Ozone ultrafine bubble water exhibits bactericidal activity against pathogenic bacteria in the oral cavity and upper airway and disinfects contaminated healthcare equipment

Fumio Takizawa; Hisanori Domon; Takumi Hiyoshi; Hikaru Tamura; Kana Shimizu; Tomoki Maekawa; Koichi Tabeta; Akiomi Ushida; Yutaka Terao

doi:10.1371/journal.pone.0284115

Peer Review History

Original SubmissionJanuary 19, 2023
7 Feb 2023 Decision Letter - Geelsu Hwang, Editor PONE-D-23-01776Ozone ultrafine bubble water exhibits bactericidal activity against pathogenic bacteria in the oral cavity and upper airway and disinfects contaminated healthcare equipmentPLOS ONE Dear Dr. Terao, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Mar 24 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. "Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. 4. Please amend your authorship list in your manuscript file to include author Dr. Satoru Hirayama. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Takizawa et al. used OUFBW to examine the sterilization effect against bacteria that inhabit the oral cavity and some bacteria that are a problem in hospital infections. And it was clarified that OUFBW accounts for the bactericidal effect against those bacteria. We also found that OUFBW has an effective bactericidal effect on toothbrushes and gauze contaminated with these bacteria, and that OUFBW has no harmful effect on gingival cells. The above results suggest that OUFBW may play an effective role in hygiene management and oral cleaning in the oral area, and are considered to be very important findings. After reviewing this manuscript, I note the following concerns. After adjusting OUFBW to various concentrations, we are investigating the bactericidal effect in a state where 100 times the amount of the culture solution containing bacteria is added. In that regard, I would like to ask the following questions. 1) There is no specific description of how to dilute OUFBW in this case. I would like you to describe it. 2) Is there any change in the effect of OUFBW compared to the OUFBW dilution method (dilution using a buffer solution) used to investigate the harmful effects of this dilution method on gingival cells? I think that this point should be clarified. 3) In 2), if unbuffered OUFBW is used for bacterial sterilization and it contains a higher amount of ozone than diluted buffered OUFBW. I think that the sterilization effect of bacteria should be performed. Reviewer #2: Terao et al. reported an assessment of using OUFBW as a disinfectant against oral pathogens. While the study results show a promising antimicrobial effect from OUFBW, my concerns are how much pathogens are present in the mixed solution of the OUFBW treatment. Line 104-107 indicated that 1ul bacteria of OD 01-0.5 was mixed with 1ml OUFBW, which is 1000 time dilution. OD 0.1-0.5 is also a wide range; how much bacteria in terms of CFU were used as a baseline? Figures 2 and 4 only reported the percentage of pathogens; reporting the actual CFU for each condition is needed to understand the potent and limit of the OUFBW comprehensively. ****** 6. 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Revision 1
15 Mar 2023 Author Response Response to Editor and Reviewers Response to Editor We thank the editor for the critical suggestions that have helped us improve our manuscript. As indicated in the responses below, we have taken into consideration all these comments and suggestions and addressed each one of them during our revision of the manuscript. <Comment #1> Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. <Response> In response to the editor’s suggestion, we ensured that our manuscript and figures complied with PLoS One's style guidlines and modified the file name. <Comment #2> We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. <Response> At the time of resubmission, the ‘Funding Information’ and ‘Financial Disclosure’ have been revised to match their descriptions. <Comment #3> In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. <Response> Figure 1, 2, 4, and 5 (in the revised version): According to the editor’s suggestion, we modified Figure 1, 2，4, and 5 to comply with PLoS One's data policy. Therefore, it became possible to read the values behind the means from these graphs. <Comment #4> Please amend your authorship list in your manuscript file to include author Dr. Satoru Hirayama. <Response> The author list has been updated after we discovered some errors in it. We apologize for this error in the authors’ electronic registration. We removed Dr. Satoru Hirayama from the author list and added Dr. Tomoki Maekawa and Prof. Koichi Tabeta (line 6 in the revised version). Accordingly, we submitted the Request for Change to Authorship and updated the ‘Funding Information’ and ‘Financial Disclosure’. Lines 329–330 (in the revised version): According to the change in author list, we updated the acknowledgement section as follows: “We thank Dr. Satoru Hirayama, Dr. Toshihito Isono, Dr. Karin Sasagawa, Dr. Rui Saito, Dr. Yoshihito Yasui (Niigata University), Mr. Tadashi Hiwatashi (Futech-Niigata LLC), and Mr. Koichi Seto (IWASE Company Limited, Niigata, Japan) for their technical support.” Response to Reviewer 1 We are grateful to Reviewer 1 for their critical comments and suggestions that have helped us improve our paper considerably. In response to your comment, we have included a supporting figure. As indicated in the following responses, we have considered all these comments and suggestions in the revised version of our paper. <Comment #1> There is no specific description of how to dilute OUFBW in this study. <Response> Lines 108–109 (in the revised version): According to the reviewer’s suggestion, we have added a description of the dilution method of OUFBW in the bactericidal assay as follows: “To prepare OUFBW containing various ozone concentrations, OUFBW containing 4–6 ppm ozone was serially diluted with distilled water.”. <Comment #2> Is there any change in the effect of OUFBW compared to the OUFBW dilution method (dilution using a buffer solution) used to investigate the harmful effects of this dilution method on gingival cells?　 <Comment #3> In 2), if unbuffered OUFBW is used for bacterial sterilization and it contains a higher amount of ozone than diluted buffered OUFBW. I think that the sterilization effect of bacteria should be performed. <Response to comments #2 and #3> Lines 151–153 and S1 figure (in the revised version): To answer the reviewer’s queries, we added a description of the preparation of OUFB-PBS in the cytotoxicity assay in lines 151–153 as follows: “For cytotoxicity assay, we prepared OUFB-phosphate-buffered saline (PBS) by diluting OUFBW with 10 × PBS (NACALAI TESQUE INC, Kyoto, Japan).” The ozone concentrations in OUFBW and OUFB-PBS used in our experiments were 1–5 ppm, as shown Figures 2, 5, and S1. We agree with the reviewer’s comments, and we performed additional experiments about bactericidal activity of OUFB-PBS three times. Thereby the new finding was brought. We are grateful for reviewer’s critical comments. We have added descriptions about bactericidal activity of OUFB-PBS to results and discussion section and supporting information to the revised manuscript as follows: “OUFB-PBS exerts bactericidal effects against gram-positive and negative bacteria. We conducted additional experiment to investigate the bactericidal activity of OUFB-PBS. For this experiment, we used S. pneumoniae strain D39 as a Gram-positive bacteria and P. aeruginosa strain RIMD 1603003 as a Gram-negative bacteria. These bacteria were exposed to OUFB-PBS containing various ozone concentrations (0.25–5 ppm) for 1 min. S1 Fig shows that exposure to ≥ 2 ppm OUFB-PBS resulted in a > 99% decrease in S. pneumoniae, and exposure to ≥ 1 ppm OUFB-PBS resulted in a > 99% decrease in P. aeruginosa. These findings suggest that OUFB-PBS also exerts bactericidal effects.” In lines 255–263, and “In addition, fig 2 and S1 showed that there was slight difference between OUFBW and OUFB-PBS on the bactericidal activity. The effects of solvents on ozone and nanobubbles have not been investigated, therefore we supposed it should be addressed in future work.” in lines 288–291, and “S1 Fig. Bactericidal effects of OUFB-PBS against S. pneumoniae strain D39 and P. aeruginosa strain RIMD 1603003. S. pneumoniae D39 and P. aeruginosa strain RIMD 1603003 were exposed to 0.4–5 ppm OUFBW for 1 min. Data are presented as the mean ± SD of triplicate experiments and were evaluated using analysis of variance with Dunnett’s multiple-comparisons test. P < 0.05 compared to the control group. ND stands for undetected and indicates below the detection limit (< 100 CFU/mL).” in lines 452–458. Response to Reviewer 2 We thank Reviewer 2 for the critical comments and suggestions that have helped us improve our paper considerably. As indicated in the following responses, we have considered the comments and suggestions in the revised version of our article. <Comment> Figures 2 and 4 only reported the percentage of pathogens; reporting the actual CFU for each condition is needed to understand the potent and limit of the OUFBW comprehensively. <Response> Table 1, Figure 2 and 4, and lines 191–195 (in the revised version): According to the reviewer’s suggestion, we modified Table 1 (the revised manuscript version on page 11) and, Figures 2 and 4 to read the values of CFU/mL of bacterial solution used in each experiment. Accordingly, we have modified the manuscript as follows: “Table 1. The number of colony-forming units of various bacterial species exposed to ozone ultrafine bubble water. The number of colony-forming units (CFU) of various bacterial species was measured after exposing to ozone ultrafine bubble water containing 4–6 ppm of ozone for 10–300 s. ND stands for undetected and indicates below the detection limit (< 100 CFU/mL).” in lines 191–195. Lines 199, 200, 238, 239, 253, and 254 (in the revised version): We modified our manuscript to include the description of the detection limit. Lines 221 and 229 (in the revised version): We modified our manuscript as follows: “Contaminated toothbrushes were immersed in approximately 5 ppm OUFBW or distilled water for 5 min. Exposure to OUFBW resulted in a > 90% decrease in the bacterial load of S. pneumoniae compared to that of the distilled water treatment group (Fig 4a).” in line 221, “Contaminated gauzes were immersed in approximately 5 ppm OUFBW or distilled water for 5 min. Exposure to OUFBW resulted in a > 90% decrease in the bacterial load of P. aeruginosa compared to that of the distilled water treatment group (Fig 4b).” in line 229. // Attachments Attachment Submitted filename: Response to reviewers.docx* https://doi.org/10.1371/journal.pone.0284115.r002
23 Mar 2023 Decision Letter - Geelsu Hwang, Editor Ozone ultrafine bubble water exhibits bactericidal activity against pathogenic bacteria in the oral cavity and upper airway and disinfects contaminated healthcare equipment PONE-D-23-01776R1 Dear Dr. Terao, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Geelsu Hwang, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors responded appropriately to questions from the reviewers and rated the quality of the manuscript as acceptable. Reviewer #2: My comments are addressed. I don't have additional comments. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0284115.r003
Formally Accepted
3 Apr 2023 Acceptance Letter - Geelsu Hwang, Editor PONE-D-23-01776R1 Ozone ultrafine bubble water exhibits bactericidal activity against pathogenic bacteria in the oral cavity and upper airway and disinfects contaminated healthcare equipment Dear Dr. Terao: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Geelsu Hwang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0284115.r004

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