Gut Microbiome Composition and Variance Are Modified by Degree of Growth Failure in Preterm Infants: A Prospective Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Journal

Nutrients (ISSN 2072-6643)

Journal

nutrients-4010918

Type

Article

Title

Gut microbiome composition and variance are modified by degree of growth failure in preterm infants

Authors

Katherine A Stumpf * , Miranda Green , Xinying Niu , Dongmei Liu , Shuheng Gan , Xiaowai Zhan , Maricel N. Maxey , Monica Boren , Sujir Pritha Nayak , Sana Jaleel , L. Steven Brown , Jane A. Foster , Julie Mirpuri *

Section

Nutrition and Metabolism

ABOUT THE MANUSCRIPT

• Based on the stamen that preterm infants often require increased caloric intake to main-tain appropriate growth while in the neonatal intensive care unit, the authors performed a prospective trial with infants less than weeks gestation with an embedded case control analysis of infants with normal or poor growth patterns. For this objective, they collected fecal samples on full enteral feeds and analyzed blindly using 16s rRNA next generation sequencing. Their results showed that the gut microbiome composition and variance were modified by the degree of growth failure.
• The authors did not include the number of pages in the manuscript.

TITLE

• The title of this manuscript is “Gut microbiome composition and variance are modified by degree of growth failure in preterm infants.

I suggest changing to “Gut microbiome composition and variance are modified by degree of growth failure in preterm infants: a prospective study”.

• It is important to include in the title the type of article

ABSTRACT

          This section is adequate.

KEYWORDS

Keywords are:

“gut microbiome; preterm; neonatal; growth; neonate”

I suggest “

 Keywords: gut microbiome; preterm; neonatal;; growth; feces;

 16s rRNA”.

INTRODUCTION

• This section has merit but also some weaknesses. In these, we can say that the literature review could be more complete. I see the need to include more recent studies, primarily published in 2025.

There is only one reference published in 2025:

Li F, Hooi SL, Choo YM, Teh CSJ, Toh KY, Lim LWZ, et al. Progression of gut microbiome in preterm infants during the first three months. Sci Rep. 2025 Apr 9;15(1):12104.

Only one published in 2024:

(Fenton TR, Merlino Barr S, Elmrayed S, Alshaikh B. Expected and Desirable reterm and Small Infant Growth Patterns. Adv Nutr Bethesda Md. 2024 Jun;15(6):100220);

Only 3 published in 2023:

Neves LL, Hair AB, Preidis GA. A systematic review of associations between gut microbiota composition and growth failure in preterm neonates. Gut Microbes. 2023 Dec 31;15(1):2190301;

Xiu W, Lin J, Hu Y, Tang H, Wu S, Yang C. Assessing multiple factors affecting the gut microbiome structure of very preterm infants. Braz J Med Biol Res Rev Bras Pesqui Medicas E Biol. 2023;56:e13186;

Mohammadi F, Green M, Tolsdorf E, Greffard K, Leclercq M, Bilodeau JF, et al. Industrial and Ruminant Trans-Fatty Acids-Enriched Diets Differentially Modulate the Microbiome and Fecal Metabolites in C57BL/6 Mice. Nutrients. 2023 Mar 16;15(6):1433.

• Another point is that the study hypothesis could be clearer than it is. How the "degree" of growth will be operationalized\/
• At the end of this section we can find that “This study provides longitudinal microbiome data from a large preterm infant population in an urban neonatal intensive care unit (NICU). We prospec-tively recruited patients and collected weekly stool samples to test this hypothesis.” These two sentences belong to the methods section.

METHODS

• In the section “Patients and Study Design:”

Please explain how was the invitation to participate of this study.

• In this the section “Consent” we can find that

This study was approved by the University of Texas Southwestern Medical Center and Parkland Hospital Institutional Review Boards and infants were enrolled after paren-tal written consent was given.” Please include the data of the approval and the country.

• Furthermore, in general in this section I felt some weakness in some points:

Sample:

Was there a sample calculation?

• Appropriately describe inclusion/exclusion criteria such as antibiotic use and diet).
• Data collection:

Was the fecal collection method (time, storage) standardized?

• Regarding sequencing: the region of the 16S rRNA gene is not specified.
• I see the need to explain how the laboratory quality control was not detailed.

RESULTS

• Please include the definitions for all the abbreviations used in Table 1;
• The same for table 2

DISCUSSION

• In this section, the authors comment on differences in the rates of Veillonella, Bifidobacterium, and Clostridium. I feel there is a lack of integration between these findings and their possible relationship with diet, metabolism, intestinal inflammation, and oxidative stress. Is it possible to briefly work on these points?
• Other points where I feel the discussion lacks depth are regarding antibiotics (very commonly used at birth). Do they influence the microbiome and growth? If comorbidities are present in the patients analyzed, is this a confounding factor?
• In the ABSTRACT (page 1) we can read that “Results: 116 infants were enrolled” (I really miss the number of lines!). In the first sentence of Methods we can read that “In this nested case-control study within a prospective cohort,, we enrolled 115 infants in the primary cohort less than 29-weeks gestation at birth and classified them based on postnatal growth outcomes.” The same in the first line of Results section and in the first line of the Discssion. Are the sample with 115 or 116 individuals?
• In some way, the authors state that fluctuations in Bifidobacterium, Clostridium, and Veillonella spp. may be related to "anti-microbial or inflammatory factors," but is there evidence in the study to support this stamen?
• On page 9, the authors include the limitations at the end of the discussion. I suggest a separate section.
•  

CONCLUSION

• Although the last sentence of this section starta to buil the “future perspectives” for this study, I suggest a separate section for this. The authors can include something like:

FUTURE PERSPECTIVES

Future studies should use standardized, longitudinal sampling to better capture microbiome dynamics in preterm infants. Approaches integrating metagenomics and metabolomics are needed to clarify how specific growth rates influence growth. Future research should also isolate the impact of important clinical factors, such as nutrition, antibiotic use, and the presence of comorbidities, on microbial composition. The development of controlled clinical trials should evaluate microbiome-modulating interventions, such as targeted probiotics, prebiotics, or personalized fortification, to determine if intentional changes in the gut ecosystem can improve growth outcomes.      

Author Response

Reviewer #1 Comments and responses:

Reviewer comment:

The title of this manuscript is “Gut microbiome composition and variance are modified by degree of growth failure in preterm infants.

I suggest changing to “Gut microbiome composition and variance are modified by degree of growth failure in preterm infants: a prospective study”.

It is important to include in the title the type of article

Response: The title has been changed as suggested.

Reviewer Comment:

 Suggest the following keywords: gut microbiome; preterm; neonatal;; growth; feces;16s rRNA”.

Response:

Keywords have been modified to include the ones suggested as well.

Reviewer comment:

This section has merit but also some weaknesses. In these, we can say that the literature review could be more complete. I see the need to include more recent studies, primarily published in 2025.

Another point is that the study hypothesis could be clearer than it is. How the "degree" of growth will be operationalized\/

At the end of this section we can find that “This study provides longitudinal microbiome data from a large preterm infant population in an urban neonatal intensive care unit (NICU). We prospectively recruited patients and collected weekly stool samples to test this hypothesis.” These two sentences belong to the methods section.

Response: 

We acknowledge that studies are limited on this topic, so references are limited as a result.  We have included the most relevant and available studies in the manuscript, and added more with the discussion, which does include studies from the previous five years. 

The degree of growth, including poor growth and appropriate growth groups, are defined within the methods section for clarity.

We have removed the statement regarding prospective recruitment and collection from the introduction as this is present in the methods section.

Reviewer comment:

Please explain how was the invitation to participate of this study.

In this the section “Consent” we can find that

This study was approved by the University of Texas Southwestern Medical Center and Parkland Hospital Institutional Review Boards and infants were enrolled after parental written consent was given.” Please include the data of the approval and the country.

Furthermore, in general in this section I felt some weakness in some points:

Sample:

Was there a sample calculation?

Appropriately describe inclusion/exclusion criteria such as antibiotic use and diet).

Data collection:

Was the fecal collection method (time, storage) standardized?

Regarding sequencing: the region of the 16S rRNA gene is not specified.

I see the need to explain how the laboratory quality control was not detailed.

Response:

We have added how patients were recruited. This was by informed consent utilizing an IRB approved consent form as an invitation to the study.

We have included the IRB study number and country. 

We include the the power calculation/sample size calculation in the methods section as follows: 

 Enrollment was done by parental informed consent using an IRB-approved consent form in the primary language of the participant. The study included infants who were born at Parkland Health and admitted to the NICU after birth between June 2019 and August 2022. Sample size was based on the assumption that the normal growth group would have an alpha diversity mean of 100 and the other groups would have a difference of 20% with a common standard deviation equal to 20. Based on the null hypothesis of no difference in alpha diversity between groups, a total of 108 infants achieves 90% power to detect differences among means versus the alternative of equal means using an F test with a 0.05 significance level.

Reviewer comment:

Appropriately describe inclusion/exclusion criteria such as antibiotic use and diet.

Response:

We describe in the methods section that we excluded infants with major congenital anomalies since these are expected to affect growth.  We did not exclude any patients based on use of antibiotics or type of diet but rather included in tables the numbers of exposures. 

Reviewer comment:

Was the fecal collection method (time, storage) standardized?

Response:

Yes it was standardized. This is clarified with this addition in the methods:

Sample collection was standardized with collection and storage of fecal samples immediately after diaper checks in the neonatal intensive care unit. 

We have previously included information on the storage at -80 degrees.

Reviewer comment:

Regarding sequencing: the region of the 16S rRNA gene is not specified.

Response:

Variable region of V4 was sequenced and is specified in the methods section.  (see paragraph under sample collection-this is stated).

Reviewer comment:

I see the need to explain how the laboratory quality control was not detailed.

Response:

This is stated under the sample collection section as follows: “Following amplification, PCR products were verified, cleaned, and normalized.”

Reviewer comment:

Please include the definitions for all the abbreviations used in Table 1 and 2.

Response:

This has been added in the legend of tables 1 and 2.

Reviewer comment:

In this section, the authors comment on differences in the rates of Veillonella, Bifidobacterium, and Clostridium. I feel there is a lack of integration between these findings and their possible relationship with diet, metabolism, intestinal inflammation, and oxidative stress. Is it possible to briefly work on these points?

Response:

We have added a paragraph in the discussion with new references to integrate the findings with diet and metabolism.

Reviewer comment:

Other points where I feel the discussion lacks depth are regarding antibiotics (very commonly used at birth). Do they influence the microbiome and growth? If comorbidities are present in the patients analyzed, is this a confounding factor?

Response: We had added a citation regarding antibiotic exposure and growth relationship (Pyle et al).  Comorbidities which may contribute to growth such as BPD and PDA were present in a portion of the population.  We have added a comment in the discussion section since these comorbidities does represent a limitation. 

Reviewer comment:

In the ABSTRACT (page 1) we can read that “Results: 116 infants were enrolled” (I really miss the number of lines!). In the first sentence of Methods we can read that “In this nested case-control study within a prospective cohort,, we enrolled 115 infants in the primary cohort less than 29-weeks gestation at birth and classified them based on postnatal growth outcomes.” The same in the first line of Results section and in the first line of the Discssion. Are the sample with 115 or 116 individuals?

Response:  Thank you for pointing this oversight out. This has been corrected in the abstract to 115, which is consistent through the entire manuscript.  

Reviewer comment:

In some way, the authors state that fluctuations in Bifidobacterium, Clostridium, and Veillonella spp. may be related to "anti-microbial or inflammatory factors," but is there evidence in the study to support this stamen?

Response: We have edited the discussion to exclude this statement since this is not supported by the study.

Reviewer comment:

On page 9, the authors include the limitations at the end of the discussion. I suggest a separate section.

Response: This has been made into a separate section as recommended.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors explored the association between gut microbiome composition and growth failure in preterm infants, with rigorous design and valuable clinical data. Some revision suggestions to improve the manuscript:

1. Please add a "Sample Screening Flowchart": Convert the textual description of sample exclusion criteria (low alpha diversity, lack of longitudinal samples) into a visual flowchart or table, clearly illustrating the process from 263 initial samples to 218 valid samples.
2. Please divide "microbiome analysis" into three independent subsections: Alpha/Beta Diversity Analysis, Differential Abundance Analysis, and Clustering Analysis, to avoid content confusion and improve readability.
3. In the "Statistical Analysis of Microbiome Data" section, supplement the specific numerical range for "Shannon diversity and read counts below 2 standard deviations of the overall mean" to enhance reproducibility.
4. Please expand variable abbreviations in the formula (e.g., “Abx status” to “Antibiotic status”, “feeding grp” to “feeding group”, “DOL_at_sample collection” to “Day of Life at sample collection” to avoid ambiguity and ensure standardized format.
5. Please maintain consistent definitions of "ASVs", "taxa", and "genus-level taxa" throughout the manuscript. Provide full annotations when they first appear (e.g., ASVs: Amplicon Sequence Variants) and avoid mixing "taxon/taxa".
6. Please standardize bacterial name formatting: Italicize bacterial genus and species names (e.g., Veillonella, Bifidobacterium) when they first appear, and abbreviate them thereafter (e.g., Veillonella to V.). 
7. Please summarize study limitations into three points: sample loss, fecal collection time deviation and feeding mode heterogeneity and provide one improvement direction for each point.

Author Response

Reviewer #2 Comments and responses:

1. Please add a "Sample Screening Flowchart": Convert the textual description of sample exclusion criteria (low alpha diversity, lack of longitudinal samples) into a visual flowchart or table, clearly illustrating the process from 263 initial samples to 218 valid samples.

Response: This has been added as a supplementary figure.

1. Please divide "microbiome analysis" into three independent subsections: Alpha/Beta Diversity Analysis, Differential Abundance Analysis, and Clustering Analysis, to avoid content confusion and improve readability.

Response: This has been completed under “Statistical Analysis of Microbiome Data”.

1. In the "Statistical Analysis of Microbiome Data" section, supplement the specific numerical range for "Shannon diversity and read counts below 2 standard deviations of the overall mean" to enhance reproducibility.

Response: This has been completed under the subheading “Alpha and Beta Diversity Analysis”.

1. Please expand variable abbreviations in the formula (e.g., “Abx status” to “Antibiotic status”, “feeding grp” to “feeding group”, “DOL_at_sample collection” to “Day of Life at sample collection” to avoid ambiguity and ensure standardized format.

Response:  This has been completed under the subheading “Differential Abundance Analysis”.

1. Please maintain consistent definitions of "ASVs", "taxa", and "genus-level taxa" throughout the manuscript. Provide full annotations when they first appear (e.g., ASVs: Amplicon Sequence Variants) and avoid mixing "taxon/taxa".

Response: This has been completed

1. Please standardize bacterial name formatting: Italicize bacterial genus and species names (e.g., Veillonella, Bifidobacterium) when they first appear, and abbreviate them thereafter (e.g., Veillonella to V.). 

Response: These have all been italicized.

1. Please summarize study limitations into three points: sample loss, fecal collection time deviation and feeding mode heterogeneity and provide one improvement direction for each point.

Response: We have edited the limitation section to include an improvement for each direction.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,
Thank you very much for performing the corrections I suggested.

With best regards.