Transcriptomic Response of Listeria monocytogenes and Salmonella enterica Typhimurium to Power Ultrasound and Chlorine Treatments

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the transcriptomic responses of two important foodborne pathogens to ultrasound and chlorine treatments using RNA-seq analysis. While the research addresses a relevant topic in food safety, several significant scientific and technical issues require major revision before publication.

L 1-34: The title mentions "S. enterica Typhimurium" but inconsistently uses "S. enterica" throughout the abstract. This creates taxonomic confusion as Salmonella enterica subsp. enterica serovar Typhimurium is the full correct designation.

L 21, 360-362: The manuscript states genes are "significantly" expressed with "-log₁₀ p-value > 1.3" but later defines significance as "adjusted P-value < 0.05 and absolute log2 fold change ≥ 1.0" (lines 269-270).

L 120-142:  The 1-minute treatment duration appears arbitrary without justification. No preliminary studies or optimization data support this specific timepoint choice.

L 151-152: Only mentioning "RIN scores >7" is inadequate for RNA-seq quality control. Missing information about RNA concentration ranges, potential degradation, and batch effects.

L 173-192: Different trimming protocols were used for the two bacterial species (Trim Galore only for L. monocytogenes vs. fastp followed by Trim Galore for S. enterica) without adequate justification.

L 305-306: The manuscript states "no multiple testing correction was applied" for GO enrichment analysis, citing the topGO manual. This approach may lead to inflated Type I error rates.

L 367-375: The conclusion that "chlorine treatment for 1 min did not trigger a significant transcriptomic response" oversimplifies the data. The treatment showed fewer DEGs but may still have biological significance.

Figures 1-2: Volcano plots lack clear indication of the significance thresholds used, and the color coding system is not consistently explained.

L 313-346: The manuscript uses different KEGG databases for the two organisms (eggNOG mapper vs. direct KEGG) which could introduce systematic differences in pathway annotations.

L 793-934: The discussion primarily describes observed patterns without sufficient mechanistic interpretation or connection to known stress response pathways in these organisms.

The manuscript addresses an important research question but requires substantial revision to meet publication standards for a scientific journal. 

 

 

Author Response

L 1-34: The title mentions "S. enterica Typhimurium" but inconsistently uses "S. enterica" throughout the abstract. This creates taxonomic confusion as Salmonella enterica subsp. enterica serovar Typhimurium is the full correct designation.

Thank you. This has been corrected.

L 21, 360-362: The manuscript states genes are "significantly" expressed with "-log₁₀ p-value > 1.3" but later defines significance as "adjusted P-value < 0.05 and absolute log2 fold change ≥ 1.0" (lines 269-270).

We understand that this may cause confusion. The negated base-10 logarithm of 0.05 is 1.3 (i.e., P < 0.05 is equivalent to -log10(P) > 1.3). We have amended the manuscript to only use the term P < 0.05.

Regarding fold change, we used the 1.0 cutoff for differentially transcribed genes, as described in the Methods (i.e., lines 269-270 of the original manuscript), but did not include this in the Results for brevity/readability. We have now included this in the Results where applicable, i.e., with the statement “p-value < 0.05 & |log2 fold change| ³ 1.0”.

L 120-142:  The 1-minute treatment duration appears arbitrary without justification. No preliminary studies or optimization data support this specific timepoint choice.

A 1-minute treatment duration was selected based on our prior studies with 2, 5, and 10 minutes of treatment. Those experiments demonstrated that even at 2 minutes, L. monocytogenes and S. enterica Typhimurium populations could be reduced by up to 2 log CFU. To ensure the treatment remained sublethal and yielded sufficient RNA for downstream transcriptomic analysis, we used a shorter duration of 1 minute. This has been added to lines 134 – 136.

L 151-152: Only mentioning "RIN scores >7" is inadequate for RNA-seq quality control. Missing information about RNA concentration ranges, potential degradation, and batch effects.

Thank you for this comment. We agree that reporting only RIN values does not fully capture RNA quality control. In addition to confirming that all samples had RIN values >7, there was an inspection of Bioanalyzer electrophoretic profiles, which showed clear rRNA bands with no smearing indicative of degradation. RNA concentrations were also quantified using Qubit, with yields ranging from 80–250 ng/µL. These concentrations were well above the minimum required for library preparation. All samples were processed in a single batch to minimize potential batch effects. This has been added to lines 155-158.

L 173-192: Different trimming protocols were used for the two bacterial species (Trim Galore only for L. monocytogenes vs. fastp followed by Trim Galore for S. enterica) without adequate justification.

As mentioned in the manuscript, S. enterica Typhimurium reads were of poor quality, particularly for trailing bases. We’ve expanded this section to explain this further (i.e., that L. monocytogenes reads were of adequate quality after trimming using Trim Galore, while S. enterica Typhimurium reads were not, hence the inclusion of the fastp pre-trimming step for S. enterica Typhimurium), and we have provided Supplemental Data files (i.e., MultiQC reports) showcasing the quality of reads at all steps in the read trimming/pre-processing pipeline (Supplemental Data S1-S6).

L 305-306: The manuscript states "no multiple testing correction was applied" for GO enrichment analysis, citing the topGO manual. This approach may lead to inflated Type I error rates.

Raw P-values produced by topGO methods that account for GO graph topology (i.e., the weight01 algorithm used here) should be interpreted as “corrected” or “not affected by multiple testing”, as the tests are not independent. This is explicitly stated, word-for-word, by the topGO developers in the topGO manual (https://bioconductor.org/packages/release/bioc/vignettes/topGO/inst/doc/topGO.pdf):

For the methods that account for the GO topology like elim and weight, the problem of multiple testing is even more complicated. Here one computes the p-value of a GO term conditioned on the neighbouring terms. The tests are therefore not independent and the multiple testing theory does not directly apply. We like to interpret the p-values returned by these methods as corrected or not affected by multiple testing.

We understand that this may be confusing, and we updated the manuscript to explain this in greater detail. Further, we only display and discuss the most significant GO terms in the main manuscript (i.e., GO terms that were significant after an additional FDR correction). We have updated the manuscript to explicitly state this.

L 367-375: The conclusion that "chlorine treatment for 1 min did not trigger a significant transcriptomic response" oversimplifies the data. The treatment showed fewer DEGs but may still have biological significance.

Thank you. Our original wording was a bit unclear, we have now edited the sentence to read “chlorine treatment for 1 min did not trigger as significant of a transcriptomic response for L. monocytogenes compared to the other two treatments that included ultrasound.”

Figures 1-2: Volcano plots lack clear indication of the significance thresholds used, and the color coding system is not consistently explained.

As mentioned in the legends, upregulated genes are denoted by red points, while downregulated genes are denoted by blue. In addition to stating this in the legend text, we have added a legend to the plot itself. Significance thresholds are stated in the main text (i.e., p-value < 0.05 & |log2 fold change| ³ 1.0), and we now include this information in the figure legends.

L 313-346: The manuscript uses different KEGG databases for the two organisms (eggNOG mapper vs. direct KEGG) which could introduce systematic differences in pathway annotations.

We used eggNOG mapper for L. monocytogenes, as our specific strain was not present in the KEGG databases. We have updated the manuscript to include this information. Given that we do not compare KEGG mappings between the two organisms (L. monocytogenes and S. enterica Typhimurium), the use of different KEGG annotation methods does not affect any of the results reported here. 

L 793-934: The discussion primarily describes observed patterns without sufficient mechanistic interpretation or connection to known stress response pathways in these organisms.

We appreciate this comment regarding mechanistic interpretation. We have revised the Discussion (line 783 – 786 and 810-814) to emphasize how the enriched pathways relate to known physiological stress adaptations in each organism. While regulator-level inferences (e.g., σ^B, PerR) are possible, our dataset did not specifically measure regulatory gene activation; thus, we focused on pathway-level interpretations directly supported by our transcriptomic findings. We have mentioned this as part of future investigations in lines 896-898.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is very relevant and addresses a topic of interest.

I suggest some corrections to better adapt the text:

Where it reads “Infefction”, change it to: “Infection”

Where it reads “Beauchat”, change it to: “Beuchat”

Where it reads “Governement”, change it to “Government”

Where it reads “widing” , change it to “window” (in the fastp trimming parameters description)

Where it reads “Each bacterial strain were subjected” , change it to  “Each bacterial strain was subjected”

Review long sentences and break them down for clarity, for example:

Lines 53–63: The sentence about the absence of a "killing step" in fresh vegetables can be divided into 2–3 short sentences. Check the verb tenses in the description of the experiment execution.

Rewrite the expression "the mode of action remains unclear"  , change it to "the molecular mechanisms remain insufficiently elucidated"

Suggestion: replace excessive repetitions of "ultrasound + chlorine" with "combined treatment" after the first mention.

Ensure that abbreviations (OD600, log-phase, CFU) are explained the first time they appear and then used consistently.

Check that the references are correctly described.

Standardize the use of hyphens in “power ultrasound-based technology” (currently appears with and without a hyphen).

Clarify the choice of these methodological parameters for applying these parameters and levels of ultrasound and chlorine.

Comments on the Quality of English Language

English grammar should be revised and the text made more fluid to better promote the readability of the manuscript.

Author Response

Where it reads “Infefction”, change it to: “Infection”

Thank you. This has been corrected.

Where it reads “Beauchat”, change it to: “Beuchat”

Thank you. This has been corrected.

Where it reads “Governement”, change it to “Government”

Thank you. This has been corrected.

Where it reads “widing” , change it to “window” (in the fastp trimming parameters description)

Thank you. This has been corrected.

Where it reads “Each bacterial strain were subjected” , change it to  “Each bacterial strain was subjected”

Thank you. This has been corrected.

Review long sentences and break them down for clarity, for example:

Lines 53–63: The sentence about the absence of a "killing step" in fresh vegetables can be divided into 2–3 short sentences. Check the verb tenses in the description of the experiment execution.

Thank you. Long sentences have been broken down between 56-59 and verb tenses have been double-checked and corrected.

Rewrite the expression "the mode of action remains unclear"  , change it to "the molecular mechanisms remain insufficiently elucidated"

Thank you for the suggestion. This has been edited in the text. 

Suggestion: replace excessive repetitions of "ultrasound + chlorine" with "combined treatment" after the first mention.

Thank you for the suggestion. We used “ultrasound + chlorine” instead of “combined treatment” because we felt that “ultrasound + chlorine” provided more clarity than “combined treatment”, especially if a reader was not reading the manuscript from start to finish and were only reading specific parts of interest.

Ensure that abbreviations (OD600, log-phase, CFU) are explained the first time they appear and then used consistently.

Thank you. Explanations have been added in lines 111-113 and 118.

Check that the references are correctly described.

Thank you. We have gone through the references to make sure that references are correctly described.

Standardize the use of hyphens in “power ultrasound-based technology” (currently appears with and without a hyphen).

Thank you. When we use the term “power ultrasound-based technology”, we do write it with a hyphen. It’s only when we use the term power ultrasound for short when we do not have a hyphen.

Clarify the choice of these methodological parameters for applying these parameters and levels of ultrasound and chlorine.

Thank you. The parameters and levels of ultrasound and chlorine have been clarified in lines 129-131, 137-139 and 144-147.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

It could be accepted in its current form.