a technically sound piece of scientific research: The study we report on was designed as a pilot to see what challenges and difficulties there might be in an infant clinical trial were we to use a dietary intervention. For a pilot study, there was no clinical endpoint measured and there was no power analysis required. We have endeavoured to better explain the reasons and rationale for the methods used and the conclusions made. We did not carry out T tests as such, but post-hoc tests from ANOVA if the results of this indicated that there was a significant (P<0.05) difference between groups. Permutation ANOVA also is a robust method not requiring the test for normality of data. We used total sum scaling and abundance because of the count changes in the number of bacteria enumerated as the infants aged from 3-12 months. We used total sum scaled taxonomy and metagenome datasets, and log-transformed metabolome data also because these methods maintain positive values, which is critical when assessing the direction of change in abundance or concentration. 1. In Fig. 1, it shows clearly n=40 infants were randomized by sex (as stated in line 101) into goups of n=30 and n=10. Thus this will lead to a higly unbalanced comparison. In addition, the final comparison is based on (valid data of) n=20 versus n=5. Personally I think it's hard to draw reasonal statistical conclusions based on n=5. Response: We acknowledge that this was a pilot study that had 40 recruited (also minimal numbers) and then we were only able to use 25. We agree with the reviewer; it is hard to draw statistical conclusions on n=5. As a pilot study we can report the results we do have so long as we do not overinterpret the results. 2. Correlation cutoffs 0.6/0.5 were used for KEGG pathway/species correlations (as mentioned in line 215/216). I understand different cutoffs for different correlations could be used. But some rationale are expected to be mentioned, i.e., why to use 0.6/0.5 as the cutoffs for KEGG pathway/species correlations? Response: We advise that we used a 0.6 cut off for the KEGG pathway correlations because the networks identified are presumed to be a stronger correlation. The 0.5 cut off was used for the species correlations to help identify potential pathways for further investigation. Minor Comments: 1. Extra "is a" at line 73. Response: corrected 2. At line 577, do we mean "Small and insignificant (FDR corrected p-value >0.05) appreant...", i.e., should be 0.05 instead of 0.5 for the "cutoff" for p-values? Response: Correct it should say 0.05 and we have amended the manuscript. Reviewer #2: 1. Sample size/power calculation should be performed when design the study. If not. clarify this is a pilot study. Response: We confirm that it is a pilot study as mentioned in the abstract and described at line 69 where we wrote “This is a secondary analysis of a pilot study. The pilot study was designed to evaluate the methodologies required for a randomized control trial (RCT).” 3. For a small sample size, it is hard to determine whether the data follows a normal distribution. Nonparametric method may be considered (e.g. Spearman correlation and Friedman test). Or consider data transformation. Response: Permutation ANOVAs were carried out and they use non-parametric methods. 4. Little details were discussed for the two intervention groups. It does not serve the research aims and no comparisons between the intervention were performed. Response: This is correct as it was a pilot study. We did not find any statistically significant differences between the dietary groups and so we reported that changes over time were the key findings. 5. Fig 3AB. What method was used for the confidence bands? Response: These are not confidence bands. The wording has been amended to describe this figure better. They indicate minimum-maximum values. 6. Fig 3 CD. T-test is not appropriate. Consider PERMANOVA/Friedman test. Response: Post-hoc testing was carried out only after ANOVA identified significant differences between the groups. We have amended the wording to reflect this. Attachments Attachment Submitted filename: response to reviewers NR .docx https://doi.org/10.1371/journal.pone.0270213.r002
7 Jun 2022 Decision Letter - yinglin xia, Editor Adaptation of the infant gut microbiome during the complementary feeding transition PONE-D-21-23857R1 Dear Dr. Mullaney, 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. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, yinglin xia, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0270213.r003
Formally Accepted
20 Jun 2022 Acceptance Letter - yinglin xia, Editor PONE-D-21-23857R1 Adaptation of the infant gut microbiome during the complementary feeding transition Dear Dr. Mullaney: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. yinglin xia Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0270213.r004

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