Dear Dr. Wells,

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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: No

Reviewer #2: Yes

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

Reviewer #1: N/A

Reviewer #2: Yes

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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: No

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1:  Dear authors, I went through your manuscript and found some changes that must be done. Here are my suggestions-

1. The definition of a new CAD lineage based solely on a cluster of ≥3 isolates lacks methodological rigor. A more quantitative threshold (e.g., allelic divergence) should be applied alongside phylogenetic clustering to robustly delineate novel lineages.

2. While 242 Canadian isolates were analyzed, the manuscript lacks a clear rationale for focusing on Canada in a globally relevant context. Adding a comparative or translational public health perspective (e.g., impact of NOVC on coastal communities or seafood industries) would improve relevance.

3. The study relies heavily on cgSNP analysis but does not clarify the rationale for SNP thresholds used to define lineages, outbreaks, and sublineages. The cgMLST cutoffs (e.g., 3, 40, 133, 1000 alleles) need to be referenced and justified within the context of V. cholerae evolution.

4. Environmental data (e.g., temperature, salinity, sample site type) are not quantitatively integrated. A generalized linear model or spatiotemporal regression could better reveal correlations between environmental factors and lineage emergence.

5. Given the known plasticity of V. cholerae genomes, especially NOVC strains, the potential role of HGT in virulence gene dissemination (e.g., tdh, T3SS, VSP islands) is underexplored and should be evaluated using tools like ICEfinder or PlasmidFinder.

6. While the authors tabulate virulence genes, they fail to conduct any comparative pan-genome or synteny analyses. A deeper annotation of pathogenicity islands (e.g., VPI-1, VPI-2) using genome browsers would provide better resolution.

7. All conclusions about virulence are based on in silico predictions. There is no transcriptomic or phenotypic validation (e.g., hemolysin assays, T3SS effector secretion), which weakens claims about clinical relevance.

8. The AMR gene analysis is described in passing with no detailed breakdown of classes (e.g., β-lactamase variants, efflux pumps). Also, no mention is made of mobile genetic elements carrying AMR genes, which is critical in surveillance.

9. The manuscript leans heavily on third-party platforms without disclosing the limitations of these tools (e.g., static databases, annotation errors). Results should be cross-validated using independent BLAST-based methods or custom HMM profiles.

10. The manuscript misses an opportunity to discuss how NOVC evolution compares with pandemic O1/O139 strains (e.g., clonal expansion vs. recombination-driven diversification). Phylogenetic trees should include time-calibration or molecular clock estimates.

11. Student’s t-test is applied to categorical variables (gene presence/absence), which violates assumptions. A more appropriate test would be Fisher’s exact or chi-square test with FDR correction.

12. There is no accessible summary table (as a figure or supplemental file) that links isolates to year, location, lineage, serogroup, and source. This omission hinders reproducibility and reader comprehension.

13. The classification of isolates as "clinical" or "environmental" is vague and lacks metadata evidence (e.g., case records, exposure history). Misclassification could bias virulence profile comparisons.

14. Recombination filtering using PhiPack is briefly mentioned, but no recombination rate estimates or visualizations (e.g., Gubbins plots) are shown. The extent of recombination should be quantified.

15. The core genome phylogenetic trees are not sufficiently annotated (e.g., bootstrap values, gene overlays). Inclusion of interactive figures (e.g., Microreact or iTOL links) is suggested but not demonstrated.

16. While climate change is a plausible driver of Vibrio emergence, the manuscript repeatedly makes unquantified claims without supporting climate models or water temperature data from Canada.

17. Genes like tdh, ctxAB, toxR are inconsistently referred to across figures and text. A consistent gene naming system should be applied (e.g., italicized for genes, capitalized for proteins).

18. The manuscript misses the chance to link Canadian findings to broader One Health or global AMR surveillance initiatives (e.g., GISAID-equivalent for Vibrio, or WHO GLASS).

19. Custom pipelines like “mikrokondo” are not well-described or linked to repositories. Readers must have access to full GitHub/DOI-archived scripts and parameters for reproducibility.

20. Although MLST types are mentioned, the manuscript doesn’t explore the evolutionary or geographic patterns of sequence types across the Canadian or global dataset using STRUCTURE or goeBURST.

Reviewer #2:  Greetings

Great work.

But there is one figure about the phylogenetic tree is not clear.

Kindly try to put more clear figure.

Kindly write the scientific names of bacteria by italic line style in whole manuscript.

Kind regards.

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Reviewer #1: Yes: Shaban Ahmad

Reviewer #2: No

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Dear Dr. Wells,

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.

Please submit your revised manuscript by Jan 26 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 applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Rahul Shubhra Mandal, Ph.D.

Academic Editor

PLOS One

Journal Requirements:

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.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: 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: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

Reviewer #1: Dear authors,

I read the revised manuscript and found that your team has resolved everything and therefore I mark it for acceptance. Congratulations.

Reviewer #3: 1. The detection of the tdh (thermostable direct hemolysin) gene in the CAD-2 lineage—which was associated with the herring egg outbreak—is a significant finding. However, the manuscript implies this gene is linked to pathogenicity similar to V. parahaemolyticus. The authors should provide a more granular analysis of this gene sequence. Specifically, what is the nucleotide or amino acid identity of this tdh allele compared to the canonical pathogenic tdh found in V. parahaemolyticus? Confirming whether this is a functional homolog or a distinct variant is crucial for substantiating the virulence potential discussion in lines 538-547.

2. In the antimicrobial resistance section (Line 725), the authors state: 'All isolates harbored at least one of the dfrA genes conferring resistance to trimethoprim.' A 100% prevalence of acquired resistance genes across diverse lineages spanning 25 years is statistically unusual for non-O1/non-O139 isolates. The authors should clarify if the specific dfrA variants detected are known intrinsic chromosomal genes in V. cholerae or legitimate acquired resistance markers. If they are intrinsic, classifying them as markers of resistance in the context of surveillance might be misleading without qualification.

3. The authors perform statistical comparisons between 'clinical' and 'environmental' isolates (Fig 3 and text). However, the 'environmental' category aggregates 'food' (seafood/retail), 'water', and 'animal'. As shown in Figure 3b, food isolates appear to share more virulence features with clinical isolates than water isolates do. Grouping retail seafood (which acts as a vector) with environmental water samples might dilute statistically significant differences regarding ecological adaptation vs. infectivity. I suggest a brief sub-analysis or discussion point distinguishing 'natural environment' (water/sediment) from 'food/retail' to refine the conclusion that clinical and environmental isolates differ.

4. The authors identify eight 'new' lineages (CAD1-8) and state they 'represent populations of NOVC local to Canada' (Line 519). While the phylogenetic clustering is sound based on the cited cgMLST scheme, the claim of locality should be tempered. Given the vast under-sampling of NOVC genomes globally (specifically from environmental sources in other nations), it is highly probable these lineages circulate elsewhere but simply haven't been sequenced. I recommend modifying the text to acknowledge that these are 'lineages currently unique to the Canadian dataset' rather than definitively local or endemic to Canada.

5. While the inclusion of Microreact links is excellent for transparency, the static versions of Figure 2, Figure 4 and Figure 5 included in the manuscript are very dense and difficult to read. In Figure 2, the heatmap columns are indistinguishable without zooming in significantly, and the tip labels are illegible. I recommend providing a simplified or 'collapsed' version of the tree for the main text that highlights the CAD lineages and major clades, while moving the fully annotated, dense trees to the Supplementary Materials.

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Reviewer #1: No

Reviewer #3: Yes: Dr. Debaki Ranjan Howlader

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Population structure and phylogenetic analysis of Vibrio cholerae non-O1/O139 by whole genome sequencing

PONE-D-25-23191R2

Dear Dr. Wells,

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,

Rahul Shubhra Mandal, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

**********

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

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

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

The PLOS Data policy

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

Reviewer #3: (No Response)

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #3: Yes: Debaki Ranjan Howlader

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