Plasma-Treated Water Retards Pellicle-like Biofilm Formation of Bacillus subtilis
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn this manuscript, the authors exposed cultures of Bacillus subtilis to plasma-treated water and examined the effects on biofilm formation in general and pellicle formation. The effectiveness of the PTW treatment on the cultures was assessed by viable counts, live-dead cell fluorescence quantification, and XTT spectrometry to assess membrane defects. Control and treated cells were cultured and serially sampled for light and SEM analysis. It is clear from the resulted presented, that PTW effectively kills B. subtilis cells, so this organism is similar in response to PTW as other species of bacteria that have been examined. On the negative side, because the treated cells are not actively growing, any conclusions regarding specific effects on biofilm and pellicle formation are not warranted. In order to form biofilms, the bacteria must replicate and subpopulation differentiate to form the various structures that have been described for biofilms. In the manuscript, the authors give their conclusions, but with a lack of detail on how these were reached. No quantitative analysis of the images was shown. It appears that the authors have categorized all bacilli that are present in the liquid phase of the cultures as being pellicle, which is unwarranted.
Specific comments:
1. Legend to Figure 2: It should be “section” 2.2.2 rather than chapter. Same situation with the legend to Table 1.
2. The figure legend for Figure 3 lacks sufficient detail to know exactly what the images in the figure convey.
3. Section 3: Details on the processing steps for the microscopy experiments are lacking. Details on the SEM processing are also lacking. According to Figure 3, the cultures were incubated in petri dishes (what diameter) in broth (presumably TSB) at what volume and starting concentration of cells (10 exp 6 is my guess, but not stated). How do you know whether cells are actually attached to the plate surface, merely on the surface but not attached, floating in the liquid medium, or at the surface of the liquid? Also, what type of microscope system was used to collect these images?
4. Lines 264-272: I am unclear how these conclusions are drawn based on the images of the control culture. For example, in the 5 hour sample, filamentous cells are clearly evident. They disappear in the 6 hour sample and in the 7 hours sample they form towers? There does appear to be a clump of floating lysed cells in the 7 hour image, so how does one definitively characterize this as a tower? The changes in the presence of the filaments over time suggest they are floating and not surface attached. That would not indicate they are pellicle components. A panel-by-panel detailed description and analysis with appropriate arrows or symbols, is needed. Because a pellicle is defined as a biofilm at the liquid-air interface, some detail on how this site was inferred would be helpful.
5. Figure 4: There is no obvious change in the images shown in the treated sample from 0 to 24 hours. Based on what can you conclude that the results would have been substantially different with a longer incubation (lines 273-275).
6. Lines 322-326: The cell aggregates of >1000 cells are not obvious in Figure 6B.
7. Lines 330 and 333: I assume this is meant to be Figure 7.
8. Line 344: What textbook?
9. Lines 362-364: PTW has been reported to be effective against Gram-positive bacteria (e.g. Staphylococcus aureus and Streptococcus mutans) so this section is misleading.
Author Response
Dear reviewer #1.
We welcomed your very helpful suggestions and improvements. Especially your improvements about our microscopically mythology helped to organize our manuscript in a more convenient way. For further information, please see statements below.
Thank you very much for your time, your patience, and the fruitful discussion.
Editing of the corrections:
- Q: Legend to Figure 2: It should be “section” 2.2.2 rather than chapter. Same situation with the legend to Table 1.
A: We corrected the legends according to the suggestions of the reviewer
- Q: The figure legend for Figure 3 lacks sufficient detail to know exactly what the images in the figure convey.
A: The legend of figure 3 has been corrected. Additionally, section 2.5. have been introduced, which summarizes the procedure of microscopic sample preparation.
- Q: Section 3: Details on the processing steps for the microscopy experiments are lacking. Details on the SEM processing are also lacking. According to Figure 3, the cultures were incubated in petri dishes (what diameter) in broth (presumably TSB) at what volume and starting concentration of cells (10 exp 6 is my guess, but not stated). How do you know whether cells are actually attached to the plate surface, merely on the surface but not attached, floating in the liquid medium, or at the surface of the liquid? Also, what type of microscope system was used to collect these images?
A: As highlighted above, section 2.5 has been introduced.
- Q: Lines 264-272: I am unclear how these conclusions are drawn based on the images of the control culture. For example, in the 5-hour sample, filamentous cells are clearly evident. They disappear in the 6-hour sample and in the 7 hours sample they form towers? There does appear to be a clump of floating lysed cells in the 7-hour image, so how does one definitively characterize this as a tower? The changes in the presence of the filaments over time suggest they are floating and not surface attached. That would not indicate they are pellicle components. A panel-by-panel detailed description and analysis with appropriate arrows or symbols, is needed. Because a pellicle is defined as a biofilm at the liquid-air interface, some detail on how this site was inferred would be helpful.
A: Thank you very much! It is a very important remark. Since we now added section 2.5 in which we explain our methodology, we now improved our explanations and the way to our conclusions. Additionally, we based a chapter in our discussion on that remark (lines 399 - 409). We also give some details about contemporary research on that topic.
- Q: Figure 4: There is no obvious change in the images shown in the treated sample from 0 to 24 hours. Based on what can you conclude that the results would have been substantially different with a longer incubation (lines 273-275).
A: Your question reflects our hopes of no biofilm formation at all after a PTW-treatment. Nevertheless, we have to admit the growing of bacteria on the surface after 24 h, which has been observed in our SEM-pictures. Of course, it is an eligible question. Even after an incubation period of 24 h, we did not see any turbidities in all scanned PTW-treated samples (petri-dishes for light microscopy).
- Q: Lines 322-326: The cell aggregates of >1000 cells are not obvious in Figure 6B.
A: Has been corrected (lines 368 - 375). An overview picture has been presented in the supplementary section.
- Q: Lines 330 and 333: I assume this is meant to be Figure 7.
A: Thank you, it has been corrected.
- Q: Line 344: What textbook?
A: Formulation error, like described in the literature. We have corrected that.
- Q: Lines 362-364: PTW has been reported to be effective against Gram-positive bacteria (e.g. Staphylococcus aureus and Streptococcus mutans) so this section is misleading.
A: Has been corrected.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe study provides new insights into the inhibition of Bacillus subtilis biofilm formation by Plasma Treated Water (PTW), which has potential application value in microbial control in the food industry and healthcare fields. The originality of the research lies in the direct comparison of the differences in B. subtilis biofilm formation before and after PTW treatment, and the exploration of possible mechanisms. The manuscript can be accepted after major revisions.
1. The authors may need to further explore the impact of specific active components in PTW on B. subtilis biofilm formation, for example, by adding or removing certain components to observe changes in biofilm formation.
2. The study mentions that B. subtilis may enter a protective state; it is suggested that the authors explore the physiological changes of the cells in this state and whether this state is reversible.
3. It is recommended that the authors consider comparing PTW treatment with other traditional food disinfection methods to assess its potential and limitations in practical applications.
4. The Python script for automated analysis of SEM images mentioned in the article is very promising; it is suggested that the authors further discuss the accuracy of this method and possible areas for improvement.
5. The authors have well outlined the formation of biofilms and their importance in the food industry. However, further emphasis could be placed on the innovation and potential advantages of Plasma Treated Water (PTW) as a disinfectant, especially against the backdrop of increasing antibiotic resistance.
6. The conclusion section should highlight the main findings of the study and suggest directions for future research, such as exploring the effects of PTW on other types of bacteria and biofilms.
Author Response
Dear reviewer #2.
We welcomed your very helpful suggestions and remarks, which helped to improve our manuscript a lot. Especially, your remarks about the counting routine helped to improve that discussion a lot. It was our intention not to deepen the discussion of the routine too much. Indeed, you are right. There are limitations, predominantly in densely populated bacterial communities. Moreover, we discuss those limitations in our appendix of the manuscript, which is also available online. For further information, please see statements below.
Thank you very much for your time, your patience, and the fruitful discussion.
Q: The authors may need to further explore the impact of specific active components in PTW on B. subtilis biofilm formation, for example, by adding or removing certain components to observe changes in biofilm formation.
A: We totally agree and it is still the subject of our research. We already described the impact of artificially composed PTW (from traceable ingredients in IC-measurements) when P. fluorescens have been used (Weihe et al. 2023, https://doi.org/10.3390/microorganisms11040932). Of course, we plan further research on biofilm formation under PTW influence.
Q: The study mentions that B. subtilis may enter a protective state; it is suggested that the authors explore the physiological changes of the cells in this state and whether this state is reversible.
A: We support your statement. We found cells with a higher connectivity for treated suspensions. This elevated connectivity strictly follows the 2n-rule. Based on that rule, which has been reliable observed in our counting routine, we expect a specific physiological answer (Kaobayashi et al., 2007). An interpretation, which is also described in the literature. Nevertheless, physiological investigations (beside the ones presented in the manuscript and the statements made in the published article described above) to answer such questions are definitely suggestive. We also interpret this behavior as a reversible stress answer to a PTW treatment. If and how it is reversible should be shown by further experiments. We discussed it now in our manuscript (line 429 – 442).
Q: It is recommended that the authors consider comparing PTW treatment with other traditional food disinfection methods to assess its potential and limitations in practical applications.
A: We introduced such a section (line 40 – 45).
Q: The Python script for automated analysis of SEM images mentioned in the article is very promising; it is suggested that the authors further discuss the accuracy of this method and possible areas for improvement.
A: We found a deeper discussion about the advantages and disadvantages of our counting routine would leading too far. Anyway, we discussed the Python script in our appendix, which you can find under the url:
- https://github.com/JanWallis/subtilis/blob/1.1/README.md
(describing the limitations of the program in greater detail)
Or
- https://doi.org/10.5281/zenodo.13908826
Q: The authors have well outlined the formation of biofilms and their importance in the food industry. However, further emphasis could be placed on the innovation and potential advantages of Plasma Treated Water (PTW) as a disinfectant, especially against the backdrop of increasing antibiotic resistance.
A: We improved the description of PTW against that backdrop. We cite research describing the PTW impact in several papers and reviews (line 40 – 45).
Q: The conclusion section should highlight the main findings of the study and suggest directions for future research, such as exploring the effects of PTW on other types of bacteria and biofilms.
A: We introduced such a section (line 488 – 492).
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsManuscript ID: microbiolres-3301996
Title: Plasma treated water retards pellicle-like biofilm formation of Bacillus subtilis
The study effectively highlights the potential of plasma-treated water (PTW) as an innovative antimicrobial agent, offering a promising alternative to traditional sanitizing agents, especially in the context of combating biofilms and antibiotic resistance. The authors thoroughly describe their methodologies for PTW production and characterization. The stepwise presentation of how PTW was generated and applied to B. subtilis suspensions offers a clear and reproducible framework for future researchers. Given the increasing importance of minimizing biofilm-related contamination and infections in healthcare and food production, this research addresses a significant challenge with practical applications.
here are some scientific comments:
- While the manuscript provides evidence of PTW's efficacy against biofilm formation, a deeper exploration into the mechanistic pathways by which PTW inhibits biofilm development, including possible disruption of quorum sensing or reactive species interactions at the cellular level, would be valuable.
- The manuscript relies heavily on qualitative observations through microscopy. The study might benefit from more quantitative data analysis, such as statistical modeling of biofilm reduction or precise measures of reactive species concentration in PTW.
- Although the focus on Bacillus subtilis is well-justified, it would enhance the manuscript's impact to include comparisons with other relevant bacterial species, especially Gram-negative bacteria, to evaluate PTW's broad-spectrum efficacy.
- The discussion regarding the potential transition of B. subtilis into a viable-but-non-culturable (VBNC) state after PTW exposure is interesting but requires additional experimental evidence to confirm this hypothesis.
- The SEM and light microscopy findings highlight changes in cell aggregation and biofilm disruption. However, more detailed images at consistent magnifications with quantitative measures (e.g., aggregate size distribution) would strengthen the manuscript's visual evidence.
- I suggest the authors investigate the long-term stability and safety of PTW in real-world applications, especially its potential use in food production.
- Also, explore synergistic effects of PTW with other antimicrobial treatments to enhance biofilm control.
Finally, this manuscript provides important insights into the utility of PTW as an antimicrobial treatment and sets a solid foundation for further exploration of plasma-based sanitation technologies.
Comments on the Quality of English LanguageThe English language quality of the manuscript is generally clear, but there are several areas where improvements can be made to enhance readability, flow, and precision.
Author Response
Dear reviewer #3.
We welcomed your very helpful suggestions and remarks, which helped to improve our manuscript a lot. Especially your remarks about further investigations to underpin our statements identified future issues. Since our observations were totally opposite to our expectations, we were keen to publish our findings. We hope to deepen our knowledge in fruitful discussions with the community and in further experiments. We improved our manuscript based on your suggestions. For further information, please see statements below.
Thank you very much for your time, your patience, and the fruitful discussion.
Q: While the manuscript provides evidence of PTW's efficacy against biofilm formation, a deeper exploration into the mechanistic pathways by which PTW inhibits biofilm development, including possible disruption of quorum sensing or reactive species interactions at the cellular level, would be valuable.
A: Of course, you are right. At the stage of our investigations, our statements are not proven and further experiments have to underpin the observations. In our manuscript, we tried not to be speculative. Although in number decreased, the predominant appearance of treated B. subtilis from the supernatant appeared intact. We also included a more detailed discussion (lines 429 - 442)
Q: The manuscript relies heavily on qualitative observations through microscopy. The study might benefit from more quantitative data analysis, such as statistical modeling of biofilm reduction or precise measures of reactive species concentration in PTW.
A: Our first approach for a more quantitative analysis of microscopically pictures is the counting routine, which can deliver data for further in silico experiments such as models for biofilm growth or detailed stress models. Please see appendix and Figure 7.
Q: Although the focus on Bacillus subtilis is well-justified, it would enhance the manuscript's impact to include comparisons with other relevant bacterial species, especially Gram-negative bacteria, to evaluate PTW's broad-spectrum efficacy.
A: We included a broad-spectrum view in our discussion (lines 399- 409 and lines 429 – 442)
Q: The discussion regarding the potential transition of B. subtilis into a viable-but-non-culturable (VBNC) state after PTW exposure is interesting but requires additional experimental evidence to confirm this hypothesis.
A: As our observations were contrary to our expectations, we were keen to publish them. Additionally, we hope the manuscript will be a foundation for further research. We added a section in our conclusions based on your remark (lines 506 - 509).
Q: The SEM and light microscopy findings highlight changes in cell aggregation and biofilm disruption. However, more detailed images at consistent magnifications with quantitative measures (e.g., aggregate size distribution) would strengthen the manuscript's visual evidence.
A: We show additional overview pictures in our appendix, which are merged pictures of the entire scan width of the microscope. These pictures compare treated and untreated B. subtilis cells from the supernatant. Aggregate sizes are solely available through the connectivity of the cells (Figure 7)
Q: I suggest the authors investigate the long-term stability and safety of PTW in real-world applications, especially its potential use in food production.
A: Such investigations have been described in Schnabel et al. 2020. https://doi.org/10.3389/fnut.2020.627483
Q: Also, explore synergistic effects of PTW with other antimicrobial treatments to enhance biofilm control.
A: Such investigations have been described in Schnabel et al. 2019. https://doi.org/10.3390/foods8020055
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors addressed the comments and concerns raised in the prior review. There are a couple of minor text edits needed in the newly added section 2.5.
1. Line 189: Missing parenthetical content.
2. Lines 193-194: Missing information at the three ??? placeholders.
Author Response
Dear Reviewer #1.
Thank you very much for your patience to repeatedly correct our flaws.
Thank you very much!
The authors
Author Response File: Author Response.pdf
