An individualized mosaic of maternal microbial strains is transmitted to the infant gut microbial community

To understand the origins of the infant gut microbial community, we have used a published metagenomic dataset of the faecal microbiome of mothers and their related infants at early (4, 7 and 21 days) and late times (6–15 months) following birth. Using strain-tracking analysis, individual-specific patterns of microbial strain sharing were found between mothers and infants following vaginal birth. Overall, three mother–infant pairs showed only related strains, while 12 infants of mother–infant pairs contained a mosaic of maternal-related and unrelated microbes. Analysis of a second dataset from nine women taken at different times of pregnancy revealed individual-specific faecal microbial strain variation that occurred in seven women. To model transmission in the absence of environmental microbes, we analysed the microbial strain transmission to F1 progenies of human faecal transplanted gnotobiotic mice bred with gnotobiotic males. Strain-tracking analysis of five different dams and their F1 progeny revealed both related and unrelated microbial strains in the mother's faeces. The results of our analysis demonstrate that multiple strains of maternal microbes, some that are not abundant in the maternal faecal community, can be transmitted during birth to establish a diverse infant gut microbial community.


Introduction
Line 61-64: Incomplete sentence Line 64-65: "Next-generation Sequencing" does not need have the capital letters.

Materials and Methods
Line 91: When were the feces samples collected from the mothers?
Line 94 or Table S1: Where is information for the 6 mother-infants from the caesarean part of the analysis?
Line 125: What happened to Donor 5 feces? Also not reported in Table S1. Table S1 -There are multiple samples from the female gnotobiotic mice post FMT with Donor 4 and Donor 5. What is the difference between these samples and were they all used when making the comparisons? It does appear the samples were merged.
Line 143: Once again, when was the mother's sample obtained?
Line 144: Was the mother's sample only compared to her infant's samples? Please clarify as when I first read this it says "any available infant samples". Upon further reading, I am guessing each mother's sample was only compared with her infant's samples.
Line 168: The authors are commended for providing the resource/link that allows for reproduction of the analyses.
Line 169, Table S2 has more than 20. Please change title to reflect the number of species reported in table. And are these the top 41 most abundant species?
Line 212: Change in font (actually there are changes in the font in several places across the manuscript). Table S3: If the comparison was only conducted between each mother and her infant, please change title to reflect that, e.g. "between each mother vs her infant". If this is not the case, then more detail is needed to describe that this reflects a mother vs all infants. Table S3: Row 2 -should this be "Fecal sample from infant"? Table S3: Includes 23 species. Table S3: What does a "NA" mean for the CO? What is the cutoff value used when CO is NA?
Line 213 -215: This sentence that describes the top 20 in Table S2 seems like it should be reported first. And then move to reporting Table S3.
Line 216-217: Once again, first sentence of paragraph seems redundant.  Table S3 and Figure 1, there seem to be several inconsistencies. For example, For Bacteroides sp 1-1-6, shouldn't the boxes for B01948, B02273, and C01917 also include blue? For Bacteroides 2 1 16, shouldn't C01994 also include blue? Perhaps you can separate the box into triangles to display two colors.
Line 220: The authors report 13 of 16 pairs had B. vulgatus. However, only 9 of 13 share related strains (as defined by > CO threshold). Perhaps the authors can further clarify that for the reader as well since the focus of the paper seems to be on the relatedness of the species. Along with that, I think having just a bit more information either in the introduction or in the materials and methods about the WSS method and the setting of the CO threshold would be helpful to the reader so they do not have to go back and find this information through reading of the references.
Line 222 -224: The authors make special note of Akkermansia muciniphila, Barnesiella intestinihominis, and Faecalibacterium prausnitzii. These seem odd to point out. And if pointing out those that are not related, why not also include Eubacterium hallii and Ruminococcus?
Line 224-225: This sentence seems very much like discussion.
Line 227-230: If using the red font as indicator of being related or not, I count 2 related between D4-21 and not at infancy, 2 that had unrelated at between D4-21 but related at infancy, 5 that had related between D4-21 and at infancy and 4 unrelated at any time point. Please address the discrepancy or where I am perhaps mistaken.
Line 231: I interpret Figure 1 as showing that C01917 mother-infant pair only shared 5 of the bacterial strains; not all detected microbial strains.
Line 236: Which 6 cases agree? Please detail for the reader.
Line 239-242: This is not described in Materials and Methods. Please add this. Table S8: Should row 2 be "Fecal sample from infant"? Table S8: Why are there only 9 species reported on?
Line 248: The authors should also discuss the limitation that only 6 mother-infant pairs were used in the caesarean analyses.
Line 251-256: Once again, these sentences seem to more appropriate for the Discussion and not in the results section.
Line 257-258: Not sure how this supports the transfer of microbes to the infants since it is only comparing strains across pregnancy and does not then compare to what is found in her infant. It is a worthwhile description as to strains that remain stable or that are more transient in nature but I do not quite see the connection that demonstrates subsequent transfer to infants. Figure 3: Individual Pre4 has a green box for Bacteroides sp. 2-1-16 indicating no strain changes during any time points of pregnancy. However, this is misleading as there were no samples available for the other two time points (both are marked as "NA" in Table S4). Therefore, I would say it is unknown whether there were any strain changes or not. Perhaps I am not understanding this figure but I would also disagree with the green box for Term6 in the Bacteroides stercoris row. If there were no strain changes during any time points of pregnancy, wouldn't there be red numbers in Table S4 for this individual for that strain? I did not go through every box but please address these inconsistences and/or clarify in the text so readers don't perhaps make the same misinterpretation that I am making.
Line 261: Why is this important?
Line 264: Please provide which 5 cases agreed.
Line 269-271: Once again, this sentence seems more like discussion, at least from looking at other articles in the journal; but perhaps I am mistaken.
Line 271-278: This seems redundant to what is in the Materials and Methods and / or any detail included here should be added to the Materials and Methods section.
Line 277: "was" should be "were" Line 287: Results from the comparisons found in Tables S5-S6 are lacking. Figure 6 does do a nice job of pulling the results together, but perhaps more of the results can be presented or detailed in this section as well. Also please direct the readers to these supplemental tables in this section.
Line 288: would recommend amending the sentence to read "maternal transmission of bacteria to the F1s…" or specify transmission of what.
Line 289: Once again, in several places in the manuscript there is a font change.
Line 298: And also caesarean birth, correct?
Line 298-301: I find this sentence somewhat misleading as very few strains were observed in early life of the infant and maintained at infancy, at least according to Table S3 and Figure 1. Line 304-306: How does appearance of strains during pregnancy suggest changes in observed strains seen postpartum for the mother and during early life of the infant?
Line 326-332: I think the authors are missing a critical component, the diet of mother and infant, that most likely plays a key role in structuring fecal bacterial communities. This should be discussed.
Line 348: This reviewer appreciates the authors acknowledging caveats of the study. In particular, it is noted that sampling of the F1 progeny's feces was done at time of weaning. Please remind the readers as to when the dams were sampled as well. I think there would have been about a 7day difference between the two sampling, correct?

Figure legends
Line 580: What was the cutoff for "abundant species"?

Recommendation?
Accept as is

Comments to the Author(s)
The analyses are elegantly done, and although the results are not groundbreaking, they elegantly confirm that engraftment in new niches brings microbes that are not observed in the source, because they were in very low load (rare). My only observation is that the text of their manuscript has inconsistent fonts. I am sure this is fixable by the journal editors.

06-Feb-2020
Dear Ms Koo, The editors assigned to your paper ("An Individualized Mosaic of Maternal Microbial Strains are Transmitted to the Infant Gut Microbial Community") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
Please submit a copy of your revised paper before 29-Feb-2020. Please note that the revision deadline will expire at 00.00am on this date. If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance. We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available, we may invite new reviewers.
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• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch.
Kind regards, Andrew Dunn Royal Society Open Science Editorial Office Royal Society Open Science openscience@royalsociety.org on behalf of Dr Ulas Tezel (Associate Editor) and Kevin Padian (Subject Editor) openscience@royalsociety.org Editor comments: As you will see we have divergent reviews of your manuscript. One reviewer still has many comments to address. Please answer them assiduously. If you need more time, please notify the editorial office. Best wishes.
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) 2020 Koo Review Summary -Understanding the maternal and environmental origins of the infant gastrointestinal (GI) is an important field of study as short-and long-term health of the infant is associated with the infant's GI microbiome. In this manuscript, the authors utilize two publicly available data sets to investigate the transfer of specific strains between mother and infant. The authors also report on a gnotobiotic mouse study that examines transfer of strains between mother and F1 progeny. Key conclusions are that there is an individualized transfer of strains as not all strains are equally transferred between mothers and their infants (this was also reflected in the gnotobiotic mouse study), there are also many differences between the strains found in feces of a mother and her infant, and many strains are related during pregnancy.

Introduction
Line 61-64: Incomplete sentence Line 64-65: "Next-generation Sequencing" does not need have the capital letters.

Materials and Methods
Line 91: When were the feces samples collected from the mothers?
Line 94 or Table S1: Where is information for the 6 mother-infants from the caesarean part of the analysis?
Line 125: What happened to Donor 5 feces? Also not reported in Table S1. Table S1 -There are multiple samples from the female gnotobiotic mice post FMT with Donor 4 and Donor 5. What is the difference between these samples and were they all used when making the comparisons? It does appear the samples were merged.
Line 143: Once again, when was the mother's sample obtained?
Line 144: Was the mother's sample only compared to her infant's samples? Please clarify as when I first read this it says "any available infant samples". Upon further reading, I am guessing each mother's sample was only compared with her infant's samples.
Line 168: The authors are commended for providing the resource/link that allows for reproduction of the analyses.
Line 169, Table S2 has more than 20. Please change title to reflect the number of species reported in table. And are these the top 41 most abundant species?
Line 176-177: I do not think you need to list Tables S3-S6 here. Instead they should be listed where they are discussed in the Results.
Line 191: This sentence seems to be missing something. Perhaps a "which" before "included 'estimated number of reads…"?

Results
Line 204 -209: These sentences seem out of place here and are more discussion than results.
Line 209-212: Redundant with information in Materials and Methods.
Line 212: Change in font (actually there are changes in the font in several places across the manuscript). Table S3: If the comparison was only conducted between each mother and her infant, please change title to reflect that, e.g. "between each mother vs her infant". If this is not the case, then more detail is needed to describe that this reflects a mother vs all infants. Table S3: Row 2 -should this be "Fecal sample from infant"? Table S3: Includes 23 species. Table S3: What does a "NA" mean for the CO? What is the cutoff value used when CO is NA?
Line 213 -215: This sentence that describes the top 20 in Table S2 seems like it should be reported first. And then move to reporting Table S3.
Line 216-217: Once again, first sentence of paragraph seems redundant.  Table S3 and Figure 1, there seem to be several inconsistencies. For example, For Bacteroides sp 1-1-6, shouldn't the boxes for B01948, B02273, and C01917 also include blue? For Bacteroides 2 1 16, shouldn't C01994 also include blue? Perhaps you can separate the box into triangles to display two colors.
Line 220: The authors report 13 of 16 pairs had B. vulgatus. However, only 9 of 13 share related strains (as defined by > CO threshold). Perhaps the authors can further clarify that for the reader as well since the focus of the paper seems to be on the relatedness of the species. Along with that, I think having just a bit more information either in the introduction or in the materials and methods about the WSS method and the setting of the CO threshold would be helpful to the reader so they do not have to go back and find this information through reading of the references.
Line 222 -224: The authors make special note of Akkermansia muciniphila, Barnesiella intestinihominis, and Faecalibacterium prausnitzii. These seem odd to point out. And if pointing out those that are not related, why not also include Eubacterium hallii and Ruminococcus?
Line 224-225: This sentence seems very much like discussion.
Line 227-230: If using the red font as indicator of being related or not, I count 2 related between D4-21 and not at infancy, 2 that had unrelated at between D4-21 but related at infancy, 5 that had related between D4-21 and at infancy and 4 unrelated at any time point. Please address the discrepancy or where I am perhaps mistaken.
Line 231: I interpret Figure 1 as showing that C01917 mother-infant pair only shared 5 of the bacterial strains; not all detected microbial strains.
Line 236: Which 6 cases agree? Please detail for the reader.
Line 239-242: This is not described in Materials and Methods. Please add this. Table S8: Should row 2 be "Fecal sample from infant"? Table S8: Why are there only 9 species reported on?
Line 248: The authors should also discuss the limitation that only 6 mother-infant pairs were used in the caesarean analyses.
Line 251-256: Once again, these sentences seem to more appropriate for the Discussion and not in the results section.
Line 257-258: Not sure how this supports the transfer of microbes to the infants since it is only comparing strains across pregnancy and does not then compare to what is found in her infant. It is a worthwhile description as to strains that remain stable or that are more transient in nature but I do not quite see the connection that demonstrates subsequent transfer to infants. Figure 3: Individual Pre4 has a green box for Bacteroides sp. 2-1-16 indicating no strain changes during any time points of pregnancy. However, this is misleading as there were no samples available for the other two time points (both are marked as "NA" in Table S4). Therefore, I would say it is unknown whether there were any strain changes or not. Perhaps I am not understanding this figure but I would also disagree with the green box for Term6 in the Bacteroides stercoris row. If there were no strain changes during any time points of pregnancy, wouldn't there be red numbers in Table S4 for this individual for that strain? I did not go through every box but please address these inconsistences and/or clarify in the text so readers don't perhaps make the same misinterpretation that I am making.
Line 261: Why is this important?
Line 264: Please provide which 5 cases agreed.
Line 269-271: Once again, this sentence seems more like discussion, at least from looking at other articles in the journal; but perhaps I am mistaken.
Line 271-278: This seems redundant to what is in the Materials and Methods and / or any detail included here should be added to the Materials and Methods section.
Line 277: "was" should be "were" Line 287: Results from the comparisons found in Tables S5-S6 are lacking. Figure 6 does do a nice job of pulling the results together, but perhaps more of the results can be presented or detailed in this section as well. Also please direct the readers to these supplemental tables in this section.
Line 288: would recommend amending the sentence to read "maternal transmission of bacteria to the F1s…" or specify transmission of what.
Line 289: Once again, in several places in the manuscript there is a font change.
Line 298: And also caesarean birth, correct?
Line 298-301: I find this sentence somewhat misleading as very few strains were observed in early life of the infant and maintained at infancy, at least according to Table S3 and Figure 1.
Line 304-306: How does appearance of strains during pregnancy suggest changes in observed strains seen postpartum for the mother and during early life of the infant?
Line 326-332: I think the authors are missing a critical component, the diet of mother and infant, that most likely plays a key role in structuring fecal bacterial communities. This should be discussed.
Line 338: Should "from caesarean birth" instead be "following caesarean birth"? Line 348: This reviewer appreciates the authors acknowledging caveats of the study. In particular, it is noted that sampling of the F1 progeny's feces was done at time of weaning. Please remind the readers as to when the dams were sampled as well. I think there would have been about a 7day difference between the two sampling, correct?

Figure legends
Line 580: What was the cutoff for "abundant species"?
Reviewer: 2 Comments to the Author(s) The analyses are elegantly done, and although the results are not groundbreaking, they elegantly confirm that engraftment in new niches brings microbes that are not observed in the source, because they were in very low load (rare).
My only observation is that the text of their manuscript has inconsistent fonts. I am sure this is fixable by the journal editors.

24-Mar-2020
Dear Dr Koo, It is a pleasure to accept your manuscript entitled "An Individualized Mosaic of Maternal Microbial Strains are Transmitted to the Infant Gut Microbial Community" in its current form for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org) and the production office (openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact --if you are going to be away, please nominate a co-author (if available) to manage the proofing process, and ensure they are copied into your email to the journal. Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Please see the Royal Society Publishing guidance on how you may share your accepted author manuscript at https://royalsociety.org/journals/ethics-policies/media-embargo/.
Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. We are returning the revised manuscript "An Individualized Mosaic of Maternal Microbial Strains are Transmitted to the Infant Gut Microbial Community" for consideration in Open Science. As per instruction, we have included a marked up version of the manuscript (changes are noted in red fonts), a clean revised version of the manuscript, and response to referees with notations to the line changes that correspond to both versions of the manuscript.
Note that as requested by RSOS editorial office that we have provided BioProject accession number (PRJNA593263) (now publicly released) and provided the data accessibility statement as "Data and relevant code for this research work are stored in GitHub: https://github.com/hkoo87/mgSNP_2 and have been archived within the Zenodo repository: https://doi.org/10.5281/zenodo.3706776".
We have incorporated all of the Reviewer 1 comments (52 in total) although we note that in several instances there appeared to be a duplication of the comments. In the revised manuscript, we have not changed the overall emphasis or conclusions and, in our opinion, the modifications suggested by Reviewer 1 have improved the flow of the manuscript.
Specifically, we have revised the original Figure 1 to include Figure 1A and Figure  1B to clarify our analysis. The text in the Results for this figure has been modified to more clearly point out the related and unrelated microbial strains in the mother-infant pairs at different times post-birth. Figure 2 in the original version has now been placed in Supplement Figure 1 (S1 Figure). Figures 2 and 3 (Figures 3 and 4 in the original manuscript) have been modified to more clearly represent related and unrelated microbial strains during days of pregnancy. We have also modified Figure 5 (Figure 6 in the original manuscript) to present our data from the individual mother (dam) and her F1's. In addition, we have now included strain-tracking analysis of Donor 5 fecal samples that were missing in the original version of the paper. Lastly, we have accordingly revised the Supplementary Tables (S1 to S8) to clearly support the revised figures. As suggested by the RSOS editorial office, we have provided a ESM Legends file which includes titles and short descriptions for each supplementary table and figure.
We believe with the incorporation of the modifications the manuscript has been improved and will be of interest to the readers of Open Science. Summary -Understanding the maternal and environmental origins of the infant gastrointestinal (GI) is an important field of study as short-and long-term health of the infant is associated with the infant's GI microbiome. In this manuscript, the authors utilize two publicly available data sets to investigate the transfer of specific strains between mother and infant. The authors also report on a gnotobiotic mouse study that examines transfer of strains between mother and F1 progeny. Key conclusions are that there is an individualized transfer of strains as not all strains are equally transferred between mothers and their infants (this was also reflected in the gnotobiotic mouse study), there are also many differences between the strains found in feces of a mother and her infant, and many strains are related during pregnancy.

Overall response:
We thank Reviewer 1 for the very thorough review of the manuscript. We have made every effort to incorporate all of the reviewer's suggestions into the revised manuscript. In doing so, we believe the manuscript has been substantially improved.
In particular, we have revised the original Figure 1 to include Figure 1A and Figure 1B to clarify our analysis of the Zhao et al., data set. The text in the Results for this figure has been modified to more clearly point out the related and unrelated microbial strains in the mother-infant pairs at different times post-birth. Figure 2 in the original version has now been placed in Supplementary Figure 1 (S1 Figure). Figures 2  and 3 (Figures 3 and 4 in the original manuscript) have also been modified to more clearly represent related and unrelated microbial strains during days of pregnancy. In our opinion, these modifications have improved the flow of the manuscript.
In the revised version, we have also modified Figure 5 ( Figure 6 in the original manuscript) to present our data from the individual dam and her F1's. In addition, we have now included strain-tracking analysis of Donor 5 fecal samples that were missing in the original version of the paper. We have accordingly reviewed all Supplementary Tables (S1 to S8) to clearly support the revised figures. Please note that the number of Supplementary Tables (S4 to S8) are changed and correspond to the revised text.
Comment 1: Line 61-64: Incomplete sentence Response 1: As suggested by the reviewer, we have corrected sentences to the revised manuscript (Line numbers: 61-64).
Comment 2: Line 64-65: "Next-generation Sequencing" does not need have the capital letters. Response 2: We have changed the word from "Next-generation Sequencing" to "nextgeneration sequencing" in the revised manuscript (Line numbers: 64).

Comment 3:
Line 91: When were the feces samples collected from the mothers? Response 3: Please note that exact sample collection times for the mothers were not reported by Shao et al. However, we have included sample collection information for the mothers in the revised manuscript (Line numbers: 96-97) based on the information that Shao et al. provided in their study.
Comment 4: Line 94 or Table S1: Where is information for the 6 mother-infants from the caesarean part of the analysis? Response 4: Thank you for pointing it out. We have included the caesarean sample information (n=6) in the revised manuscript (Line numbers: 99-101) as well as Tables S1(A) and S2(A).
Comment 5: Line 125: What happened to Donor 5 feces? Also not reported in Table  S1. Response 5: Thank you for pointing it out. We have included Donor 5 information in the revised manuscript (Line number: 113, 128, 604-607) as well as Table S1(C). We have also revised the Tables S2(C), S6, S7, S8 and Figure 5 by including Donor 5 information.
Comment 6: Table S1 -There are multiple samples from the female gnotobiotic mice post FMT with Donor 4 and Donor 5. What is the difference between these samples and were they all used when making the comparisons? It does appear the samples were merged. Response 6: Thank you for pointing it out. In the revised manuscript, we have included new information for only one female gnotobiotic mouse and her corresponding infants for Donor 3, 4, and 5. Accordingly, we have revised Tables S1(C), S2(C), S6, S7, and Figure 5 (Line numbers: 128, 604-607). We also changed the word "breeder" to "dam" throughout the manuscript.
Comment 7: Line 143: Once again, when was the mother's sample obtained? Response 7: As we already mentioned in Response 3, we have included sample collection information for the mothers in the revised manuscript (Line numbers: 96-97).
Comment 8: Line 144: Was the mother's sample only compared to her infant's samples? Please clarify as when I first read this it says "any available infant samples". Upon further reading, I am guessing each mother's sample was only compared with her infant's samples. Response 8: Please note that each mother's sample was only compared with her infant's samples. We have clarified this sentence in the revised manuscript (Line numbers: 145-146).
Comment 9: Line 168: The authors are commended for providing the resource/link that allows for reproduction of the analyses. Response 9: Thank you. Please note that the metagenomics sequencing FASTQ files from the gnotobiotic mice used in this study were deposited in NCBI under accession number PRJNA593263.
Comment 10: Line 169, Table S2 has more than 20. Please change title to reflect the number of species reported in table. And are these the top 41 most abundant species? Response 10: Please note that the value in the Table S2 represents the number of sample pairs for each species after filtering any low sequence coverage (<30%) and low sequence depth (<3.5) samples against their given reference sequences via WSS. We have already reported these parameters in the Materials and Methods section of the previous version of the manuscript.
Comment 12: Line 191: This sentence seems to be missing something. Perhaps a "which" before "included 'estimated number of reads…"? Response 12: As suggested by the reviewer, we have modified the sentence by including "which" before "included 'estimated number of reads..". (Line number: 190) Comment 13: Line 204 -209: These sentences seem out of place here and are more discussion than results. Response 13: We agree and have modified this section in the revised manuscript (Line numbers: 204-206).

Comment 14:
Line 209-212: Redundant with information in Materials and Methods.
As suggested by the reviewer, we have clarified the sentences by including detailed results observed from the revised Figure 1 as well as Table S3 (Line numbers  214 -225, 228-237). Additionally, we have revised the Introduction by including detailed information regarding WSS and cut-off value (Line numbers: 71-74).
Comment 24: Line 222 -224: The authors make special note of Akkermansia muciniphila, Barnesiella intestinihominis, and Faecalibacterium prausnitzii. These seem odd to point out. And if pointing out those that are not related, why not also include Eubacterium hallii and Ruminococcus? Response 24: Thank you for pointing it out. As suggested by the reviewer, we have revised the manuscript by including only those species that did not have a related strain at all examined time points that were available in the data sets (Line numbers: 220-222).
Comment 25: Line 224-225: This sentence seems very much like discussion. Response 25: As suggested by the reviewer, we have moved this sentence to the Discussion section of the revised manuscript (Line number: 379-381).
Comment 28: Line 236: Which 6 cases agree? Please detail for the reader. Response 28: As suggested by the reviewer, we have revised the manuscript accordingly by including detailed information for comparison analysis between WSS and StrainPhlAn (Line numbers: 240-241).
Please also note that we have changed " Figure 2" to "S1 Figure" since we changed Figure 1 as suggested by the reviewer.
Comment 29: Line 239-242: This is not described in Materials and Methods. Please add this. Response 29: As we mentioned in Response 4, we have included the caesarean sample information in the revised manuscript (Line numbers: 99-101) as well as Tables S1(A) and S2(A). Table S8: Should row 2 be "Fecal sample from infant"? Response 30: As we mentioned in Response 17, we have revised Row 2 in Table S4 along with Columns C, D, E, and F to clarify sample information.

Comment 30:
Comment 31: Table S8: Why are there only 9 species reported on? Response 31: Please note that the species that we reported in Table S4, as well as  Tables S2, S3, S5, S6, and S7 were selected based on sequence coverage and depth against reference sequences. Moreover, based on metadata provided by Shao et al., there were only two samples (B00075, BBS0070) that had a normal Bacteroides profile and the remaining samples had low Bacteroides profile. We believe this may lower the number of species that can be compared for the caesarean section birth data set.
Comment 32: Line 248: The authors should also discuss the limitation that only 6 mother-infant pairs were used in the caesarean analyses. Response 32: As suggested by the reviewer, we have included a caveat of sample size for the caesarean section birth samples in the revised manuscript (Line numbers: 255-256).
Comment 33: Line 251-256: Once again, these sentences seem to more appropriate for the Discussion and not in the results section. Response 33: We have modified this section in the revised manuscript (Line numbers: 259-262).
Comment 34: Line 257-258: Not sure how this supports the transfer of microbes to the infants since it is only comparing strains across pregnancy and does not then compare to what is found in her infant. It is a worthwhile description as to strains that remain stable or that are more transient in nature but I do not quite see the connection that demonstrates subsequent transfer to infants. Response 34: We believe our Response 46 is related to the reviewer's concern. Comment 35: Figure 3: Individual Pre4 has a green box for Bacteroides sp. 2-1-16 indicating no strain changes during any time points of pregnancy. However, this is misleading as there were no samples available for the other two time points (both are marked as "NA" in Table S4). Therefore, I would say it is unknown whether there were any strain changes or not. Perhaps I am not understanding this figure but I would also disagree with the green box for Term6 in the Bacteroides stercoris row. If there were no strain changes during any time points of pregnancy, wouldn't there be red numbers in Table S4 for this individual for that strain? I did not go through every box but please address these inconsistences and/or clarify in the text so readers don't perhaps make the same misinterpretation that I am making. Response 35: Thank you for pointing it out. To avoid any inconsistency matches that can be caused by an unknown factor such as "NS = No Score", we have thoroughly revised Figure 2 as well as Table S5.
As we already mentioned in the previous version of the manuscript, gestational days for 9 women were different. Out of 9 women, 3 women (Pre 1, Pre 2, and Pre 4) gave birth prematurely, therefore a total number of samples for these women are different as compared to the remaining 6 women who gave birth after 37 weeks. This was why only three women had "NA" values in Table S5. Thus, we still believe no strain changes observed for Pre 4 sample during pregnancy, since her gestational day ended before 150 days. We believe the revised Figure 2 will satisfy the reviewer. Comment 38: Line 269-271: Once again, this sentence seems more like discussion, at least from looking at other articles in the journal; but perhaps I am mistaken. Response 38: We have modified this section in the revised manuscript (Line numbers: 276-278).
Comment 39: Line 271-278: This seems redundant to what is in the Materials and Methods and / or any detail included here should be added to the Materials and Methods section.
Response 39: As suggested by the reviewer, we have removed redundant sentences (Line numbers: 278-280) and revised the Materials and Methods section by including any missed information (Line numbers: 120-124).
Comment 40: Line 277: "was" should be "were" Response 40: This sentence has removed as suggested by the reviewer in Comment 39.
Comment 41: Line 287: Results from the comparisons found in Tables S5-S6 are lacking. Figure 6 does do a nice job of pulling the results together, but perhaps more of the results can be presented or detailed in this section as well. Also please direct the readers to these supplemental tables in this section. Response 41: As suggested by the reviewer, we have included detailed results for Figure 5 as well as Tables S6-S7 in the revised manuscript (Line numbers 295-306, 311-319).
Comment 42: Line 288: would recommend amending the sentence to read "maternal transmission of bacteria to the F1s…" or specify transmission of what. Response 42: As suggested by the reviewer, we have changed the sentence from "maternal transmission to the F1s in all of the.." to "maternal transmission of microbes to the F1s in all of the.." in the revised manuscript (Line number: 306-307).
Comment 43: Line 289: Once again, in several places in the manuscript there is a font change.
Response 43: As we mentioned in Response 15, we have made every effort to maintain the same font in the revised version of the manuscript.
Comment 44: Line 298: And also caesarean birth, correct? Response 44: Thank you for pointing it out. We have revised the sentence by including "caesarean section birth" in the revised manuscript (Line number: 323).
Comment 45: Line 298-301: I find this sentence somewhat misleading as very few strains were observed in early life of the infant and maintained at infancy, at least according to Table S3 and Figure 1. Response 45: Thank you for pointing it out. Since Figure 1 and Table S3 have been revised as requested by the reviewer, we have accordingly included this information in the Discussion section of the revised manuscript (Line numbers: 324-326).