Identification of Antibiotic Resistance Gene Hosts in Treatment Wetlands Using a Single-Cell Based High-Throughput Approach
and Jochen A. Müller 1,3,*Round 1
Reviewer 1 Report
I find this article very interesting and very important. The article provides a good review of the literature, which is presented in a very good introduction. In my opinion, the manuscript is well organized. The wording of the problems agrees with the title and is meaningful. The methods used to collect the data for this article are clearly explained. The quality of citations is good, the authors refer to interesting and new works in this field of research. The topic is interesting and the results are concrete and useful to the scientific community. I don't really have any critical comments about the article. However: 1. The research results were presented in a very interesting way, but I would suggest to refine the quality of these figures. In my opinion, this is a very valuable publication, based on the most modern research methods and is a valuable source of information and is the basis for further analyzes and implementation of new solutions. Thank you for considering my opinion. I encourage the authors to continue working on improving the manuscript.
Author Response
We do appreciate these positive comments from the reviewer. The resolution of the illustrations has been improved and is now hopefully of sufficient quality.Reviewer 2 Report
The manuscript ‘Identification of antibiotic resistance gene hosts in treatment wetlands by a single-cell based high-throughput approach’ by Camila A. Knecht and co-authors used epicPCR (emulsion, paired isolation, and concatenation PCR) as a cultivation-independent method to reveal host profiles of the AMR indicator genes intI1, sul1, sul2, and dfrA1 in two constructed wetlands treating municipal wastewater. The epicPCR analysis revealed a profile of AR indicator gene hosts that is consistent with literature data from cultivation-based approaches. This is an interesting study and the authors have found several novel hosts for the indicator genes that were widely distributed in the wetlands, including the genera Legionella and Ralstonia. However, there are a few shortcomings and including them will improve the manuscript. I also find that the authors have not stressed on the novelty of the method used in this study enough.
Introduction:
A more extensive and careful use of literature should be used along the manuscript since there are some sentences that does not include a reference. For instance, ‘The classical cultivation-dependent approach is subject to bias as many bacteria do not grow readily in the laboratory’ (line 64-66). Include reference for classical cultivation-dependent approach. It would be right to include more references related to the cultivation-independent metagenomics.
Add more about other methods currently used for high throughput in situ AMR gene host profiling with relevant references.
Methods:
Although materials and methods section are described in detail and well-outlined but it’s not well written.
Line 202, Illumina sequencing and data analysis heading should be 2.3. instead of 2.2.
Results and discussion:
EpicPCR approach used in this study seems to be of medium throughput due to the cumbersome multi-steps involved. Using this method as a high-throughput approach need more clarification. In the discussion section please stress on the novelty of this study more! Explain how this is a novel finding using this high-throughput platform and how it can help change the field to move towards similar technologies.
Another point to consider is to add correlation network analysis between the bacterial community and ARGs, taxonomic assignment, phylogenetic tree, and cluster linkage representation since it is difficult for the reader to make an interpretation and get any conclusion from figure 1 only.
Author Response
Reviewer 2
Comments and Suggestions for Authors
The manuscript ‘Identification of antibiotic resistance gene hosts in treatment wetlands by a single-cell based high-throughput approach’ by Camila A. Knecht and co-authors used epicPCR (emulsion, paired isolation, and concatenation PCR) as a cultivation-independent method to reveal host profiles of the AMR indicator genes intI1, sul1, sul2, and dfrA1 in two constructed wetlands treating municipal wastewater. The epicPCR analysis revealed a profile of AR indicator gene hosts that is consistent with literature data from cultivation-based approaches. This is an interesting study and the authors have found several novel hosts for the indicator genes that were widely distributed in the wetlands, including the genera Legionella and Ralstonia. However, there are a few shortcomings and including them will improve the manuscript. I also find that the authors have not stressed on the novelty of the method used in this study enough.
We grateful for the positive as well as critical comments by this reviewer. Regarding the novelty of the method, we would prefer not to stress that aspect since a description of the method has been published in 2016.
Introduction:
A more extensive and careful use of literature should be used along the manuscript since there are some sentences that does not include a reference. For instance, ‘The classical cultivation-dependent approach is subject to bias as many bacteria do not grow readily in the laboratory’ (line 64-66). Include reference for classical cultivation-dependent approach. It would be right to include more references related to the cultivation-independent metagenomics.
We would like to consider the number of references in the Introduction as sufficiently extensive for a research article (in the original manuscript there were 28, now there are 30 references). Regarding the specific examples mentioned by the reviewer: the statement on the classical cultivation is, we think, by now standard microbiology knowledge; and regarding metagenomics we provide now the reference Stalder et al. 2019, which was used already in the original manuscript as reference for Hi-C metagenomics, and added Martinez et al., 2015 and Pärnänen et al., 2016 as references for the challenges of metagenomics for AMR host profiling. Specifically, metagenomics can be very powerful to elucidate hosts of chromosomally located ARGs, but is hampered in discovering hosts of plasmid-located ARGs (like the genes in this study often are).
Add more about other methods currently used for high throughput in situ AMR gene host profiling with relevant references.
The above-mentioned methodological challenge in assigning in situ hosts of mobile genetic elements with metagenomics applies to any high throughput method that does not include a physical linkage step between the AMR gene indicator and a DNA fragment of the host that allows phylogenetic identification. Other than epicPCR, we are not aware of high throughput approaches other than proximity ligation-based methods such as Hi-C (which we cite, i.e. Stalder et al., 2019 as prime example).
Methods:
Although materials and methods section are described in detail and well-outlined but it’s not well written.
Thank you, we corrected punctuation and other language issues in several sentences in the hope to improve readability.
Line 202, Illumina sequencing and data analysis heading should be 2.3. instead of 2.2.
Thank you, it has been corrected.
Results and discussion:
EpicPCR approach used in this study seems to be of medium throughput due to the cumbersome multi-steps involved. Using this method as a high-throughput approach need more clarification. In the discussion section please stress on the novelty of this study more! Explain how this is a novel finding using this high-throughput platform and how it can help change the field to move towards similar technologies.
We agree that the term “high throughput” is somewhat subjective and relative. Compared to cultivation-based investigations, epicPCR is indeed a high throughput approach.
We do appreciate the reviewer’s call for stressing the novelty of the study. However, since epicPCR has been introduced in Spencer et al., 2016, and was applied for the determination of AMR gene hosts several times, we feel that the present text is a good balance between acknowledging previous work and novel results obtained in the present study.
Another point to consider is to add correlation network analysis between the bacterial community and ARGs, taxonomic assignment, phylogenetic tree, and cluster linkage representation since it is difficult for the reader to make an interpretation and get any conclusion from figure 1 only.
Thank you, we fully agree with the general rationale of this suggestion and had actually considered performing this analysis prior to submission of the original manuscript, but decided against it for the following reason (and still believe that the earlier decision is valid). Because a correlation network analysis would reveal a statistical relationship, it would only be an advance over the present results if the epicPCR data were quantitatively more reliable. In this case, the important question of whether a particular bacterial phylotype was over- or under-represented as a carrier of a particular gene in the wetlands could be adequately answered. However, because the epicPCR results are only semiquantitative, the proposed analysis would be subject to a level of uncertainty that we do not believe is present in the present manuscript.
