Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses an interesting topic; however, several issues should be addressed before it can be considered for publication.

1. The words “antibacterial” and “antimicrobial” convey very similar meanings and are redundantly used in the title. The authors are advised to revise the title to avoid repetition and improve clarity.
2. A similar issue is present in the keywords section, where terms such as “antibacterial effect” and “antimicrobial alternative” appear redundant. Please revise the keywords to avoid overlapping terminology.
3. The Introduction section lacks adequate discussion of existing literature on the antibacterial activity of sulfated chitosan (ChS). Relevant and recent studies should be incorporated to better establish the scientific background and novelty of the work.
4. The Discussion section appears overly expanded and descriptive. It is recommended that the authors make this section more concise and focused on key findings and their scientific significance.
5. The methodology for calculating the Degree of Sulfation (DS) is not clearly described. Please provide the calculation method along with an appropriate reference to support the approach used.
6. FTIR analysis is mentioned in the methodology; however, the corresponding results and discussion are not presented in the Results section. The authors should include the FTIR spectra along with proper interpretation.
7. If available, the authors are encouraged to include XRD spectra to further support the structural characterization of the synthesized material.
8. The conclusion section should be rewritten into a single, well-structured paragraph highlighting the major findings, significance, and possible applications of the study.
9. Appropriate references should be cited for the Propidium Iodide (PI) assay used to demonstrate red fluorescence indicating membrane damage in bacterial cells. Authors may also refer to related work such as: Topologically Zn²⁺ hybridized ZnS nanospheres (Zn²⁺/nZnS) efficiently restrained the infection of Fusarium verticillioides in rice seeds by hyphal disorganization and nutritional modulation, which could strengthen the discussion on antimicrobial mechanisms.

Author Response

Response to Reviewer 1 Comments

Comments and Suggestions for Authors: The manuscript addresses an interesting topic; however, several issues should be addressed before it can be considered for publication.

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Point 1: The words “antibacterial” and “antimicrobial” convey very similar meanings and are redundantly used in the title. The authors are advised to revise the title to avoid repetition and improve clarity.

 

Response 1: We thank the Reviewer for this helpful comment. The title was adjusted. Revised and done

 

Point 2: A similar issue is present in the keywords section, where terms such as “antibacterial effect” and “antimicrobial alternative” appear redundant. Please revise the keywords to avoid overlapping terminology.

 

Response 2: We thank the Reviewer for this observation. The key word was adjusted. Revised and done

 

Point 3: The Introduction section lacks adequate discussion of existing literature on the antibacterial activity of sulfated chitosan (ChS). Relevant and recent studies should be incorporated to better establish the scientific background and novelty of the work.

Response 3: We thank the Reviewer for this insightful remark. Revised and done: A greater number of current references were incorporated, and it was better suited to the discussion.

 

Point 4. The Discussion section appears overly expanded and descriptive. It is recommended that the authors make this section more concise and focused on key findings and their scientific significance.

 

Response 4: We thank the reviewer for this excellent comment. Revised and done: Some paragraphs were summarized, rewriting with greater focus, while still explaining the relevant or significant findings.

 

Point 5: The methodology for calculating the Degree of Sulfation (DS) is not clearly described. Please provide the calculation method along with an appropriate reference to support the approach used.

 

Response 5:  We thank the reviewer for this excellent comment. Revised and done: The suggested modification was made with reference support and improved wording and explanation of the procedure, in section 4.1. Degree of Sulfation (DS) Estimation. On lines 715-726

 

Point 6: FTIR analysis is mentioned in the methodology; however, the corresponding results and discussion are not presented in the Results section. The authors should include the FTIR spectra along with proper interpretation.

Response 6: We thank the Reviewer for raising this important point. Revised and done: The suggested modification was made with the support of figure (figure 2) in the results section on page 4 lines: 122-138.

 

Point 7: If available, the authors are encouraged to include XRD spectra to further support the structural characterization of the synthesized material.

Response 7: We thank the reviewer for this excellent comment. Revised and done: The suggested modification was made with the support of figure XRD (figure 3) in the results section on page 4 y 5 lines: 140-152.

 

Point 8: The conclusion section should be rewritten into a single, well-structured paragraph highlighting the major findings, significance, and possible applications of the study.

Response 8: We thank the Reviewer for raising this important point. Revised and done

 

Point 9: Appropriate references should be cited for the Propidium Iodide (PI) assay used to demonstrate red fluorescence indicating membrane damage in bacterial cells. Authors may also refer to related work such as: Topologically Zn²⁺ hybridized ZnS nanospheres (Zn²⁺/nZnS) efficiently restrained the infection of Fusarium verticillioides in rice seeds by hyphal disorganization and nutritional modulation, which could strengthen the discussion on antimicrobial mechanisms.

Response 9: We thank the Reviewer for raising this important point. Revised and done: The suggested modification was made with the support of the reference you indicated and others, in the discussion section on page 15 lines: 458-465.

 

Finally, we sincerely thank the reviewer for their constructive and insightful comments, which have greatly helped us to improve the clarity and quality of the manuscript. All suggestions have been carefully considered and addressed.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses an important topic and presents promising findings on the antibacterial and antibiofilm effects of sulfated chitosan against P. salmonis. However, some methodological and presentation issues need to be addressed to strengthen the reliability of the conclusions.

First, the MIC determination is based primarily on OD measurements, which may not accurately reflect bacterial viability, especially in the presence of aggregation caused by chitosan. Validation using plate count (CFU) methods would improve the robustness of the results.

Second, the fluorescence imaging (Figure 5) requires clarification. The images appear to be presented as separate channels rather than merged LIVE/DEAD overlays, which limits accurate interpretation of membrane integrity. Additionally, the similarity between images across time points should be clarified to ensure they represent independent observations. These figures seem to be the same.

Overall, the study is valuable, but addressing these points will significantly improve the scientific rigor and clarity of the work.

Author Response

Response to Reviewer 2 Comments

Comments and Suggestions for Authors: The manuscript addresses an important topic and presents promising findings on the antibacterial and antibiofilm effects of sulfated chitosan against P. salmonis. However, some methodological and presentation issues need to be addressed to strengthen the reliability of the conclusions.

 

Point 1: First, MIC determination is based primarily on OD measurements, which may not accurately reflect bacterial viability, especially in the presence of aggregation caused by chitosan. Validation using plate count (CFU) methods would improve the robustness of the results.

Response 1: We thank the Reviewer for this helpful comment. Revised and done: Although in lines 706-710 and lines 719-720 of the manuscript (original for your review), it was described that bacterial growth was evaluated (visual reading of turbidity of wells, reading of OD values ​​and confirmation by re-inoculation in culture medium), we improved the wording of this explanation considering your valuable comment. The suggested modification was incorporated in the methodology section, subheading: c. Minimum Inhibitory Concentration (MIC), on page 22, lines: 805-812.

Point 2: Second, the fluorescence imaging (Figure 5) requires clarification. The images appear to be presented as separate channels rather than merged LIVE/DEAD overlays, which limits accurate interpretation of membrane integrity. Additionally, the similarity between images across time points should be clarified to ensure they represent independent observations. These figures seem to be the same.

Response 2: We thank the reviewer for this valuable observation. Revised and done: Figure 5 became Figure 7 in the results section. In the revised version, merged LIVE/DEAD images (green/red overlays) have been included (Figure 7 A.3-C.3) to allow accurate visualization of membrane integrity and signal co-localization. Additionally, we clarified in the legend (figure 7) and Methods section (Subheading 5.6. and paragraph 3: Bacterial Viability and Membrane Permeability by Fluorescence Microscopy (LIVE/DEAD Imaging), that images correspond to independent fields obtained from separate samples at each time point. Representative images were selected to illustrate consistent observations across replicates.

 

Point 3: Overall, the study is valuable, but addressing these points will significantly improve the scientific rigor and clarity of the work.

Response 3: We thank the Reviewer for this insightful remark and we totally agreeing.

 

 

Finally, we sincerely thank the reviewer for their constructive and insightful comments, which have greatly helped us to improve the clarity and quality of the manuscript. All suggestions have been carefully considered and addressed.

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

Introduction

- In the first part of introduction section, the authors may include information on the scale of the Chilean salmon industry and mention Chile’s position in the global salmon market. This will help strengthen the introduction and highlight the significance of the study.

Results

2.2. Antibacterial Activity

- Line 102, what is ChC? Please provide the full term when it is first mentioned, followed by the abbreviation in parentheses

2.2.1. Bacterial Viability and Bacterial Membrane Permeability

- The authors mentioned ethanol as an effective positive control, inducing a rapid and sustained increase in bacterial membrane alteration; however, this is not described in the Methods section. Please include it.

2.3. Fluorescence Microscopy Analysis

- The authors used ethanol as a positive control for membrane disruption; however, this is not mentioned in the Methods section. Please include it.

Materials and methods

The Methods section is well described. However, several points still need to be addressed to improve clarity.

- Line 687, The authors stated that “Bacterial suspensions were then adjusted to approximately (0.5 McFarland standard, 1.5 × 10⁻¹/mL)”. However, a 0.5 McFarland standard typically corresponds to approximately 1.5 × 10⁸ CFU/mL. Please carefully verify this value and check for consistency throughout the manuscript.

- Line 696 and 714, for MIC determination, the authors incubated the samples for 144 h (6 days). However, for MBC determination, the incubation period was extended to 14 days. Please clarify the rationale for using different incubation periods.

- Line 717, “Biofilm Inhibition Assay” should be revised to “Biofilm Formation Inhibition Assay” for clarity.

Conclusion

Please consider shortening the conclusion for the clarity and focus on the key findings of the study. Avoid repeating detailed results.

Other comments

- The scientific name Piscirickettsia salmonis and P. salmonis should be italicized throughout the manuscript.

 

Author Response

Response to Reviewer 3   Comments

Comments and Suggestions for Authors:

Introduction

Point 1: In the first part of introduction section, the authors may include information on the scale of the Chilean salmon industry and mention Chile’s position in the global salmon market. This will help strengthen the introduction and highlight the significance of the study.

Response 1: We thank the Reviewer for this helpful comment. Revised and done: in the first paragraph of the introduction.

 

 

Results

2.2. Antibacterial Activity

Point 2: Line 102, what is ChC? Please provide the full term when it is first mentioned, followed by the abbreviation in parentheses

 

Response 2: We thank the Reviewer for this observation. Revised and done

 

 

2.2.1. Bacterial Viability and Bacterial Membrane Permeability

Point 3: The authors mentioned ethanol as an effective positive control, inducing a rapid and sustained increase in bacterial membrane alteration; however, this is not described in the Methods section. Please include it.

Response 3: We thank the Reviewer for this observation. Revised and done: The suggested modification was incorporated into the methodology section, subheading 5.6, "Bacterial Viability and Membrane Permeability by Fluorescence Microscopy (LIVE/DEAD Imaging)," on page 23, lines 869-873.

 

Point 4: 2.3. Fluorescence Microscopy Analysis

- The authors used ethanol as a positive control for membrane disruption; however, this is not mentioned in the Methods section. Please include it.

 

Response 4: We thank the Reviewer for this observation. Revised and done

 

Materials and methods

The Methods section is well described. However, several points still need to be addressed to improve clarity.

Point 5:  Line 687, The authors stated that “Bacterial suspensions were then adjusted to approximately (0.5 McFarland standard, 1.5 × 10⁻¹/mL)”. However, a 0.5 McFarland standard typically corresponds to approximately 1.5 × 10⁸ CFU/mL. Please carefully verify this value and check for consistency throughout the manuscript.

 

Response 5: We thank the Reviewer for this observation and detect the printing error. Revised and done: This typographical error was corrected in the Methodology section each time it was detected, thanks to your valuable observation. Your modification is highlighted in yellow.

 

Point 6: Line 696 and 714, for MIC determination, the authors incubated the samples for 144 h (6 days). However, for MBC determination, the incubation period was extended to 14 days. Please clarify the rationale for using different incubation periods.

 

Response 6: We thank the Reviewer for this observation. Revised and done. The manuscript was better worded to explain (sección de metodologia,  subtitulo: d.  Minimum Bactericidal Concentration (MBC), en pagina 22, lineas: 869-873.), that 14 days is not the experimental incubation period (144 hours). 14 days correspond to an incubation period as an additional control in samples re-cultured after the experimental incubation period (144 hours), as recommended by the references cited in the original manuscript for revision.

 

Point 7: Line 717, “Biofilm Inhibition Assay” should be revised to “Biofilm Formation Inhibition Assay” for clarity.

 

Response 7: We thank the Reviewer for this observation. Revised and done

 

 

Conclusion

Point 8: Please consider shortening the conclusion for the clarity and focus on the key findings of the study. Avoid repeating detailed results.

 

Response 8: We thank the Reviewer for this observation. Revised and done:

 

 

Other comments

Point 9: The scientific name Piscirickettsia salmonis and P. salmonis should be italicized throughout the manuscript.

 

Response 9: We thank the Reviewer for this observation. Revised and done: It was summarized and the most important points were highlighted.

 

 

 

Finally, we sincerely thank the reviewer for their constructive and insightful comments, which have greatly helped us to improve the clarity and quality of the manuscript. All suggestions have been carefully considered and addressed.

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The author made improvements to the manuscript. Now it can be accepted for publication. N.B.: Can you please check the reference no. 1? Is that ok?

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

The authors have addressed my comments. Thank you for your revision.

Best regards