An initial genomic blueprint of the healthy human oesophageal microbiome
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Background. The oesophageal microbiome is thought to contribute to the pathogenesis of oesophageal cancer. However, investigations using culture and molecular barcodes have provided only a low-resolution view of this important microbial community. We therefore explored the potential of culturomics and metagenomic binning to generate a catalogue of reference genomes from the healthy human oesophageal microbiome, alongside a comparison set from saliva.
Results. Twenty-two distinct colonial morphotypes from healthy oesophageal samples were genome-sequenced. These fell into twelve species clusters, eleven of which represented previously defined species. Two isolates belonged to a novel species, which we have named Rothia gullae . We performed metagenomic binning of reads generated from UK samples from this study alongside reads generated from Australian samples in a recent study. Metagenomic binning generated 136 medium or high-quality metagenome-assembled genomes (MAGs). MAGs were assigned to 56 species clusters, eight representing novel Candidatus species , which we have named Ca . Granulicatella gullae, Ca . Streptococcus gullae, Ca . Nanosynbacter quadramensis, Ca . Nanosynbacter gullae, Ca . Nanosynbacter colneyensis, Ca . Nanosynbacter norwichensis, Ca . Nanosynococcus oralis and Ca . Haemophilus gullae. Five of these novel species belong to the recently described phylum Patescibacteria . Although members of the Patescibacteria are known to inhabit the oral cavity, this is the first report of their presence in the oesophagus. Eighteen of the metagenomic species were, until recently, identified only by hard-to-remember alphanumeric placeholder designations. Here we illustrate the utility of a set of recently published arbitrary Latinate species names in providing user-friendly taxonomic labels for microbiome analyses.
Our non-redundant species catalogue contained 63 species derived from cultured isolates or MAGs. Mapping revealed that these species account for around half of the sequences in the oesophageal and saliva metagenomes. Although no species was present in all oesophageal samples, 60 species occurred in at least one oesophageal metagenome from either study, with 50 identified in both cohorts.
Conclusions. Recovery of genomes and discovery of new species represents an important step forward in our understanding of the oesophageal microbiome. The genes and genomes that we have released into the public domain will provide a base line for future comparative, mechanistic and intervention studies.
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This is a study that would be of interest to the field and community.
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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. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
Site specificity is a hallmark of the human microbiome. It is known that the microbial community of the mouth is constitutionally unique from that of the skin, lung and the gut, even though these body sites are contiguous. Little is known about the resident microbiota of the esophagus. Importantly, it is not known if the esophagus has a resident microbiota or only transiently hosts microbes from the oral cavity swallowed with saliva. Here, Gilroy and colleagues performed a combined metagenomic and culturomics study to identify the esophageal microbiome. The authors used recently developed tools for de novo assembling metagenome assembled genomes (MAGs). Authors also isolated organisms and sequenced whole genomes to build an esophageal microbiome reference database and combined their data with another …
Comments to Author
Site specificity is a hallmark of the human microbiome. It is known that the microbial community of the mouth is constitutionally unique from that of the skin, lung and the gut, even though these body sites are contiguous. Little is known about the resident microbiota of the esophagus. Importantly, it is not known if the esophagus has a resident microbiota or only transiently hosts microbes from the oral cavity swallowed with saliva. Here, Gilroy and colleagues performed a combined metagenomic and culturomics study to identify the esophageal microbiome. The authors used recently developed tools for de novo assembling metagenome assembled genomes (MAGs). Authors also isolated organisms and sequenced whole genomes to build an esophageal microbiome reference database and combined their data with another recent survey. Using these methods, the authors identified a new candidate species of Rothia by culturing and 8 novel candidate species from MAGs. The conclusions of the authors are consistent with their data as presented. This is a novel work that contributes to our understanding of a newly recognized human microbiome. What follows are minor suggestions to improve the text. L92 - 95 Authors should reference recent skin MAG work Kashaf et al. PMID: 34952941 L166 -171 Saliva samples were stored at 4C for minimum 24 hours. What was maximum storage time? In Same paragraph authors statement all samples were processed within 3 hours. How does that reconcile with the minimum 24 hour statement previous? Authors should include Colony morphology descriptions, especially for new Rothia isolate. Maybe I missed it. What fraction of contigs were not assembled into either medium or high quality MAGs? L189 -192 mags with less then 500kbs were removed. How chose this number? Ref? L217 which ref here? L223-225 did the authors make new Latinate names for their species? L243-251 used ribosomal proteins to make tree. This is standard in field. How did the authors choose the subset of dozens of ribosomal proteins? Reference needed for this. L255-265 some samples have less than 25% of taxa in phylum level see fig 4C. Is this because many reads didn't map? Is this why the authors constructed multiple 2mb pseudogenomes? Am I understanding this? Is there a reference for doing this? Do the authors have plans to make the esophageal microbiome sequences available to other researchers, as well as their bioinformatics pipelines?
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very 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
1. Methodological rigour, reproducibility and availability of underlying data: These parameters appear satisfactory. Please see list of comments below. 2. Presentation of results: Good; The presentation and figures are clear. Please see comments listed below. 3. How the style and organization of the paper communicates and represents key findings. This paper describes the genomes that were recovered from culturing and metagenomic methods applied to esophogeal samples. 4. Literature analysis or discussion: Discussion satisfactory, albeit brief. 5. Any other relevant comments: a) Methods: Line 102- Please include a study design description. e.g. How was the sample size determined? Line 125- Only two growth media were used. Please describe how this range meets a culturomic study design. Line 180- …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data: These parameters appear satisfactory. Please see list of comments below. 2. Presentation of results: Good; The presentation and figures are clear. Please see comments listed below. 3. How the style and organization of the paper communicates and represents key findings. This paper describes the genomes that were recovered from culturing and metagenomic methods applied to esophogeal samples. 4. Literature analysis or discussion: Discussion satisfactory, albeit brief. 5. Any other relevant comments: a) Methods: Line 102- Please include a study design description. e.g. How was the sample size determined? Line 125- Only two growth media were used. Please describe how this range meets a culturomic study design. Line 180- How were the 50 healthy patient metagenomes selected from the 59 normal subjects in the Deshpande et al. study. Results: Line 290- ANI and phylogeny differences are important findings. It would help to show the reader the discrepancy details (isolate ANI data alongside Fig. 2). A supplemental figure is encouraged to show the additional segments in the cultured genomes. Line 350- Please add Figure S1. It does not appear in the submission documents. General- Are there virulence factors present in any of the bacterial isolates or MAGs?
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Very 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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