Lower respiratory tract microbiome composition and community interactions in smokers
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The lung microbiome impacts on lung function, making any smoking-induced changes in the lung microbiome potentially significant. The complex co-occurrence and co-avoidance patterns between the bacterial taxa in the lower respiratory tract (LRT) microbiome were explored for a cohort of active (AS), former (FS) and never (NS) smokers. Bronchoalveolar lavages (BALs) were collected from 55 volunteer subjects (9 NS, 24 FS and 22 AS). The LRT microbiome composition was assessed using 16S rRNA amplicon sequencing. Identification of differentially abundant taxa and co-occurrence patterns, discriminant analysis and biomarker inferences were performed. The data show that smoking results in a loss in the diversity of the LRT microbiome, change in the co-occurrence patterns and a weakening of the tight community structure present in healthy microbiomes. The increased abundance of the genus Ralstonia in the lung microbiomes of both former and active smokers is significant. Partial least square discriminant and DESeq2 analyses suggested a compositional difference between the cohorts in the LRT microbiome. The groups were sufficiently distinct from each other to suggest that cessation of smoking may not be sufficient for the lung microbiota to return to a similar composition to that of NS. The linear discriminant analysis effect size (LEfSe) analyses identified several bacterial taxa as potential biomarkers of smoking status. Network-based clustering analysis highlighted different co-occurring and co-avoiding microbial taxa in the three groups. The analysis found a cluster of bacterial taxa that co-occur in smokers and non-smokers alike. The clusters exhibited tighter and more significant associations in NS compared to FS and AS. Higher degree of rivalry between clusters was observed in the AS. The groups were sufficiently distinct from each other to suggest that cessation of smoking may not be sufficient for the lung microbiota to return to a similar composition to that of NS.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. Please deposit the data underlying the work in the Society’s data repository Figshare account here: https://microbiology.figshare.com/submit. Please also cite this data in the Data Summary of the main manuscript and list it as a unique reference in the References section. When you resubmit your article, the Editorial staff will post this data publicly on Figshare and add the DOI to the Data Summary section where you have cited it. This data will be viewable on the Figshare website with a link to the preprint and vice versa, allowing for greater discovery of your work, and the unique DOI of the data means it can be cited independently.
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Comments to Author
I thank the authors for addressing all of my concerns, comments, and suggestions and I am happy for this work to be published in it's current form. Below are some comments in response to changes and questions that arose during the review. - The changes to OTU filtering during the quality control step of 16S rRNA data is a method that I lean towards using in my own work as it increases data quality by removing low abundance/prevalence OTUs while also retaining some biological signals such as the example used by the authors. - Regarding my request for individual p-values, Some journals request that a p-value is provided for each comparison referred to in the text or used to draw conclusions, but this varies between journals and I wasn't sure what the policy was here. It is acceptable to state that …
Comments to Author
I thank the authors for addressing all of my concerns, comments, and suggestions and I am happy for this work to be published in it's current form. Below are some comments in response to changes and questions that arose during the review. - The changes to OTU filtering during the quality control step of 16S rRNA data is a method that I lean towards using in my own work as it increases data quality by removing low abundance/prevalence OTUs while also retaining some biological signals such as the example used by the authors. - Regarding my request for individual p-values, Some journals request that a p-value is provided for each comparison referred to in the text or used to draw conclusions, but this varies between journals and I wasn't sure what the policy was here. It is acceptable to state that these were below a threshold which is logical and clearly stated. - It is unfortunate that a gradient-like relationship or intermediary lung microbiome (for the former smoker samples) wasn't supported by the variables used here. I agree that this would be a very interesting avenue of investigation that would require a lot of work and is beyond the scope here.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
I thank the authors for their responses to my comments. I am satisfied with the changes they have made
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Comments to Author
I thank the authors for their responses to my comments. I am satisfied with the changes they have made
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Firstly, thank you to both peer reviewers for their time and expertise assessing this manuscript- it is appreciated. Having read through the reviewers comments there are several questions about the approaches used to analyse the data, questions about the statistic analysis performed, and image quality issues with some of the figures. Can authors please address the reviewers comments below.
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Comments to Author
Summary and Overall Impression The authors present a compelling profiling of the lung microbiome structure in active, former, and never smokers. The analysis methods themselves are reasonably robust but require a few clarifications regarding quality control metrics and statistical methods. I have also suggested a few areas where their observations should be supported by statistical analysis before publication. I look forward to reading the revised manuscript. Major Issues Throughout: Lacking appropriate statistical testing to support observations. L55: DESeq2 has been previously shown to have a relatively large false discovery rate compared to other, more conservate, microbiome-tailored approaches (such as ANCOM and ALDEx2) when applied to 16S microbiome data as the underlying statistics are more …
Comments to Author
Summary and Overall Impression The authors present a compelling profiling of the lung microbiome structure in active, former, and never smokers. The analysis methods themselves are reasonably robust but require a few clarifications regarding quality control metrics and statistical methods. I have also suggested a few areas where their observations should be supported by statistical analysis before publication. I look forward to reading the revised manuscript. Major Issues Throughout: Lacking appropriate statistical testing to support observations. L55: DESeq2 has been previously shown to have a relatively large false discovery rate compared to other, more conservate, microbiome-tailored approaches (such as ANCOM and ALDEx2) when applied to 16S microbiome data as the underlying statistics are more suited to RNA-Seq gene expression data [https://www.nature.com/articles/s41467-022-28034-z]. I would urge the authors to consider reanalysing their data using one of these approaches. L109: The authors note that the study by Morris et al. is limited because it featured imbalanced group sizes. It is worth noting that the current study also featured an imbalanced design (9 Never Smokers vs. 24 Former Smokers and 22 Active Smokers). If the authors feel that an imbalanced design hinders the validity of results, then I ask them to please explain how their study is not impacted. L648: It is good practice to remove OTUs from a dataset if they fail a defined inclusion criteria (such as zero in > 50% of samples or
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
This paper describes a 16S rRNA based microbiome analysis of samples collected from the lower respiratory tract of people who have never smoked, are former smokers and are current smokers. The work is an improvement upon previous analyses as it is based on BAL samples, which should limit the amount of contamination from the upper respiratory tract. Overall the paper is well clear and the approach seems sound, but I have a few comments: 1) The authors imply that their study is an improvement upon others as they have more balanced group sizes. However in this paper they had 9 never smokers compared to 24 former and 22 active smokers. The authors need to discuss this in the discussion, and whether they think they had enough data from never smokers to make robust conclusions. 2) In the results section the …
Comments to Author
This paper describes a 16S rRNA based microbiome analysis of samples collected from the lower respiratory tract of people who have never smoked, are former smokers and are current smokers. The work is an improvement upon previous analyses as it is based on BAL samples, which should limit the amount of contamination from the upper respiratory tract. Overall the paper is well clear and the approach seems sound, but I have a few comments: 1) The authors imply that their study is an improvement upon others as they have more balanced group sizes. However in this paper they had 9 never smokers compared to 24 former and 22 active smokers. The authors need to discuss this in the discussion, and whether they think they had enough data from never smokers to make robust conclusions. 2) In the results section the authors often discuss OTUs such as "Ralstonia.03" - these designations are unhelpful for the reader, so a more intuitive way of referring to these OTUs needs to be devised. What species most closely match these OTUs? 3) I understand why the bacterial co-occurrence networks were analysed separately in the different cohort groups - but I also think it be more powerful to analyse them as a combined dataset Minor comments: Line 155 "composing a whopping 75%" - I don't think whopping is appropriate for an academic manuscript Line 295: please explain the cross-validation errors more - what does this mean? Fig -6 needs bigger legends Line 553 - DNA itself isn't metagenomic, the approach is. Please remove "metagenomic"
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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