Codon Bias Variation in Staphylococcus aureus

PLOS One

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Reviewers' comments:

Reviewer’s Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?-->?>

Reviewer #1: I Don't Know

Reviewer #2: No

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Greetings

Goid work. But edit the points below:

Major Comments

The study shows clear variation in CAI across genomes, but the biological significance is not fully explained. The association with submitting institutions is interesting, yet it remains unclear whether this reflects true biological adaptation or potential biases (e.g., sampling, sequencing, or submission practices). This point needs further clarification.

The work is entirely computational and lacks functional validation. Without linking codon bias to gene expression, virulence, or fitness, the findings remain largely descriptive.

The use of publicly available metadata may introduce bias. Institutional differences could reflect sequencing pipelines or clonal outbreaks rather than true biological variation. This limitation should be more clearly discussed.

The interpretation of institutional clustering may be overstated, especially given sample imbalance and lack of control for clonality. Additional supporting analysis would strengthen this conclusion.

Minor Comments

The Introduction should better justify the relevance of codon bias to Staphylococcus aureus biology.

Some results are overly descriptive and could be more concise.

Figures need clearer labeling and interpretation.

Methods should more clearly define inclusion/exclusion criteria.

Kind regards.

Reviewer #2: This study examines codon usage bias (CUB) in the human pathogen Staphylococcus aureus through genome-wide computational analyses using publicly available data. It explores variations both within and among strains derived from diverse sources. Overall, the work offers valuable insights into the evolutionary and functional significance of codon usage patterns in this clinically important bacterium. Given its relevance, I recommend this manuscript for publication, subject to revisions addressing the following points:

1. Calculating codon specific values, e.g. CAI for non-coding sequences such as tRNAs are inappropriate. The codon bias analysis should be restricted to codon sequences; therefore, only protein-coding genes should be retained, and all other gene types should be filtered out from the downloaded dataset. Accordingly result section need to be updated.

2. Most of the comparative analyses presented in the manuscript are primarily qualitative and are not supported by numerical data or statistical significance testing. For example, the difference in average CAI scores mentioned in the first paragraph of the Results section is not quantitatively substantiated within the text.

3. It would be valuable to include biological interpretation of the differences in codon usage bias observed among strains obtained from different laboratories; numerical differences alone do not provide sufficient insight or merit.

Reviewer #3: The authors of Codon Bias Variation in Staphylococcus aureus present a descriptive analysis of the variation in codon usage bias of many samples collected across geographic regions and clinical settings. They identify differences in codon usage, as measured by CAI, associated with samples from one institution.

I have major concerns with the manuscript that the authors must address prior to publication to ensure scientific rigor.

First, the authors do not sufficiently test for alternative methodological influences on the observed variation in CAI. Sequencing technology and amplification steps are known to induce GC bias in sequencing data. It is critical that the authors examine the role of sequencing method, assembly method, and other non-biological factors when examining closely related species.

Second, the authors do not sufficiently present their underlying data or methods in a way that would enable replication or ongoing analysis. Essential data that is missing from the supplemental data includes:

- The metadata associated with every sample analyzed. This should include the abbreviations, analyzed metadata, and any metrics generated during the analysis, such as the total number of genes annotated.

- All the genome annotations generated in this analysis.

- All of the raw CAI statistics for every gene

- Table S1 & S3– should report all the calculated loadings, not just the ones presented

- The CAI means, percentages, and interquartile ranges should all be reported.

Third, the methods lack details that should be included in the written description. While all of the code is available, the essential details should be included in the main text. This includes:

- The annotation step should include details such as whether protein or DNA sequences were used for blast, what metric was used to establish similarity, what the metric cutoff was, and how duplicate genes were handled.

- The general steps of the PCA and sPLS-DA, including how missing data was handled and any non-standard parameters

Finally, I agree with the authors that a more compelling analysis would include additional codon bias metrics and analysis of tRNA diversity. There are codon usage metrics that are designed to identify the amount of selection acting on a genome overall, and on specific genes within a genome. The authors, at times, are clear that the variation observed cannot be linked to selection (line 266), but also freely speculate that there is a role for codon optimization in infection (Lines 276-277) or selection on codon usage for expression (Lines 291-293).

There are other minor concerns the authors should address

Line 25-26 – “it must maximize.” The wording of this sentence anthropomorphizes the role of selection.

Results “Variation in average CAI between genomes” – the authors should report the amount of missing data in the genomes.

Line 108 – the evidence for a misannotation of 50S ribosomal protein S15 should be reported.

Line 110 – “it is apparent that genes used in transcription…” The authors should conduct a statistical overrepresentation analysis to confirm the observation.

Line 120 – PCA – The PCA loadings would provide interesting information about the drivers of the separation seen on the PCA. If it is GC bias, this could provide additional interpretations.

Line 225 – The analysis of the ribosomal genes starting on lines 225 would be better described first (before the other analyses). This was a lingering question I had while reading the manuscript and seeing the results. Addressing it after the fact is not as effective.

Line 278 – What “sub-Saharan genomes” are being referenced? Are these also S. aureus?

Line 318 – What does “dehydrated closed contig” mean?

Line 334 – Do the authors mean “dplyr” and not “deplyr”?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Dear Dr. Patel,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Dear Dr. Patel,

Thank you for submitting your revised manuscript, which has now been reviewed by the original reviewers. While the reviewers think that most of the comments have been adequately addressed, there are still several lingering issues that need to be addressed before we can consider the manuscript further. Please respond to the reviewers' comments and submit a point-by-point response, together with the revised manuscript. We look forward to receiving your revisions!

==============================

Please submit your revised manuscript by Aug 14 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Jordy Evan Sulaiman, Ph.D.

Academic Editor

PLOS One

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If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Dear Dr. Patel,

Thank you for submitting your revised manuscript, which has now been reviewed by the original reviewers. While the reviewers think that most of the comments have been adequately addressed, there are still several lingering issues that need to be addressed before we can consider the manuscript further. Please respond to the reviewers' comments and submit a point-by-point response, together with the revised manuscript. We look forward to receiving your revisions!

Best regards,

Jordy

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Reviewers' comments:

Reviewer’s Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The authors of “Codon Bias Variation in Staphylococcus aureus” have made substantial changes to the manuscript to address the comments of the reviewers.

I was unable to assess Figure 5 as it was not included in the submission.

There are remaining issues to be addressed prior to publication.

Line 28-29: Fitness may increase during replication, but that depends on the relative forces of drift and selection.

Line 50-51: There is some evidence that codon bias acts during transcription and is not exclusively post-transcriptional. I’m not sure how it can control protein product after transcription but before translation.

Line 53: A gene with high CAI is hypothesized to be translated at a high rate

Line 54: “may not be translated” – this phrase is used throughout. What biologically does this mean? Are you referring to pseudogenes, RNAs, etc?

Fig1A: Based on the scale it is hard to see how the high and low genomes differ. It would be good to report the mean of the mean CAIs for the top and bottom 10. Are the genomes sorted in any way in Fig1A and B? It may be useful to sort them by mean.

Line 111-113 – When comparing the number of genes within each institution, is this the total number of genes regardless of the number of genomes? It is difficult to understand how these absolute values compare without knowing what percent they are. This is purely observational, a statistical test could be done on the count data to support the conclusions drawn.

Line 125-126: Is the proportional number of ribosomal proteins with high CAI scores reported anywhere? Are they statistically indistinguishable or observationally similar?

Figure 3B: Why are there two data points associated with “metal ion binding”?

Line 188: The subheadings in this section are confusing.

Figure 4G-I: These are not cited in the manuscript.

Line 252: The correlation referred to in this section, is that a slope or an R2?

Line 299: It is unsurprising that the lowest CAI would be in genes that are hypothetical and therefore less likely to be true protein coding genes. It would be more biologically relevant to present the lowest CAI of non-hypothetical proteins.

Line 361: When the authors exclude likely non-aureus isolates, is this done only with the annotation number or were other methods employed to remove non-aureus isolates?

Line 433: What type/method of p-value adjustment (multiple testing correction) was done?

Linear mixed modeling methods are not included in the methods section.

Line 475: Why was mean CAI across the genomes used for the comparison to proteomics? Would you expect that all genomes would fit the proteomics data equally well?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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Codon Bias Variation in Staphylococcus aureus

PONE-D-26-16137R2

Dear Dr. Patel,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Jordy Evan Sulaiman, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Dear Dr. Patel,

Thank you for submitting the revised manuscript, which is now suitable for publication in PLOS One. Once again, thank you for your patience and efforts throughout the review process.

Reviewers' comments: