-->PONE-D-26-01626-->-->Genomic characterization of Salmonella enterica isolates causing typhoid among Ghanaian patients-->-->PLOS One

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: This is a fairy well written manuscript that provides insightful information on S. enterica isolates causing Typhoid fever. It is quite interesting that typhoid toxins genes were found in Non typhoidal isolates. Please find my comments below to improve on the manuscript.

Abstract

The authors should consider adding a statement on the existing research gap this study aimed to fill just before the statement on the research objective in lines 17-19.

The authors should consider qualifying the statement 'Plasmid analysis showed limited diversity. Antibiotic resistance genes were detected at low frequencies, including blaTEM variants (beta-lactam resistance), qnr (quinolone resistance), sul1/sul2 (sulfonamide resistance), aminoglycoside-modifying enzymes (aadA1, aph(6)-Id, aac(3)-IIa), tet(A) (tetracycline resistance), dfrA1 (trimethoprim resistance), and catA1 (chloramphenicol resistance), highlighting limited antimicrobial resistance in the populations sampled' in lines 35-40 by including numbers and proportions of the same.

From the Results section of the abstract, it's not clear, what was the distribution of the S. enterica serotypes after sequencing.

Materials and Methods

The statement 'S. enterica isolates were obtained from patients presenting with typhoid fever in selected health

facilities in Ghana' is not accurate and should be revised to read that the patients presented with typhoid fever symptoms and not typhoid fever unless a typhoid fever positive diagnosis was made at the time of presentation.

In line 101, it's unclear if the S. enterica isolates confirmation was through culture on salmonella shigella agar and if this is the case, this may be inaccurate as colonies on salmonella-shigella are not sufficient enough to give a positive identification of Salmonella spp without further identification using Serology/biochemical testing (API 20E) or molecular techniques such as PCR and sequencing. It would be ideal to refer to the isolates as suspect colonies for Salmonella and not confirmed ones.

Reviewer #2: This manuscript presents WGS-based characterization of 28 Salmonella enterica isolates recovered from stool and blood cultures from patients described as clinically diagnosed with typhoid fever in Ghana. The topic is relevant and the genomic methods are broadly appropriate, but several issues require major revision before the conclusions are reliable.

Major issues

Case definition and sampling frame are unclear

The manuscript frames the work as typhoid fever, yet the dataset includes multiple non-typhoidal Salmonella serovars (e.g., Typhimurium, Enteritidis, Virchow, Saintpaul, etc.). Please clarify:

The clinical definition used to enrol “suspected/diagnosed typhoid” cases (symptoms, duration, exclusion criteria, and whether any diagnostic test beyond culture was used).

The clinical syndrome associated with stool isolates (enteric fever workup vs diarrhoeal illness workup) and whether stool isolates were from the same patients as blood isolates.

A clear breakdown of isolate counts by specimen type (blood vs stool), serovar, and sequence type, ideally in a single table.

If the cohort is “patients suspected of typhoid/enteric fever” rather than culture-confirmed typhoid, this should be reflected consistently in the title, abstract, and conclusions.

Internal inconsistencies in S. Typhi counts and ST reporting

The manuscript reports S. Typhi ST2 as n=7 (24%). However, the supplementary isolate list (Table S1) shows six S. Typhi isolates, all ST2. The seventh ST2 isolate appears to be a non-Typhi serovar (Bangui). This needs to be corrected throughout the text, tables, and figures. Please ensure that all summaries (percentages and n values) match Table S1 and that GenoTyphi lineage assignment is applied to the correct number of S. Typhi genomes.

Virulence gene interpretation is overextended without validation

The report of typhoid toxin genes (cdtB, pltA, pltB) and Vi-related genes outside S. Typhi is potentially interesting, but gene presence calls require more detail and validation because partial hits and fragmented assemblies can produce misleading patterns. In Table S1, several isolates show incomplete toxin gene patterns (e.g., cdtB present without pltA/pltB or vice versa). Please provide:

Identity and coverage thresholds used for calling a gene “present”.

Whether the genes are full-length and intact (no frameshifts or truncations).

Locus context (contig location and surrounding genes, or read-mapping confirmation across the locus).

Without this, discussion implying these non-typhoidal serovars may produce typhoid-like clinical disease should be toned down.

AMR conclusions need phenotypic confirmation or more cautious framing

The manuscript draws strong inferences about susceptibility from genomic AMR gene detection alone. This is not sufficient, especially for fluoroquinolones and other agents where chromosomal mutations and regulatory mechanisms are important. If phenotypic AST was performed, please include it and compare genotype versus phenotype. If AST was not performed, please revise the AMR section to clearly state that findings represent genomic predictions only and avoid clinical treatment implications.

Reproducibility details need strengthening

Please provide software versions and key parameters for the major tools used (read QC and trimming, assembly, annotation, Panaroo settings, IQ-TREE model selection, and thresholds for AMR/virulence detection). Also include basic assembly QC metrics (coverage estimates, N50, contamination checks) and a brief statement on how low-quality assemblies were handled.

Public genome selection for comparative analyses requires justification

The manuscript combines study isolates with public genomes from BV-BRC and Pathogenwatch. Please state explicit inclusion criteria (geography, date range, clinical source, quality filters) and how duplicates or biased sampling were avoided. This affects interpretability of the phylogeographic conclusions.

Minor issues and editorial suggestions

Ensure serovar naming is consistent across the manuscript and supplementary material (avoid spacing/format variants that can affect counts).

Improve figure legends so a reader can interpret rings/labels without referring back to methods.

Consider adding a simple flow diagram of recruitment and isolate selection (patients enrolled, cultures done, positives, exclusions, final 28 isolates).

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Reviewer #1: No

Reviewer #2: Yes: Nubwa Medugu

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<p>Genomic characterization of Salmonella enterica isolates causing typhoid among Ghanaian patients

PONE-D-26-01626R1

Dear Dr. Duodu,

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,

Gabriel Trueba, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #2: Yes

Reviewer #3: Yes

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-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #2: The authors have satisfactorily addressed my previous comments. The revised manuscript is substantially improved, and the main concerns raised during the earlier round of review have been resolved. The manuscript is technically sound, the analyses are adequately described, and the presentation is clear. I consider the manuscript acceptable for publication in its current form.

Reviewer #3: (No Response)

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #2: Yes: Nubwa Medugu

Reviewer #3: Yes: Samuel Kariuki

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