Plasma-Treated Water Effect on Sporulating Bacillus cereus vs. Non-Sporulating Listeria monocytogenes Biofilm Cell Vitality

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents an insightful comparative study on the effects of plasma-treated water (PTW) on biofilms formed by sporulating (Bacillus cereus) and non-sporulating (Listeria monocytogenes) foodborne pathogens.   The study is structured, and the experimental design is sound with well-chosen methods. The authors provide a compelling analysis of the physicochemical changes in PTW and their biological consequences. However, several areas require improvement in clarity, scientific clarity, data interpretation, and presentation to ensure maximum impact and transparency.

Major comments

1. The manuscript makes several mechanistic claims—such as spore germination in Bacillus cereus and entry into a VBNC state in Listeria monocytogenes—without providing direct experimental validation. These interpretations must be presented as hypotheses and should not be stated as conclusions unless supported by appropriate assays such as microscopy, gene expression, or resuscitation experiments.
2. Figures and tables lack consistent formatting and labeling. Several key results (e.g., in Figures 2–5) are not visually annotated with statistical significance, error bars, or clear treatment labels, making interpretation difficult. Improve figure legends and ensure all plots are self-explanatory, with consistent units and scales across comparable panels.
3. The statistical methods are generally sound but insufficiently described in parts. The manuscript uses non-parametric tests with Bonferroni corrections, but normality testing is not reported. Effect sizes, confidence intervals, and the rationale for choosing specific sample sizes or treatment conditions should be included to improve transparency and reproducibility.
4. The Conclusions section lacks specificity. It fails to clearly state which PTW treatments were most effective, how the two bacteria responded differently, and what the practical implications are for food safety. A more focused and data-driven conclusion is necessary to reflect the study’s findings and potential applications.

Minor comments

1. Abstract (Lines 9–28): Rewrite the first sentence. “Millions of people fall ill due to various foodborne illnesses” is vague and non-scientific. Be more specific: e.g., “Foodborne pathogens are a major global health concern, contributing to millions of infections annually.”
2. Line 11–13: The phrase “processing stages before being distributed including sanitation steps” to improve clarity.
3. Line 20–22: Clearly state the research objective in the abstract.
4. Lines 24–28: The conclusion of the abstract is weak. End with a clear summary of the significance of the findings.
5. The abstract should include a concise sentence on the broader application or implication of the findings in food safety or sanitation.
6. Clarify what is meant by “compounding effects on the quality of the product” be specific about whether this refers to taste, texture, or nutritional loss.
7. Instead of stating "bacteria commonly found to cause foodborne illness," directly mention their prevalence in specific foodborne outbreaks with references.
8. Avoid stating “although being significantly reduced” without quantifying the reduction or stating under what conditions.
9. Clearly state how thermal treatments negatively affect food quality—e.g., "leading to nutrient degradation and undesirable sensory changes"—rather than using vague terms like "quality."
10. Add specific evidence about B. cereus resistance to processing methods such as pasteurization or radiation.
11. Line 53–55: Introduce “non-thermal plasma” more clearly, as it is a key term in the study. Define what it is and distinguish between direct and indirect applications.
12. ine 58: The sentence "while preserving the quality of the food product" is too generic. Be specific: Does it preserve texture, nutritional value, or shelf-life?
13. Line 70–72: Add a definition of biofilms as "structured communities of bacteria enclosed in a self-produced polymeric matrix that adheres to surfaces."
14. Line 73–76: Specify that B. cereus can grow under refrigeration and resist pasteurization, adding a recent food outbreak example if possible.
15. Line 95–98: Be more concise.
16. Line 98–99: The study objective is not clearly stated.
17. Line 104–105: Specify the quality and composition of the tap water used (e.g., filtered, deionized, or municipal source), as water chemistry can affect PTW output.
18. Line 105–106: Describe how “cold boiled tap water” was prepared and cooled to 5 °C—was it cooled on ice or at room temperature?
19. Line 114–117: Indicate whether flasks were shaken or left static during the overnight culture incubation at 30 °C.
20. Line 118–119: Justify the use of different OD600 values for the two organisms (0.1 for B. cereus, 0.2 for L. monocytogenes)—was this empirically optimized for similar biomass?
21. Line 124–125: Mention whether biofilm growth periods (48 h for B. cereus, 52 h for L. monocytogenes) were chosen based on saturation curves or literature precedent.
22. Line 297–299: The control CFU values for L. monocytogenes are higher than B. cereus by ~1 log. Explain whether biofilm biomass or initial OD differences account for this.
23. Line 301–303: Clarify whether proliferation reduction was due to killing or VBNC state. These results alone cannot distinguish this without viability/metabolic assays.
24. The significant difference between species (Table 4) is interesting, but more commentary is needed in Results before jumping to mechanistic discussion.
25. In Figure 3, include direct indicators of statistical significance (e.g., asterisks) above boxplots. Coding alone is insufficient.

Author Response

Dear reviewer,

please see the attachment for our answers to your comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This work revealed the Plasma-treated water effect on Sporulating Bacillus cereus vs. non-Sporulating Listeria monocytogenes biofilm cell vitality. This topic is interesting. However, this quality of this manuscript need be further improved. More characterizations need be provided. Major revision is suggested:

1. The cortex and inner membrane damage need be characterized.

2. Morphological alterations can be characterized by atomic force microscopy, TEM, Raman spectra, etc.

3. The changes before and after Plasma treatment need be well summarized, and related mechanism should be well proposed via Plasma treatment. Related scheme need be provided in the text.

4. In "4. Discussion", the novelty of this work need be well highlighted. In-depth discussion need be made.

5. "5. Conclusions" is too general, and important results and conclusions need be given.

 

Author Response

Dear reviewer,

please see the attachment for our reply to your comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript effectively addresses all previous concerns, and the updates have improved clarity. 
The manuscript is suitable for publication in its current form, and I recommend that it be accepted.

Reviewer 2 Report

Comments and Suggestions for Authors

This revised version can be accepted.