Meta-analysis reveals the predictable dynamic development of the gut microbiota in commercial pigs

ABSTRACT Understanding the mechanisms of microbiome assembly during host development is crucial for successful modulation of the gut microbiome to improve host health and growth. However, results from previous microbial intervention studies to improve swine growth have largely been inconsistent due to the constantly changing nature of the gut microbiota and limited longitudinal sampling. Detailed characterization of the swine gut microbiota through meta-analysis allows us to understand the dynamics of microbial community succession, as well as the transient and natural variations between time points and animals. A total of 3,313 fecal microbial communities from over 349 pigs covering 60 time points (from birth to market age) from 14 publications were included in this meta-analysis. Despite differences in animal breeds and management, generalizable patterns of community assembly were identified. Alpha diversity continuously increased during early stages of animal growth and more slowly in the later stages. Beta regression analysis revealed that more microbial taxa were enriched, while fewer were excluded by the gut microbiota. The microbial community structure also changed significantly between days at early ages and became more similar as pigs aged, as revealed by dissimilarity and distance metrics. Dirichlet multinomial mixtures analysis supported gradient microbial clustering in longitudinal pig fecal samples, and we found that the early samples spread to more clusters than those from older pigs. Random forest regression identified 30 operational taxonomic units as potential microbial biomarkers for modeling swine gut microbiota development. External validation showed this model could be generalized to future microbiome studies conducted in suckling and weaning pigs and adds to the toolkit to quantify community succession. IMPORTANCE The swine gut microbiome undergoes an age-dependent assembly pattern with a developmental phase at early ages and a stabilization phase at later ages. Shorter time intervals and a wider range of data sources provided a clearer understanding of the gut microbiota colonization and succession and their associations with pig growth and development. The rapidly changing microbiota of suckling and weaning pigs implies potential time targets for growth and health regulation through gut microbiota manipulation. Since swine gut microbiota development is predictable, swine microbiota age can be calculated and compared between animal treatment groups rather than relying only on static time-matched comparisons.

o The names of the phyla are not according to the most recent guidelines.Please adapt this throughout the manuscript.
-DMM clustering o DMM clustering is performed using the control and treated pigs.The treatments appear often to have a maturating effect on the gut microbiota community.Also, weaning day differs from study to study and also weaning is associated with the maturation of the gut microbiota, as described by .Have you assessed whether both factors influenced the DMM clustering and/or introduced artificial clusters in the transition area?Please clarify.
-Figure 1: o Figure 1C is not referred to in the text.Please also add a timeline indicating the growth stages of the pig and from which day to which day these stages go, to further clarify the figure.Since days and growth stages are used interchangeably in the article, this illustration will make it easier to follow.o The pie charts in figure 1B overlap.Please adapt this.
-Figure 3 o Figure 3 is counterintuitive now as it seems that early-day samples cluster better together than late-day samples.Would it be possible to visualize the clusters (to the example of figure S7) to improve this? (suckling vs finishing) o Also, there is a typo in the figure description (Figure 3 and S6).Panel A-B and C-D need to be switched in the description of the figures.
-Figure 4 o Figure 4B is rather complex since many colors are used and not everything on the figure has an added value.As clustering of the phyla is not further discussed, this can be left out.To further clarify the figure, the names of the cluster (letters) on the other hand could be added on top and on the left to their respective color for legibility.
Reviewer #2 (Comments for the Author): In the manuscript "Meta-analysis reveals the predictable dynamic development of the gut microbiome in commercial pigs", Wenxuan Dong, et al. used a total of 3,313 fecal microbiota sampling from over 349 pigs covering 60 time points (from birth to market age) from 14 publications to make meta-analysis.The authors' detailed characterization of gut microbiome through metaanalysis enables an understanding of the dynamics of microbial community succession, as well as transient and natural variation between timepoints and animals.Major 1. Please be clear with the concept of "microbiome", it is defined as ""a characteristic microbial community occupying a reasonably well-defined habitat which has distinct physio-chemical properties."The term thus not only refers to the microbial community composition, but also encompasses their activity and function.In the current study, it is generally the microbiota analyzed than microbiome.In addition, the manuscript mentioned "microbial taxa were recruited" several times, I assume it was talking about bacterial succession, but how exactly microbes are "recruited"?From where?Please explain.
2. The significance of the paper wasn't clear.What is the new?The dynamics of microbial community succession?Yes, however, it is more or less known."Potential microbiota biomarkers"?Please specify what biomarkers are identified.The manuscript also mentioned that they identified core microbiota.These core microbiotas are shared and constant in life time of pigs.Isn't that they are the opposite of the dynamics, and how they can be manipulated?Please elaborate.
3. The link between pig study and human research wasn't solid.In introduction, it was mentioned that in human study, highfrequency sampling and analysis were conducted.Therefore, it is necessary to do the same in pig study.So, could the already done analysis apply directly in the current study, or the methodology developed in the current study could promote human study?Please explain.S1 and Figure 1 should be combined.Figure 1 A doesn't show sampling frequency clearly.Figure 1C (plus legend) does not explain the time range and the scheme clearly.

Table
5. The manuscript accurately predicted the microbiota age of pigs (up to 80 days of age) in the external validation dataset, what does "80 days " mean and why did you choose this point in time? 6.The manuscript used "DMM" model to predict whether the gut microbiota clusters with age, but the gut microbiota can be influenced by many factors, such as diet and environment, so how to avoid interference from these factors？ Minor 1. Figure 1 should be revised, the pie chart in Figure 1B is too tightly laid out and should be properly spaced 2. Figure 2 should be revised, the layout of the images is too messy, the P value in the legend of Figure 2A should be italicized (none of the significant P-value in the article are in italics), the font of the legend on the right side of Figure 2B is squeezed together, and the fonts of the X and Y axes are not very clear in grey, which is not consistent with the font of the images.3. Figure 4 should be revised, why is there a letter "C" in the background at the bottom of Figure 4B, and the placement of the legend is a bit confusing.

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To the Authors
In this paper 'Meta-analysis reveals the predictable dynamic development of the gut microbiome in commercial pigs', Dong and colleagues present their re-analysis of published datasets on pig gut microbiome development.Using their RF model, they predict the age of the piglets based on their gut microbiota.Overall, the paper is clearly written and contains important messages for the scientific community, but some points that need further clarification are raised below: -Overall comment o The names of the phyla are not according to the most recent guidelines.Please adapt this throughout the manuscript.-DMM clustering o DMM clustering is performed using the control and treated pigs.The treatments appear often to have a maturating effect on the gut microbiota community.Also, weaning day differs from study to study and also weaning is associated with the maturation of the gut microbiota, as described by .Have you assessed whether both factors influenced the DMM clustering and/or introduced artificial clusters in the transition area?Please clarify.-Figure 1: o Figure 1C is not referred to in the text.Please also add a timeline indicating the growth stages of the pig and from which day to which day these stages go, to further clarify the figure.Since days and growth stages are used interchangeably in the article, this illustration will make it easier to follow.o The pie charts in figure 1B overlap.Please adapt this.
-Figure 3 o Figure 3 is counterintuitive now as it seems that early-day samples cluster better together than late-day samples.Would it be possible to visualize the clusters (to the example of figure S7) to improve this? (suckling vs finishing) o Also, there is a typo in the figure description (Figure 3

and S6). Panel A-B and C-D
need to be switched in the description of the figures.-Figure 4 o Figure 4B is rather complex since many colors are used and not everything on the figure has an added value.As clustering of the phyla is not further discussed, this can be left out.To further clarify the figure, the names of the cluster (letters) on the other hand could be added on top and on the left to their respective color for legibility.
We would like to thank the reviewers for their helpful critiques of our manuscript.We have utilized and addressed all comments to improve the quality of our manuscript.Please see below for an item-by-item response.

Reviewer #1 (Comments for the Author):
To the Authors In this paper 'Meta-analysis reveals the predictable dynamic development of the gut microbiome in commercial pigs', Dong and colleagues present their re-analysis of published datasets on pig gut microbiome development.Using their RF model, they predict the age of the piglets based on their gut microbiota.Overall, the paper is clearly written and contains important messages for the scientific community, but some points that need further clarification are raised below: • Overall comment: The names of the phyla are not according to the most recent guidelines.Please adapt this throughout the manuscript.Response: We gratefully appreciate for your valuable suggestion.We have changed the names of the phyla according to the most recent guidelines.See L222-223, Figure 2.
• DMM clustering is performed using the control and treated pigs.The treatments appear often to have a maturating effect on the gut microbiota community.Also, weaning day differs from study to study and also weaning is associated with the maturation of the gut microbiota, as described by .Have you assessed whether both factors influenced the DMM clustering and/or introduced artificial clusters in the transition area?Please clarify.Response: Thank you for your comments.The objective of DMM analysis was to determine the age-dependent pattern of the gut microbiota.We hoped to improve the robustness of clustering selections by adding samples from treated pigs, which could provide more biological variation (1,2).We completed additional analysis to determine if known animal treatments caused any specific clustering patterns, and we found there were no treatment-specific clusters, so we only focused on the control samples downstream as in other analyses in this manuscript.We added this information to the results: Line 179-182: "Various animal treatments (fecal microbiota transplant, antibiotic treatment, and feed restriction) were equally represented in the DMM clusters, so we concluded that age was the primary factor in community clustering patterns."Figure 1: Figure 1C is not referred to in the text.Please also add a timeline indicating the growth stages of the pig and from which day to which day these stages go, to further clarify the figure.Since days and growth stages are used interchangeably in the article, this illustration will make it easier to follow.The pie charts in figure 1B overlap.Please adapt this.Response: Thank you for your comments.We decided to remove Figure 1C because it contributes little to the presentation of the research work in this manuscript, and may actually cause confusion due to its brevity.Although days and growth stages can be used interchangeably, different feeding regimes were used in different papers re-analyzed here.There is no clearly separation of growth stages based on the age of pigs world-wide.Instead, we added a brief introduction of the time span of the swine production cycle in the first paragraph of discussion, which will provide more information than was conveyed in Figure 1C and can help readers have a better understanding.. Also, Figure 2B has been revised to avoid overlaps.
• Figure 3: Figure 3 is counterintuitive now as it seems that early-day samples cluster better together than late-day samples.Would it be possible to visualize the clusters (to the example of figure S7) to improve this? (suckling vs finishing).Also, there is a typo in the figure description (Figure 3  Regarding if the clustering pattern is counter-intuitive.We assume the reviewer is saying this because of the patterns observed in the distance matrices (Fig. 3E-F).It is important to remember when comparing the PCoAs and the distance matrix, that the distance matrix represents the actual pairwise distances, while the PCoA only conveys a portion of the variability of the distance matrix (about 17% in the case of Bray-Curtis).Thus the relationships between samples are more accurate in the distance matrix.Given the number of samples in this dataset, we decided to include the distance matrix to convey more accurate distances.In the distance matrix, there is a limited degree of stability between suckling samples (there is some white and light blue in the lower left corner).However, we feel that this degree of stability is low.It is an interesting pattern and one we want to explore in future experiments.
The typo in the figure legends has been fixed.We switched the description of panels (AB) and (CD).
• Figure 4: Figure 4B is rather complex since many colors are used and not everything on the figure has an added value.As clustering of the phyla is not further discussed, this can be left out.To further clarify the figure, the names of the cluster (letters) on the other hand could be added on top and on the left to their respective color for legibility.Response: Thank you for your nice suggestions.Figure 4 has been revised to reduce its complexity.Unnecessary legends has been removed.We removed the phylum legends since it is not further discussed.We also removed the DMM legend by adding the names on the top and on the left to their respective colors.We also removed the letter "C" in the background at the bottom of Figure 4B.

Reviewer #2 (Comments for the Author):
In the manuscript "Meta-analysis reveals the predictable dynamic development of the gut microbiome in commercial pigs", Wenxuan Dong, et al. used a total of 3,313 fecal microbiota sampling from over 349 pigs covering 60 time points (from birth to market age) from 14 publications to make meta-analysis.The authors' detailed characterization of gut microbiome through meta-analysis enables an understanding of the dynamics of microbial community succession, as well as transient and natural variation between timepoints and animals.

Major
1. Please be clear with the concept of "microbiome", it is defined as ""a characteristic microbial community occupying a reasonably well-defined habitat which has distinct physio-chemical properties."The term thus not only refers to the microbial community composition, but also encompasses their activity and function.In the current study, it is generally the microbiota analyzed than microbiome.In addition, the manuscript mentioned "microbial taxa were recruited" several times, I assume it was talking about bacterial succession, but how exactly microbes are "recruited"?From where?Please explain.Response: Thank you for your thoughtful comments.We have changed the term "microbiome" to "microbiota" when it refers to only the microbial component of the microbiome but not to other features of the gut environment.We also replaced the word "recruited" to "enriched" since it is describing the relative abundances of the microbial taxa.(L25, 133) 2. The significance of the paper wasn't clear.What is the new?The dynamics of microbial community succession?Yes, however, it is more or less known."Potential microbiota biomarkers"?Please specify what biomarkers are identified.The manuscript also mentioned that they identified core microbiota.These core microbiotas are shared and constant in life time of pigs.Isn't that they are the opposite of the dynamics, and how they can be manipulated?Please elaborate.Response: Thank you for your comments.We have tried to make the significance more clear in the last sentence of the abstract, "Despite differences in animal breeds and management, external validation showed this model could be generalized to future microbiome studies conducted in suckling and weaning pigs to quantify community succession."Primarily, we think the most significant piece of this work is the generalizable pattern of swine microbial community succession, that we can quantify community succession by determining microbiota age, and there are bacterial biomarkers of specific ages, as identified by random forest analysis.
For individual studies, it is challenging to quantify the microbial development using single data set with limited sample size and time points.Our meta-analysis provided new facets of the developmental swine microbiota using the largest data set and concluded that generalized age-dependent microbial developmental patterns exist, independent of specific animal management practices.
The presence of core microbiotas does not indicate that the community is not dynamic.The core microbiotas and the dynamic nature of the community reflect two important aspects of the swine gut microbiota.The core microbiotas reflect the stability of the gut microbiota.The core microbiotas are important in maintaining the homeostasis of gut microbiome.They provide potential targets in future manipulation.However, dynamics, and we would add, predictable dynamics, of the community reflects the path of the microbiota to a relatively stable state.One way to manipulate the gut microbiome would be to regulate the host-microbe and microbe-microbe interactions.The dynamics enable us to better explore the above interactions and achieve successful manipulations.

The link between pig study and human research wasn't solid. In introduction,
it was mentioned that in human study, high-frequency sampling and analysis were conducted.Therefore, it is necessary to do the same in pig study.So, could the already done analysis apply directly in the current study, or the methodology developed in the current study could promote human study?Please explain.

Response: Thank you for your comments. 1. The analysis already completed in humans have provided numerous in-depth insights in disease diagnosis and treatment, and health interventions. Swine production is like-wise dealing with issues related to gut health, particularly those brought on by dysbiosis of the gut microbiota.
We anticipate that a more precise, human-like comprehension will enable us to make advancements in the swine industry.2. The human studies revealed the importance of large sample sizes and repeated measurements in gut microbiome research, which triggered us to do the current analysis.3. We borrowed some analysis approaches from humans to help us better understand the development of swine gut microbiota, e.g.microbiota age, and DMM analysis.The first QIIME2 based closed-reference OTU picking pipeline in this paper could be directly used to facilitate human studies.S1 and Figure 1 should be combined.Figure 1 A doesn't show sampling frequency clearly.Figure 1C (plus legend) does not explain the time range and the scheme clearly.Response: Thank you for your comments.Table S1 serves as supplementary material for Figure 1. Figure 1 generally introduces the locations, sampling days, sample sizes, and sequencing information.Readers will know why we complete the next steps of the analysis based on knowledge on Figure 1, e.g., why sub-sampling was completed to avoid bias introduced by uneven sample sizes, why we choose closed reference OTU picking based on sequence region, how our data sets represents global sampling, etc.However, readers may be more interested in one specific paper, or the genetic background of pigs used in this analysis, etc. Readers may get more information from the Table S1 if they would like to know anything else that is not directly associated with the current analysis.We decided to remove Figure 1C because it contributes little to the presentation of the research work in this manuscript, and may actually cause confusion due to its brevity.Although days and growth stages can be used interchangeably, different feeding regimes were used in different papers re-analyzed here.There is no clearly separation of growth stages based on the age of pigs world-wide.Instead, we added a brief introduction of the time span of the swine production cycle in the first paragraph of discussion, which will provide more information than Figure 1C and can help readers have a better understanding.

Table
5. The manuscript accurately predicted the microbiota age of pigs (up to 80 days of age) in the external validation dataset, what does "80 days " mean and why did you choose this point in time?Response: Thank you for your comments.Samples with an age of less than 80 days represent 85% (2818 out of 3313) of all samples and 84% (1910 out of 2261) of control samples collected in the current analysis.Based on most feeding regimes in the collected data, samples with ages less than 80 days covered both suckling, weaning, and part of the growing stages of pigs' life.A previous study also observed that after postnatal day 77, microbiota age is no longer significantly positively correlated with chronological age (Chang et al, PNAS, 2021, Figure S7).The choice of 80 days was based on our observation that the correlation between microbiota age and chronological age was reduced when samples older than 80 days were included in the correlation.The exact day as to when the prediction is accurate is not as important as understanding the performance of the random forest regression model was robust in younger pigs.6.The manuscript used "DMM" model to predict whether the gut microbiota clusters with age, but the gut microbiota can be influenced by many factors, such as diet and environment, so how to avoid interference from these factors?Response: Thank you for your comments.The objective of DMM analysis was to determine the age-dependent pattern of the gut microbiota.We hoped to improve the robustness of clustering selections by adding samples from treated pigs, which could provide more biological variation (1,2).We completed additional analysis to determine if known animal treatments caused any specific clustering patterns, and we found there were no treatment-specific clusters, so we only focused on the control samples downstream as in other analyses in this manuscript.We added this information to the results: Line 179-182: "Various animal treatments (fecal microbiota transplant, antibiotic treatment, and feed restriction) were equally represented in the DMM clusters, so we concluded that age was the primary factor in community clustering patterns."4 should be revised, why is there a letter "C" in the background at the bottom of Figure 4B, and the placement of the legend is a bit confusing.Response: Thank you for your helpful suggestions.Figure 4 has been revised to reduce its complexity.Unnecessary legends has been removed.We removed the phylum legends since it is not further discussed.We also removed the DMM legend by adding the names on the top and on the left to their respective colors.We also removed the letter "C" in the background at the bottom of Figure 4B.

Figure
Thank you for submitting your manuscript to Microbiology Spectrum.As you will see your paper is very close to acceptance.Please modify the manuscript along the lines recommended by reviewer #1.As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner.If your manuscript was reviewed, you will find the reviewers' comments below.
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Thank you for the privilege of reviewing your work.Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.The extra analysis regarding the influence of treatments on DMM clustering was helpful.However, I would also add this analysis in Figure S8 and refer to it in the extra paragraph added in the text.
The adaptions to the figures have made them much clearer and easier to understand.Especially the addition of 95% confidence intervals and adjustments to the colors in Figure 3 have increased the intelligibility of the figure.
Reviewer #2 (Comments for the Author): The authors have provided a detailed response to the comments and have revised the figures and tables accordingly.The study reveals changes of the porcine gut microbial community structure with age by analyzing a large amount of data and identifies potential microbial biomarkers that influence the development of the porcine gut microbiota, which can help to understand the mechanisms of microbiome assembly during host development.

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To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex.Go to Author Tasks and click the appropriate manuscript title to begin the revision process.The information that you entered when you first submitted the paper will be displayed.Please update the information as necessary.Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file.For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript." Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
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To the Authors
The overall comments raised before were addressed properly.
The extra analysis regarding the influence of treatments on DMM clustering was helpful.However, I would also add this analysis in Figure S8 and refer to it in the extra paragraph added in the text.
The adaptions to the figures have made them much clearer and easier to understand.Especially the addition of 95% confidence intervals and adjustments to the colors in Figure 3 have increased the intelligibility of the figure.
and S6).Panel A-B and C-D need to be switched in the description of the figures.Response: Thank you for your nice suggestions.The reviewer is correct that there appears to be some clustering of the samples from suckling animals in Fig. 3.We improved the aesthetics in Fig. 3 to visualize the community shifts more clearly.Panels A and B (Figures 3 and S6) now include 95% confidence interval ellipses for each stage.The ellipses indicate that late stages (growing and finishing) are generally smaller than early stages (suckling and weaning) according to all four measures of beta diversity.Suckling samples have more dispersal in PCo1 axis and finishing samples have more dispersal in PCo2 axis.We also increased the number of colors in panels C and D in efforts to more clearly indicate different ages.It seems the community shift occurs in large extent by day 30, indicated by yellow points.
, Hao-Wei, et al. "Gut microbiome contributions to altered metabolism in a pig model of undernutrition."Proceedings of the National Academy of Sciences 118.21 (2021): e2024446118.

1 . 2 ,
Stewart, Christopher J., et al. "Temporal development of the gut microbiome in early childhood from the TEDDY study."Nature 562.7728 (2018): 583-588.2. Holmes, I., Harris, K. & Quince, C. Dirichlet multinomial mixtures: generative models for microbial metagenomics.PLoS ONE 7, e30126 (2012).Minor 1. Figure 1 should be revised, the pie chart in Figure 1B is too tightly laid out and should be properly spaced Response: Thank you for your nice suggestions.It has been revised.There are no overlaps after re-organization.2. Figure 2 should be revised, the layout of the images is too messy, the P value in the legend of Figure 2A should be italicized (none of the significant P-value in the article are in italics), the font of the legend on the right side of Figure 2B is squeezed together, and the fonts of the X and Y axes are not very clear in grey, which is not consistent with the font of the images.Response: Thank you for your helpful suggestions.It has been revised.P values in this manuscript (including figures and the main text) has been italicized.See Fig.Fig.S2, Fig.S3, Fig.S5, Fig.S9, Fig.S10, L114, L198, L203.The legend of Figure 2B has been revised to avoiding overlaps.The fonts of X and Y axes has been modified from grey to black.

1 (
Comments for the Author):To the Authors The overall comments raised before were addressed properly.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred

Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. "
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.