Detection, Genomic Characterization, and Antibiotic Susceptibility of Salmonella Anatum SPBM3 Isolated from Plant-Based Meat

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study by Phayakka et al. concerns of  detegtion and genomic of Salmonella Isolated from plant-based meat

• The methodology section is well prepared.

• The data analysis are correct.

• - The English of the text is well written and well readable.

Comments and questions:

• The study focused only on Salmonella isolates from plant-based products; however, other microorganisms present in the samples could also serve as reservoirs of resistance genes.

• Section 2.4. I did not find the control in the experiment (AST). Control samples should be. Please add it.

• In Table 3, it would be useful to replace the column containing only “interpretation” with two columns: “diameter zone (mm)” and “interpretation.”

Author Response

1. The study focused only on Salmonella isolates from plant-based products; however, other microorganisms present in the samples could also serve as reservoirs of resistance genes.

Response 1: We thank the reviewer for this valuable comment. We fully agree that PBM products may harbor various microorganisms, some of which could serve as reservoirs of antimicrobial resistance genes. However, the focus of this study was specifically on Salmonella, given its global importance as one of the leading foodborne pathogens and its established role in public health threats. the occurrence of Salmonella in PBM products is relatively rare and is most likely linked to contamination during production, handling, or packaging processes. For this reason, we designed our study to investigate the genomic features and antimicrobial susceptibility of Salmonella as a representative pathogen in PBM. We believe that focusing on Salmonella provides an important first step toward highlighting the potential food safety risks associated with these novel products. Other microorganisms present in plant-based meat products, such as Escherichia coli, Bacillus cereus, Listeria monocytogenes, Staphylococcus spp. may also act as reservoirs of antimicrobial resistance genes. Future investigations should broaden the scope to include these additional microbial groups.

2. Section 2.4. I did not find the control in the experiment (AST). Control samples should be. Please add it.

Response 2: We appreciate the reviewer’s careful observation. In our antibiotic susceptibility testing (AST), quality control was performed in accordance with CLSI 2024 guidelines using the reference strain Escherichia coli ATCC 25922 as a control. This strain was included to ensure the accuracy and reliability of the Kirby–Bauer disk diffusion method, as recommended by CLSI for Gram-negative bacterial testing (Section 2.4).

3.In Table 3, it would be useful to replace the column containing only “interpretation” with two columns: “diameter zone (mm)” and “interpretation.”

Response 3: We thank the reviewer for this helpful suggestion. In the revised manuscript, Table 3 has been reformatted to present the antibiotic susceptibility results in two separate columns: “diameter zone (mm)” and “interpretation (S/I/R),” instead of combining them in a single column. This change improves clarity and transparency of the experimental results.

Reviewer 2 Report

Comments and Suggestions for Authors

This article studies the situation of Salmonella in plant-based meat products collected from retail markets in Bangkok, Thailand. They discovered Salmonella and also conducted whole-genome sequencing and antibiotic susceptibility tests，Although this article has certain research significance, the following problems still need to be solved。

1.The article collected only 63 samples from a market in Bangkok, among which only one was found to contain Salmonella (S. Anatum). The small sample size and single geographical source may limit the representativeness and statistical power of the results. It is suggested that this limitation be clearly stated in the discussion, and it is recommended that future research expand the sample size and sampling range.

2. The full text is based on all advanced genomic and phenotypic analyses of a single isolate (SPBM3), but attempts to draw general conclusions. The lack of comparison among multiple isolates makes it impossible to determine whether the strain is typical or representative. Moreover, no sampling and tracking were conducted on raw materials, processing environments, operators, etc., making it impossible to determine whether the pollution originated from the raw materials, cross-contamination or the processing stage, which seriously weakened the practical guiding significance of the research.
3. A phylogenetic tree was constructed using 220 ST64 genomes, but it was not specified whether the screening criteria, time, and geographical distribution of these strains were representative. There might be selection bias, which could affect the credibility of evolutionary inferences.
4. Relying solely on the presence of virulence genes and AMR genes in the genome to suggest "potential risks", without actual pathogenicity experiments (such as cell invasion, animal models) or dose-response assessment, the conclusion is overly speculative.
5. The detection of Salmonella in plant-based meat is not the first time, and genomic analysis is only a routine process. The research failed to propose new mechanisms, new risk points or new prevention and control strategies, and its academic contribution was relatively limited.

Author Response

This article studies the situation of Salmonella in plant-based meat products collected from retail markets in Bangkok, Thailand. They discovered Salmonella and also conducted whole-genome sequencing and antibiotic susceptibility tests. Although this article has certain research significance, the following problems still need to be solved.

1. The article collected only 63 samples from a market in Bangkok, among which only one was found to contain Salmonella ( Anatum). The small sample size and single geographical source may limit the representativeness and statistical power of the results. It is suggested that this limitation be clearly stated in the discussion, and it is recommended that future research expand the sample size and sampling range.

Response 1: We fully agree that the relatively small sample size (n = 63) and the fact that all samples were collected from a single metropolitan area (Bangkok) may limit the representativeness and statistical robustness of our findings. As this study was designed as an initial investigation into the occurrence and genomic characteristics of Salmonella in plant-based meat (PBM) products, the sample size was necessarily limited.

Nevertheless, this first report of S. Anatum in PBM highlights the potential risk of contamination, even if rare, and underscores the importance of continued surveillance. Following the reviewer’s suggestion, we have now clearly stated this limitation in the Discussion section and emphasized the need for future studies to expand both the sample size and geographic range to strengthen the generalizability of the findings.

2. The full text is based on all advanced genomic and phenotypic analyses of a single isolate (SPBM3), but attempts to draw general conclusions. The lack of comparison among multiple isolates makes it impossible to determine whether the strain is typical or representative. Moreover, no sampling and tracking were conducted on raw materials, processing environments, operators, etc., making it impossible to determine whether the pollution originated from the raw materials, cross-contamination or the processing stage, which seriously weakened the practical guiding significance of the research.

Response 2: Regarding the limitation of analyzing only a single isolate and the absence of upstream sampling (raw materials, processing environments, or operators). We fully agree that these limitations restrict the generalizability of our findings and weaken the ability to trace the exact source of contamination. In our study, we aimed to report an initial detection and genomic characterization of S. Anatum from PBM, which to our knowledge is among the first such reports worldwide. Although based on one isolate, the comprehensive genomic and phenotypic data we provide (including WGS-based pangenome placement, AMR/virulence profiling, and antibiotic susceptibility) offer an important proof-of-concept and highlight the potential risks of Salmonella in PBM products, which have been considered lower risk compared to conventional meat. We agree that larger-scale studies involving multiple isolates, sampling of raw ingredients and processing environments, and source tracking along the production chain are necessary to determine whether SPBM3 is representative and to establish contamination routes. We have now added a clear statement of these limitations in the Discussion/Conclusions section of the revised manuscript, and emphasized the need for expanded surveillance under a One Health framework to strengthen the practical implications of this work.

 

3. A phylogenetic tree was constructed using 220 ST64 genomes, but it was not specified whether the screening criteria, time, and geographical distribution of these strains were representative. There might be selection bias, which could affect the credibility of evolutionary inferences.

Response 3: We focused on all S. Anatum genomes available in the NCBI database to ensure precise and comprehensive data comparisons. The year of isolation and geographical distribution of each strain are listed in Supplemental Table 1. Based on our analysis, these ST64 strains represent S. Anatum, although some S. Anatum genomes were annotated in NCBI as S. enterica. This misannotation likely occurred before the recognition and proper classification of S. Anatum. These genomes are in fact S. Anatum, as demonstrated by their clustering within the S. Anatum clade in Figures 1A and 1B. For example, the Thai strains S. enterica A4, A2, D8, and C76 all clustered with S. Anatum strains. This observation indicates that all ST64 strains included in this study are S. Anatum. If any strain were not closely related to S. Anatum, the phylogenetic analysis of the 3,756 core genes would have identified them as outliers, clustering instead with the S. Oranienburg strain, which we used as an outgroup.

4. Relying solely on the presence of virulence genes and AMR genes in the genome to suggest "potential risks", without actual pathogenicity experiments (such as cell invasion, animal models) or dose-response assessment, the conclusion is overly speculative.

Response 4: We appreciate the reviewer’s thoughtful comment. We agree that the presence of virulence and AMR genes in the genome does not by itself confirm pathogenicity, and that further functional studies (e.g., cell invasion assays, animal infection models, or dose–response assessments) would be required to directly establish the pathogenic potential of the isolate. Our primary aim was to highlight the occurrence of Salmonella in PBM products and to provide genomic insights that may indicate potential public health risks. We believe that presenting these genomic features offers an important first step in assessing the safety of PBM products. We acknowledge this limitation more clearly in the revised Discussion section and emphasize that future research, including functional and pathogenicity studies, will be necessary to validate these findings. Such studies would likely be better suited for publication in journals focusing on microbial pathogenesis and host–pathogen interactions. We have added the response in the Discussion section.

5. The detection of Salmonella in plant-based meat is not the first time, and genomic analysis is only a routine process. The research failed to propose new mechanisms, new risk points or new prevention and control strategies, and its academic contribution was relatively limited.

Response 5: We understand that this is not the first discovery. However, its occurrence in plant-based meat is unusual and has been rarely documented, particularly in developing countries. We believe this research emphasizes the risk of Salmonella contamination in alternative foods and highlights the importance of implementing the same monitoring and safety checks used for animal-derived products.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript "Detection, Genomic Characterization, and Antibiotic Susceptibility of Salmonella Isolated from Plant-Based Meat" is well-written and important study in the field of AMR. It highlighted the detection of Salmonella serotype Anatum from raw pork meat. However, the study needs minor revisions in the content of the manuscript which can be found the attached PDF document. 

Comments for author File: Comments.pdf

Author Response

1. Title: It should be “Salmonella Anatum SPBM3”

Response 1: Corrected.

2. Abstract

How many sample?

Also write types of samples.

How was assessed?

Which Salmonella species? Mention them here

Write prevalence on total sample basis too. write in percentage and numbers both.

Response 2: Corrected all of them.

3. Introduction: and other pathogens such as S. haemolyticus. Please refer and cite relevant content. https://doi.org10.3389/fcimb.2023.1183390

Response 3:  Corrected as per suggestion.

4. Why you decided to choose following parameters? As you have done Illumina sequencing.

Response 4: We apologize for our mistake, we used default parameters. After quality control and trimming, 113 de novo assembly of the remaining paired-end reads was performed using CLC Genomics 114 Workbench v20.0.4, with default parameters.

5. Why did you not included S. anatum from NCBI? Why you included specifically ST64 genomes?

Response 5: The reviewer is correct that our focus should be on S. Anatum. However, in practice, some S. Anatum genomes were annotated in NCBI as S. enterica. This likely occurred before the recognition and proper classification of S. Anatum. These genomes are in fact S. Anatum, as demonstrated by their clustering within the S. Anatum clade in Figure 1A and 1B. For example, the Thai strains S. enterica A4, A2, D8, and C76 were all clustered with S. Anatum strains, indicating that they represent S. Anatum. We therefore included all S. Anatum isolates to ensure precise and comprehensive data comparisons.

This observation suggests that all ST64 strains included in this study are S. Anatum. If any strain were not closely related to S. Anatum, the phylogenetic analysis of the 3,756 core genes would have revealed them as outliers, clustering instead near the S. Oranienburg strain, which we used as an outgroup. All strain details are provided in Supplemental Data 1.

6. Did you performed AST after WGS or before WGS. if before than shift before WGS.

Response 6: In our study, WGS of the isolate was performed first, followed by the AST. The AST was conducted according to the Clinical and Laboratory Standards Institute (CLSI, 2024) guidelines specific for Enterobacteriaceae (specific for Salmonella).

7. Table S3: I think this table should be included in main article content instead of supplementary data.

Response 7: Some of the details originally presented in Table S3 have been integrated into the main text according to the reviewer’s suggestion, while the remaining information is provided in Table S3.

8. Conclusion: It needs to be revised under following points; Objective, key findings, potential threats, future implications, and recommendations based on this study.

Response 8: We thank the reviewer for this suggestion. The conclusion has been revised to clearly state the objective, key findings, potential threats, future implications, and recommendations.