Bacteriophages isolated from mouse feces attenuates pneumonia mice caused by Pseudomonas aeruginosa

Nuttawut Sutnu; Wiwat Chancharoenthana; Supitcha Kamolratanakul; Pornpimol Phuengmaung; Uthaibhorn Singkham-In; Chiratchaya Chongrak; Sirikan Montathip; Dhammika Leshan Wannigama; Tanittha Chatsuwan; Puey Ounjai; Marcus J. Schultz; Asada Leelahavanichkul

doi:10.1371/journal.pone.0307079

Peer Review History

Original SubmissionMarch 22, 2024
31 May 2024 Decision Letter - Yung-Fu Chang, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-24-09029Bacteriophages isolated from mouse feces attenuates pneumonia mice caused by Pseudomonas aeruginosaPLOS ONE Dear Dr. Chancharoenthana, 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. Your manuscript has been reviewed by an expert in this field. However, a minor revision is suggested and I agree. Please follow the comments and make the necessary revision. Please submit your revised manuscript by Jul 15 2024 11:59PM. 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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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript offers an in-depth investigation into the isolation and characterization of bacteriophages from mouse feces and their ability to treat Pseudomonas aeruginosa pneumonia. The observation is methodologically sound and explores a novel technique that could decorate the remedy of bacterial infections, mainly in the context of growing antibiotic resistance. However, several critical factors require attention before it is considered for publication. Introduction: - The usage of phages isolated from the mice and how helpful they are as compared with the other animal or environmental sources can be cited to highlight its importance. Also, it would be helpful to intricate on why this technique is predicted to be advanced to present strategies. - The mortality records cited are useful but may be impactful if connected directly to the need for alternative therapies like bacteriophage therapy against antibiotics or others. Providing more inputs from a broader geographical context may also be relevant. Methods: - The strategies used from the isolation, and characterization, to applications/treatment, can be summarized in a flow chat to have an overall understanding of the study conducted. - The criteria for selecting the specific bacterial strains (PA1, PA2, and many others.) as well as phages ought to be clarified. Were these cell lines chosen totally on their clinical relevance, resistance profiles, or any other particular attributes? Also, the phage selection criteria must be explained in detail. - The phage purification using cesium chloride density gradient must be explained in detail. Results: - The effects of isolating phages from mouse feces and the effectiveness of those phages in treating pneumonia have to be explicitly stated. - The effect of isolated phages on different host stains and their lysis can be better presented with the inclusion of additional comparative analyses, possibly through graphical representations like bar charts or heatmaps. To demonstrate its significance. - All the studies have been properly conducted but the significance of the animal model, histology, as well as in-vitro studies with neutrophils must be explained in detail with proper justification for their findings! To provide further importance to the observed results. Discussion: - The specificity of phages isolated from feces is well reported, but potential solutions or future directions to such studies concerning effective solutions of antibiotic-resistant strains must be explained with limitations. - The ability to translate these findings to human clinical settings has to be discussed with possible hurdles and potential future studies. Conclusion: - A call to action for additional research or collaboration may also add significance to the manuscript. ****** 6. 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: Yes: Dr. Swapnil Ganesh Sanmukh ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0307079.r001
Revision 1
14 Jun 2024 Author Response Response to the Reviewers Reviewer #1: The manuscript offers an in-depth investigation into the isolation and characterization of bacteriophages from mouse feces and their ability to treat Pseudomonas aeruginosa pneumonia. The observation is methodologically sound and explores a novel technique that could decorate the remedy of bacterial infections, mainly in the context of growing antibiotic resistance. However, several critical factors require attention before it is considered for publication. Introduction: - The usage of phages isolated from the mice and how helpful they are as compared with the other animal or environmental sources can be cited to highlight its importance. ANS: We appreciate the reviewer's feedback and would like to share our thoughts as follows: "We chose mouse feces for the proof of concept because i) humans are mammals and phages isolated from mammals may be more similar to their use in humans, and ii) experimental mice are small enough to use as an initial recovery experiment and future experiments for improved phage isolation in mice are feasible and easier than the larger mammals." Also, it would be helpful to intricate on why this technique is predicted to be advanced to present strategies. ANS: We appreciate the reviewer's feedback and would like to share some of our ideas: "Since phage isolation doesn't necessitate a significant amount of feces, fecal collection and other manipulations on mice are more straightforward than in larger animals; for instance, administering certain molecules that could potentially enhance the target phage. Additionally, phages targeting specific bacteria may exhibit greater specificity in certain mouse strains that have undergone genetic modifications, a prospect for future research. " - The mortality records cited are useful but may be impactful if connected directly to the need for alternative therapies like bacteriophage therapy against antibiotics or others. Providing more inputs from a broader geographical context may also be relevant. ANS: We appreciate the reviewer's feedback and have included a sentence in the introduction that highlights the potential of using phages as an alternative treatment as “Therefore, we suggest that bacteriophage therapy could serve as an alternative therapy or an adjuvant strategy to combat bacterial infections.” Methods: - The strategies used from the isolation, and characterization, to applications/treatment, can be summarized in a flow chat to have an overall understanding of the study conducted. ANS: We thank the reviewer for their comments and have added a flow chart to the method section. - The criteria for selecting the specific bacterial strains (PA1, PA2, and many others.) as well as phages ought to be clarified. Were these cell lines chosen totally on their clinical relevance, resistance profiles, or any other particular attributes? Also, the phage selection criteria must be explained in detail. ANS: We thank the reviewer for comments. We just use all bacterial strain that are available in our lab and the best phage that cover the highest number of PA. Because we just try to prove the concept of phase isolation from samples of living animal. Then, only the simple criteria were used. Then, we added in the method section as following; “As such, the isolated stains of all currently available bacteria, including”, and added in the result section as following “Different from the isolation from environmental sources, there were only 4 different bacteriophages from our results (Table 1) and pseudomonas phage vB_PaeM-AL was selected for further test in vivo and in vitro due to the coverage in most of the P. aeruginosa strains used in our experiments. Also, the table of host range determination is expanded to other phages that were not selected to test (a new table 1) and some sentences are also added in the new discussion as following; “Despite possible complex criteria of phage selection in the real clinical setting; for example, high coverage on bacterial strains without antibiotic resistant genes), vB_PaeM-AL (a lytic phage) was selected here just for a proof of concept on phage isolation using animal sources. More studies on phage selection or phage combination therapy are interesting.”. - The phage purification using cesium chloride density gradient must be explained in detail. ANS: We thank the reviewer for comments and add more details in the new method section. Results: - The effects of isolating phages from mouse feces and the effectiveness of those phages in treating pneumonia have to be explicitly stated. ANS: We thank the reviewer for comments. Due to the limitation of animal study protocol, we can teste in mice only with a single phage, despite a good matching between in vitro and in vivo. Then, we discuss the possibly correlation between the in vitro and in vivo and the lack of test in other non-selected phages as a limitation of our study. - The effect of isolated phages on different host stains and their lysis can be better presented with the inclusion of additional comparative analyses, possibly through graphical representations like bar charts or heatmaps. To demonstrate its significance. ANS: We apologize for the unclear presentation. We use only PACL, a strain that previously successfully activate diseases in mice, and the selected phage is an only strain that can neutralize PACL (as shown in the new table1). Hence, we more clearly mentioned the use of PACL in the new result section as following; “Although Pseudomonas phage vB_PaeM-AL can neutralize several pseudomonas strains (Table 1), PACL, a strain with previously demonstrated pathogenicity in an animal model (wound model) [6], might also be able to induce mouse pneumonia was selected to use for further experiment”. - All the studies have been properly conducted but the significance of the animal model, histology, as well as in-vitro studies with neutrophils must be explained in detail with proper justification for their findings! To provide further importance to the observed results. ANS: We thank the reviewer for the comment and add more explanation in the new result section as following “Without phage treatment, the lethal pneumonia of our model was indicated by approximately 90% mortality rate within 7 days after bacterial administration (Fig 2A) with high bacterial abundance (BALF and in blood), elevated inflammatory cytokines (Fig 2B - I), and lung consolidation (the presence of exudate in the airways and alveoli) (Fig 3A, B), which was compatible with severe lobar pneumonia in patients.” for our pneumonia model. For the in vitro findings, we rewrite all of the result of this section and conclude as following; “Although bacterial abundance between bacteria + phage + PMN and bacteria + phage was similar (Fig 5A), lower phages in the latter group was required for this similar reaction (Fig 5B) and PMN could recognize phages as indicated by increased IL-6 (Fig 5D on phage + PMN group), but not NET activation (Fig 6A-D). Thus, bactericidal activity of our phage might, at least in part, be through the activation of neutrophils (cytokine production but not NETs). Discussion: - The specificity of phages isolated from feces is well reported, but potential solutions or future directions to such studies concerning effective solutions of antibiotic-resistant strains must be explained with limitations. ANS: We thank the reviewer for the comment and add more discussion as following; “Although a tremendous phage library and preparation pipeline for emergency phage therapy are mentioned to use as a personalized treatment against antibiotic resistant strains [39], laboratory tests to select the specific phage is still difficult, the newly extracted phages against the bacteria that are cultured from patient specimens using several methods, including mice, to accelerate the process will be another strategy of phage personalized medicine in the future. More studies are interesting.”. - The ability to translate these findings to human clinical settings has to be discussed with possible hurdles and potential future studies. ANS: We thank the reviewer for the comment and add more discussion as following; “Nevertheless, we demonstrated a proof of concept to extract phages from mouse feces, implying that there are some phages against human pathogenic bacteria in mouse feces. For the future direction, rapid extraction and enhancement of the specific phages against bacteria, especially the antibiotic resistant strains, that are cultured from the patients through the administration of these bacteria into the mice might be able to use as a personalized medicine.”. Conclusion: - A call to action for additional research or collaboration may also add significance to the manuscript. ANS: We thank the reviewer for the comment and add a few sentences as following; “The international collaboration working on several aspects; for example, enhancing the possibility of phage discovery (specific mouse strains, bedding, and probiotics), rapid phage propagation methods, prevention of phage incorporation into the targeted bacteria, and product preparation for human use, might accelerate the progression of phage therapy. More studies are warranted.” Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0307079.r002
1 Jul 2024 Decision Letter - Yung-Fu Chang, Editor Bacteriophages isolated from mouse feces attenuates pneumonia mice caused by Pseudomonas aeruginosa PONE-D-24-09029R1 Dear Dr. Chancharoenthana, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Yung-Fu Chang 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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 author's have addressed most of the comments and the arricle is significantly improved from its previous version. ****** 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: Yes: Swapnil Ganesh Sanmukh ******** https://doi.org/10.1371/journal.pone.0307079.r003
Formally Accepted
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