The Oral Microbiome in Children with Black Stained Tooth

Round 1

Reviewer 1 Report

Summary:

Please mention at the beginning that you refer to humans and briefly what is black stain.

line 25: do you mean black pigment- producing bacteria?

line 26-27: please rephrase, metagenome is not just the 16S. 

Line 29: Referring to "an identified" heatmap at the abstract is not very informative. Please rephrase. 

Line 30: please be more specific than just 'had more' ... It is not scientifically accurate.

Line 32: Please rephrase for accuracy: just by identifying taxa at a certain niche it does not provide evidence of causality. Please definitely rephrase this.

Line 39: what is the cervical third?

Please place the full stop after the reference.

Line 46-48: Please rephrase the whole sentence bcs it has a confusing message.

Line 49-50: Please give more details, this is not informative enough.

Line 51: references?

Line 53: Please rephrase bcs 'simple ... composition' is not informative and accurate enough.  

Line 58: please specify PCRs do you refer to?

Line 51-62: Overall this is lacking specific information, try make it more detailed but still keep it short, refer to tested cohorts, techniques, taxonomic groups etc.

Line 63-66: Please revise with references. Is pyrosequencing the best sequencing technique? Line 66: please be more accurate in this phrase. 

Line 67-69: Why refer to why we use sequencing and its benefits? this is common knowledge. This space should be used for BS related information and not for stating sth known. 

Lines 69-72: Please show with references how sequencing has advanced the study of oral microbiota.

Also, is pyrosequencing so much better than the other methods of next generation sequencing?

Line 73: please state in the first line that you refer to children. 

MATERIALS AND METHODS

Line 80:what is DMFT?

Was there an ethics approval for the study? It should be mentioned please.

Line 91: was there mechanical lysis? it is shown that it improves the yield. Could you please specify and justify why not.

Line 97: could you please provide references on why choosing these primers/V regions?

Lines 112-126: Please give more details on this section. It is very brief and lacks adequate information. 

What about the statistics? This must be included please in order to evaluate the results.

Line 127: Is this Result section? Please correct. 

Line 148: please provide information on other alpha diversity indexes such as Observed Species.

Line 157: Please do not show these data, this is part of the pipeline that you must describe in details in the relevant section. If you wish take this table in supplementary.

Line 160: Instead of 'comparison...' that should be detailed in the materials and methods statistic analysis anyway, why don't you choose a title that would summarise the significant results you found?

Table 3: Are these numbers abundances? It is not clear.

Lines 170-171: Please rephrase the whole sentence with accuracy.

The whole section from lines 76-215 must be significantly reorganised. First, Results section is missing. The Methods are not adequately described with significant issue being the missing statistics part. The Results are not clearly presented and must be so with Results sections title summarising the significant findings.

Discussion

Line 246, 249, 263, 271, 281: reference please.

Author Response

Reviewer 1

Please mention at the beginning that you refer to humans and briefly what is black stain.

• Thank you. I corrected it as you guided.

line 25: do you mean black pigment- producing bacteria?

• Yes, thank you.

line 26-27: please rephrase, metagenome is not just the 16S. 

• Thank you. We corrected it as you guided.

Line 29: Referring to "an identified" heatmap at the abstract is not very informative. Please rephrase. 

• We rephrased the sentence.

Line 30: please be more specific than just 'had more' ... It is not scientifically accurate.

• We rephrased the sentence.

Line 32: Please rephrase for accuracy: just by identifying taxa at a certain niche it does not provide evidence of causality. Please definitely rephrase this.

• We rephrased the sentence.

 

Line 39: what is the cervical third?

• Cervical third is defined as the third of the tooth closest to the cervix and BS is usually occurred on the cervical third of the teeth.
•  

Please place the full stop after the reference.

• Yes, thank you.

Line 46-48: Please rephrase the whole sentence bcs it has a confusing message.

• We rephrased the sentence.

Line 49-50: Please give more details, this is not informative enough.

• We rephrased the sentence.

Line 51: references?

• We added the references.

Line 53: Please rephrase bcs 'simple ... composition' is not informative and accurate enough.  

• We rephrased the sentence.

Line 58: please specify PCRs do you refer to?

• We rephrased the sentence.

Line 51-62: Overall this is lacking specific information, try make it more detailed but still keep it short, refer to tested cohorts, techniques, taxonomic groups etc.

• We rephrased the sentence.

 

Line 63-66: Please revise with references. Is pyrosequencing the best sequencing technique?

• We revised with reference. There are many better NGS methods now, but at the time we planned this study, pyrosequencing was the most suitable method for this research.

 Line 66: please be more accurate in this phrase. 

• We rephrased the sentence.

Line 67-69: Why refer to why we use sequencing and its benefits? this is common knowledge. This space should be used for BS related information and not for stating sth known. 

• We deleted the sentence. The sentence were for the dentist who are not familiar with the microbiology.

Lines 69-72: Please show with references how sequencing has advanced the study of oral microbiota.

• We rephrased the sentence.

Also, is pyrosequencing so much better than the other methods of next generation sequencing?

• Pyrosequencing is known to be more effective than other methods for identification of oral microbiome due to its short read length.

Line 73: please state in the first line that you refer to children. 

• At the first sentence of abstract and introduction, we stated that BS occurs particularly in primary dentition (baby teeth) of human.
•  

MATERIALS AND METHODS

Line 80:what is DMFT?

• DMFT is Decayed-missing-filled teeth in permanent dentition and dmft is same abbreviation in primary dentition. The abbreviation was used because it is a term commonly used in the dental field, but we modified to full name.

Was there an ethics approval for the study? It should be mentioned please.

• We mentioned the ethics approval in the manuscript and attached the document.

Line 91: was there mechanical lysis? it is shown that it improves the yield. Could you please specify and justify why not.

• Since the DNA extraction process was left to the company`s protocol, the exact reason is unknown.

 

Line 97: could you please provide references on why choosing these primers/V regions?

• The process after sampling was entrusted to a pyrosequencing company and they chose it.

Lines 112-126: Please give more details on this section. It is very brief and lacks adequate information. 

What about the statistics? This must be included please in order to evaluate the results.

• We added an independent statistical analysis section.

Line 127: Is this Result section? Please correct. 

• We corrected it.

Line 148: please provide information on other alpha diversity indexes such as Observed Species.

• We calculated the alpha diversity totally.

Line 157: Please do not show these data, this is part of the pipeline that you must describe in details in the relevant section. If you wish take this table in supplementary.

• We deleted the table 1.

Line 160: Instead of 'comparison...' that should be detailed in the materials and methods statistic analysis anyway, why don't you choose a title that would summarise the significant results you found?

• We corrected the title.

Table 3: Are these numbers abundances? It is not clear.

• These numbers are OTUs.

Lines 170-171: Please rephrase the whole sentence with accuracy.

• We rephrased the sentence.

The whole section from lines 76-215 must be significantly reorganised. First, Results section is missing. The Methods are not adequately described with significant issue being the missing statistics part. The Results are not clearly presented and must be so with Results sections title summarising the significant findings.

• Thank you for your advice. We corrected it with you and other reviewers.
•  

Discussion

Line 246, 249, 263, 271, 281: reference please.

• References were attached to the referred sentences.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

 The Oral Microbiome in Children with Black Stained Tooth.

 

 

This experimental study had the aim to identify the oral microbiome in black stained tooth using pyrosequencing, since it is not clear which bacterial species is directly involved in this causative process.

 

The paper is well written, and its main strength is that it shows the first species-level analysis of the pyrosequencing data in BS formation.  

 

On the contrary, the main bias is that the sample size is too small to draw definite conclusions, especially taking into consideration that the authors cite other papers in which thousands of subjects were included. This bias makes impossible to draw definite conclusions, since the oral microbiome is made up of a wide variety of bacterial species, and a causal relationship between a bacterial species and any condition cannot be analyzed without the accurate assessment of the sample size.

 

For this reason, the article is not suitable for publication in this present form.

 

Some major changes are required:

 

• The materials and methods section is not clear, and the results are included in the methods section. I kindly ask to the authors to rewrite the two section, in order to better distinguish between the two, also including a paragraph clearly stating the statistical analysis that has been performed.

 

 

Author Response

Reviewer 2

 

This experimental study had the aim to identify the oral microbiome in black stained tooth using pyrosequencing, since it is not clear which bacterial species is directly involved in this causative process.

 

The paper is well written, and its main strength is that it shows the first species-level analysis of the pyrosequencing data in BS formation.  

• Thank you for your kind review. We did our best to revise it according to your review.

 

On the contrary, the main bias is that the sample size is too small to draw definite conclusions, especially taking into consideration that the authors cite other papers in which thousands of subjects were included. This bias makes impossible to draw definite conclusions, since the oral microbiome is made up of a wide variety of bacterial species, and a causal relationship between a bacterial species and any condition cannot be analyzed without the accurate assessment of the sample size.

• We totally agree with you. We are well aware of recent NGS studies on large popuations. However, at the time we planned the study, there were not many NGS studies, and our study was also in its initial stage, so we could not target a large number of them. We thought it was a study that would serve as a springboard for future research. Also, since there has been no research on Korean children, we think it is possible to have racial originality.

For this reason, the article is not suitable for publication in this present form.

 

Some major changes are required:

 

• The materials and methods section is not clear, and the results are included in the methods section. I kindly ask to the authors to rewrite the two section, in order to better distinguish between the two, also including a paragraph clearly stating the statistical analysis that has been performed.

=> We separate the M &M and Results, and added the statistical analysis section in M&M.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript proposed by the Authors describes the Oral Microbiome in Children with Black Stained 2 Tooth. While the overall idea for this engineering and research effort is good, the presentation of it in the paper minor substantial improvement.

 

1. list 282 The title of the conclusion should be added.

 

2. I think each paragraph needs to increase the previous number. For example 2.MATERIAL AND METHODS  2.1 Sampling  2.2 Genomic DNA extraction.

 

3. “Ten children were recruited as study subjects.” The author used human specimens, but I did not see any IRB statements. The approval documents of the IRB must be added to ensure compliance with the relevant research specifications of human trials.

 

Author Response

Reviewer 3

The manuscript proposed by the Authors describes the Oral Microbiome in Children with Black Stained 2 Tooth. While the overall idea for this engineering and research effort is good, the presentation of it in the paper minor substantial improvement.

• Thank you so much for your review.

 

1. list 282 The title of the conclusion should be added.

• We added the title of the conclusion.

 

1. I think each paragraph needs to increase the previous number. For example 2.MATERIAL AND METHODS  2.1 Sampling  2.2 Genomic DNA extraction.

• We corrected our manuscript as you guided.

 

1. “Ten children were recruited as study subjects.” The author used human specimens, but I did not see any IRB statements. The approval documents of the IRB must be added to ensure compliance with the relevant research specifications of human trials.

• We mentioned about the IRB in the manuscript (2.1 Sampling) and attached an approval document of IRB.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Please recognise in your text any flaws like the small sample size, that bigger samples size is needed for safe experiments and the potential confouding factor that the pyrosequencing may present.

You do not need to state that you read the Helsinki Declaration, it goes without saying that you have read it. Also, there is no need to attach the actual document.

If I understand well, all sequencing was done by a seq. company. Please clearly state which procedures were done in your lab and which by the company. 

Please mention in what software were the stats performed. Also, state that you did not correct for multiple testing or FDR correction and why you did not.

There is no need to show the summary of he sequencing in main text. Please transfer to supplementary.

Table 2: what are these numbers? Please specify with units.

Please do not use the word 'flora' that is dated and use the word microbiome.

The Discussion is rather big and in some parts it reads like a review e.g. the info about Streptococcus must be more succinct lines 299-306, and lines 312-317, 326-332.

Lines 318-322: please revise in Discussion format, this reads like results. 

Author Response

Thank you very much for your review. We modified it according to the your recommendation line by line. Thank you.

Reviewer 1

Please recognise in your text any flaws like the small sample size, that bigger samples size is needed for safe experiments and the potential confouding factor that the pyrosequencing may present.

• Thank you. We agree with your opinion, and we well design a study by reflecting this in the next study. This point is mentioned in the text as a limitation of the study.

You do not need to state that you read the Helsinki Declaration, it goes without saying that you have read it. Also, there is no need to attach the actual document.

• Yes, we deleted that sentence about the Helsinki Declaration.

If I understand well, all sequencing was done by a seq. company. Please clearly state which procedures were done in your lab and which by the company. 

• Yes, it was clarified what processes were carried out in our laboratory and in the company. In our laboratory, sampling and post-data analysis were performed. The sequencing company performed DNA extraction, pyrosequencing, and data analysis in the preceding section.

Please mention in what software were the stats performed. Also, state that you did not correct for multiple testing or FDR correction and why you did not.

• The software used is the CL community program(ChunLab, Inc., Seoul, Korea). Alpha diversity was determined using the “phyloseq” R package. A heatmap analysis was performed based on microbial abundance in the “heatplus” R package. Principal component analysis (PCA) was performed using “devtools” and “ggbiplot” R packages.

There is no need to show the summary of the sequencing in main text. Please transfer to supplementary.

• We transfer it to supplementary file.

Table 2: what are these numbers? Please specify with units.

• The number means the number of OTUs.

Please do not use the word 'flora' that is dated and use the word microbiome.

• Yes, we exchange the ‘flora’ to the ‘microbiome.

The Discussion is rather big and in some parts it reads like a review e.g. the info about Streptococcus must be more succinct lines 299-306, and lines 312-317, 326-332.

• As you said, we have organized unnecessary content.

Lines 318-322: please revise in Discussion format, this reads like results. 

• As you said, we have organized unnecessary content.

Reviewer 2 Report

The authors have fulfilled the requested changes, so the article is now suitable for publication.

Author Response

The authors have fulfilled the requested changes, so the article is now suitable for publication.

• Thank you very much for your help.

Reviewer 3 Report

The author modified the manuscript to be more complete.

I think this article is acceptable.

Wish you happiness in work！

Author Response

The author modified the manuscript to be more complete.

I think this article is acceptable.

Wish you happiness in work！

• Thank you very much for your help.

Round 3

Reviewer 1 Report

Please clearly state which of the molecular and sequencing procedures were performed outside your lab in lines 79-102. It is not clear as it is now.

Please state why there was no FDR correction, lines 117-124.

Lines 104-116: please mention where were all these actions performed?  QIIME, mothur? the description is still cryptic. 

Please address my previous comment about Table 2. You have to explain to your readership on the table not just to me.

Author Response

Please clearly state which of the molecular and sequencing procedures were performed outside your lab in lines 79-102. It is not clear as it is now.

• We clearly stated that molecular and sequencing procedures were performed outside our lab.

 

Please state why there was no FDR correction, lines 117-124.

• We are very sorry for not answering your sentence. We missed it while focusing on the front sentence.
• We know that sampling is very important in pyrosequencing studies. While doing the research plan, we thought about three times multiple testing. However, first, the portion of the tooth that could be sampled was very small. Secondly, the cost of pyrosequencing was high. Thirdly, in my previous research experience, multiple testing did not yield unusually different results from sample to sample. Therefore, it was decided to perform one sampling more accurately than the multiple test, and the plaque from the sampling site was well collected.

 

Lines 104-116: please mention where were all these actions performed?  QIIME, mothur? the description is still cryptic. 

• We are very sorry for not being able to explain clearly. We didn`t use QIIME or mothur. For microbiome analysis, we used the CL community program developed by ChunLab as mentioned in line 142.

Please address my previous comment about Table 2. You have to explain to your readership on the table not just to me.

• Yes, we corrected our manuscript as you guided.

Thank you very much for your help.