The Effectiveness of Nafion-Coated Stainless Steel Surfaces for Inhibiting Bacillus Subtilis Biofilm Formation
Round 1
Reviewer 1 Report
The authors report a facile way to modify the stainless steel to be negatively charged via Nafion coating. The surface properties were characterized by contact angle measuring instrument, laser scanning microscope and scanning electron microscopy. The antibacterial properties of the Nafion coated stainless steel surfaces have also been studied.
Research leads to interesting conclusions confirmed by appropriate measurements. It is very valuable that the results can be used in practice.
I have some minor comments:
1. “The cleaned discs were coated with 1% (v/v) Nafion solution” - what type of solution was used (what solvent)?
2. References should be consistently formatted according to journal guidelines (year - bold, volume - italics)
3. I wonder if “dd water” is double-distilled water?
Author Response
Dear reviewer,
We appreciate the insightful comments and are grateful for the time you have spent for reviewing our manuscript. The questions have been repeated and line-by-line response follows. All of the modifications are highlighted (red color) in the revised manuscript. These do not affect our interpretation of the result. Thank you.
Best regards
Lijuan Zhong
The authors report a facile way to modify the stainless steel to be negatively charged via Nafion coating. The surface properties were characterized by contact angle measuring instrument, laser scanning microscope and scanning electron microscopy. The antibacterial properties of the Nafion coated stainless steel surfaces have also been studied
Research leads to interesting conclusions confirmed by appropriate measurements. It is very valuable that the results can be used in practice.
I have some minor comments:
1. “The cleaned discs were coated with 1% (v/v) Nafion solution” - what type of solution was used (what solvent)?
Response: The solvent used to dilute Nafion solution was absolute ethanol. We have modified the description in the manuscript as line 109.
2. References should be consistently formatted according to journal guidelines (year - bold, volume - italics)
Response: Thank you for pointing this, we have checked and revised the references.
3. I wonder if “dd water” is double-distilled water?
Response: Sorry for the unclear abbreviation, the“dd water” is double-distilled water, we have revised “dd water” to double-distilled water in the manuscript as line 108.
Author Response File: 
Author Response.docx
Reviewer 2 Report
See attachment.
Comments for author File: 
Comments.pdf
Author Response
Dear reviewer,
We appreciate the insightful comments and are grateful for the time you have spent for reviewing our manuscript. The questions have been repeated and line-by-line response follows. All of the modifications are highlighted (red color) in the revised manuscript. These do not affect our interpretation of the result. Thank you.
Best regards
Lijuan Zhong
This manuscript focuses on the application of a Nafion coated stainless-steel substrate to prevent the formation of biofilm of the gram positive bacterium B. subtilis. The manuscript is clear and well written, the data are solid and properly described. The authors honestly report that they adopted this surface modification procedure on the same material on a gram negative bacteria (E. coli) in a previous publication (https://doi.org/10.1016/j.colsurfb.2013.05.039). This of course reduces the novelty of this work. However, I am confident that they can provide additional data to make the manuscript more solid and suitable for publication on “MDPI Applied Sciences”
Major comments:
- It is important highlighting the structural difference between gram positive and gram negative bacteria. However, since it is a well-established basic concept in microbiology, I think it is too much devoting a figure (Fig. 2) to that. The paper focuses on gram positive bacteria and their biofilm formation, the comparison and data on gram negative are reported elsewhere (publication from the same authors).
Response: Thank you for the comments, we have deleted figure 2 in the revised manuscript.
- The introduction can be extended considering how surface material and patterning help in preventing bacterial biofilm assembly. Here some examples:
- https://doi.org/10.1021/la700650q
- https://doi.org/10.1021/acsomega.9b03540
- https://doi.org/10.1097/01.brs.0000147801.63304.8a
- https://doi.org/10.1093/ps/79.12.1839
Response: We have revised the introduction. The influence of surface properties on biofilm formation is briefly introduced as line 44-60 of the introduction (in red color).
- To make the results stronger, the authors could provide a comparison of the anti-biofilm activity on different gram positive microorganisms. B. subtilis is definitely an excellent case study, but it will be much more interesting working with pathogenic gram positive bacteria like S. aureus or C. difficile. These microorganisms are extremely important in medicine and there is a great interest in finding approaches to prevent their adhesion.
Response: Thank you for the kind suggestions. It will be important to inhibit pathogenic bacterial biofilm formation since a variety of diseases even some cancers are related to them. We will take this kind of bacteria as one of research objects for our future researches.
- In section 3.2 the authors report the number of living cells on the Nafion-coated steel plate and compare it with the non-treated surface. I wonder if they can provide any data on possible toxic effect of the Nafion on the bacteria. The Nafion coating is “repelling” the bacteria because of the negative charges or it is killing the cells which get closer to the surface?
Response: Thank you. The toxicity of Nafion solution to B.subtilis was investigated and the results was shown as Figure 2. The results indicate that Nafion has no obvious inhibition on the growth of Bacillus subtilis.
Minor comments
Line 54. Remove the colored text.
Response: Thank you, the colored text is removed in the revised manuscript.
Figure 3 and 4 can be combined.
Response: Figure 4 was deleted and the roughness data was presented in words as line 150-152 in the revised manuscript.
Author Response File: Author Response.docx
Reviewer 3 Report
Lines 44, 48, 63, 68 etc. The complex multicomponent structure covering the bacterial cell is termed the envelope and not the membrane. (see e.g. Silhavy TJ, Kahne D, Walker S. The bacterial cell envelope. Cold Spring Harb Perspect Biol. 2010;2(5):a000414. doi:10.1101/cshperspect.a000414)
Line 86. dd -?
Line 100. 150 rpm -?
Figure 2 has nothing to do with the experimental data and their interpretation.
Line 98. The concentration of the cells should be indicated.
Line 104. The model of the sonicator should be indicated.
There is no need in Figure 4. The results could be presented in words. However the method of roughness assessment in μm (lines 94-95) should be clarified.
Figures 6 and 8 are impossible to understand without comments on “what is what”.
Author Response
Dear Reviewer,
We appreciate the insightful comments and are grateful for the time you have spent for reviewing our manuscript. The questions have been repeated and line-by-line response follows. All of the modifications are highlighted (red color) in the revised manuscript. These do not affect our interpretation of the result. Thank you.
Best regards
Lijuan Zhong
Lines 44, 48, 63, 68 etc. The complex multicomponent structure covering the bacterial cell is termed the envelope and not the membrane. (see e.g. Silhavy TJ, Kahne D, Walker S. The bacterial cell envelope. Cold Spring Harb Perspect Biol. 2010;2(5):a000414. doi:10.1101/cshperspect.a000414)
Response: Thank you for pointing this, the word “membrane” was replaced by “envelope” in the revised manuscript.
Line 86. dd -?
Response: Sorry for the unclear abbreviation, the“dd water” is double-distilled water, we have revised “dd water” to double-distilled water in the manuscript as line 108.
Line 100. 150 rpm -?
Response: 150 rpm was replaced by 150 min-1 in the revised manuscript as line 127.
Figure 2 has nothing to do with the experimental data and their interpretation.
Response: Thank you for the comments, we have deleted figure 2 in the revised manuscript.
Line 98. The concentration of the cells should be indicated.
Response: The concentration of the cells was added as line 125 in the revised manuscript.
Line 104. The model of the sonicator should be indicated.
Response: The model of the sonicator was added as “KUDOS, SK8200GT, China” in the revised manuscript as line 131.
There is no need in Figure 4. The results could be presented in words. However the method of roughness assessment in μm (lines 94-95) should be clarified.
Response: Figure 4 was deleted and the method for roughness assessment was added as line 118-120.
Figures 6 and 8 are impossible to understand without comments on “what is what”.
Response: Thank you for pointing this, We have revised the figure captions of Figures 6 and 8. As shown in Figure 5 and 7 in the revised manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Briefly, the authors are stating:
"The questions have been repeated and line-by-line response follows. All of the modifications are highlighted (red color) in the revised manuscript"
But the updated manuscript reports no red text at all! The new sections are not highlighted, and only minimal part of the modifications are reported.
The answers to the questions raised during the review process have been only partially answered and require significant further explanations.
In the current state the manuscript is still far from the quality requested to be accepted.
Author Response
Dear Reviewer,
Many thanks for your time and your comments, I am sorry for my mistake about the explanation as "All of the modifications are highlighted (red color) in the revised manuscript". When we modified this manuscript, we use the function of Microsoft Word as "show all tags" in "Track change", thus all the modifications were shown and in red, as shown in the attached PDF file we uploaded previously. We are sorry for the confusing. We revised the manuscript and highlighted the modifications in red in the new revised manuscript.
We are grateful for the time you have spent for reviewing our manuscript. Many thanks.
Following is our response to your comments:
This manuscript focuses on the application of a Nafion coated stainless-steel substrate to prevent the formation of biofilm of the gram positive bacterium B. subtilis. The manuscript is clear and well written, the data are solid and properly described. The authors honestly report that they adopted this surface modification procedure on the same material on a gram negative bacteria (E. coli) in a previous publication (https://doi.org/10.1016/j.colsurfb.2013.05.039). This of course reduces the novelty of this work. However, I am confident that they can provide additional data to make the manuscript more solid and suitable for publication on “MDPI Applied Sciences”
Major comments:
- It is important highlighting the structural difference between gram positive and gram negative bacteria. However, since it is a well-established basic concept in microbiology, I think it is too much devoting a figure (Fig. 2) to that. The paper focuses on gram positive bacteria and their biofilm formation, the comparison and data on gram negative are reported elsewhere (publication from the same authors).
Response: Thank you for the comments, we have deleted figure 2 in the revised manuscript.
- The introduction can be extended considering how surface material and patterning help in preventing bacterial biofilm assembly. Here some examples:
- https://doi.org/10.1021/la700650q
- https://doi.org/10.1021/acsomega.9b03540
- https://doi.org/10.1097/01.brs.0000147801.63304.8a
- https://doi.org/10.1093/ps/79.12.1839
Response: We have revised the introduction. The influence of surface properties on biofilm formation is briefly introduced as line 44-60 of the introduction (in red color).
- To make the results stronger, the authors could provide a comparison of the anti-biofilm activity on different gram positive microorganisms. B. subtilis is definitely an excellent case study, but it will be much more interesting working with pathogenic gram positive bacteria like S. aureus or C. difficile. These microorganisms are extremely important in medicine and there is a great interest in finding approaches to prevent their adhesion.
Response: Thank you for the kind suggestions. It will be important to inhibit pathogenic bacterial biofilm formation since a variety of diseases even some cancers are related to them. We will take this kind of bacteria as one of research objects for our future researches.
- In section 3.2 the authors report the number of living cells on the Nafion-coated steel plate and compare it with the non-treated surface. I wonder if they can provide any data on possible toxic effect of the Nafion on the bacteria. The Nafion coating is “repelling” the bacteria because of the negative charges or it is killing the cells which get closer to the surface?
Response: Thank you. The toxicity of Nafion solution to B.subtilis was investigated and the results was shown as Figure 2. The results indicate that Nafion has no obvious inhibition on the growth of Bacillus subtilis.
Minor comments
Line 54. Remove the colored text.
Response: Thank you, the colored text is removed in the revised manuscript.
Figure 3 and 4 can be combined.
Response: Figure 4 was deleted and the roughness data was presented in words as line 150-152 in the revised manuscript.
Best regards
Lijuan Zhong
------------------------------
Assitant professor
Department of Bioengineering and Biotechnology
College of Chemical Engineering Huaqiao University
668 Jimei Avenue Xiamen, Fujian 361021, China
Author Response File: Author Response.pdf
Round 3
Reviewer 2 Report
See attachment
Comments for author File: Comments.pdf
Author Response
Dear Reviewer,
Many thanks for your time and your comments, Those comments are all valuable and very helpful for revising and improving our paper. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion for the 1st round and 2nd round are marked in red and blue respectively in the revised manuscript. The main corrections in the paper and the responds to the reviewer’s comments are showed as detached file.
Once again, thank you very much for your comments and suggestions.
Best regards
Lijuan Zhong
------------------------------
Assitant professor
Department of Bioengineering and Biotechnology
College of Chemical Engineering
Huaqiao University, 668 Jimei Avenue Xiamen
Fujian 361021, China
Author Response File: Author Response.docx
Round 4
Reviewer 2 Report
The authors addressed the requested points.
