Inter-species diversity and functional genomic analyses of closed genome assemblies of clinically isolated, megaplasmid-containing Enterococcus raffinosus Er676 and ATCC49464
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Enterococcus raffinosus is an understudied member of its genus possessing a characteristic megaplasmid contributing to a large genome size. Although less commonly associated with human infection compared to other enterococci, this species can cause disease and persist in diverse niches such as the gut, urinary tract, blood and environment. Few complete genome assemblies have been published to date for E. raffinosus . In this study, we report the complete assembly of the first clinical urinary E. raffinosus strain, Er676, isolated from a postmenopausal woman with history of recurrent urinary tract infection. We additionally completed the assembly of clinical type strain ATCC49464. Comparative genomic analyses reveal inter-species diversity driven by large accessory genomes. The presence of a conserved megaplasmid indicates it is a ubiquitous and vital genetic feature of E. raffinosus . We find that the E. raffinosus chromosome is enriched for DNA replication and protein biosynthesis genes while the megaplasmid is enriched for transcription and carbohydrate metabolism genes. Prophage analysis suggests that diversity in the chromosome and megaplasmid sequences arises, in part, from horizontal gene transfer. Er676 demonstrated the largest genome size reported to date for E. raffinosus and the highest probability of human pathogenicity. Er676 also possesses multiple antimicrobial resistance genes, of which all but one are encoded on the chromosome, and has the most complete prophage sequences. Complete assembly and comparative analyses of the Er676 and ATCC49464 genomes provide important insight into the inter-species diversity of E. raffinosus that gives it its ability to colonize and persist in the human body. Investigating genetic factors that contribute to the pathogenicity of this species will provide valuable tools to combat diseases caused by this opportunistic pathogen.
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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.
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This study would be a valuable contribution to the existing literature. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments. 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
This article covers the genomic sequencing, assembly, and analysis of Enterococcus raffinosus, an understudied species within the clinically significant Enterococcus genus. This article is largely well written but leaves out some key features (e.g. parameters, citations, and citations of "helper" tools used by wrapper tools (e.g. Prokka using Barrnap) etc.) of their bioinformatic tools. I have some concerns regarding the phylogenetic analyses performed. The authors mention using the GTR+Γ model of nucleotide evolution, however it is not mentioned how they selected this, perhaps it was the default setting, but if so, this should be stated as the choice of model can have quite dramatic implications for phylogeny construction. Secondly, Roary does not perform sequence trimming prior to alignment …
Comments to Author
This article covers the genomic sequencing, assembly, and analysis of Enterococcus raffinosus, an understudied species within the clinically significant Enterococcus genus. This article is largely well written but leaves out some key features (e.g. parameters, citations, and citations of "helper" tools used by wrapper tools (e.g. Prokka using Barrnap) etc.) of their bioinformatic tools. I have some concerns regarding the phylogenetic analyses performed. The authors mention using the GTR+Γ model of nucleotide evolution, however it is not mentioned how they selected this, perhaps it was the default setting, but if so, this should be stated as the choice of model can have quite dramatic implications for phylogeny construction. Secondly, Roary does not perform sequence trimming prior to alignment concatenation, which can also have a serious impact on phylogenetic reconstruction (although probably not for a phylogeny this small). Finally, the authors use midpoint rooting for their phylogeny. While this is commonly used, I strongly advise against it. In a recent study of vancomycin resistant Enterococcus isolates from Ireland (see https://www.microbiologyresearch.org/content/journal/jmm/10.1099/jmm.0.001590), this group produced two phylogenies, one from an amino acid superalignment with an outgroup (Staphylococcus aureus) and one from the output of Roary (without S. aureus). This group used the outgroup containing tree to infer which VRE could be the outgroup of their Roary tree. This approach as it found a robust root for a Roary tree. I strongly suggest that the authors also root their phylogenies in a more robust manner than the way they presented. Another option is MinVar rooting. It might also be of interest if the authors included some other Enterococcus representatives (e.g. three per species) on their phylogeny to highlight where E. raffinosus sits amongst its relatives. This is optional however, and I just think it would be interesting as this is an underrepresented species. On line 159, the authors mentioned using BUSCO for genome completion assessment. Could the authors please state what taxonomic database they used for this? Also, use of CheckM using the Enterococcus (genus) database may enhance their findings here (https://pubmed.ncbi.nlm.nih.gov/25977477/). I suggest the authors perform a CheckM analysis to confirm their BUSCO result. I am suggesting both as E. raffinosus is underrepresented in genome repositories so relying on just one method may yield spurious results. On line 175, the authors mention using ResFinder. For an underrepresented species, I recommend using ABRicate with the CARD database (using a 50% pident cutoff (--mincov 50)). I also recommend using the VFDB database with ABRicate. On line 181, the authors describe their statistical approach to gene enrichment using a Fisher's exact test. While a hypergeometric distribution is commonly used to determine enrichment, the strong relationship between Fisher's exact test and the hypergeometric distribution probably means that the statistical results would be identical. I am confused about why the authors normalised their data beforehand. If the authors mean that they used the COG data counts as proportions of the total pangenome size, this needs to be stated more clearly. If a procedure was performed (e.g. normalisation), could the authors please explain why to me? I am also concerned about the author's choice of a critical α
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Very 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
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Comments to Author
The authors of this paper conducted a thorough genotyping of two separate strains of enterococcus raffinosus, one clinical and one environmental. The methodology of the study was extremely sound, the results were well presented, clear and interesting, and I can see no problem with accepting this manuscript for publication. Excited to see where these results lead in the future. I only have 2 very minor comments, that I do not think impact the publishing of this manuscript to a large degree. Firstly, I did not understand the significance of raffinose metabolism in these species. If it is just a characteristic that marks it from other strains, then that is fine - I thought that there is maybe some biological significance that I may be missing. Secondly, there is one spelling identified - pairwise …
Comments to Author
The authors of this paper conducted a thorough genotyping of two separate strains of enterococcus raffinosus, one clinical and one environmental. The methodology of the study was extremely sound, the results were well presented, clear and interesting, and I can see no problem with accepting this manuscript for publication. Excited to see where these results lead in the future. I only have 2 very minor comments, that I do not think impact the publishing of this manuscript to a large degree. Firstly, I did not understand the significance of raffinose metabolism in these species. If it is just a characteristic that marks it from other strains, then that is fine - I thought that there is maybe some biological significance that I may be missing. Secondly, there is one spelling identified - pairwise sequence IDENTIFY should be pairwise sequence IDENTITY (Line 315).
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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