Genomic screening of eight antibiotic-resistant Pseudomonas isolated from rainbow trout (Oncorhynchus mykiss)
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Open aquaculture systems represent a strategic environment for the study of antibiotic resistance dynamics, given the interactions between bacteria from humans, animals and the environment. The genomic investigation of eight Pseudomonas isolated from rainbow trout in a previous study demonstrated that non-wild-type and resistance phenotypes to several antibiotics (oxytetracycline, sulphonamides and florfenicol) were predominantly attributable to the presence of related genes [ tet(Y) , sul2 and floR ]. Phylogenetic analyses revealed that several species of Pseudomonas iridis harboured these genes on mobile genetic elements, including integrative conjugative elements (ICE) on the chromosome or plasmids with a high degree of sequence similarity (>99%) between the genetic structures. Furthermore, comparisons between isolates with low and high MIC values to colistin showed mutations in the amino acid sequences of the PhoP/Q two-component system and a lack of the pmrA/B system. A wide diversity of known LPS-modifying genes involved in colistin resistance was also detected in resistant isolates. This study provided insights into the dynamics of antibiotic resistance in aquaculture systems. It demonstrated the presence of genes located on an ICE inserted into a chromosome or plasmid in P. iridis , which was isolated from healthy rainbow trout in different farms within the same watershed. Our study raises questions about the ability of environmental Pseudomonas bacteria to spread their antibiotic resistance genes to other bacterial species that are of interest in terms of human or animal surveillance.
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I am pleased to tell you that your article has now been accepted for publication in Access Microbiology. This research will be of interest to the community, and I thank you for addressing both sets of reviewer comments.
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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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Please make sure you address the comments raised by the reviewers carefully. Some wording seems ambigious, so please make sure that you clarfiy those comments regarding what you are referring to and include more details on the methods and clarify some of the result presentations. Please address the comments regarding your conclusions, these need to be better supported (eg regarding the colistin resistance and including more species). Make sure all data including the raw reads are added to the bioproject and your scripts are available.
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Comments to Author
Overall, an interesting paper that attempts to address antimicrobial resistance dynamics in aquaculture, an area that is of importance due to a One Health aspect. I think the work carried out is appropriate, though some interpretation could be improved. The work carried out to obtain complete genomes of a robust nature is admirable. I would have liked more acknowledgement of the nature of the resistance genes identified e.g. the frequency of tet resistance is discussed but not really for the others. It would also improve the manuscript to work at identifying the species of the isolates the authors have obtained, especially since they carried out the phylogenetic analysis. There are some sections that could be rewritten for clarity, it isn't always clear what the authors are trying to say. I …
Comments to Author
Overall, an interesting paper that attempts to address antimicrobial resistance dynamics in aquaculture, an area that is of importance due to a One Health aspect. I think the work carried out is appropriate, though some interpretation could be improved. The work carried out to obtain complete genomes of a robust nature is admirable. I would have liked more acknowledgement of the nature of the resistance genes identified e.g. the frequency of tet resistance is discussed but not really for the others. It would also improve the manuscript to work at identifying the species of the isolates the authors have obtained, especially since they carried out the phylogenetic analysis. There are some sections that could be rewritten for clarity, it isn't always clear what the authors are trying to say. I think adding in figures that illustrate the points they would like to make would be very useful. Below are specific points based on the manuscript. Line 1: I am not sure I understand the use of this title. Atypical to what? For example, the type strain for Pseudomonas edaphica has the same predicted AMR genes as your isolate B19125 (which I have determined is the same species), which would imply this is typical for that species. For many species of Pseudomonas they have a lot of antimicrobial resistances. Line 18: Please also include the raw reads, I am aware the reads have been deposited elsewhere, but it increases accessibility if they are added to the bioproject. In addition, the isolate identifiers on NCBI/ENA/DJJB are not the same as those used in this paper e.g. A03RPs3-08 is listed as A0308 within the databases. Please change the names of the isolates in the paper to correspond with the name in the databases. Line 48: Good to see scripts have been shared and additional place for associated data. Line 61: Would be nice to include examples of other Pseudomonas species that cause infections in humans and animals. Also, could include plant pathogens too, increasing the value of monitoring Pseudomonas species. The genus Pseudomonas is quite diverse. Line 86: type "ans" should be "and" Line 99: Again, I am unsure as to why we are referring to these isolates as atypical. Also, was the original number of Pseudomonas isolates found to be 125, then 51 confirmed with MALDI-TOF? Line 126: What does it mean "one month interval"? Do you mean that DNA extraction was performed twice, one month apart from each other? Or do you mean they were grown for a period of a month and DNA extracted at the start and again after growing for a month? I think the former but as written it isn't clear. 151: If default settings were used for all tools listed here, please add a line to say this e.g. "defaults setting used for all bioinformatic analyses unless otherwise stated". Line 230: Did you use Genbank format or fasta for ICEFinder analysis? Line 236: There seems to be no description of methods here, just explaining thought process. Think this can be added to results for the colistin resistance section. Line 249: Like the inclusion of this table. Line 267: The phylogenetic analysis is really good, therefore I am confused as to why you didn't use that information to identify the species of your isolates. For example, A1230 is Pseudomonas iridis based on ANI (96.99%). Please look to define the specific species, rather than leaving as sp within the databases. This will help other researchers. Line 269: Tool you can use for ANI analysis: https://www.ezbiocloud.net/tools/ani Line 269: B19125 is Pseudomonas edaphica (ANI 98.80 %) Line 270: Looking at your phylogenetic tree it is possible B22129 is a novel species of Pseudomonas. Line 278: This table could be more informative. Would be good to perhaps use black to indicate resistance genes detected and phenotype shows resistance, grey for resistance gene detected but no phenotype information and white for no gene and no resistance. OR something similar, main thing would be to make it clearer whether there ought to be a resistance gene detected or not e.g. to correspond to phenotype. That way a reader can clearly see why you began investigating further for the phenotypes that didn't correspond to your geneotypes. Line 295: I find it odd that you determined A1230 did not have an ICE just because ICEFinder didn't find it. Given the degree of homology it is likely this is an ICE that has been missed by the tool. I searched using ICEFinder using the nucleotide sequence deposited to NCBI and it gave no hits. I then reverse complemented the sequence and uploaded it, which resulted in 2 hits. I think that highlights a flaw in the software. Line 296: Material and Methods states use of the ICEberg database 2.0, rather than 3.0. Note, I have also found disparities between using ICEFinder with the database 3.0 and 2.0 e.g. your AMR ICE isn't detected using 3.0 but is found using 2.0 for isolate A0308. Line 297: Please shared the region location within the genome e.g. region in A1230: 3808532..3835602, region in A0308: 4690511..4722653 (this regions might not be correct, this is what I could determine by manual examination). Having this information will make it easier for other researchers to search for similar regions in other isolates. Line 298: Typo, think it is meant to say A1437, rather than A1230. Figure 2: Just a note, interestingly this Tn5393 is also found in pseudomonas syringae B728a and has aminoglycoside genes too (accession GCF_000012245.1), but none of the others you have identified. Figure 2: This could be much more informative. Please add the size information, are these comparable? I would recommend using a tool that includes the percentage nucleotide identity/homology shared between the regions (such as EasyFig or DiGAlign). Would make what you are trying to illustrate much easier. Line 306: Please include locus tag information e.g. for A0308 this is annotated as AB4P92_21885. Again, this makes it much easier for others to use what you have found/repeat your work. Line 305-309: This could be rewritten for clarity. I believe the authors are trying to say the region containing the AMR genes are co-located with genes involved in horizontal transfer, trbJKL and traIJK, as well as with Tn5393. There is also an intA gene present. Line 316: There would be added value if you identifying the specific species of these isolates, as you could then highlighting having found a plasmid that can move between species. Line 327-329: ICE can integrate into plasmids, therefore this region could be the same ICE as found for the other isolates. Would make sense given the sequence similarity. Figure 4: Not sure what value this figure has, it doesn't seem to show anything that wasn't already written. Again, having a figure showing the shared homology and nucleotide identity would be helpful if you would like to include the operon. Line 399-401: Or you could say you found this within an ICE that has either integrated into the genome or a plasmid? Line 418: What is the type 1 toxin-anti-toxin system, trbL?? Line 418-419: Plasmid sequence, rather than plasmid genome?? I think a figure comparing these regions would be very helpful to highlight what you mean. Line 421-433: Good to link to the literature. What role could efflux pumps play in this resistance? I am just curious; I have no idea. Line 439: What genetic events? Line 438-441: High frequency…when it was 8 out of an original 51 (16 %), or more accurately 8 out of 125 isolates? I would not call that a high frequency. Also, of the isolates you looked at 4 are from the same species, so perhaps it is a species-specific event for the chromosome-based ICE?
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?
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 work described by Oberle et al. consists of a descriptive analysis of the genomes of eight Pseudomonas strains isolated from rainbow trout, focusing on antimicrobial resistance determinants and their associations with their phenotypes, which have been previously described. The main objection to this work is that the data on colistin resistance determinants are mostly weak or incorrectly addressed. Therefore, the mere observation of the presence of the arnCADTEF operon on the chromosome of three bacterial strains cannot be associated with their colistin resistance, even if the remaining five strains are colistin-susceptible and lack those sequences. Both groups of strains are relatively distant, phylogenetically speaking, and the role of the arnBCADTEF genes is unknown in most Pseudomonas species. …
Comments to Author
The work described by Oberle et al. consists of a descriptive analysis of the genomes of eight Pseudomonas strains isolated from rainbow trout, focusing on antimicrobial resistance determinants and their associations with their phenotypes, which have been previously described. The main objection to this work is that the data on colistin resistance determinants are mostly weak or incorrectly addressed. Therefore, the mere observation of the presence of the arnCADTEF operon on the chromosome of three bacterial strains cannot be associated with their colistin resistance, even if the remaining five strains are colistin-susceptible and lack those sequences. Both groups of strains are relatively distant, phylogenetically speaking, and the role of the arnBCADTEF genes is unknown in most Pseudomonas species. To my knowledge, most of the work has been done on P. aeruginosa, the most clinically relevant one. In this case, the arnBCADTEF operon performs a function similar to that of Enterobacteriaceae, modifying LPS with L-Ara4N. Most strains carry arnBCADTEF in their chromosomes and are susceptible to colistin (Olaitan et al., 2014), demonstrating that being positive for arn genes does not imply resistance, at least when it is chromosomally encoded. A different situation may arise when the arnT operon is encoded on a plasmid, such as the Kluyvera-like genes identified in colistin-resistant E. coli (Gallardo et al., 2021). Therefore, I suggest the following: 1. Remove any mention in the abstract of the finding of arn genes on the chromosome of the three colistin-resistant strains, as this is likely a random association. 2. Lines 333-346 should be rewritten to better explain the identification (or lack thereof) of the eptA, eptC, cptA, and rna genes in the different strains. It would be advisable to include a table. In addition to the mcr genes, the most common mechanism of colistin resistance is the constitutive activation by mutation of two-component systems, such as PmrAB or PhoPQ, in Enterobacteriaceae or P. aeruginosa, among many other Gram-positive bacteria (Olaitan et al., 2014). The authors could review these sequences in the analyzed genomes and mention their polymorphisms. 3. Lines 421-433 should be rewritten, taking the above into account. 4. Figure 2 could easily be simplified, showing only a map indicating the two minimal changes between the two strains. The inversion could also be explained in the legend, better than in the actual image. 5. Figure 4 should be removed as it does not provide valuable information.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
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?
No: I have no concern to this respect
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