Quantitative Differences in the Human Intestinal Microbiota Through the Stages of Life: Infants, Children, Adults and the Elderly

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The work is of interest, particularly in relation to its purpose, however, it is submitted that the introduction would benefit from greater emphasis on the motivation, given its focus on a RT-PCR technique rather than on sequencing systems that are now standardised and utilised routinely.It is recommended that the point be clarified that sequencing systems are not yet ubiquitous in diagnostic laboratories and that molecular systems based on PCR continue to be useful for the purpose. It is imperative to elucidate this aspect to avoid the technique's potential limitations from becoming apparent.In the materials and methods section, it would be beneficial to provide a clarification of the age-related categories, such as infants, children, adults, and elderly, by including a definition and a reference to support the categorization.Additionally, the indications concerning diet on lines 107-110 require further elucidation to ensure clarity.

Author Response

microbiolres-3462669

Manuscript: Quantitative differences in human intestinal microbiota through arc of life: infants, children, adults and elderly

We thank the reviewers for their valuable feedback, which has significantly improved our manuscript. Below, please find our responses to your questions and remarks. The changes in the text are marked in yellow.

Reviewer 1

1. The work is of interest, particularly in relation to its purpose, however, it is submitted that the introduction would benefit from greater emphasis on the motivation, given its focus on a RT-PCR technique rather than on sequencing systems that are now standardised and utilised routinely.It is recommended that the point be clarified that sequencing systems are not yet ubiquitous in diagnostic laboratories and that molecular systems based on PCR continue to be useful for the purpose. It is imperative to elucidate this aspect to avoid the technique's potential limitations from becoming apparent.

Answer: The text  has been changed and  rephrased:

’’The quantitative real-time PCR (qPCR) method is a well-established and widely used approach for the quantification of different microbial groups within the intestinal microbiota. The next-generation sequencing has advanced microbiome research by enabling absolute quantification and deeper taxonomic resolution. However, this method is not yet universally adopted for diagnosis of dysbiosis in routine diagnostic laboratories. Given the complementary nature of these techniques, it is crucial to evaluate the continued relevance of qPCR in microbiome studies. Additionally, some recent studies have demonstrated the necessity of applying absolute quantification alongside whole genome sequencing to obtain a comprehensive understanding of microbiome dynamics and interactions [34,35]. The use of specific primers and probes enables accurate and sensitive quantification of bacterial populations [25,33,35] as well as the ability to compare correlations between detected bacteria across different age groups.’’

2. In the materials and methods section, it would be beneficial to provide a clarification of the age-related categories, such as infants, children, adults, and elderly, by including a definition and a reference to support the categorization.

Answer: The age-related categories and references have been added.

’’The study groups were defined based on age categories as follows: infants (0–12 months), children (1–12 years), adults (18–64 years), and elderly (65 years and older), according to established demographic and clinical criteria. [36,37] (Table 1). Fecal samples were collected from all individuals.’’

3. Additionally, the indications concerning diet on lines 107-110 require further elucidation to ensure clarity.

Answer: In this article, we did not specifically analyze the effect of nutrition on the quantitative gut microbiota. Instead, we provided an average statistical description of the participants' nutrition.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors set out to map changes in the gut microbiota with aging, from newborns to the elderly. Changes in diversity and quantitative composition at different ages were examined. This study is timely because there are no comprehensive and comparable studies at different ages.

My comments about the manuscript

1. The study design, materials, and methods are sufficiently detailed and suitable for reproduction.

2. The sex ratio of the adult population sampled is not equal, with considerably more women being sampled. It is well known that, in general, women consume more fiber-rich foods than men. Could this male-female ratio have resulted in differences in the resulting bacterial composition? Could such a difference have modified the final results?

3. Line 350, typographical error

4. Line 508, missing DOI

5. Line 514 missing DOI

6. Line 517. DOI is missing

8. delete ENTER at lines 541-542

Overall, we consider the manuscript well written and, after some minor revisions, suitable for publication.

 

Author Response

Reviewer 2

The authors set out to map changes in the gut microbiota with aging, from newborns to the elderly. Changes in diversity and quantitative composition at different ages were examined. This study is timely because there are no comprehensive and comparable studies at different ages. My comments about the manuscript

1. The study design, materials, and methods are sufficiently detailed and suitable for reproduction.
2. The sex ratio of the adult population sampled is not equal, with considerably more women being sampled. It is well known that, in general, women consume more fiber-rich foods than men. Could this male-female ratio have resulted in differences in the resulting bacterial composition? Could such a difference have modified the final results?

Answer: The reason is that women are more involved in clinical trials. The differences between the male and female gut microbiomes have been published (Kim et al., 2019). Naturally, eating a diet with greater fiber consumption can alter the gut microbiota. However, we did not specifically analyze the effect of nutrition on the quantitative aspects of the gut microbiota. Instead, we provided an average statistical description of the participants' nutrition.

3. Line 350, typographical error: Corrected
4. Line 508, missing DOI : this reference has no DOI
5. Line 514 missing DOI: this reference has no DOI
6. Line 517. DOI is missing: this reference has no DOI
7. delete ENTER at lines 541-542: Corrected

Overall, we consider the manuscript well written and, after some minor revisions, suitable for publication

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript addresses a significant topic in the microbiome field, comparing the gut microbiota of healthy individuals across various age groups using quantitative qPCR to analyze fecal samples. The study highlights notable differences in microbiota composition, particularly emphasizing the Bacteroidetes/Firmicutes ratio, bacterial diversity, and the presence of specific bacterial groups throughout the lifespan. While the study presents valuable data and thorough analysis, a few suggestions and questions could help improve its clarity and further contribute to the field. Additionally, I recommend an English revision for greater precision.

 

1. Please revise the term 'facultative anaerobes' to 'anaerobes' throughout the entire manuscript. This is a recurring error made by several authors. The correct terminology for bacterial respiration should be aerobic, anaerobic, and facultative.
2. Lines 58-62: Please rewrite this sentence as follows: "However, there are fewer studies comparing the intestinal microbiota of healthy individuals across all age groups (infants, young and preschool children, adults, and the elderly)."
3. The manuscript notes significant individual variation in the microbiome of the elderly. Does this variation suggest a decline in microbiota resilience with age? Would interventions, such as probiotics or dietary modifications, effectively restore balance in older individuals?

Comments on the Quality of English Language

General English revision to make some sentences more concise.

Author Response

Reviewer 3

The manuscript addresses a significant topic in the microbiome field, comparing the gut microbiota of healthy individuals across various age groups using quantitative qPCR to analyze fecal samples. The study highlights notable differences in microbiota composition, particularly emphasizing the Bacteroidetes/Firmicutes ratio, bacterial diversity, and the presence of specific bacterial groups throughout the lifespan. While the study presents valuable data and thorough analysis, a few suggestions and questions could help improve its clarity and further contribute to the field. Additionally, I recommend an English revision for greater precision.

1. Please revise the term 'facultative anaerobes' to 'anaerobes' throughout the entire manuscript. This is a recurring error made by several authors. The correct terminology for bacterial respiration should be aerobic, anaerobic, and facultative.

 

Answer: The word ’’facultative’’ has been removed.

Lines 58-62: Please rewrite this sentence as follows: "However, there are fewer studies comparing the intestinal microbiota of healthy individuals across all age groups (infants, young and preschool children, adults, and the elderly)."

Answer: The sentence has been rewritten:

’’However, there are fewer studies comparing the intestinal microbiota of healthy individuals across all age groups, including infants, young children, preschool children, adults, and the elderly.

 

2. The manuscript notes significant individual variation in the microbiome of the elderly. Does this variation suggest a decline in microbiota resilience with age? Would interventions, such as probiotics or dietary modifications, effectively restore balance in older individuals.’’

 

Answer: The significant individual variation in the intestinal microbiota of the elderly can be attributed to several factors: dietary differences, health status, life style, age–related diseases, using medication use. Of course consuming of probiotics and dietary modifications may be beneficial for restoring balance in the gut microbiota of older individuals. The effectiveness of these interventions depends on the specific health context of the elderly.

 

The English language has been improved.