Antimicrobial Resistance Elements in Coastal Water of Llanquihue Lake, Chile

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Abstract should be re-written giving importance to major findings of the study.

Even though AMR was detected by genomic methods, why only few samples were subjected to ABST should be justified and discussed.

Comments on the Quality of English Language

Some sentences need to be corrected or re-phrased. 

Author Response

Response to reviewer  (our answers are in red)
Abstract should be re-written giving importance to major findings of the study.

Re: Thank you for your consideration of our work. We rewrote the abstract according to your suggestion.

Even though AMR was detected by genomic methods, why only few samples were subjected to ABST should be justified and discussed.

Re: We thank the reviewer for raising this observation. We tested the sensibility to antibiotics for 14 strains because the number of the species confirmed by BLAST that were close to the ATCC strains that we had available for use as a control. We agree that the number is low, and it will be desirable to test ABST in a wide range of microbial species recovered from the lake. We expect to do that in a next work with the aim to dilucidated whether our genomic finding related with ARGs identified had a functional impact over the microbiology associated to the lake, using ABST and qRT-PCR to test ARGs gene expression. However, we need to apply for funds that will allow us to finance these experiments as scientific projection of this work.

 

Comments on the Quality of English Language: Some sentences need to be corrected or re-phrased.

Re: We appreciate your suggestion. The new version of the work was reviewed by an expert translator to improve the English and made modifications for a better presentation of our article.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is in fact an interesting story, but the results are not well presented and the authors could have made more use of all the data they have generated. 

In the supplementary material provided, only the file with fasta result and the three supplementary tables 1 - 3 were provided. The supplementary figures 1 and 2 which are referred to in the manuscript were not provided. Therefore, this reviewer cannot evaluate whether some of the critic points raised below would have been elucidated in those figures.

F.ex., the sampling sites are not well described (maybe it is explained in the supplementary figures). Given the differences in the composition between sampling sites, it would have been interesting to know whether there is any outlet from the city (sewage outlets), total water depth at the sampling sites, is there any water flow in the lake in direction to or from the city? Or is there anything else to explain the differences in microbiota composition? As far as I can see on Google Earth, there is no efflux from rivers around Puerto Varas City, is that correct? 

Also the sampling scheme is somewhat confusing and must be better explained, I get back to that later.

Figures need to be in a better resolution and with better legends, more about that later.

In the results, there are several references. However, the results section should only present the results and not refer to references. This belongs in the discussion.

Specific comments:

Line 27: antibiotic resistance genes

L. 29: unsure what you mean by AMR ability. Maybe susceptibility pattern would be a better expression or resistance pattern.

L. 30-32: You have chosen to focus this study very much on antibiotic resistance factors, and I acknowledge that we do not know so much about AMR in the environment. However, the major part of your data is on the composition of the microbiota and there are so many interesting details in this that have not been properly addressed. Personally, I doubt that the resistance factors you have found in this study constitutes any health hazard for humans but some of the bacteria you have found definitely may. More about that later.

L. 42: resistance mechanism

L. 42-44: We don't really know what the role of AMR in the environment plays. I would not call it an imperative threatening but agree that it is something we need to look into.

L. 46: Unsure what you mean by "overall economic burden". Please expand on this.

L. 65: Insert a space between resistance and the bracket. The same applies to several places further down the manuscript, e.g., in line 68, 320, 321, 332, 324326, 329, and 330.

L. 84: used

L. 84: metagenomics

L. 101: indicates

L. 104 and Figure 1: I have difficulties interpreting Figure 1. What do the colours mean? And also, in each of the sampling points, there are certain bacteria that seem to dominate. Which are they, and what does it tell about the origin of the microbiota? F.ex. in EC_5m, EC_Sup, PV2, PV2.1 and PV3.1 there is a brown/beige coloured group that dominates. Which group of bacteria is that? Similarly, in PV1B.1 and PV3 a red/orange group of bacteria dominate. What are thy? And why is the composition of the microbiota in PV1B and PV1B.1, which I assume is the same sampling point (?) so totally different? And which group of bacteria is the violet dominant group in PV1B?

L. 106-120: The results of the microbiota could be much better described and discussed. I have taken a closer look at Supplementary Table 1. You specifically mention Mycoplasma, but only a single mycoplasma sequence occurs in Supplementary Table 1, namely a M. pulmonis, which to my knowledge only is found in mice and rats. You may say that it has veterinary interest, but probably only from a wildlife perspective. 

You mention several genera belonging to the human intestinal microbiota. However, many of these also belong to the intestinal microbiota of animals, so they may in fact originate from animals rather than humans? It is interesting that apparently only a single E. coli sequence was found (according to Suppl. Table 1). Normally we consider E. coli an indicator for faecal contamination, so considering all the sequences of Bacteroides, Prevotella, Faecalibacterium, and others, it may seem strange that not more E. coli were found.

Some of the sequences belong to avian specific bacteria, such as Jeotgalibaca ciconiae, Ornithobacterium rhinotracheale (ORT), Pasteurella multocida subsp. multocida (fowl cholera), and Riemerella anatipestifer. Are there many birds living at the lake, maybe close to the city, which could be the source of these bacteria?

Similarly, some of the bacteria are fish specific, such as Aeromonas salmonicida (furunculosis in salmonids), Vibrio anguillarum (classic vibriosis), Flavobacterium columnare (columnare disease), Flavobacterium psychrophilum (and many other Flavobacterium species of unknown significance), Renibacterium salmoninarum (BKD, bacterial kidney disease in salmonids), Aliivibrio fischeri, and Tenacibaculum maritimum. Is the lake full of fish or is there aquaculture in the lake?

Then there are some bacteria, that we mostly associate with water, such as Aeromonas, Synechoccus or Pseudomonas (really many), Burkholderia, so they may be indigenous to the lake. Other bacteria, such as Rhizobium, Streptomyces and Mesorhizobium are mostly associated with soil. So, how did they end up in the lake? Had there been any recent rainfall that could have flushed soil bacteria out in the lake? 

You also found several Vibrio species, V. anguillarum (mentioned above), but also V. vulnificus, V. parahaemolyticus and even V. cholerae, which all are pathogenic. However, vibrios - except for V. cholerae - are halophilic and will not live in fresh water. Why are they present in a lake? Is there any influx from the sea, can the bacteria have been brought by birds foraging in the sea, seagulls, f.ex.? Your finding of some Salinibacterium species and the above mentioned Tenacibaculum maritimum, which causes disease - ulcers - in marine fish species, may also suggest a certain salinity.

And finally, you mention a few veterinary and human pathogenic bacteria, but there are actually many more that could be emphasized, such as the various Campylobacter species, Clostridioides difficile, various Clostridium, but e.g. tetani and botulinum, Francisella, Legionella pneumophila, Moraxella bovis, many streptococci, e.g. suis, pneumoniae and pyogenes, Yersinia enterocolitica, Coxiella burnetti, Rickettsia pravazekii (epidemic typhus).

L. 107: You mention Leprospira, which would not be unexpected, but I cannot find it in Suppl. Table 1.

Figure 2: The quality of this figure is not sufficiently good.  

L. 138: penicillins are also beta-lactams

L. 143, 144, and 146: names of genes in italics, bla, dfr, cat

L. 145: found

L. 148-150: This needs to be described in more detail.

L. 181-182: names of genes in italic, mph, msr, mef, erm, etc. (not the bracket)

Table 2: Susceptibility testing was performed on only 14 bacterial isolates. This is a very small sample size compared to the vast number of OTUs recovered by sequencing

L. 230, 231, 232: bla in italics

L. 233-234: This sentence needs to be rephrased.

L. 238: mcr in italics. However, the finding of mcr genes is not described in the results section.

L. 289: 3 must be spelled out after full stop.

L. 294: streaked

L. 295: BHI, TSA and EMB must be spelled out.

L. 295: Mueller-Hinton, MacConkey

L. 294-296: It is uncertain how colonies were selected and from which plates. This is not nearly well enough described.

L. 335: It is ciprofloxacin, not norfloxacin that is mentioned in Table 2. Which is right?

L. 335: vancomycin and erythromycin is not mentioned in Table 2, but cefotaxime is.

L. 346: phenoms? Do you mean phenomena?

References: The references need to be revised according to journal style. F.ex. species names in italics, and he species should not be with capital letter, e.g., Pasteurella multocida, not Pasteurella Multocida. Names of genes in italics. 

Reference 64: not with capital letters in title.

Reference 71 and 73: mention all authors, not "et al."

Reference 90: Title not in capital letters. And this reference should be cited with CLSI as author.

 

 

Author Response

Response to reviewer  (our answers are in red)

This is in fact an interesting story, but the results are not well presented, and the authors could have made more use of all the data they have generated. 

Re: We thank you for your consideration and extensive review of our manuscript. This has allowed us to significantly improve our work. We also appreciate the positive assessment of our results. We carefully read each point and made the suggested modifications in this new version of the manuscript.

In the supplementary material provided, only the file with fasta result and the three supplementary tables 1 - 3 were provided. The supplementary figures 1 and 2 which are referred to in the manuscript were not provided. Therefore, this reviewer cannot evaluate whether some of the critic points raised below would have been elucidated in those figures. 

F.ex., the sampling sites are not well described (maybe it is explained in the supplementary figures). 

Re: Thank you for highlighting this issue. We are sorry about this shameful mistake. In the new revised version, we enclosed the missing supplementary figures.

 

Given the differences in the composition between sampling sites, it would have been interesting to know whether there is any outlet from the city (sewage outlets), total water depth at the sampling sites, is there any water flow in the lake in direction to or from the city? Or is there anything else to explain the differences in microbiota composition? As far as I can see on Google Earth, there is no efflux from rivers around Puerto Varas City, is that correct? 

 

Re: We thank the reviewer for their thoughtful comments. Puerto Varas city lacks direct wastewater sewage outlets to Llanquihue lake. However, several rain outlets come from the city and discharge the fallen water to the lake. The differences in microbiota composition can be explained by rain outlets, streams of water that reach the lake and the presence of hydrodynamic phenomena that move the waters around of the Puerto Varas riverbank. The latter has not been published in scientific literature, but there are reports indicating currents that move the lake waters. We include this lately observations in the Discussion section.

Also the sampling scheme is somewhat confusing and must be better explained, I get back to that later. Figures need to be in a better resolution and with better legends, more about that later.

Re: Thanks for pointing out this observation. The sampling scheme was improved in the Material and Methods section. Unfortunately, the figures are pixeled because of their inclusion in the word document. However, we already submitted the figures in high resolution at 300 dpi to be used in the final PDF version of the article by MDPI editors.

In the results, there are several references. However, the results section should only present the results and not refer to references. This belongs in the discussion.

Re: Thank you for this suggestion. We reduced the number of citations in the Results section to use them in the Discussion, but we consider it important to preserve some references that reinforce our observations or define some of the concepts presented.

Specific comments:

Line 27: antibiotic resistance genes

Re: Thanks for the correction. The change has been made.

29: unsure what you mean by AMR ability. Maybe susceptibility pattern would be a better expression or resistance pattern.

Re: We thank you for your suggestion. We rephrase the sentence for a better understanding.

30-32: You have chosen to focus this study very much on antibiotic resistance factors, and I acknowledge that we do not know so much about AMR in the environment. However, the major part of your data is on the composition of the microbiota and there are so many interesting details in this that have not been properly addressed. Personally, I doubt that the resistance factors you have found in this study constitutes any health hazard for humans but some of the bacteria you have found definitely may. More about that later.

Re: We appreciate your comment. It is an interesting point you raise. From our perspective and according to the global trend of studying population health under the concept "One Health" understanding that the determinants of this are associated with 3 pillars: human, animal and environmental health. Is highly relevant to address the problem of antibiotic resistance through environmental surveillance of microbial communities, because the impairment environment may act as reservoirs of resistant bacteria or be the medium in which resistance genes are exchanged between different species. We also include references that support these arguments.

42: resistance mechanism

Re: Thank you for the correction. The change has been made.

42-44: We don't really know what the role of AMR in the environment plays. I would not call it an imperative threatening but agree that it is something we need to look into.

Re: Thanks for raising this point. We made changes to the entire paragraph.

46: Unsure what you mean by "overall economic burden". Please expand on this.

Re: We thank the revisor for point attention to this. We extend the information including the causes of economic burden regard to a systematic review and meta-analysis.

65: Insert a space between resistance and the bracket. The same applies to several places further down the manuscript, e.g., in line 68, 320, 321, 332, 324326, 329, and 330.

Re: We reviewed this point, and the changes were made in accordance to your suggestion.

84: used

Re: Thanks for the correction. The change has been made.

84: metagenomics

Re: Thanks for the correction. The change has been made.

101: indicates

Re:Thanks for the correction. The change has been made.

104 and Figure 1: I have difficulties interpreting Figure 1. What do the colours mean? And also, in each of the sampling points, there are certain bacteria that seem to dominate. Which are they, and what does it tell about the origin of the microbiota? F.ex. in EC_5m, EC_Sup, PV2, PV2.1 and PV3.1 there is a brown/beige coloured group that dominates. Which group of bacteria is that? Similarly, in PV1B.1 and PV3 a red/orange group of bacteria dominate. What are they? And why is the composition of the microbiota in PV1B and PV1B.1, which I assume is the same sampling point (?) so totally different? And which group of bacteria is the violet dominant group in PV1B?

Re: We thank the reviewer for this comment. This kind of stacked barplot is used to shows the taxonomical patterns between conditions, allowing us to represent a high density of information to contrast differences visuality. We improve the description of the figure to explain the meaning of the colors and the sample description. Also, we include a description about the dominate bacteria in the figure description. We remade the figure to reorder the representation of the samples.

 

106-120: The results of the microbiota could be much better described and discussed. I have taken a closer look at Supplementary Table 1. You specifically mention Mycoplasma, but only a single mycoplasma sequence occurs in Supplementary Table 1, namely a M. pulmonis, which to my knowledge only is found in mice and rats. You may say that it has veterinary interest, but probably only from a wildlife perspective. 

Re: We thank the reviewer for their thoughtful comments. Microbiota description is not the focus of this work, because we are preparing a work with the taxonomical description of several other sites using metagenomic approaches. For that reason, we do not describe in deep the taxonomical findings in the sequences used for this work. We focus on the taxonomical patterns and the findings in general terms, which can support our findings related to the presence of AMRg. We agree that the genus Mycoplasma is relevant from a wildlife perspective. Our sequences include the species Mycoplasma pulmonis, which causes murine respiratory mycoplasmosis in rats, and some authors considered it with potentially zoonotic because some reports show the prevalence and seropositivity of M. pulmonis in humans who have had direct contact with rats (DOI: 10.1089/vbz.2016.2104; DOI: 10.1111/j.1863-2378.2008.01122.x). Considering the One Health approach, Puerto Varas is located near the habitat of wild stock species, and the presence of this kind of bacteria may constitute a potential health risk.

 

You mention several genera belonging to the human intestinal microbiota. However, many of these also belong to the intestinal microbiota of animals, so they may in fact originate from animals rather than humans? It is interesting that apparently only a single E. coli sequence was found (according to Suppl. Table 1). Normally we consider E. coli an indicator for faecal contamination, so considering all the sequences of Bacteroides, Prevotella, Faecalibacterium, and others, it may seem strange that not more E. coli were found.

Re: Thank you for your observation, we remove the word “human”. It is necessary to clarify that supplementary table 1 has the species taxa's identifies and the relative abundances for each sample, not the number of sequences. In that sense, Escherichia coli was presented in all the samples, in relative abundances ranging between 0.019 to 0.25 %, which can be considered high among the hundreds of taxas identified.

Some of the sequences belong to avian specific bacteria, such as Jeotgalibaca ciconiae, Ornithobacterium rhinotracheale (ORT), Pasteurella multocida subsp. multocida (fowl cholera), and Riemerella anatipestifer. Are there many birds living at the lake, maybe close to the city, which could be the source of these bacteria?

Re: Thanks for pointing out this observation. We include these observations in our results. In fact, as you mention, there are several wild avian species living in the lake, and in the forest located around the lake and Puerto Varas city. Some species are native species and others are introduced species. The most present species are Xolmis pyrope, Megaceryle torquata, Falco sparverius, Vanellus chilensis, Theristicus melanopis, Cinclodes patagonicus, and Milvago chimango. Also, the lake is close to the sea, hence there is presence of sea birds like Larus dominicanus and Phalacrocorax brasilianus.

Similarly, some of the bacteria are fish specific, such as Aeromonas salmonicida (furunculosis in salmonids), Vibrio anguillarum (classic vibriosis), Flavobacterium columnare (columnare disease), Flavobacterium psychrophilum (and many other Flavobacterium species of unknown significance), Renibacterium salmoninarum (BKD, bacterial kidney disease in salmonids), Aliivibrio fischeri, and Tenacibaculum maritimum. Is the lake full of fish or is there aquaculture in the lake?

Re: Thank you for your observation, we include this description in the results. In fact, although many sites have closed, the lake still preserves several places related to fish farming. The main activity in the region is the salmonid culture, that includes a growth cycle on fresh water and salt water. Also, the lake supports the life of different species of fish, including introduced fish such as salmonids and trout.  It is highly possible that metagenomic sequences capture the microbial mark of both.

Then there are some bacteria, that we mostly associate with water, such as Aeromonas, Synechoccus or Pseudomonas (really many), Burkholderia, so they may be indigenous to the lake. Other bacteria, such as Rhizobium, Streptomyces and Mesorhizobium are mostly associated with soil. So, how did they end up in the lake? Had there been any recent rainfall that could have flushed soil bacteria out in the lake? 

Re: Thank you for highlighting this. In fact, Puerto Varas shore has several rainy outlets which discharge almost continuously water that comes from the rain. The average precipitation is close to 1100 mm of rain per year, between 56 to 176 mm per month, raining around 11 to 20 days per month (https://www.weather-atlas.com/es/chile/puerto-varas-clima).

You also found several Vibrio species, V. anguillarum (mentioned above), but also V. vulnificus, V. parahaemolyticus and even V. cholerae, which all are pathogenic. However, vibrios - except for V. cholerae - are halophilic and will not live in fresh water. Why are they present in a lake? Is there any influx from the sea, can the bacteria have been brought by birds foraging in the sea, seagulls, f.ex.? Your finding of some Salinibacterium species and the above mentioned Tenacibaculum maritimum, which causes disease - ulcers - in marine fish species, may also suggest a certain salinity.

Re. Thanks for pointing out this observation. In fact, the lake is close to the sea, and several sea birds come to the lake. Also, several aquaculture activities are still performed around the lake, which may explain this microbial observations.

 

And finally, you mention a few veterinary and human pathogenic bacteria, but there are actually many more that could be emphasized, such as the various Campylobacter species, Clostridioides difficile, various Clostridium, but e.g. tetani and botulinum, Francisella, Legionella pneumophila, Moraxella bovis, many streptococci, e.g. suis, pneumoniae and pyogenes, Yersinia enterocolitica, Coxiella burnetti, Rickettsia pravazekii (epidemic typhus).

Re. We appreciate your suggestion. We will focuses it in a next work.

107: You mention Leprospira, which would not be unexpected, but I cannot find it in Suppl. Table 1.

Re: Thank you for your observation. We remove this genus from the text.

Figure 2: The quality of this figure is not sufficiently good.  

Re: Thank you for highlighting this issue, we incorporate the high-resolution image at 300 dpi as separate file. Also, we remade the figure to improve visualization and representation.

138: penicillins are also beta-lactams

Re: Thank you for your observation. We remove the word penicillins.

143, 144, and 146: names of genes in italics, bla, dfr, cat

Re: Thank you for your observation. We perform the changes.

145: found

Re: We are sorry for the misspelling. We perform the correction.

148-150: This needs to be described in more detail.

Re: We appreciate your suggestion. In this regard, we will incorporate more information to precise the description.

181-182: names of genes in italic, mph, msr, mef, erm, etc. (not the bracket)

Re: Thank you for your observation. We perform the indicates changes.

Table 2: Susceptibility testing was performed on only 14 bacterial isolates. This is a very small sample size compared to the vast number of OTUs recovered by sequencing

Re: Thank you for highlighting this We use this bacteria number because we only have a disposition one ATCC bacteria as control. We have a microbial environmental isolated ceparium with more than 500 strains, but only 100 of these strains are 16s/PCR/sequenced identified already, and only 14 of these strains were close to the ATCC used as CLSI control. We are really sorry and ashame about it, but was the resourced available at the moment of perform the experiments.

 

230, 231, 232: bla in italics

Re: Thanks for pointing out this misspelled. We already performed this correction.

233-234: This sentence needs to be rephrased.

Re: Thank you for your observation, we rephrased the sentence.

238: mcr in italics. However, the finding of mcr genes is not described in the results section.

Re: Thank you for this suggestion. We reviewed our findings, and the suggested gene was added to the result. Also, we correct the discussion regarding the observation of this specific gene.

289: 3 must be spelled out after full stop.

Re: Thank you for highlighting this. We rewrote the paragraph.

294: streaked

Re: Thanks for pointing out this misspelled. We already performed this correction.

295: BHI, TSA and EMB must be spelled out.

Re: Thank you for highlighting this. We write the entire name of the culture media.

295: Mueller-Hinton, MacConkey

Re: Thanks for pointing out this typo error. We write the entire name of the culture media.

294-296: It is uncertain how colonies were selected and from which plates. This is not nearly well enough described.

Re: Thank you for your observation, we rephrased the sentence.

335: It is ciprofloxacin, not norfloxacin that is mentioned in Table 2. Which is right?

Re: We regret the error, thank you for pointing it out. The change was made.

335: vancomycin and erythromycin is not mentioned in Table 2, but cefotaxime is.

Re: We regret the error, thank you for pointing it out. The change was made.

346: phenoms? Do you mean phenomena?

Re: Thanks for pointing out this misspelled. We already performed this correction.

References: The references need to be revised according to journal style. F.ex. species names in italics, and the species should not be with capital letter, e.g., Pasteurella multocida, not Pasteurella Multocida. Names of genes in italics. 

Re: Thank you for your observation. We already performed this correction in references.

Reference 64: not with capital letters in title.

Re: Thanks for pointing out this misspelled. We already performed this correction.

Reference 71 and 73: mention all authors, not "et al."

Re: Thanks for pointing out this misspelled. We already performed this correction.

Reference 90: Title not in capital letters. And this reference should be cited with CLSI as author.

Re: Thank you for your observation. We already performed this correction.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The paper is good but concerns raised as comments to authors need to be satisfactorily addressed before the manuscript can reach a publishable value

Comments to authors

1. Title: I think the word “identified” is redundant and could be deleted from the title
2. Line 42: What do the authors mean by “increment in antimicrobial resistant mechanism”? Do you mean “increment in the rate of antimicrobial resistance”?
3. Lines 42-44: Revise for clarity, please.  
4. Line 49-51: You can check the suggested paper below and cite it to back up this categorical statement that requires at least one citation

Detection of multidrug-resistant Campylobacter Species from food-producing animals and humans in Nigeria: public health implications and One Health control measures. Comparative Immunology, Microbiology and Infectious Disease, https://doi.org/10.1016/j.cimid.2023.102083

5. Line 55: Consider replacing “pharmacotherapy” with ”practice”
6. Line 61-62: Antimicrobial Resistance Genes (ARGs) – just use the already defined acronym instead
7. Line 62-63: Is virions a mechanism of HGT or should we be talking of Transduction here?
8. Line 70-83: This should be a separate section under M&M
9. Line 84: A few lines on shotgun metagenomic could be helpful to the journal readership in this introduction part of the paper
10. Line 103: change “pointing” to “suggesting”
11. Line 108: Veterinary clinical practice” instead of “veterinary health”
12. Line 110: Is Ruminococus part of the human microbiota as claimed?
13. Table 1: The resistance mechanisms are not strictly as suggested. A bacteria pathogen may deploy multiple resistance mechanisms to express resistance against an antimicrobial agent. For example, Enzymatic inactivation and efflux pump meaganisms may be deployed to express AMR against beta-lactams (bla gene). I suggest you list or chart up the AMR genes found and their corresponding frequency instead.  
14. Is there any ethical approval for this work?
15. I suggest Moderate English language editing for this manuscript.

Comments on the Quality of English Language

Moderate English editing required

Author Response

Response to reviewer  (our answers are in red)

The paper is good but concerns raised as comments to authors need to be satisfactorily addressed before the manuscript can reach a publishable value.

Re: We appreciate your review and suggestions; we address each of the points requested. Point-by-point responses are provided below.

 

Title: I think the word “identified” is redundant and could be deleted from the title 

 Re: Thanks for the suggestion, the change was made.

Line 42: What do the authors mean by “increment in antimicrobial resistant mechanism”? Do you mean “increment in the rate of antimicrobial resistance”?

Re: We appreciate to the reviewer to raise this point. We change the sentence to better describe the idea about AMR.

Lines 42-44: Revise for clarity, please.  

Re: We appreciate to the reviewer to raise this point. We rewrite this sentence regard AMR concern.

 

Line 49-51: You can check the suggested paper below and cite it to back up this categorical statement that requires at least one citation

Re: Thank you for your suggestion. We reviewed the reference, and we thought it was pertinent to add it. The change was made.

Line 55: Consider replacing “pharmacotherapy” with ”practice”

Re: We accept your suggestion. The change was made.

Line 61-62: Antimicrobial Resistance Genes (ARGs) – just use the already defined acronym instead

Re: Thank you for bringing this error to our attention. The change was made

Line 62-63: Is virions a mechanism of HGT or should we be talking of Transduction here?

Re: Thanks for the suggestion, we change the sentence to correct it.

Line 70-83: This should be a separate section under M&M

Re: We thanks to the reviewer to suggest this point. We move this information to material and methods section.

Line 84: A few lines on shotgun metagenomic could be helpful to the journal readership in this introduction part of the paper.

Re: We appreciate to the reviewer to raise this point. We include a brief description of shotgun metagenomics to introduce to the reader to this sequencing methodology.

Line 103: change “pointing” to “suggesting”

Re: Thanks for the suggestion, we made the change.

Line 108: Veterinary clinical practice” instead of “veterinary health”

Re: Thanks for the suggestion, we made the change.

Line 110: Is Ruminococus part of the human microbiota as claimed?

Re: Thank you for bringing this point to our attention. According to our information some species of the genus Ruminococus are considered part of the human microbiota. Please find enclosed a reference.

Henke MT, Kenny DJ, Cassilly CD, Vlamakis H, Xavier RJ, Clardy J. Ruminococcus gnavus, a member of the human gut microbiome associated with Crohn's disease, produces an inflammatory polysaccharide. Proc Natl Acad Sci U S A. 2019 Jun 25;116(26):12672-12677. doi: 10.1073/pnas.1904099116. Epub 2019 Jun 10. PMID: 31182571; PMCID: PMC6601261

Table 1: The resistance mechanisms are not strictly as suggested. A bacteria pathogen may deploy multiple resistance mechanisms to express resistance against an antimicrobial agent. For example, Enzymatic inactivation and efflux pump meaganisms may be deployed to express AMR against beta-lactams (bla gene). I suggest you list or chart up the AMR genes found and their corresponding frequency instead. 

 

Is there any ethical approval for this work?

Re: Thank you for your concern on this point. In the country where this study was carried out, Chile, under the current legal framework, the review by an ethics committee is not considered for environmental studies on this scale. Only work which involves human or animal should be approved by ethics committee. Specifically, in this study, neither people nor animals were involved. The University where this study was carried out has a human ethics committee and another ethics committee for the use and care of experimental animals, neither of these committees reviews projects related to environmental nature.

Re: We thanks to the reviewer to suggest this point. We include bibliographic support and change the column descriptor of the table.

I suggest Moderate English language editing for this manuscript.

Re: We welcome your suggestion. We asked an expert translator to review the English version and made modifications for a better presentation of our article.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Considerable modifications have been made, which have improved the manuscript. I would have liked to se more description of the microbiota composition as well, but I understand that the authors are currently preparing a separate manuscript on this, so maybe we shall just leave it there, then.

I still have some comments, though:

Line 27: antibiotic-resistance genes

L. 29: I still do not quite know what is meant by AMR ability. Please rephrase.

Figure 1: The colours have changed from version 1 to the revised version. I assume the computer programme assigns the colours automatically, so it may change between versions. Therefore, the explanatory text under the figure (line 124-131) does not fit with the colours of the actual figure. Thus, the gold colour in PV2, PV2.1, PV3.1 and EC is not gold but blue in the version I have, and the purple colour in PV1B is green in my version. Make sure that the text matches the actual figure.

L. 107: A figure legend is missing for Supplementary Figure 1. 

Figure 1: beta-lactams instead of betalactamics, and trimethoprim instead of trimetoprim

L. 189, 203, 204, 209: sp. and spp. should not be in italics.

L. 193: Do you mean Supplementary Figure 2 here? And this figure also needs a figure legend.

Table 2: I assume you mean sulfamethoxazole 

L. 304-310: I also still think the sampling points need more explanation. What characterises each sampling point, such as is it near a beach, near an outlet from the city, near an industrial area, etc., and also what is the actual water depth at each site.

L. 309: Do you mean Supplementary Figure 1 here? 

L. 358: Do you mean sulfamethoxazole?

References: You need to go through all references again and adjust to journal style. F.ex., species names must be in italics, e.g., line 448 Escherichia coli - and coli not with capital C, Same for line 456, multocida not with capital m, line 465, Streptomyces ambofaciens in italics and not capital a. Same in line 463, 517, 520, 534, 574, and maybe others.

Similarly, names of genes must be in italics and not beginning with a capital letter, e.g., line 460 mcr-7.1 in italics and not capital m, line 468 lnu in italics and not capital l. Same in line 471 478, 531 and maybe others.

All author names must be mentioned, and not as et al., as in reference 35, 46, 74, 76  and maybe others.

In reference 27 there needs a couple of spaces between words.

Reference 67 and 94: not capital letters throughout.

Author Response

Response to Reviewer 2, Round 2 (our answers are in red)

Considerable modifications have been made, which have improved the manuscript. I would have liked to se more description of the microbiota composition as well, but I understand that the authors are currently preparing a separate manuscript on this, so maybe we shall just leave it there, then.

Re: We thank the reviewer for their thoughtful comments. We have made a second round of updates to our manuscript, and we think that it has been improved based on the reviewer’s feedback. Below, please find a point-by-point response to each of the reviewer's comments. All the corrections have been revised and highlighted in sky-blue in the text, keeping the first round of corrections highlighted in yellow.

Line 27: antibiotic-resistance genes

Re: Thanks for pointing this. We use the term “antimicrobial resistance genes” because is the actual term used for these kind of genes which confers resistance to antibiotics. We prefer to keep this term instead your suggestion.

Some literature that used “antimicrobial resistance genes” term for ARGs can be foun on:

https://doi.org/10.1016/j.cofs.2021.02.019

doi: 10.1080/10495390600957092

doi: 10.1016/j.giec.2020.06.004.

doi: 10.1016/j.jgar.2021.01.007

https://doi.org/10.1016/j.ijfoodmicro.2024.110634

https://doi.org/10.3390/antibiotics13070639

L29: I still do not quite know what is meant by AMR ability. Please rephrase.

Re: We thank you for your suggestion. We remove “AMR ability” to avoid misunderstanding and change it to “antimicrobial susceptibility”.

Figure 1: The colours have changed from version 1 to the revised version. I assume the computer programme assigns the colours automatically, so it may change between versions. Therefore, the explanatory text under the figure (line 124-131) does not fit with the colours of the actual figure. Thus, the gold colour in PV2, PV2.1, PV3.1 and EC is not gold but blue in the version I have, and the purple colour in PV1B is green in my version. Make sure that the text matches the actual figure.

Re: Thank you for highlighting this issue and we are sorry about this mistake, in fact, the colours changed after re-made the figure. We already updated the colours in the description of the figure.

L107: A figure legend is missing for Supplementary Figure 1. 

Re: Thank you for your observation. We include titles with a bried decription for Supplementary Figures 1 and 2 at the end of the article.

Figure 1: beta-lactams instead of betalactamics, and trimethoprim instead of trimetoprim

Re: Thank you for your observation. We perform the changes suggested in Table 1.

L. 189, 203, 204, 209: sp. and spp. should not be in italics.

Re: We are sorry for the misspelling. We perform the correction.

L. 193: Do you mean Supplementary Figure 2 here? And this figure also needs a figure legend.

Re: Thank you for your observation. We include titles with a bried decription for Supplementary Figures 1 and 2 at the end of the article and correct the Sup. Figures names.

Table 2: I assume you mean sulfamethoxazole 

Re: Thanks for pointing out this misspelled. We already performed this correction.

L. 304-310: I also still think the sampling points need more explanation. What characterises each sampling point, such as is it near a beach, near an outlet from the city, near an industrial area, etc., and also what is the actual water depth at each site.

Re: Thank you for your observation, we rephrased the sentence for a better description including your suggestions.

L. 309: Do you mean Supplementary Figure 1 here? 

Re: Thank you for your observation. We perform the indicated correction in the Supplementary Figure file names.

L. 358: Do you mean sulfamethoxazole?

Re: Thanks for pointing out this typo error. We already performed this correction.

References: You need to go through all references again and adjust to journal style. F.ex., species names must be in italics, e.g., line 448 Escherichia coli - and coli not with capital C, Same for line 456, multocida not with capital m, line 465, Streptomyces ambofaciens in italics and not capital a. Same in line 463, 517, 520, 534, 574, and maybe others.

Re: Thanks for pointing out this observation. We perform the indicated correction in taxonomic names included in the reference section

Similarly, names of genes must be in italics and not beginning with a capital letter, e.g., line 460 mcr-7.1 in italics and not capital m, line 468 lnu in italics and not capital l. Same in line 471 478, 531 and maybe others.

Re: Thank you for your observation. We perform the pointed out correction.

All author names must be mentioned, and not as et al., as in reference 35, 46, 74, 76  and maybe others.

Re: We appreciate your suggestion, but this suggestion was not addressed. The citation journal style indicates in their “Reference List and Citations Style Guide for MDPI Journals”:

For documents co-authored by a large number of persons (more than 10 authors), you can cite the first ten authors, then add a semicolon and add ‘et al.’ at the end: Author 1; Author 2; Author 3; Author 4; Author 5; Author 6; Author 7; Author 8; Author 9; Author 10; et al.

The indicated references include more than 10 authors.

In reference 27 there needs a couple of spaces between words.

Re: Thank you for your observation. We include the spaces.

Reference 67 and 94: not capital letters throughout.

Re. We appreciate your correction. We performed the indicated changes.

Author Response File: Author Response.pdf