Genomic analysis of Oceanotoga teriensis strain UFV_LIMV02, a multidrug-resistant thermophilic bacterium isolated from an offshore oil reservoir
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Bacteria of the species Oceanotoga teriensis belong to the family Petrotogaceae , are Gram-negative bacilli, are moderately thermophilic and are included in the group of thiosulfate-reducing bacteria, being capable of significantly accelerating corrosion in metallic structures. However, no in-depth study on the genome, antibiotic resistance and mobile elements has been carried out so far. In this work, the isolation, phenotypic and genotypic characterization of the multi-resistant O. teriensis UFV_LIMV02 strain was carried out, from water samples from an offshore oil extraction platform in Rio de Janeiro (Brazil). We determined that the isolate has a genome of 2 812 778 bp in size, with 26 % GC content, organized into 34 contigs. Genomic annotation using Rapid Annotation using Subsystem Technology revealed the presence of genes related to resistance to antibiotics and heavy metals. By evaluating the antimicrobial resistance of the isolate using the disc diffusion technique, resistance was verified for the classes of antibiotics, beta-lactams, fluoroquinolones, aminoglycosides, sulfonamides, lincosamides and rifamycins, a total of 14 antibiotics. The search for genomic islands, prophages and defence systems against phage infection revealed the presence of five genomic islands in its genome, containing genes related to resistance to heavy metals and antibiotics, most of which are efflux pumps and several transposases. No prophage was found in its genome; however, nine different defence systems against phage infection were detected. When analysing the clustered regularly interspaced short palindromic repeat (CRISPR) systems, four CRISPR arrays, classified as types I-B and III-B, with 272 spacers, can provide the strain with immunity to different mobile genetic elements and bacteriophage infection. The results found in this study show that the isolate UFV_LIVM02 is an environmental bacterium, resistant to different classes of antibiotics, and that the proteins encoded by the predicted genomic islands may be associated with the development of greater resistance to antibiotics and heavy metals. They provide evidence that environmental bacteria found in offshore oil exploration residues may pose a risk for the spread of antibiotic resistance genes. More comprehensive studies on the microbial community present in oil waste are needed to assess the risks of horizontal gene transfer.
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The work presented is clear and the arguments well formed.
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Comments to Author
I am happy that the authors have responded adequately to all of the points raised.
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
The authors have addressed all comments from the first round of revisions and have rewritten the manuscript which now follows a logical, easy to follow narrative. I have no further revision requests.
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 …
Comments to Author
The authors have addressed all comments from the first round of revisions and have rewritten the manuscript which now follows a logical, easy to follow narrative. I have no further revision requests.
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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The paper is poorly structured and written, which has prevented a proper assessment of the research done.
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Comments to Author
Do Carmo Santos and colleagues perform genomic analysis of a multidrug resistant strain of Oceanotoga teriensis, a thermophile that can accelerate metal corrosion. The work is of importance in its presentation of a new strain of this understudied species. The Introduction is on the whole a well-referenced summary of the background to the work presented. Overall, I have several concerns about the manuscript. I have specific concerns about the figures that need to be addressed, detailed below. The narrative of the Results is confused and lacks focus which makes it very difficult to follow. There is a particularly confused section on antibiotic sensitivity testing that, in its current iteration, makes it difficult to assess whether their claims of multidrug resistance and genotype/phenotype mismatch are …
Comments to Author
Do Carmo Santos and colleagues perform genomic analysis of a multidrug resistant strain of Oceanotoga teriensis, a thermophile that can accelerate metal corrosion. The work is of importance in its presentation of a new strain of this understudied species. The Introduction is on the whole a well-referenced summary of the background to the work presented. Overall, I have several concerns about the manuscript. I have specific concerns about the figures that need to be addressed, detailed below. The narrative of the Results is confused and lacks focus which makes it very difficult to follow. There is a particularly confused section on antibiotic sensitivity testing that, in its current iteration, makes it difficult to assess whether their claims of multidrug resistance and genotype/phenotype mismatch are valid. My specific comments are below. Major comments: Fig 1A - Please include error bars. I would also prefer to see the individual data points, particularly given there are only three measurements in a 24 hour period which means there is a lot of estimation when only a line is drawn. Related - Lines 250-255 - I am not confident that I can agree with this without seeing the individual data points. Fig 1B/Lines 276-277 - I am not convinced that I can see the flagellum mentioned. Please could this be made clearer. The authors might consider a second, zoomed in box to highlight this. The narrative of the Results section as a whole is quite confused. It jumps between results, introduction sentences, and discussion points in a manner which makes it difficult to follow. I strongly recommend the authors re-write this with a clear focus on the point that each individual section is trying to make. Lines 328-340 - There is very little comparative genomic insight here. The authors do not for example mention whether the resistance genes, MFS transporters and mobile elements identified in their strain are also present in the reference. There is some information elsewhere but with this section being headed 'Comparative Genomics' it makes the narrative confusing. Lines 383-394 - These data do not appear to be presented in the manuscript. Additionally, the authors say 'UFV_LIMV02 was resistant to most of the antibiotics evaluated' with a list of beta lactams provided. The authors then say 'Only six antibiotics were able to inhibit growth' with no beta lactams listed. Please can the authors clarify this point. Line 398-400 - I have concerns about using the 'loose' parameter for extracting hits from CARD with only weak similarity. Given very little is know about this species, how are the authors confident that these are true resistance genes? As a related point, the CARD database contains genes that are not established resistance genes. I would recommend the authors re-run this analysis with ResFinder or the NCBI database. Lines 408-409 - Ciprofloxacin resistance is more commonly associated with point mutations. Can the authors provide context for their assertion that patA and patB are ciprofloxacin resistance genes? Line 409-410 - It is not clear to me that the authors have shown this with the data as presented currently. Lines 411-443 - I am not clear what the aim of this text is and how it relates to the results being presented. It belongs more as a discussion, particularly as it is disproportionately weighted against the result and the other results section. 486-487 - The authors should comment on which specific phenotype/genotypes did not match, and why they think this is the case. Minor comments: How are the authors defining MDR? The manuscript would benefit from a thorough proofread as there are a considerable number of grammatical errors throughout. The abstract is long and verbose, particularly when describing the results. It would benefit from a re-write to highlight the key information. Line 35-36 - I am not clear on what conclusion the authors are making by this statement. Line 110-116 - This is interesting but not relevant as the authors do not conduct any experimental or computation investigation into carbon source utilisation in this strain. Line 127-130 - I agree with this statement but I am not convinced that that is what the authors are showing, given they are investigating a sample from an offshore oil rig which will be producing contaminants. Line 203-205 - These are not optimal conditions for antibiotic sensitivity testing for E. coli. Can the authors comment on how relevant this is as a control strain? Line 256-259 - I cannot find any data presented in the manuscript to support this. Please could the authors clarify. Line 304 - How many genes were identified? Line 306-209 - Which genes were identified? If the authors have gene names available, please provide them. Fig 2 - Could the inversions be highlighted as they are not immediately obvious. Lines 437-443 - I am not clear on this point because the authors have not presented any work on intestinal bacteria or on gene transfer.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
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
In this manuscript, the authors carry out phenotypic and genotypic analysis of a novel strain of Oceanotoga teriensis. Transmission electron microscopy and hydrogen sulphide production are used to demonstrate phenotypic similarities to the O. teriensis strain DMS 24906. Genomic comparisons between these strains were then utilised to assess differences in antibiotic resistance genes, CRIPR loci and prophages encoded in these strains of O. teriensis. Finally, disc diffusion assays were used to determine the resistance profiles of the novel O. teriensis isolate. Whilst, in general, the scientific methods of this paper are sound, there is a disconnect in the narrative that makes it difficult for the reader to understand the authors rationale behind some lines of inquiry. For example, the manuscript jumps …
Comments to Author
In this manuscript, the authors carry out phenotypic and genotypic analysis of a novel strain of Oceanotoga teriensis. Transmission electron microscopy and hydrogen sulphide production are used to demonstrate phenotypic similarities to the O. teriensis strain DMS 24906. Genomic comparisons between these strains were then utilised to assess differences in antibiotic resistance genes, CRIPR loci and prophages encoded in these strains of O. teriensis. Finally, disc diffusion assays were used to determine the resistance profiles of the novel O. teriensis isolate. Whilst, in general, the scientific methods of this paper are sound, there is a disconnect in the narrative that makes it difficult for the reader to understand the authors rationale behind some lines of inquiry. For example, the manuscript jumps from H2S production to electron microscopy to genomic identification of resistance genes, prophages and CRISPR elements and then back to phenotypic characterisation of antimicrobial resistance. Detailed comments are listed below, however I would suggest some rewriting of the manuscript to add clarity and cohesion to the manuscript. Major: Fig 1a - No controls appear to be included in the H2S experiment. Would it be possible to repeat this experiment to include eg. a media only control or a bacterium that cannot produce H2S. This would also add context to the importance of H2S production, eg if it is produced at a higher rate in O. teriensis than in other bacteria or if other bacteria simply cannot produce it. Fig 1b - The importance of the TEM image is not clear. It seems to show a similar phenotype as the previously characterised DSM strain. Does this provide any additional information or is this here to provide further evidence that this novel isolate is similar to the DSM strain? Either way, could the authors address this in text. Line 329 - Why was the strain DSM 24906 used? Is this the type strain or is this the only other strain of O. teriensis isolated to date? In a paper which sequenced an Oceanotoga sp. They suggest that the DSM strain sequence is contaminated (PMID: 36856423). Can the authors comment on why the DSM strain specifically was used and if additional O. teriensis sequences are available, is it possible for the bioinformatic analysis be expanded to encompass these strains? Line 340 - In addition to the above comment regarding the DSM strain, the authors state that the novel O. teriensis isolate should be considered a subspecies of O. teriensis. Is it possible that this is due to the DSM sequence contamination referenced? Also if this is to be considered a subspecies should it be given the nomenclature as such? Line 343-348 - There is no rationale given for the identification of prophages on the genome of O. teriensis. It is implied that mobile elements are important in the horizontal transfer of resistance gene, however the importance of this is not addressed when introducing the data. A figure of these prophage elements or of alignments with the referenced prophage from NCBI would add some context for the reader as to what the similarities between these prophage are. In addition, resistance genes can be transferred on other mobile elements including chromosomal islands and plasmids. Have the authors tried to identify either of these in this strain of O. teriensis? Fig 3 - Whilst the data provide for CRISPR identification is thorough, again there is a disconnect in the narrative as to why this was investigated. The authors mention elsewhere the importance of bacterial immunity to phages etc., however, other antiphage defence systems are commonly found in the bacterial genome but have not been addressed. Online programs such as defencefinder and PADLOC can be used to identify known antiphage systems on the genome. If the authors don't want to include other phage defence systems, could they expand in text as to why CRISPR in particular was selected to study? Overall I think the manuscript would benefit from rewriting to improve continuity of the narrative and providing some rationale as to why each experiment was performed and how each finding leads on/links to the next. Minor: All uses of de novo and in silico etc. should be italicised. Line 165-168 - The list of media components is very hard to follow. A summary table might help the reader here. Line 183 - "After the incubation' implies that samples were taken, starting at the end of the 6 day incubation period. Could the authors clarify in text if this is the case or if sample collection was ongoing throughout the incubation? Lines 223-224 - Could the authors clarify why only odd k-mers were selected? Having not used this program I'm not sure if this is common practice or not, and the rationale isn't included in the methods section. Line 253 - Can the authors clarify what they mean by a 'top change'? This isn't mentioned in the methods and it is not clear if this refers to changing the media or something else. Line 292 - 297 - A summary table for the groups of genes might make this data more accessible to the reader and allow for a more concise summary and importance of the data to be addressed in text. Line 315 - 'co-sealing of genes' - could the authors expand on what co-sealing of genes means? Lines 437-443 - Some rewording of this paragraph describing the horizontal gene transfer of ARGs between environmental bacteria and enteric pathogens would add clarity for the reader. Spelling and grammar: There is inconsistency throughout the manuscript with the use of beta-lactam. Sometimes it is Beta, sometimes the symbol, and sometimes B. One of the first two should be used throughout. Line 32 - 'the man phenotypic' - should be 'main' Line 204-205 - the sentence regarding the reference strain is worded in a confusing way. Perhaps remove the word 'test'? Line 236 - 'The predict prophage regions was performed…' is confusing. Perhaps: 'To predict prophage regions, the web server…' Line 340 - 'subspecie' should be 'subspecies' Line 417-418 - the sentence doesn't make sense at the end, it seems to be missing a word? Line 418 - 'Wang et al.,' doesn't appear to be referenced
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Poor
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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