Characterization of the Microbiome and Virulence and Resistance Genes in the Howler Monkey (Alouatta seniculus) in Colombian Andean Forests

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

It is an interesting paper about characterization of the microbiome, virulence and resistance genes of the howler monkey in Colombian Andean Forests. The manuscript us clear written and features sound experimental results. The paper may be optimized with following suggestions:

 

Line 54-56 - please specify which antibiotics or classes of antibiotics can be named here as examples

Line 99 - suggest to move the figure 1 to supplementary

Line 384-390 - were any transporters of monoamines like ethanolamine or polyamines found? Ethanolamine in particular has been described as abundand compound in the gut, and its utilization by E. coli as a part of virulence mechanism

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper by Florez et al. explores the microbiome of howler monkeys living in the Colombian Andean forests. The authors conducted 16S rRNA gene sequencing using next-generation technologies and complemented this with metagenomic analyses, including genome reconstruction and the identification of antibiotic resistance and virulence genes, along with their genomic context (e.g.,plasmid-associated or located near mobile elements).

Overall, the study is well-conceived and clearly presented. The bioinformatic tools used for sequence assembly and analysis are modern, and the databases referenced are up to date. All sequence data have been deposited in publicly accessible GenBank repositories. The figures effectively illustrate the findings, and the authors appropriately discuss the study’s limitations and the need for further research.

Comments:

1)The Discussion section is overly descriptive and lengthy, it would benefit from being more concise and focused on the main findings.

2)The page formatting should be corrected, as the margins (top, bottom, and sides) appear misaligned.

Minor issues:

1)Line 67: remove the extra “in” (“inhabit Colombia without in”).

2)Line 122: specify which Illumina platform was used.

3)Update phylum names according to current taxonomic conventions (e.g., Pseudomonadota instead of Proteobacteria), check the LPSN website for reference.

4)Line 344: correct “poccible” to “possible.”

5)Line 362: replace “resistant” with “resistance.”

6)Lines 398, 424, 452: add the missing “e” in “gene.”

7)Line 506: complete the phrase “similar environmental…” (a word appears to be missing).

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Abstract well-written, but includes a clearer statement of the exploratory nature of the study.

Only six fecal samples (from three individuals per site) were analyzed, with metagenomic data derived from pooled DNA.

This extremely small, non-replicated design prevents statistical inference or ecological comparisons between sites.

The manuscript should emphasize that the results are preliminary and hypothesis-generating, not definitive.

Include information about the sequencing depth for each sample.

Pooling samples masks individual variation and prevents associations between ARGs/VFs and specific microbes. The authors should more clearly justify this choice and discuss its limitations on data interpretation.

The PERMANOVA results are not statistically significant (p = 0.2), but the discussion sometimes refers to environmental differences.
The lack of statistical significance should be stated explicitly and not over-interpreted.

Detection thresholds (>80% identity) for ARGs and VFs may lead to false positives. Authors should consider more stringent cutoffs (e.g., ≥90%) or discuss potential overestimation due to database limitations.

The manuscript suggests the possibility of horizontal transmission, but lacks supporting evidence.

These claims should be tempered or reframed as mere speculation.

While the detection of E. coli, Enterococcus, and Citrobacter is noteworthy, there is no indication of an actual risk of infection.

The discussion should better separate environmental presence from pathogenic potential.

Figures 4–7 are useful, but lack clear explanatory phrases and statistical indicators (such as confidence intervals).

Precision and labeling should be improved to ensure publication quality.

Quantify the proportion of unclassified taxa in 16S analysis.

Identify genera carrying plasmid-associated resistance genes.

Some sections (4.1–4.4) are overly detailed and could be shortened.

Avoid paraphrasing results; focus more on interpretation and implications.

The English is generally good, but minor grammatical adjustments are needed to improve the flow of writing.
Some sentences are too long and could be broken up for clarity. Remove unnecessary repetition of gene names in multiple sections.

 

 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The revised manuscript has significantly improved clarity, methodological transparency, and scientific integrity. The authors have comprehensively addressed almost all previous comments and made helpful changes to the text and figures, enhancing the exploratory scope of the study. The study is still limited by its small sample size, but now clearly acknowledges this limitation and appropriately categorizes the results as preliminary.

 

Although the annotations have been improved, slightly larger font sizes could be used in Figures 4–7 for easier readability. Please ensure that all abbreviations (e.g., ARG, VF, VFDB) are defined within each figure annotation to ensure clarity.

 

Some reference citations remain duplicated and inconsistent (e.g., references are numbered consecutively but are duplicated in Sections 2.1–2.5). A final review of the references is recommended before acceptance.

 

Please ensure that all additional figures and tables referenced in the main text are clearly numbered.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf