Review Reports

Round 1

Reviewer 1 Report

This study aimed to combine primers (V1-V2 and ITS2) to detect both bacteria and fungi using NGS assay which can be cost-effective.

Please refer to the comments and suggestions given in the pdf document. The Introduction should be written in a relevant flow to accurately reflect the topic and aim of the study.

Otherwise, it is a good study.

Comments for author File: Comments.pdf

Author Response

<Please see the attachment>

Response to Reviewer 1 Comments

This study aimed to combine primers (V1-V2 and ITS2) to detect both bacteria and fungi using NGS assay which can be cost-effective.

Please refer to the comments and suggestions given in the pdf document. The Introduction should be written in a relevant flow to accurately reflect the topic and aim of the study.

Otherwise, it is a good study.

Response : Thank you for your comment. As recommended in PDF file, we revised the manuscript as listed below.

To Pdf reviewer response:

It is too general. Human oral samples of? The location of sampling?

Response : Thank you for the comment. "human oral sample" is "oral samples from intubated patients in intensive care unit"

Just to be clear. Is it "Intubated patients" or "Patient intubated" in ICU? It carries different meaning.

Response : Thank you. "Patients intubated" has been changed to "intubated patients".

Suggest to end the sentence and start a new one. So remove "and".

Response : Thank you. Modified as recommended.

using what assay?

Response : Thank you. Analysis method has been added.

This paragraph does not flow well with the second paragraph where authors explain about oral microbes of intubated patients.

Response : Thank you. We agree with the reviewers comment. We have removed and revised this paragraph.

These microbes should be listed down.

Response : Thank you. Specific microbes have been listed in the manuscript. : Pseudomonas aeruginosa, Enterobacteriacea, Acinetobacter, S. aureus, and Streptococcus spp.

remove "by"

Response : Thank you. It was removed.

and?

Response : Thank you. corrected as "and"

Measured

Response : Thank you. Corrected as "measured"

How about the length of time patients in ICU? This is important information that should be included.

Response : Thank you. We agree that this is very import. As shown in Fig5, the length of time patients in ICU varied very much. Thus, length of time could not be included as Inclusion criteria. However, We have described the length of time in the oral sample preparation section: during ICU treatment (day 0 - day 12)

Swab

Response : Thank you. “swap” was removed.

This needs to be more clear. Multiple times during each sampling or multiple times over 1 day? Please be specific with details.

Response : Thank you. "Multiple times" was intended to mean "samples were collected once in a day but multiple times in total during the ICU treatment for each patients". It was corrected as : during ICU treatment (day 0 – day 12; shown as Figure)

Please describe why these bacteria and fungus were chosen for the validation.

Response : These are one the most frequently found pathogens that are related with VAP in ICU patients. Changed as : species-specific primers for VAP-associated pathogens including Acinetobacter baumannii

Authors need to to describe the adjustment conducted briefly.

Response : The adjustment was conducted by reducing the taxa assigned to the fungi kingdom by factor of 20. For instance, if read count for bacteria kingdom was 100,000 and fungi kingdom was 20,000, the corresponding taxa assigned to fungi kingdome was reduced to 1,000. Following the adjustment, we could get predicted relative abundance closely matching theoretical overall abundance. Reduction sample was described as : For example, if the initial read count for the fungi kingdom was 20,000 while the read count for the bacteria kingdom was 100,000, the taxa assigned to the fungi kingdom was reduced to 1,000.

What sort of adjustment? Reader should be able to understand this.

Response : Thank you. Adjustment was included in the legend as : Read counts of taxa assigned to the fungi kingdom was reduced by a factor of 20 for adjustment.

Please be consistent with the use of terminology such as microbiome and mycobiome through out the manuscript. In this finding, there is result related to fungus/ fungivwhere previously the authors have referred fungi as mycobiome.

Response : Thank you. It was one of the issues that was very hard for us. In general, microbiome is used to describe microbiota including bacteria, archea, fungi, and algae. Mycobiome specifically focus on fungal community. Thus, we used microbiome to both include bacteria and fungi in this study.

Author Response File: Author Response.docx

Reviewer 2 Report

very interesting and novative study, written in a perfect english.

However i have two minors remarks :

your number of patients is very small and i understand it's not easy to recruit in ICU but i think yuo should explain it in a better way in your introduction and conclusio.

In a same way, you have only elderly patients with variuos and really different diseases responsible for their presence in IUC. Please explain the possible risks of biais in the results due to these facts

Author Response

<Please see the attachment.>

Response to Reviewer 2 Comments

very interesting and novative study, written in a perfect english.

However i have two minors remarks :

Point 1: your number of patients is very small and i understand it's not easy to recruit in ICU but i think yuo should explain it in a better way in your introduction and conclusion.

Response 1: Thank you for your comment. As recommended, we described in the discussion (line 293-294). In the discussion section, we have discussed about the small number of the study and further recommended well designed longitudinal study in future. We have also tested our condition in healthy subjects but very low count of fungus was only detected. Thus, the results were not reported in this study.

Point 2: In a same way, you have only elderly patients with variuos and really different diseases responsible for their presence in IUC. Please explain the possible risks of biais in the results due to these facts

Response 2: Thank you for your comment. As recommended, we described in the discussion (line 294-296).

Author Response File: Author Response.docx

<Please see the attachment>

Response to Reviewer 1 Comments

This study aimed to combine primers (V1-V2 and ITS2) to detect both bacteria and fungi using NGS assay which can be cost-effective.

Please refer to the comments and suggestions given in the pdf document. The Introduction should be written in a relevant flow to accurately reflect the topic and aim of the study.

Otherwise, it is a good study.

Response : Thank you for your comment. As recommended in PDF file, we revised the manuscript as listed below.

To Pdf reviewer response:

It is too general. Human oral samples of? The location of sampling?

Response : Thank you for the comment. "human oral sample" is "oral samples from intubated patients in intensive care unit"

Just to be clear. Is it "Intubated patients" or "Patient intubated" in ICU? It carries different meaning.

Response : Thank you. "Patients intubated" has been changed to "intubated patients".

Suggest to end the sentence and start a new one. So remove "and".

Response : Thank you. Modified as recommended.

using what assay?

Response : Thank you. Analysis method has been added.

This paragraph does not flow well with the second paragraph where authors explain about oral microbes of intubated patients.

Response : Thank you. We agree with the reviewers comment. We have removed and revised this paragraph.

These microbes should be listed down.

Response : Thank you. Specific microbes have been listed in the manuscript. : Pseudomonas aeruginosa, Enterobacteriacea, Acinetobacter, S. aureus, and Streptococcus spp.

remove "by"

Response : Thank you. It was removed.

and?

Response : Thank you. corrected as "and"

Measured

Response : Thank you. Corrected as "measured"

How about the length of time patients in ICU? This is important information that should be included.

Swab

Response : Thank you. “swap” was removed.

This needs to be more clear. Multiple times during each sampling or multiple times over 1 day? Please be specific with details.

Response : Thank you. "Multiple times" was intended to mean "samples were collected once in a day but multiple times in total during the ICU treatment for each patients". It was corrected as : during ICU treatment (day 0 – day 12; shown as Figure)

Please describe why these bacteria and fungus were chosen for the validation.

Response : These are one the most frequently found pathogens that are related with VAP in ICU patients. Changed as : species-specific primers for VAP-associated pathogens including Acinetobacter baumannii

Authors need to to describe the adjustment conducted briefly.

What sort of adjustment? Reader should be able to understand this.

Response : Thank you. Adjustment was included in the legend as : Read counts of taxa assigned to the fungi kingdom was reduced by a factor of 20 for adjustment.

Please be consistent with the use of terminology such as microbiome and mycobiome through out the manuscript. In this finding, there is result related to fungus/ fungivwhere previously the authors have referred fungi as mycobiome.

Response : Thank you. It was one of the issues that was very hard for us. In general, microbiome is used to describe microbiota including bacteria, archea, fungi, and algae. Mycobiome specifically focus on fungal community. Thus, we used microbiome to both include bacteria and fungi in this study.

<Please see the attachment.>

Response to Reviewer 2 Comments

very interesting and novative study, written in a perfect english.

However i have two minors remarks :

Point 1: your number of patients is very small and i understand it's not easy to recruit in ICU but i think yuo should explain it in a better way in your introduction and conclusion.

Point 2: In a same way, you have only elderly patients with variuos and really different diseases responsible for their presence in IUC. Please explain the possible risks of biais in the results due to these facts

Response 2: Thank you for your comment. As recommended, we described in the discussion (line 294-296).