Round 1

Reviewer 1 Report

1. On Line 24 instead of “staffs” use “staff” for better clarity.
2. On Line 29 instead of ”underwent” use “undergoing” for better clarity.
3. On Line 30 please delete the verb “were” for better clarity.
4. On Line 31 instead of “By the next generation sequence” use “Using next generation sequencing techniques” for better clarity.
5. On Line 32 and 33 please delete “To predict the incidence of pneumonia after cancer operation” for better clarity.
6. On Line 34 insert the word “Also” before “Atopobium parvulum” for better clarity.
7. On Line 35 instead of “was” use “increased” for better clarity.
8. On Line 51 please delete the word “including” for better clarity.
9. On Line 52 instead of “risks of” use “a risk for” for better clarity.
10. On Line 53 please delete the word “be” for better clarity.
11. On Line 54 please delete the phrasing “opportunistic respiratory pathogens and it can” for better clarity.
12. On Line 57 instead of “staffs” use “staff” for better clarity.
13. At Study Design, you mention the type of cancer only for 8 out of 10 patients enlisted in the study, please add info about these 2 patients also.
14. On Line 80 instead of “On” use “Our” for better clarity.
15. On Line 107 instead of “with” use “without”, because otherwise those 5 patients could not be control cases and all 10 patients had pneumonia from beginning.
16. On Line 172 instead of “theoral” use “the oral” for better clarity.
17. On Line 203 regarding the analysis of biofilm you need to mention the possible future use of MALDI-TOF MS on a selected case series of patients: “Matrix-Assisted Laser Desorption - Ionisation-Time of Flight Mass Spectrometry (MALD-TOF MS) is a novel technique used for visualizing biofilm, that could permit identifying the pathogens responsible for cases with acquired postoperative pneumonia.” And reference it to Jeican, I.I.; Barbu Tudoran, L.; Florea, A.; Flonta, M.; Trombitas, V.; Apostol, A.; Dumitru, M.; Aluaș, M.; Junie, L.M.; Albu, S. Chronic Rhinosinusitis: MALDI-TOF Mass Spectrometry Microbiological Diagnosis and Electron Microscopy Analysis; Experience of the 2nd Otorhinolaryngology Clinic of Cluj-Napoca, Romania. J. Clin. Med.2020, 9, 3973. https://doi.org/10.3390/jcm9123973
18. On Line 205 instead of “predator” use “predictor” for better clarity.
19. On Line 216 please correct the references “[D6,7,8]”.
20. On Line 227 you need to mention one limitation of the study regarding the possible associated pathology of the patients included in the study group: “For cases included in our study we did not take into consideration the possible associated pathology of respiratory allergies and asthma that can modify the commensal flora at the level of the aerodigestive tract and increase the risk of perioperative pneumonia.” And reference it to Costache A, Berghi ON, Cergan R, Dumitru M, Neagos A, Popa LG, Giurcaneanu C, Vrinceanu D. Respiratory allergies: Salicaceae sensitization (Review). Exp Ther Med. 2021 Jun;21(6):609. doi: 10.3892/etm.2021.10041. Epub 2021 Apr 14. PMID: 33936266; PMCID: PMC8082596.
21. On Line 229 instead of “of” use “for” for better clarity.
22. On Line 246 instead of “was” use “increased” for better clarity.
23. On Line 247 instead of “be the predictor of the incidence” use “predict the incidence”
24. On Line 295 delete “10I10”.
25. Renumber the reference after inserting the 2 articles suggested in order to improve the clarity of the manuscript.

Author Response

Reviewer 1

 

Thank you for your valuable comments and pointing out our mistakes to improve the quality of our manuscript. We reply to your comments point by point. The changes for your comments are highlighted in green (sky blue for reviewer 2, and yellow for reviewer 4).

 

• At Study Design, you mention the type of cancer only for 8 out of 10 patients enlisted in the study, please add info about these 2 patients also.

<Reply>

Thank you for pointing out our mistake.

Esophageal cancer was four patients.

The manuscript was revised.

 

• On Line 203 regarding the analysis of biofilm you need to mention the possible future use of MALDI-TOF MS on a selected case series of patients: “Matrix-Assisted Laser Desorption - Ionisation-Time of Flight Mass Spectrometry (MALD-TOF MS) is a novel technique used for visualizing biofilm, that could permit identifying the pathogens responsible for cases with acquired postoperative pneumonia.”

<Reply>

Following sentence was inserted in manuscript and reference was inserted.

MALDI-TOF mass spectrometry made it possible to identify the pathogens in bio-film[A28].

 

• On Line 227 you need to mention one limitation of the study regarding the possible associated pathology of the patients included in the study group: “For cases included in our study we did not take into consideration the possible associated pathology of respiratory allergies and asthma that can modify the commensal flora at the level of the aerodigestive tract and increase the risk of perioperative pneumonia.”

<Reply>

Following sentence was inserted in manuscript and reference was inserted.

 

For cases included in our study we did not take into consideration the possible associated pathology of respiratory allergies and asthma that can modify the commensal flora at the level of the aerodigestive tract and increase the risk of perioperative pneumonia[41]. It is one of the limitations of this study.

 

• On Line 51 please delete the word “including” for better clarity.
• On Line 52 instead of “risks of” use “a risk for” for better clarity.
• On Line 53 please delete the word “be” for better clarity.
• On Line 54 please delete the phrasing “opportunistic respiratory pathogens and it can” for better clarity.

<Reply>

According to the advice from your advice and reviewer 3’s, the paragraph was revised.

Revised paragraph

Periodontal pocket harbors pathogenic anaerobes for periodontal tissue [3]. These pathogens also affect on respiratory disease [4] by causing inflammatory response to the endotoxin of the periodontal pathogens. Poor oral hygiene causes the dysbiosis of oral microbiome. For dysbiosis, Porphyromonas gingivalis and Streptococcus mutans are emphasized because pf their bacterial metabolism and virulence, community development, and bacteria-host interactions [5]. Oral microbiome is the main source of lung microbiome [6]. Aspiration of opportunistic respiratory pathogens can cause pneumonia [7]. Increased anaerobe including periodontal pathogens in the oral microbiome are suggested to be a risks for the multiple respiratory diseases.

 

Following errors were revised.

• On Line 24 instead of “staffs” use “staff” for better clarity.
• On Line 29 instead of ”underwent” use “undergoing” for better clarity.
• On Line 30 please delete the verb “were” for better clarity.
• On Line 31 instead of “By the next generation sequence” use “Using next generation sequencing techniques” for better clarity.
• On Line 32 and 33 please delete “To predict the incidence of pneumonia after cancer operation” for better clarity.
• On Line 34 insert the word “Also” before “Atopobium parvulum” for better clarity.
• On Line 35 instead of “was” use “increased” for better clarity.
• On Line 57 instead of “staffs” use “staff” for better clarity.
• On Line 80 instead of “On” use “Our” for better clarity.
• On Line 107 instead of “with” use “without”, because otherwise those 5 patients could not be control cases and all 10 patients had pneumonia from beginning.
• On Line 172 instead of “theoral” use “the oral” for better clarity.
• On Line 205 instead of “predator” use “predictor” for better clarity.
• On Line 216 please correct the references “[D6,7,8]”.
• On Line 229 instead of “of” use “for” for better clarity.
• On Line 246 instead of “was” use “increased” for better clarity.
• On Line 247 instead of “be the predictor of the incidence” use “predict the incidence”
• On Line 295 delete “10I10”.

 

Thank you again for your comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

This generally acceptable research paper should be language-edited. Otherwise there are no criticisims.

Author Response

Reviewer 2

Comments and Suggestions for Authors

This generally acceptable research paper should be language-edited. Otherwise there are no criticisims.

 

<Reply>

Thank you for considering our manuscript for publication in Applied science.

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear Authors, 
Congratulations on your work.

However, I would suggest some changes to the text, which is as follows: 

I suggest to define the purpose of the work in the abstract and in the introduction

From line 46 to line 54 the text is not very fluid and I suggest a change; also at line 48 other pathologies related to oral dysbiosis should be mentioned.

In the materials and methods section, it should be specified how patients were chosen for the study; also, please mention the inclusion criteria and exclusion criteria. Have the patients signed informed consent?

Line 85: It is unclear how the plaque was removed; please define "Professional Mechanical Tooth Cleaning"

Line 93: is Pneumonia and not Pneumonia

In Figure 1 is Others and non Oters; it is not clear which figure 1 is. 

Line 121 Firmicutes would go in italics 

From line 122 to 125: what do the authors mean by "Clear difference between two groups". Which groups? Specify better. 

Line 208, 223, 225, 231, 235 the names of the mentioned bacteria should be in italics. 

Line 211: There is a repeat of Infectious pneumonia. Is that a mistake?

It might also be useful to mention these articles for the management of the oral microbiome: DOI: 10.3390/ microorganisms9010069; DOI: 10.3390/ microorganisms10020337

Author Response

Reviewer 3

Thank you for your valuable comments and pointing out our mistakes to improve the quality of our manuscript. We reply to your comments point by point. The changes for your comments are highlighted in sky blue (green for reviewer 1, and yellow for reviewer 4).

 

• I suggest to define the purpose of the work in the abstract and in the introduction

<Reply>

Following sentences were inserted in the manuscript.

Abstract

The purpose of this study was to find out the candidate of bacterial species for the postoperative pneumonia.

Introduction

The purpose of this study was to find out the bacterial species that can be the candidate of postoperative pneumonia by applying case control study design.

 

 

• From line 46 to line 54 the text is not very fluid and I suggest a change; also at line 48 other pathologies related to oral dysbiosis should be mentioned.

<Reply>

The paragraph was revised.

Previous paragraph

Periodontal pocket harbors pathogenic anaerobes for periodontal tissue [3]. These pathogens also affect on respiratory disease [4], coronary heart disease [5], and Alzheimer's disease[6] by causing inflammatory response to the endotoxin. Therefore, oral microbiome is the second most diverse microbial community in the human body. Poor oral hygiene causes the dysbiosis of oral microbiome. Increased anaerobe including periodontal pathogens are suggested to be a risks for the multiple respiratory diseases. Oral microbiome is the main source of lung microbiome [7]. Aspiration of opportunistic pathogens colonized in dental plaque can be opportunistic respiratory pathogens and it can cause pneumonia [8].

 

Revised paragraph

Periodontal pocket harbors pathogenic anaerobes for periodontal tissue [3]. These pathogens also affect on respiratory disease [4] by causing inflammatory response to the endotoxin of the periodontal pathogens. Poor oral hygiene causes the dysbiosis of oral microbiome. For dysbiosis, Porphyromonas gingivalis and Streptococcus mutans are emphasized because pf their bacterial metabolism and virulence, community development, and bacteria-host interactions [5]. Oral microbiome is the main source of lung microbiome [6]. Aspiration of opportunistic respiratory pathogens can cause pneumonia [7]. Increased anaerobe including periodontal pathogens in the oral microbiome are suggested to be a risk for the multiple respiratory diseases.

 

 

• In the materials and methods section, it should be specified how patients were chosen for the study; also, please mention the inclusion criteria and exclusion criteria. Have the patients signed informed consent?

<Reply>

Following paragraph was inserted in Study design.

 

The inclusion criterion was having undergone surgery under general anesthesia with epidural anesthesia All patients were extubated in the operating room. Edentulous patients were excluded. Among them, patients who had post operative pneumonia were selected. Type of cancer was used for matching. Informed consent was obtained from all the patients.

 

 

• Line 85: It is unclear how the plaque was removed; please define "Professional Mechanical Tooth Cleaning"

<Reply>

Following sentences were inserted.

Dental plaque was removed by the dentist by using contra-angle handpiece with polishing paste (MERSSAGE, SHOFU, Kyoto, Japan) until no stained plaque was observed. Sponge brush was used to clean up the oral mucosa. When dental calculous was detected, scaling was performed.

 

 

• From line 122 to 125: what do the authors mean by "Clear difference between two groups". Which groups? Specify better.

<Reply>

As the check points of Figure 1. were described, "Clear difference between two groups" was removed.

 

 

• It might also be useful to mention these articles for the management of the oral microbiome: DOI: 10.3390/ microorganisms9010069; DOI: 10.3390/ microorganisms10020337

<Reply>

Following reference was inserted in Discussion

To control these species, antimicrobials or probiotics should be applied [49].

49.　Butera, A.; Gallo, S.; Maiorani, C.; Molino, D.; Chiesa, A.; Preda, C.; Esposito, F.; Scribante, A. Probiotic Alternative to Chlorhexidine in Periodontal Therapy: Evaluation of Clinical and Microbiological Parameters. Microorganisms. 2020, 9, 69. DOI: 10.3390/microorganisms9010069 PMID: 33383903

 

However, DOI: 10.3390/ microorganisms10020337, could not find out by Pubmed. OPlease check the DOI or give us more information of this reference.

 

• Line 211: There is a repeat of Infectious pneumonia. Is that a mistake?

<Reply>

Thank you for pointing out our mistake.

The phrase was revised.

infectious pneumonia and noninfectious pulmonary disease

 

• Following errors were revised.
  • Line 208, 223, 225, 231, 235 the names of the mentioned bacteria should be in italics.
  • Line 93: is Pneumonia and not Pneumonia
  • In Figure 1 is Others and non Oters; it is not clear which figure 1 is.
  • Line 121 Firmicutes would go in italics

 

 

 

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Thank you for providing this manuscript. I think the idea behind it has some interest to the wider community. Pneumonia is still a major concern, particularly with hospitalisation and post-operative acquisition.

This study aimed to characterise changes in the microbiomes between patients with and without pneumonia after cancer-related operations.

Although somewhat interesting, the paper has many substantial flaws and in its current state is not near suitable for publication. Please see below for my detailed comments.

Incidence of Post Operative Pneumonia and Oral Microbiome for the Patients with Cancer Operation

Comments:

Minor

Need English language formatting throughout – phrasing and extra words that aren’t necessary. I’ve listed some of these below.

 

Remove ‘the’ from title

 

Abstract:

Line24: remove ‘as well as’ or ‘and’

L24: ‘staff’ is singular (repeated throughout)

L25: perioperative means during the operation, do the authors mean during pre- or post-operative care?

L26: “However, <a> high percentage….

L30: remove ‘were’

L30: before the operation would be ‘preoperative’

L31: sentence needs re-phrasing, e.g. Using next generation sequencing technologies…”

Be careful using ‘predictors’ with a sample size of 10. Associations maybe, but not strong enough to predict. At best, it will give an indication that it is a higher risk

 

L43: duplication of ‘oral’

L44: consists

L47: ‘These pathogens affect on’ needs to be re-phrased (impact on would read better)

L49: Therefore, <the> oral…. Needs a reference too

L56: Is ‘

It would be beneficial early on to refer to the fact you are looking at ventilator associated pneumonia, rather than other types of pneumonia.

L63: perioperative – not sure this is the right term here and throughout, it seems as though you are looking at pre-operative and post-operative care

L80: One? Not ‘On’

L89: remove ‘for’. Rephrase next sentence

L91: Sampling was

L106: 5 patients with postoperative pneumonia, and 5 patients with postoperative pneumonia – is this correct? Need to define your patient groups. It looks like they all underwent surgery but matched to someone that then went on to develop pneumonia – is that correct?

Patient matching needs to be much more stringent. There are numerous factors that would render them an unsuitable match in my opinion, detailed in the major comments. Can you please elaborate on how the patients were matched before analysis?

L121: clarify that Firmicutes were decreased in all cases after the operation, not any specific group

L136-137: I disagree that the groups are distinct. There is quite a lot of overlap in the groupings

L138: ‘specific oral bacterial speci<e>s’

Figure 2: needs more description in the legend – is the percentage referring to the actual relative presence of the bacteria in the samples, or is it something else?

 

Fig 3: Describe this method a little more and explain what the relationships/positioning shows for each cluster/species

L184: remove ‘the’

L185-186: This isn’t a particularly surprising finding – community oral care is always going to be different to that of oral professionals. And oral care will remove a lot of microorganisms from the mouth, which may be why you’re seeing differences here.

L188/189/190: Capital letter for Gram

L195-196: There may well be a relationship between the respiratory and oral microbiomes, but would it be more likely to be oral that then affects respiratory rather than reverse?

L196: No need for italics for streptococci (not latin)

L197: italicise Neisseria

Careful with English language in the second half of the paragraph starting at L204

L211: check phrasing - duplicate?

L216: What is this reference?

L218: Isn’t this what your study is looking at? You should know if there is an increased prevalence of this in your samples..

L225/226: italics

L228: too small a sample size to say they are predictors of disease, can you do a power calculation to show the sample sizes required for this kind of study?

L230/231/235: italics

L238-240: Not really within the scope of this study

L241: Not sure what this provides to the manuscript – it is more of a method statement than in the discussion, little relevance.

 

Conclusion is logical, more pathogens = more risk of disease, but the data isn’t that supportive of the statement. Associations are there, but weak in power.

 

Major

Need more information on the methods:

Sample DNA extraction, processing, sequencing (instrument, runs, reads etc), pipeline for bioinformatic analysis as well as any assumptions/specific parameters that are unique to this study, and interpretation of the findings

 

16S sequencing (which you haven’t specified in the methods, but I assume is the method of use here, as typical with these studies) is renowned to be hit and miss for species-level determination. How confident are you in your species level identification? Would it be better to stick with genus level as there may be associations here?

 

Supplementary Table S1 – three of your patients had diabetes which is a known risk factor for pneumonia. You should reconsider your statistical analysis of medical history factors; 0 vs 3 for diabetes is clearly a significant difference. If you consider all factors collectively (which you shouldn’t for this, because it is a variable you are looking at), it wil give you a false negative, because overall there are similar numbers.

 

Chemotherapy is also going to have a significant impact on the bacterial microbiome, and isn’t controlled for in this study.

 

Looking at the raw data in reads, there should be a cutoff at which the number of reads is not considered to be ‘by chance’ or through something other than actual bacterial cells. Having reads of for example, less than 10, removed from the analysis will give your data a bit more confidence that the numbers you are seeing are representative of the community present.

Otherwise, changes from 1 read to 9 reads will come across as statistically significant, whereas it actually probably has no real biological significance, if the member of that community is even there in real proportions. I would suggest a re-analysis with a threshold that you determine to be suitable.

 

Table 1 shows the likelihood of fever with the presence of specific bacterial species, but these are colonising the mouth, not the airway, and without sampling from bronchial lavage or aspirates, this study doesn’t determine the causative agents of the pneumonia, merely weak correlations from a different anatomical site – correlation does not mean causation.

There are a number of studies that have looked at the link between the oral microbiome and VAP, e.g. DOI: 10.1099/jmm.0.000212 that would be directly relevant to this study.

Author Response

Reviewer 4

Thank you for your valuable comments and pointing out our mistakes to improve the quality of our manuscript. We reply to your comments point by point. The changes for your comments are highlighted in yellow (green for reviewer 1, and sky blue for reviewer 3).

 

• Line24: remove ‘as well as’ or ‘and’

<Reply>

“as well as” was removed.

 

 

• L25: perioperative means during the operation, do the authors mean during pre- or post-operative care?
• L63: perioperative – not sure this is the right term here and throughout, it seems as though you are looking at pre-operative and post-operative care

<Reply>

In Japan, perioperative oral care management is officially recommended by the Ministry of Health, Labour and Welfare. For the patients with cancer operation, perioperative oral care management include dental treatment by family dentist before hospitalization, oral care during hospitalization including immediately before cancer operation, and after cancer operation, and maintenance by family dentist.

Throughout the manuscript, we checked the word “perioperative”.

Revised

“post operative pneumonia”

“preoperative oral care”

“preoperative oral care management” was remained.

 

• Following mistakes were revised according to your advice.
  • Remove ‘the’ from title
  • L24: ‘staff’ is singular (repeated throughout)
  • L26: “However, <a> high percentage….
  • L30: remove ‘were’
  • L30: before the operation would be ‘preoperative’
  • L44: consists
  • L196: No need for italics for streptococci (not latin)
  • L197: italicise Neisseria
  • L47: ‘These pathogens affect on’ needs to be re-phrased (impact on would read better)
  • L80: One? Not ‘On’
  • L91: Sampling was
  • L188/189/190: Capital letter for Gram
  • L138: ‘specific oral bacterial speci<e>s’
  • L184: remove ‘the’
  • Careful with English language in the second half of the paragraph starting at L204
  • L211: check phrasing - duplicate?
  • L216: What is this reference?
  • L225/226: italics
  • L230/231/235: italics

 

 

• L31: sentence needs re-phrasing, e.g. Using next generation sequencing technologies…”

<Reply>

The sentence was re-phrased.

Previous manuscript

By the next generation sequence, oral microbiome of these patients was analyzed at first visit, the day before and after cancer operation.

Revised

Using the next generation sequence, oral microbiome of these patients was analyzed at the time of their first visit, the day before and after cancer operation.

 

 

• Be careful using ‘predictors’ with a sample size of 10. Associations maybe, but not strong enough to predict. At best, it will give an indication that it is a higher risk

<Reply>

All the word predictor in manuscript were replaced for high risk.

 

 

• L43: duplication of ‘oral’

<Reply>

Both “oral microbiome” and “oral” cavity are technical term. Please give us advice how to revise.

 

 

• L49: Therefore, <the> oral…. Needs a reference too

<Reply>

According to the advice from other reviewers, the paragraph was revised, and the sentence was removed.

 

 

• L89: remove ‘for’. Rephrase next sentence

<Reply>

“for” was removed.

The sentence was revised

Previous manuscript

Coat of tongue attached to the mucosal brush was suspended in ice cold phosphate buffer saline and immediately stocked in the freezer (-20℃) until the microbiome analysis.

Revised manuscript

The tongue coat attached to the mucosal brush was suspended in ice-cold phosphate buffered saline and immediately stocked in the freezer (-20℃) until the microbiome analysis.

 

 

• L106: 5 patients with postoperative pneumonia, and 5 patients with postoperative pneumonia – is this correct? Need to define your patient groups. It looks like they all underwent surgery but matched to someone that then went on to develop pneumonia – is that correct?

<Reply>

The mistake was revised.

Five patients with postoperative pneumonia as a case and 5 patients without postoperative pneumonia as a control participated.

 

 

• L121: clarify that Firmicutes were decreased in all cases after the operation, not any specific group

<Reply>

The sentence was revised.

For Phylum levels of oral microbiome, Firmicutes was decreased in all cases after cancer operation when compared with baseline.

 

 

• L136-137: I disagree that the groups are distinct. There is quite a lot of overlap in the groupings

<Reply>

Following sentence was removed.

Patients with pneumonia and control were separately located.

 

 

• Fig 3: Describe this method a little more and explain what the relationships/positioning shows for each cluster/species

<Reply>

In the method following paragraph was inserted.

Inserted paragraph

For the ordination analysis, tSNE was used. t-SNE is a tool to visualize high-dimensional data. It converts similarities between data points to joint probabilities and tries to minimize the Kullback-Leibler divergence between the joint probabilities of the low-dimensional embedding and the high-dimensional data. It is highly recommended to use another dimensionality reduction method to reduce the number of dimensions to a reasonable amount if the number of features is very high. This method suppress some noise. It plots the similar data points on the same map as close as possible. Therefore, tSNE is often used for microbiome analysis as a popular new ordination technique. [21-24]

 

 

• L185-186: This isn’t a particularly surprising finding – community oral care is always going to be different to that of oral professionals. And oral care will remove a lot of microorganisms from the mouth, which may be why you’re seeing differences here.

<Reply>

This sentence was an intro of Discussion. The sentence was changed.

Previous manuscript

By the decision analysis, pathogens were clearly different between initial visit and after oral care management.

Revised manuscript

By decision analysis, pathogens that can be predict the incidence postoperative pneumonia were presented.

 

 

• L195-196: There may well be a relationship between the respiratory and oral microbiomes, but would it be more likely to be oral that then affects respiratory rather than reverse?

<Reply>

The sentence “The respiratory microbiome may affect on the oral microbiome. “ was removed.

 

 

• L218: Isn’t this what your study is looking at? You should know if there is an increased prevalence of this in your samples.

<Reply>

The sentences concerning Streptococcus anginosus was removed.

 

 

• L228: too small a sample size to say they are predictors of disease, can you do a power calculation to show the sample sizes required for this kind of study?

<Reply>

This study is exploratory research to find out the candidate of the predictor of postoperative pneumonia. By the post-hoc analysis P. gingivalis at baseline and Atopobium parvulum at the day before operation were statistically significant by Fisher’s exact test (P=0.024) using the cut off presented in Figure 2. The result of post-hoc sample size calculation using alfa 0.05, power 0.8 balance 1:1 was n=4 for each group.

 

 

• L238-240: Not really within the scope of this study
• L241: Not sure what this provides to the manuscript – it is more of a method statement than in the discussion, little relevance.

<Reply>

The sentence was removed.

Removed sentence.

These evidences suggest that increase of these bacteria in gut microbiome may be associated with stomach cancer, esophageal cancer, or pancreatic cancer. In addition, there may be the limitation for controlling oral microbiome without using antimicrobials. In this study, preoperative oral care management was performed by mechanical tooth cleaning, not using antimicrobials.

 

 

• Conclusion is logical, more pathogens = more risk of disease, but the data isn’t that supportive of the statement. Associations are there, but weak in power.

<Reply>

Conclusion was revised.

Previous manuscript

Poor oral hygiene increased the risk of incidence of postoperative pneumonia. In-creased periodontal pathogens can predict the incidence of postoperative pneumonia. In addition, increased intestinal bacteria after oral care management can also be the predictor for the incidence of postoperative pneumonia.

Revised

Poor oral hygiene increased the risk of incidence of postoperative pneumonia. Increased periodontal pathogens can be a high risk for incidence of postoperative pneumonia. In addition, increased intestinal bacteria after oral care management can also be a high risk for the incidence of postoperative pneumonia.

 

 

• Need more information on the methods:
  • Sample DNA extraction, processing, sequencing (instrument, runs, reads etc), pipeline for bioinformatic analysis as well as any assumptions/specific parameters that are unique to this study, and interpretation of the findings
  • 16S sequencing (which you haven’t specified in the methods, but I assume is the method of use here, as typical with these studies) is renowned to be hit and miss for species-level determination. How confident are you in your species level identification? Would it be better to stick with genus level as there may be associations here?

<Reply>

Following section was inserted in Method

2.4 Microbial DNA Extraction

Tongue surface samples suspended in PBS were collected by a centrifuge at 3000 rpm for 10 min. DNA extraction was performed by a Maxwell 16 LEV Blood DNA Kit (Promega KK, Tokyo, Japan)according to the manufacturer‘s instructions. DNA concentration was measured by a NanoDrop ND-2000 (Thermo Fisher Scientific KK, Tokyo, Japan). Degradation of DNA was visually checked by electrophoresis on 1% agarose gel. Degradation of DNA and contamination of RNA were checked by a Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific KK, Tokyo, Japan).　Samples meeting the following criteria were used for further sequence analysis: conc > 20 ng/mL,　volume ³ 20 mL, A260/280 ³ 1.8, and A260/230 > 1.5. In this study, all samples met the criteria.

 

2.5. Microbial Community Analysis

Extracted DNA was analyzed in a laboratory (Chun Lab, Seoul, Korea). Polymerase chain reaction　PCR) amplification was performed using primers specific to the V3–V4 region pyrosequencing tags of the 16S rRNA gene in the extracted bacterial DNA. Taxonomic classification of each read was assigned based on a search of the EzBioCloud 16S database [15，16], which contains the 16S rRNA genes of type strains that have valid published names and representative species-level phylotypes of both cultured and uncultured entries in the GenBank database, with complete hierarchical taxonomic classification from the phylum to species level [17].

 

• Patient matching needs to be much more stringent. There are numerous factors that would render them an unsuitable match in my opinion, detailed in the major comments. Can you please elaborate on how the patients were matched before analysis?
• Supplementary Table S1 – three of your patients had diabetes which is a known risk factor for pneumonia. You should reconsider your statistical analysis of medical history factors; 0 vs 3 for diabetes is clearly a significant difference. If you consider all factors collectively (which you shouldn’t for this, because it is a variable you are looking at), it wil give you a false negative, because overall there are similar numbers.

<Reply>

We do our best for the matching. However, complete matching including medical history was impossible. Therefore, following sentence was inserted as a limitation.

Inserted sentence

We could set up the medical history completely. Diabetes is a risk for postoperative pneumonia[42]. It is one of the limitations of this study.

 

 

• Chemotherapy is also going to have a significant impact on the bacterial microbiome, and isn’t controlled for in this study.

<Reply>

All the patients underwent chemotherapy. Following sentence was inserted in the manuscript.

Inserted sentence

All the patients underwent chemotherapy after cancer operation.

 

 

• Looking at the raw data in reads, there should be a cutoff at which the number of reads is not considered to be ‘by chance’ or through something other than actual bacterial cells. Having reads of for example, less than 10, removed from the analysis will give your data a bit more confidence that the numbers you are seeing are representative of the community present.
• Figure 2: needs more description in the legend – is the percentage referring to the actual relative presence of the bacteria in the samples, or is it something else?
• Otherwise, changes from 1 read to 9 reads will come across as statistically significant, whereas it actually probably has no real biological significance, if the member of that community is even there in real proportions. I would suggest a re-analysis with a threshold that you determine to be suitable.

 

<Reply>

As described in Result 3.2., number of reads varied between samples. Therefore, actual account of itself has no meaning to characterize the samples (Range: 4273-47149). In addition, zero count of pathogenic species is desirable condition. If species less than 10 reads cut for analysis, we can not investigate desirable conditions.

 

• Table 1 shows the likelihood of fever with the presence of specific bacterial species, but these are colonising the mouth, not the airway, and without sampling from bronchial lavage or aspirates, this study doesn’t determine the causative agents of the pneumonia, merely weak correlations from a different anatomical site – correlation does not mean causation.

<Reply>

Following sentences were included in Discussion.

Inserted sentences

In this study, we could not determine the causative agents of the pneumonia. Sampling from bronchial lavage or aspirates is necessary to determine the causative agents. However, pathogenic bacteria in oral microbiome can be a risk for postoperative pneumonia after cancer surgery.

 

 

• It would be beneficial early on to refer to the fact you are looking at ventilator associated pneumonia, rather than other types of pneumonia.
• There are a number of studies that have looked at the link between the oral microbiome and VAP, e.g. DOI: 10.1099/jmm.0.000212 that would be directly relevant to this study.

<Reply>

Following three sentences were included in Discussion.

A "microbial shift" occurred in dental plaque, with colonization by potential VAP pathogens and reverted back to having a predominantly normal oral microbiota after extubation[27,28].

 

A transition to a lung microbiome enriched with gut flora is found in patients with acute respiratory distress syndrome with an increased inflammatory response[47,48].

 

The high similarity between the microbiomes of dental plaque, non-directed bronchial lavages, and endotracheal tube biofilms in mechanically ventilated patients[51].

 

27. Sands, K.M.; Twigg, J.A.; Lewis, M.A.O.; Wise, M.P.; Marchesi, J.R.; Smith, A.; Wilson, M.J.; Williams D.W. Microbial profiling of dental plaque from mechanically ventilated patients. J Med Microbiol. 2016, 65, 147-159. DOI: 10.1099/jmm.0.000212 PMID: 26690690
28. Sands, K.M.; Wilson, M.J.; Lewis, M.A.O.; Wise, M.P.; Palmer, N.; Hayes, A.J.; Barnes, R.A.; Williams D.W. Respiratory pathogen colonization of dental plaque, the lower airways, and endotracheal tube biofilms during mechanical ventilation. J Crit Care. 2017, 37, 30-37.DOI: 10.1016/j.jcrc.2016.07.019. PMID: 27621110
29. Fromentin, M.; Ricard, J.D.; Roux, D. Respiratory microbiome in mechanically ventilated patients: a narrative review. Intensive Care Med. 2021, 47, 292-306. DOI: 10.1007/s00134-020-06338-2. PMID: 33559707
30. Yin, Y.; Hountras, P.; Wunderink, R.G. The microbiome in mechanically ventilated patients. Curr Opin Infect Dis. 2017, 30, 208-213. DOI: 10.1097/QCO.0000000000000352. PMID: 28067677
31. Marino P.J.; Wise M.P.; Smith A.; Marchesi J.R.; Riggio M.P.; Lewis M.A.O.; Williams D.W. Community analysis of dental plaque and endotracheal tube biofilms from mechanically ventilated patients. J Crit Care. 2017, 39, 149-155. DOI: 10.1016/j.jcrc.2017.02.020. PMID: 28259058

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Dear authors,

The work has been improved.

However, I report in this comment the article in question (which was not found): Butera A, Gallo S, Pascadopoli M, Maiorani C, Milone A, Alovisi M, Scribante A. Paraprobiotics in Non-Surgical Periodontal Therapy: Clinical and Microbiological Aspects in a 6-Month Follow-Up Domiciliary Protocol for Oral Hygiene. Microorganisms. 2022; 10(2):337. https://doi.org/10.3390/microorganisms10020337

Author Response

Reply to Reviewers

Reviewer 3

Thank you for your comment to add valuable information in our manuscript.

The reference was included as reference 50.

 

 

The work has been improved.

However, I report in this comment the article in question (which was not found): Butera A, Gallo S, Pascadopoli M, Maiorani C, Milone A, Alovisi M, Scribante A. Paraprobiotics in Non-Surgical Periodontal Therapy: Clinical and Microbiological Aspects in a 6-Month Follow-Up Domiciliary Protocol for Oral Hygiene. Microorganisms. 2022; 10(2):337. https://doi.org/10.3390/microorganisms10020337