PONE-D-20-26471

Shotgun metagenome sequencing identification of a set of genes encoded by Actinomyces specific to medication-related osteonecrosis of the jaw

PLOS ONE

Dear Dr. Yahara,

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.

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Both reviewers have positive attitudes to your study. Reviewer #2, however, has pointed out that the statistical reliability of the results is minimally OK and that additional samples should be analyzed.  I, not expert in the field can imagine that additional samples could be obtained  and analyzed to test the frequency of the correlation.

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PLOS ONE

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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: This is a really well realized paper.

This paper analyzes an interesting and modern aspect of the pathogenesis of MRONJ.

The presence of actinomyces is a frequent finding in BRONJ.

Previously it was believed to be present only in osteonecrosis related to the use of bisphosphonates (BRONJ), recently it has also been detected in osteonecrosis related to denusumab (MRONJ), (as in the series that my group has just published Cerrato A, Zanette G, Boccuto M, Angelini A , Valente M, Bacci C. Actinomyces and MRONJ: A retrospective study and a literature review [published online ahead of print, 2020 Aug 20]. J Stomatol Oral Maxillofac Surg. 2020; S2468-7855 (20) 30175-0. Doi: 10.1016 / j.jormas.2020.07.012 which the Authors can quote if they want).

The present paper further supports the infectious theory of MRONJ bringing an important contribution to the knowledge of the pathology.

Reviewer #2: The manuscript by Yahara et al focusses on potential bacterial causes of MRONJ by deep shotgun metagenome sequencing of saliva samples. With the method they used, they could overcome the limitations in previous studies, such as unculturable taxa in human oral microbiome or lacking the quantitative information on abundance of specific taxa. They analyzed saliva samples collected from 5 MRONJ patients and 4 healthy volunteers, and revealed that the genus Actinomyces was 10.1% more abundant in MRONJ patients than in healthy volunteers. They also determined the 119 bacterial genes specifically found in these patients, and showed that all of them were encoded by Actinomyces genus. Furthermore they also described the most abundant eight Actinomyces genes in MRONJ patients.

In general, the data presented in this study is interesting and would shed light on an unknown field which is the molecular connection between Actinomyces and MRONJ. Nevertheless, the manuscript needs a major revision.

Major issues

• A clear difference in bacterial composition between the samples from MRONJ patients and healthy donors could be demonstrated; however, the cohort is too small to draw any generalizable conclusions. I would suggest that at least a total of 20 samples per group should be evaluated. By taking the fact in account that MRONJ is a rare disease, the cohort can be enlarged by establishing collaborations with other centers. Still, the authors should discuss the limitations of the study, particularly because of small sample number and present their data as a pilot study in this area which needs to be confirmed further.

• Having the reads from only two MRONJ patients presented in figure S2 compromises the reliability. The authors should collect the same data from all MRONJ patients and then recreate the figure for reliable interpretation. When all these requisites have been fulfilled, Figure S2 may be one of the main figures since determination of Actinomyces-specific genes found predominantly in MRONJ patients is an important research question in this study.

• The manuscript is well-written and easy to follow except from the discussion part. This section should be more compactly structured to lead the reader to follow the story depicted in Figure 4.

 

Minor issues

• It would be easier to follow if the figure legends were presented with the figures in the manuscript.

• There’re typos in Figure S2 axis labeling.

• Lines 278-280: …orange and purple indicate presence and absence of the genes. Missing “, respectively”?

• Line 285: “except” instead of “outside”

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Reviewer #1: Yes: Christian Bacci

Reviewer #2: No

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Shotgun metagenome sequencing identification of a set of genes encoded by Actinomyces associated with medication-related osteonecrosis of the jaw

PONE-D-20-26471R1

Dear Dr. Yahara,

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. The comments of the previous reviews have been more than adequately addressed. However: line 349 should be”its’ rather than “is.

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.

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Kind regards,

Ulrich Melcher

Academic Editor

PLOS ONE

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Reviewers' comments: