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All remaining concerns of the reviewer were addressed, and the revised manuscript is acceptable now.
[# PeerJ Staff Note - this decision was reviewed and approved by Valeria Souza, a PeerJ Section Editor covering this Section #]
Please address remaining concerns of the reviewer and amend manuscript accordingly.
**PeerJ Staff Note**: Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
The article improved in comparison to its initial version. The topic is relevant, the results obtained are interesting, and the analyses were adequately performed. However, I would suggest a professional English revision, in order to improve the readability and make the reading less exhaustive. Also, the Discussion session is long, and authors could make an effort to reduce redundant and unnecessary text.
Methods were well described and performed.
The results obtained were adequately informed and displayed in general. Few changes must be done in some fields of the Results session and in tables and supplementary tables to make them more adequate.
Specific comments:
Line 30: if resistance was found for more antimicrobials than “tigecycline, azithromycin, colistin and ciprofloxacin”, perhaps you could replace “with” for “including”.
Lines 32-33: this percentage/proportion is confusing. Consider removing it.
Line 42: remove the extra period.
Line 71: remove “The causative agent”
Line 85: standardize “S.” or “Salmonella” in this field and others.
Line 117: remove “further”
Lines 133-134: I’m still confused by how the authors managed to collect “one colony of each serovar per positive sample”, if the vast majority of Salmonella serovars is indistinguishable by colony morphology, and serovars can only be assigned phenotypically by serum agglutination.
Lines 172-173: Authors should make clear that the five strains selected for WGS harbored SIMULTANEOUSLY the three plasmid-associated genes searched
Line 228: something seems missing after “were”.
Line 239: remove “concurrently”.
Line 275: write “genes” instead of “gene”
Line 276: change “Five (1.37%) serovar Enteritidis” for “Five strains (1.37%) of Salmonella Enteritidis”. Also, reinforce here that they were the only strains harboring simultaneously the three genes searched.
Lines 283-285: this sentence seems out of context here. Consider moving it to a more appropriate place.
Line 289: remove “island”.
Line 290: include the accession number of this unnamed SPI.
Lines 291-293: replace this for “Three plasmid incompatibility (Inc) groups were identified: IncFIB and IncFII, present in all five isolates, and IncX1, specifically identified in SA615, SA616, and SA617.”
Lines 295-322: authors could simplify this fragment by keeping information focused on the genes/mutations found and their frequencies. Also explain why all these genes found are not displayed in Table 4.
Lines 296-297: please remove “conferring resistance to various antibiotic classes and contributing to MDR”.
Line 335: remove “with a unique function”.
Lines 366-368: replace this for “No transconjugants were obtained under ampicillin selective pressure for plasmid transference”, or something similar.
Lines 409-410: remove “antibiotics”
Line 434: write “predominant” or “predominantly found” in this field
Lines 441-443: please check this study (https://doi.org/10.1093/molbev/msg074). As I mentioned on my first review, this is a chromosomal gene, with little or no effective resistance against aminoglycoside drugs.
Line 443: I would suggest for you to mention gyrA as a gene, and not a protein, here and in other fields.
Table 4: accession numbers could also be included here.
Tables S1, S2 and S3: authors should display the data of each strain separately, and not as a summary.
Please address the concerns of all reviewers and amend the manuscript accordingly.
**PeerJ Staff Note:** Please ensure that all review and editorial comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
**Language Note:** The review process has identified that the English language must be improved. PeerJ can provide language editing services - please contact us at [email protected] for pricing (be sure to provide your manuscript number and title). Alternatively, you should make your own arrangements to improve the language quality and provide details in your response letter. – PeerJ Staff
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General Comments
This manuscript investigates antimicrobial resistance (AMR) and plasmid-associated virulence genes in Salmonella enterica isolates collected from pigs, pork, and humans across Thailand and neighboring countries. Out of 366 isolates screened via PCR for three plasmid-associated virulence genes (spvC, pefA, rck), only five human-derived S. Enteritidis isolates were positive. These were subjected to whole genome sequencing (WGS), revealing clonal dissemination of ST11 S. Enteritidis strains carrying IncFIB-IncFII virulence plasmids and, in some cases, IncX1 resistance plasmids.
1. Limited scope of key findings: Although the study aims to highlight the coexistence of virulence and resistance plasmids in S. Enteritidis, the core findings are based on just five closely related isolates. These represent only 1.37% of the total isolates and exhibit high genetic similarity. As such, the conclusions should be more conservatively framed as preliminary findings rather than indicative of widespread plasmid dissemination.
2. Lack of discussion on plasmid significance in S. Enteritidis: The virulence plasmid is well known in MDR S. Typhimurium, particularly those with the ACSSuT resistance phenotype, and S. Choleraesuis. Its detection in S. Enteritidis may have specific implications worth discussing—whether related to host adaptation, evolutionary origin, or pathogenic potential. The manuscript currently lacks this context, which could enrich the scientific value of the study.
3. Zoonotic linkage cannot be assessed given the sample composition: No virulence plasmids were detected in isolates from pigs or pork, which is not surprising since S. Enteritidis is generally associated with poultry rather than swine. Therefore, the apparent lack of linkage between human and non-human isolates may reflect the serovar distribution among sources rather than the true absence of transmission. This point should be clarified to avoid misinterpretation.
Minor Comments
1. Table 2: The use of the total isolate count (n=366) as the denominator across diverse sources (pigs, pork, humans) and countries is misleading. These subgroups differ significantly in origin and sample size. It would be clearer to report percentages within each subgroup separately to avoid misrepresentation of prevalence data.
2. Limited informative value of Table 3: Table 3 presents resistance patterns stratified by serovar, country, and sample source (host). However, the total number of isolates (n=366) is too small for such detailed subgrouping to yield meaningful insights. The over-stratification leads to fragmented data with limited interpretive value. A more concise presentation, such as focusing on resistance patterns by serovar alone, may improve clarity and epidemiological relevance.
3. Conclusion overstatement: As stated in lines 38–40, “Phylogenetic analysis of whole genome sequence and virulence plasmids revealed the clonal dissemination of the isolates and the horizontal transfer of virulence plasmids, suggesting the circulation of Salmonella and their plasmids in the region,” and in lines 470–471, “These findings suggest the clonal dissemination of the isolates and the horizontal transfer of virulence plasmids.” However, the conjugation assays showed no evidence of horizontal gene transfer, and the pSEVT plasmids appear structurally incapable of conjugative transfer due to missing tra regions. These statements should be adjusted or more cautiously interpreted to reflect the experimental findings.
4. English usage: The manuscript is mostly readable but would benefit from a light professional edit to improve fluency, especially in the Results and Discussion sections.
5. The term “Lao PDR” is used throughout the manuscript, but its full name (“Lao People's Democratic Republic”) is not spelled out upon first mention.
6. Lack of methodological detail for SPI Identification: The manuscript states that 13 types of Salmonella Pathogenicity Islands (SPIs) were identified (lines 267–270), but does not describe how these were detected (e.g., tools, databases, or criteria used). This information should be provided in the Methods section to ensure reproducibility.
7. Unclear or misleading phylogenetic placement in Figure 4B: The phylogenetic tree in Figure 4B shows Salmonella Typhimurium strain LT2 positioned between S. Enteritidis isolates, which contradicts expected evolutionary relationships. This raises concerns about the accuracy or rooting of the tree. The authors should clarify the input data and methods used for tree construction and consider revising the figure or analysis to reflect more biologically plausible relationships.
8. In the Results section (lines 28–31), the authors state that five S. Enteritidis isolates (5/366, 1.36%) carried virulence plasmids. However, this denominator includes all Salmonella isolates (n=366) rather than just the S. Enteritidis subgroup (n=7). This significantly underrepresents the actual proportion of S. Enteritidis isolates harboring virulence plasmids (5/7, ~71%). The statement should be revised to reflect the correct context and avoid misinterpretation.
Language is clear and correct, but modifications are necessary. Some background information is necessary in the Introduction and Abstract. References are sufficient, and the structure of the paper is adequate.
The manuscript is suited for the journal. The topic is relevant for OneHealth, Food Safety, and Public Health researchers. Methods were sufficiently detailed, although a few corrections are necessary. The investigation performed was adequate.
The impact of the research and findings is recognized. Data is, mostly, robust, controlled, and clear, although some points need to be raised and clarified. Conclusions are adequate but can be improved to be more suitable.
Specific comments to the authors:
The author should make public the database where these genomes were deposited and their accession numbers.
If the manuscript is analyzing samples from Thailand and other countries, this should be taken into consideration throughout the manuscript, including the Title and a better contextualization of these countries in the first paragraph.
Abstract – although abbreviations have been defined in the main text, they must also be defined at least for the first time in the Abstract.
Line 24 – provide quick background for the study
Line 24 – “drug-resistant” instead of “resistant”
Lines 37-43 – please check and restructure this fragment. Information about the transference or non-transference of plasmids is confusing, and the conclusion of the abstract needs to be improved.
Introduction – A major issue of the Introduction is the lack of information on non-typhoid Salmonella serovars, in particular S. Enteritidis. The audience must know why you are studying it, why it is relevant for AMR, etc. Also, authors must define the concept of non-typhoid serovars before they even begin to talk about any Salmonella serovar, and mention the abbreviation of Salmonella enterica subspecies enterica as “S.” properly before any abbreviation.
Line 49 – The authors should consider something broader, like “poor basic sanitation” instead of “unhygienic public utility”.
Line 55 – “increases” instead of “increasing”
Line 60 – consider “developed” or “acquired” instead of “evolved”
Line 62 – If authors mean to say “resistance plasmids” by “R plasmids”, please include this abbreviation.
Line 68 – Instead of “plasmid gene,” consider “plasmid-borne gene.”
Lines 81-82: replace “leading to severe and life-threatening conditions” with “favoring the development of more severe infections”
Line 84 – “Sequences” instead of “Sequence”
Line 86 – If you mean “AMR” when you say “resistance”, please change it. Resistance is too broad if you’re referring to antimicrobial resistance (AMR). Change this throughout the manuscript as well.
Line 87 – Please change “S. enterica serovar Dublin” to “S. Dublin” only.
Lines 93-96 – Please review this fragment for a simpler, more grounded, and scientific version. Also, keep in mind that a strain harboring virulence-associated factors (genes, SPIs, plasmids, phages) will not necessarily have a virulent phenotypic profile, in vitro or in vivo, despite the strong correlation.
Line 98 – define what “LMICs” are
Line 144 – “were” instead of “was”
Lines 157-158 – Please clarify that these five strains selected for WGS harbored all three genes searched.
Line 159 – “integrity” instead of “degradation”
Lines 180-192 – Authors should mention the parameters used for their searches.
Line 191-192 – please review this sentence.
Lines 203-204 – check if “One colony of each serovar was collected from each positive sample.” matches with what you meant on lines 157-158
Lines 242-246 – please check the writing for the isolation sources
Lines 254-257 – authors should consider mentioning how many isolates and serovars harbored one or two of the three genes. It is impressive how only five S. Enteritidis of 366 samples from diverse serovars were positive for the three genes together.
Lines 266-267 – Did all three distinct isolates show the same number of bp for genome and chromosome?
Lines 269-270 – for more clarity, please cite all SPIs found, consecutively.
Line 276 – please write the chromosomal mutation in its correct form: Asp87→Tyr, and not D87Y
Lines 281-282 – this could be integrated into the text or as a small table.
Line 289 – “All were with 100% sequence identity”. Please clarify.
Line 344-345 – Authors affirm here that the five genomes analyzed were closely related to the S. Enteritidis reference included. However, Figure 4B shows that they were more closely related to S. Typhimurium than S. Enteritidis. This is concerning. If these isolates are in fact S. Enteritidis, why are they more closely related to S. Typhimurium (when they should NOT be)? Non-typhoid Salmonella serovars are genetically close, but not close enough to make a genome from one serovar cluster with genomes from others, and not with one from their own. This divergence should be carefully checked. They must also reconfirm in silico by tools like SeqSero if these genomes are, in fact, S. Enteritidis.
Line 362-363 – please detail and clarify this information: Given the dynamics of AMR, antibiotic use and AMR development may not occur simultaneously.
Line 376 – please check, but I believe these antimicrobials are drugs-of-choice for salmonellosis treatment when hydration only is not sufficient, and not only for invasive infections.
Lines 388-395 – consider removing or reducing this fragment.
Lines 403-404 – This should be better demonstrated in the Results.
Lines 404-407 - aac(6')-Iaa is, in fact, an aminoglycoside resistance gene. However, its presence in Salmonella is not surprising or impacting. It is considered a cryptic/conserved gene, with limited or no influence on phenotypic resistance to this class of drugs.
Line 408 – Correct how the QRDR mutation is written. Also, include the gene where this mutation was found. This information is necessary.
Line 414 – use “the occurrence of mcr-independent” instead of “the mcr-independent”
Line 426 – The authors could provide more details on this
Line 438 – remove the period after the parenthesis
Line 445-446 – remove this final sentence
Table 2 – Authors must include a line “Others” in the serovar column, so readers can comprehend the number of serovars that were not detailed because they were less frequently found.
Table 4 – Genome sequencing metadata could be more suited (in more detail, in fact) in the “Raw data of WGS analysis” file.
Figure 4 – If I understood correctly, Figure 4B was created with the genomes, and not the plasmids of these samples. Authors are also affirming that “The close genetic relatedness emphasizes a shared recent ancestor of the isolates. However, they diverge from the two reference Salmonella strains, S. Typhimurium LT2 and S. Enteritidis.”. This is concerning. If these isolates are in fact S. Enteritidis, why are they more closely related to S. Typhimurium (when they should NOT be)? This divergence should be carefully checked. They must also reconfirm in silico by tools like SeqSero if these genomes are, in fact, S. Enteritidis.
Table S1 – Please check if this table is correct, because it looks like the output of a single genome submitted at SPIFinder, and not the data of the five genomes. I also think that this data is not necessary for the article.
Table S2 – Please provide a more suitable title. Also, check if this table is correct, because it looks like the output of a single genome submitted at CARD, and not the data of the five genomes.
Table S3 – Check if this table is correct, because it looks like the output of a single genome submitted, and not the data of the five genomes.
Raw data of WGS analysis – the file is confusing to read. It can be better organized and displayed. Also, since these genomes were sequenced for the first time in this study, genomic data (accession numbers, CG content, size, coverage, etc) should be included in this file.
The study aimed the characterize serovars found in Thailand and its borders. The methodology used is robust, however, the study design seems random and at the mercy of the available data. Very few common parameters to compare in terms of location, period, and source of samples. The title announces a specific finding, which is the coexistence of 2 plasmids in 5 strains of human source in Thailand. While the paper gives a lot of details not related to the title. I suggest reviewing the content and not focusing on a comparison of the sites, since the study lacks common parameters that justify these statistics.
An additional effort in writing is needed to make it more fluid and more concise.
The accession number of genomic data should also be added in the text.
Most methods are well described, but need some additions:
Lines 110-111. The sentence (they were previously isolated and stored as our bacterial stock) is not relevant for the paper.
Lines 112-114. Authors seem to give importance to the geographical sites of strain collection. But it’s hard for the reader to locate these sites correctly. A map containing the geographical sites as a supplementary figure will help.
Line 131. The reference for clinical breakpoints used in this study should be added.
Line 182. We have observed in our studies discrepancies between AMR prediction using different tools. I recommend using starAMR, which was specifically validated for Salmonella and is publicly available.
The finding is not new worldwide, and the study design shows some weaknesses, as already highlighted.
Paragraph 3.1. The prevalence comparison is biased by the fact that locations and collection dates are different. You can compare locations for the same years, or the same period for different locations. We can also expect that the epidemiology of Salmonella can change over time depending on many factors, including animals. I am really confused, how to interpret this finding.
Table 2 should also contain collection dates (years), as this is not a real comparison between locations.
Paragraph 3.2. Please add the MDR definition used in this study and the reference
Please also add a reference for the antibiotics used clinically.
The paper content should reflect the title or change the title, but harmonize the comparison between sites and sources.
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