Genotypic and Phenotypic screening of agriculturally based antimicrobial resistant E. coli.
This article has been Reviewed by the following groups
Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Antimicrobial resistance is undoubtedly one of the most significant threats to human health. Whilst finding alternative antimicrobials proves valuable, understanding how resistance is spread remains a necessity. The link between antimicrobial resistance in agriculture and the effect on human medicine is unclear, however the contribution and dissemination of resistance should not be overlooked. Escherichia coli (E. coli) are of particular concern for human health as the most common Gram-negative pathogen and are often used as a representative indicator of antimicrobial resistance. Sheep hold a unique role in agriculture, as large numbers are often transported between multiple farms to make use of seasonal grazing. We present a robust analysis of multi-antimicrobial resistance profiles, using short read metagenomics and long read sequencing, to identify plasmid borne resistance as well as chromosomal and mutational resistances. The analysis of these data highlighted putative plasmids in 85% of the isolates with putative plasmid borne resistance genes against the most commonly used antimicrobials, such as streptomycin, tetracycline, sulphonamide, licosamide, betalactam, macrolide, kanamycin, fosfomycin, chloramphenicaol, cephalosporin and aminoglycoside.
Article activity feed
-
-
Please make sure you address the reviewers concerns, it is clear that the current version is difficult to follow- there are specific suggestions given by the reviewers how this may be done. It is also that you need to clarify the methodolgy. Please provide more detail in the Methods section and ensure that software is consistently cited and its version and parameters included.
-
Comments to Author
The work presented by Marshall et al. investigates the incidence of antimicrobial resistance on the hoofs of sheep using phenotypic and genotypic methods. They discover a number of drug-resistant isolates and identify that some resistance elements are encoded on conjugatable plasmids. Their work contributes to our understanding of antimicrobial resistance in agriculture. However, I have some concerns on the methodology and overall clarity. Comments: The authors state in their abstract, and keywords, that they use a short-read metagenomic approach to study AMR in sheep, yet the methods and results are from single isolate sequencing. I am also unclear on how the authors arrived at 72 bacterial isolates for sequencing. The methods state that 12 sheep were sampled and up to 2 colonies were selected. …
Comments to Author
The work presented by Marshall et al. investigates the incidence of antimicrobial resistance on the hoofs of sheep using phenotypic and genotypic methods. They discover a number of drug-resistant isolates and identify that some resistance elements are encoded on conjugatable plasmids. Their work contributes to our understanding of antimicrobial resistance in agriculture. However, I have some concerns on the methodology and overall clarity. Comments: The authors state in their abstract, and keywords, that they use a short-read metagenomic approach to study AMR in sheep, yet the methods and results are from single isolate sequencing. I am also unclear on how the authors arrived at 72 bacterial isolates for sequencing. The methods state that 12 sheep were sampled and up to 2 colonies were selected. Were all 4 hoofs sampled? With 2 colonies per hoof? If so, why were there not 96 isolates? More information around how the isolates were obtained is needed. If multiple isolates per sheep were collected how do those isolates relate to each other? Was the same AMR profile found on the same sheep? Or were there differences? Presenting data at the level of the host sheep would be beneficial. Bioinformatic methods are insufficient. Software versions of the bioinformatic tools used are missing, these are required as differing versions can affect results. Database versions are also required. I am also unclear how bacterial genomes were assembled for short-read only data. Using ABRicate to detect AMR genes requires genome assemblies yet the methods section only describes how hybrid genome assemblies were made. Furthermore, the methods section states that ABRicate with the NCBI AMR Finder database was used yet the results section states that the RESFinder database was used. I am also unclear on how plasmids were identified in the dataset, the methods state that plasmids were identified manually from hybrid assemblies. But how were they identified in the 55 short-read only genomes, I presume from the output of plasmidSPAdes? It is not clear why the 20 isolates were selected for long read sequencing. Why were those resistance profiles chosen? Did all 12 sheep have at least one isolate long-read sequenced? I found the structure of section 7.2 difficult to follow, the results start discussing hybrid results, then short read only results, then hybrid results again. Restructuring would aid clarity. The citation for plasmid KK11 being a mega-plasmid is a review of HGT in E. coli that makes no mention of plasmid KK11 nor mega-plasmids. Figure 4: the names and sizes of the plasmids are too small to be able to read clearly. I also do not understand how the phylogenetic tree in panel B was made, there is no section in the methods describing it.
Please rate the manuscript for methodological rigour
Poor
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
-
Comments to Author
The manuscript submitted by Masrhall et al. describes the isolation and characterisation of antibiotic-resistant E. coli strains from sheep. This type of work is relevant as the monitoring of environmental antimicrobial resistance is an important pillar to address the AMR problem from a One Health perspective. Although the results are interesting, the manuscript is not very well written and it's very difficult to follow, understand and evaluate the results presented. In my opinion, the manuscript needs to be completely revised and rewritten in a more precise way. Here are some suggestions that might improve understanding: - The reason for looking for Tc-resistant E. coli should be explained in the introduction. Why tetracycline resistance in particular? What are the implications of this resistance for …
Comments to Author
The manuscript submitted by Masrhall et al. describes the isolation and characterisation of antibiotic-resistant E. coli strains from sheep. This type of work is relevant as the monitoring of environmental antimicrobial resistance is an important pillar to address the AMR problem from a One Health perspective. Although the results are interesting, the manuscript is not very well written and it's very difficult to follow, understand and evaluate the results presented. In my opinion, the manuscript needs to be completely revised and rewritten in a more precise way. Here are some suggestions that might improve understanding: - The reason for looking for Tc-resistant E. coli should be explained in the introduction. Why tetracycline resistance in particular? What are the implications of this resistance for human health? - For the isolation of E coli strains: o Did you grow bacterial colonies only in the presence of the antibiotic? o How many colonies did you obtain in total? o How do you know that all isolates are different strains? It is possible that the same bacterial strain is present in different sheep on the same farm. o These results should also be discussed. - It is not clearly stated how many isolates were selected for long read sequencing and on what basis the selection was made. Why are two different DNA extraction protocols used? In addition, the reference Quick et al. (2018) is missing in the bibliography section. - Regarding section 7.1: o The results must be put in context and a brief introduction about how the bacteria were isolated is needed. o Regarding the antibiotic resistance profile, this section is very difficult to understand as it is written. The result should be summarised in a table showing the antibiotic resistance profile of each isolate. In addition, it is very difficult to interpret the results in Figure 2. The presence or absence of resistance genes should be separated from the phenotype to allow interpretation of the results. The same colour should not be used for two different things (genotype and phenotype). - Regarding section 7.2: o There are some sentences that seem to be written incorrectly. In addition, the section should be written more clearly and all results concerning the presence of ARGs on the chromosome, plasmid or transposon should be presented in some way (e.g. in a table). o It is not clear what is KK11, a method ("Every isolate assembled with using the hybrid method bar KK11 possessed a copy of sul1 ") or a plasmid. o Have plasmids KK06 and KK11 been described before? o Does the antibiotic resistance profile of each strain carrying these plasmids match its genotype? o For the conjugation experiment some result should be shown, for example: Did you try to conjugate both plasmids (KK11 and KK06) or just the megaplasmid? What is the frequency of conjugation for KK11? why KK11 transfer does not confer Km resistance to the recipient bacterium? o Figure 4 is of very poor quality and the plasmid maps are impossible to read. In addition, the figure legend should indicate that panel b) corresponds to plasmid KK06 and the phylogenetic tree numbers are impossible to read. Furthermore, these results need to be discussed: is it a unique plasmid present in different isolates, what is the percentage of identity and coverage within the group of KK06-like plasmids? - Regarding discussion: o How can it be distinguished whether they are different isolates, or the same strain isolated several times? Does each isolate come from different sources? Have the complete genomes of the isolates been compared? o According to the text, the sequence data from 65 isolates showed that 55 of them contain plasmids associated with antibiotic resistance, but you only analysed KK11 and KK06. What about the rest of the plasmids? o You have selected 20 samples representing 10 resistance profiles for the long read sequence. Why these 10 resistance profiles and not others? o Have KK1 and KK06 plasmids been described before? If so, this should be discussed. If not, you should compare the sequence of these plasmids with the database to try to determine their origin or phylogeny. o If the KK11 plasmid is conjugative ...., it is curious that this plasmid was only present in a single isolate, whereas KK06 is more widespread among the isolates ...., this should also be discussed.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Poor
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
-