Characterization of Antimicrobial Effects of Plasma-Treated Water (PTW) Produced by Microwave-Induced Plasma (MidiPLexc) on Pseudomonas fluorescens Biofilms

Round 1

Reviewer 1 Report

In this work, the authors describe the effect of plasma treated water on Pseudomonas fluorescens biofilms. They employed many techniques to study different aspects of treated biofilms, compared to the control: CFU analysis, fluorescence assays, XTT assays, fluorescence microscopy, confocal laser scanning microscopy and AFM. The exposure time of PTW to plasma (pre-treatment time) and of the biofilms to PTW (post-treatment time) are varied in order to observe eventual trends in the results.

The topic of this work is of high interest for the scientific community and the authors addressed it in a quite complete and detailed way. The analysis of the state of the art is good, the aim of the work is quite clearly presented, the methods are adequately described and the results are clear and pertinent to the scope. Some figures and captions would benefit of some small improvements (see later).

The only important thing that is missing in my opinion is a brief description of the PTW used for the treatment (in terms of pH, conductivity and reactive species) and a more detailed discussion of the results in terms of the chemical difference between PTW and control water. In other words, how the reactive species present in PTW cause the effect that the authors observed in the biofilm?

Here a list of some other minor comments:

• It is better to avoid the use of acronyms in the abstract.
• Abstract, lines 32-33 “Morphologically, the fusion of cell walls into a uniform dense cell mass” something is missing.
• Recently, a review on Application of Non-Thermal Plasma on Biofilm was published on Applied Sciences. Authors should mention this work. Appl. Sci. 2019, 9(17), 3548; 10.3390/app9173548

• I was not able to find ref 30. If the work is already published, please write the full reference. In any case, some more information about the plasma source in section 2.1 would be useful for the readers.
• Section 2.5. Why is the dilution of the control samples different from the treated samples?
• Figure 1. What causes the variability in the controls? Aren’t they supposed to be the same?
• Caption for Figs. 1, 2 and 3 can be improved. I suggest to invert the order of the sentence. This helps the reader in understanding immediately the content of the figures.
• It would be better to use present tense in the captions.
• Section 3.2, line 224. Should be “slight decrease” instead of “slight increase”.
• 4. I suggest re-organizing this figure to emphasize changes in pre- and post-treatment times. For example single images could be organized in such a way that pre-treatment time increases by going from left to right and post-treatment time by going from top to bottom (or vice versa).

• Section 3.4. Some reference to the different parts of figure 4 in the text are wrong. Please check them.
• Figure 6. Labels with the pre/post-treatment times close/in the AFM images would increase readability.

Author Response

In this work, the authors describe the effect of plasma treated water on Pseudomonas fluorescens biofilms. They employed many techniques to study different aspects of treated biofilms, compared to the control: CFU analysis, fluorescence assays, XTT assays, fluorescence microscopy, confocal laser scanning microscopy and AFM. The exposure time of PTW to plasma (pre-treatment time) and of the biofilms to PTW (post-treatment time) are varied in order to observe eventual trends in the results.

The topic of this work is of high interest for the scientific community and the authors addressed it in a quite complete and detailed way. The analysis of the state of the art is good, the aim of the work is quite clearly presented, the methods are adequately described and the results are clear and pertinent to the scope. Some figures and captions would benefit of some small improvements (see later).

1. The only important thing that is missing in my opinion is a brief description of the PTW used for the treatment (in terms of pH, conductivity and reactive species) and a more detailed discussion of the results in terms of the chemical difference between PTW and control water. In other words, how the reactive species present in PTW cause the effect that the authors observed in the biofilm?

1. Answer: We have added a new section to the introduction where we go into the features of the PTW.

Here a list of some other minor comments:

• It is better to avoid the use of acronyms in the abstract.
• Answer: we have changed it in the abstract

• Abstract, lines 32-33 “Morphologically, the fusion of cell walls into a uniform dense cell mass” something is missing.
• Answer: done

• Recently, a review on Application of Non-Thermal Plasma on Biofilm was published on Applied Sciences. Authors should mention this work. Appl. Sci. 2019, 9(17), 3548; 10.3390/app9173548
• Answer: We thank the reviewer for the reference to the paper. However, we have decided not to cite the paper as it is more related to the medical aspects of plasma application and we are feeling well positioned in terms of citations within our area of expertise.

• I was not able to find ref 30. If the work is already published, please write the full reference. In any case, some more information about the plasma source in section 2.1 would be useful for the readers.
• Answer: We have added the appropriate DOI to the citation. It is an accepted paper which has the same DOI as the paper published in the future. It can therefore already be cited. In the paper you will find detailed instuctions about the plasma source with images and instructions about the plasma source. Depsite that fact, we think we had given the readers all important informations about the plasma source we used in the text. We showed the gas flow, the kind of gas we used and the power we used.

• Section 2.5. Why is the dilution of the control samples different from the treated samples?
• Answer: This is due to the reduction of CFU by plasma treatment. For the control, the sample was diluted up to 1:1.000.000 until it was in a range where you could count between 20-100 cells in the dilution. Because there is a significant reduction in the Plasma treatment, no more cells could be counted at a dilution of 1:1.000.000 as in the control. The range of 1:1.000 was perfect for the plasma treated biofilms, because here, between 20-100 cells could be counted per dilution.

• Figure 1. What causes the variability in the controls? Aren’t they supposed to be the same?
• Answer:No. As indicated in 2.5, the experiments were carried out on 4 different days per treatment or control with 3 biofilms per day. This means that we tested 12 different biofilms for the control or each post-treatment time within the pre-treatment times. Due to the heterogeneity of the biofilms, even if they are grown on the same plate under the same conditions, slight differences in the CFU number are observed. Especially since we have tested each varainte on different days as already mentioned.

• Caption for Figs. 1, 2 and 3 can be improved. I suggest to invert the order of the sentence. This helps the reader in understanding immediately the content of the figures.
• Answer: done

• It would be better to use present tense in the captions.
• Answer: done

• Section 3.2, line 224. Should be “slight decrease” instead of “slight increase”.
• Answer: done

• 4. I suggest re-organizing this figure to emphasize changes in pre- and post-treatment times. For example single images could be organized in such a way that pre-treatment time increases by going from left to right and post-treatment time by going from top to bottom (or vice versa).
• Answer: In principle we agree with the reviewer. No matter how you set it up, the problem with the control would always be that it would stand alone. It is of course a subjective feeling, but we found it optically more beautiful and it fits better into the text, if the images as a whole would form a square and not a single image of the control on top. Of course with the 5-series we have the effect that you have to look exactly which picture represents which pre-treatment and post-treatment time. However, after much consideration we have subordinated this problem to the overall optics. We hope you can agree.

• Section 3.4. Some reference to the different parts of figure 4 in the text are wrong. Please check them.
• Answer: done

• Figure 6. Labels with the pre/post-treatment times close/in the AFM images would increase readability.
• Answer: done

Reviewer 2 Report

This manuscript presents the  characterization of antimicrobial effects of plasma-treated water produced by microwave-induced plasma on Pseudomonas fluorescens biofilms

 The introduction is well written and explains the need for these studies. Chapter materials and methods is described in detail. Discussion and summary chapters are written clearly and correctively.

As a reviewer, I suggest making the following changes:

In line 101  - P. fluorescens (strain ATCC13525) can be change to P. fluorescens ATCC 13525 (ATCC- American Type Culture Collection)

In line 107, 110, 112,131, 197 - add "under aerobic conditions"

In line 108 – 0,050 OD or 0,5 OD?

In line 176  - CLSM can be change to Confocal Laser Scanning Microscopy (CLSM)

In line 185 - Atomic-force microscopy can be change to Atomic-force microscopy (ATF)

In line 186  - (ALM) can be remove

In line 277 – remove line

In line 330 – RONS can be change to RONS (reactive oxygen and nitrogen species)

References should be described in accordance with the instructions for the authors.

Author Response

This manuscript presents the  characterization of antimicrobial effects of plasma-treated water produced by microwave-induced plasma on Pseudomonas fluorescens biofilms

 The introduction is well written and explains the need for these studies. Chapter materials and methods is described in detail. Discussion and summary chapters are written clearly and correctively.

As a reviewer, I suggest making the following changes:

In line 101  - P. fluorescens (strain ATCC13525) can be change to P. fluorescens ATCC 13525 (ATCC- American Type Culture Collection)

Answer: done

In line 107, 110, 112,131, 197 - add "under aerobic conditions"

Answer: done

In line 108 – 0,050 OD or 0,5 OD?

Answer: It was indeed 0.05. The pathogen is characterized by a very fast biofilm growth. Since we are conducting these experiments in further studies with other pathogens and we wanted to achieve an approximate continuity in the CFU numbers due to the comparability, we decided to use this dilution. As the CFU results show, we were able to achieve a sufficient cell count with this dilution.

In line 176  - CLSM can be change to Confocal Laser Scanning Microscopy (CLSM)

Answer: done

In line 185 - Atomic-force microscopy can be change to Atomic-force microscopy (ATF)

Answer: We prefer the already accepted abbreviation AFM for atomic force microscopy. Nevertheless, we thank the reviewers for their comments.

In line 186  - (ALM) can be remove

Answer: done

In line 277 – remove line

Answer: done

In line 330 – RONS can be change to RONS (reactive oxygen and nitrogen species)

Answer: done

References should be described in accordance with the instructions for the authors.

Answer: We have revised the references and sorted them according to the text passages that were added.