The Role of Aquatic Ecosystems (River Tua, Portugal) as Reservoirs of Multidrug-Resistant Aeromonas spp.

Round 1

Reviewer 1 Report

The authors present the AMR of Aeromonas spp. isolated from the Tua river in Portugal. The results presented in this manuscript is of interest. The World Health Organization (WHO) requires developing programs for monitoring antimicrobial resistance in zoonotic bacteria, animal pathogens, and indicator bacteria derived from food-producing animals.

The language in some parts needs revising. For instance, l. 36-39 should be "Agriculture, urban, and animal wastes, often characterized by numerous toxic and carcinogenic chemicals, and pathogenic bacteria and antibiotics, loaded with microflora can contaminate water and enter the food chain, posing a considerable danger to public health."; l. 46-47 should be "Bacteria in the environment are frequently exposed to selective pressures from all types of sources that promote ARG transfer"; l. 49 the overuse; l. 52 exhibits; l. 63-66 hard to understand; l. 80-81 should be "Tua River is one of the Douro River's main tributaries (affluent of the right bank) and results from the merge of the Tuela and Rabaçal Rivers, both born in Spain."  etc. This will improve the manuscript's general readability and make it easier for the reader to interpret the results and discussion. The authors should check the whole manuscript carefully for English mistakes.

Here are my comments:

1. Please include in the title the name of the country and even the river.

2. Please include in the whole manuscript the numbers with two decimals.

3. The Abstract:

• l. 21 should be Antibiotic resistance rates
• l. 24 multiple resistance - please include numbers

4. Keywords - please include multidrug resistance

5. The introduction section: l. 47-50 needs a reference

6. Material and Methods: l. 133-136 needs a reference

please specify the definition of MDR used in this manuscript

7. Section 3 Results and Discussion should be transformed into 2 different sections: 3. Results, 4. Discussion

l. 191-192, can you please explain how it was effective in reducing resistance?

l. 194-195 needs a number

l. 208-209, please removed the references; it is data from this study

l. 255-258 needs a reference

Please try to compare your data with data from other studies regarding AMR resistance of Aeromonas spp.

Please include the limitations of your study.

8. The conclusion sections need to be revised. The conclusion should be more definitive rather than an extension of the discussion section.

9. Please include a list of abbreviations in alphabetical order.

 

 

 

 

Author Response

Response to Reviewer #1

Dear reviewer:

Thank you for your comments concerning our manuscript entitled “The role of aquatic ecosystems (River Tua, Portugal) as reservoirs of multidrug-resistant Aeromonas spp.)” (Ref: water-1094035). Those comments are all valuable and helpful for revising and improving our paper, as well as important for guiding significance to our studies.

 

We carefully revised the manuscript and all changes were marked by the red font (with the track changes tool), and addressed each comment in the point-by-point reply. We appreciate for your warm work earnestly, and hope that the correction will meet with approval. Once again, thank you for the careful review of our manuscript and the opportunity to respond to your comments.

 

Review Report Form #1

Open Review

(x) I would not like to sign my review report
( ) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required
(x) Moderate English changes required
( ) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style

 

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	( )	(x)	( )	( )
Is the research design appropriate?	(x)	( )	( )	( )
Are the methods adequately described?	(x)	( )	( )	( )
Are the results clearly presented?	( )	(x)	( )	( )
Are the conclusions supported by the results?	( )	( )	(x)	( )

 

 

 

Comments and Suggestions for Authors

The authors present the AMR of Aeromonas spp. isolated from the Tua river in Portugal. The results presented in this manuscript is of interest. The World Health Organization (WHO) requires developing programs for monitoring antimicrobial resistance in zoonotic bacteria, animal pathogens, and indicator bacteria derived from food-producing animals.

• Thank you for the comment that this article is adequate and of interest.
• We also appreciate the remaining comments that we believe have improved the article.
• Below we will try to answer all the questions that will be translated into changes in the article, which will be properly marked using the track changes.

The language in some parts needs revising. For instance, l. 36-39 should be "Agriculture, urban, and animal wastes, often characterized by numerous toxic and carcinogenic chemicals, and pathogenic bacteria and antibiotics, loaded with microflora can contaminate water and enter the food chain, posing a considerable danger to public health."; l. 46-47 should be "Bacteria in the environment are frequently exposed to selective pressures from all types of sources that promote ARG transfer"; l. 49 the overuse; l. 52 exhibits; l. 63-66 hard to understand; l. 80-81 should be "Tua River is one of the Douro River's main tributaries (affluent of the right bank) and results from the merge of the Tuela and Rabaçal Rivers, both born in Spain."  etc. This will improve the manuscript's general readability and make it easier for the reader to interpret the results and discussion. The authors should check the whole manuscript carefully for English mistakes.

Answer: The suggestions for changing the sentences was taken into account and as such corrections were made and duly noted in the text. Also, the whole manuscript was carefully reviewed for this kind of mistakes.

 

Here are my comments:

1. Please include in the title the name of the country and even the river.

Answer: As suggested, the title includes river name and country.

2. Please include in the whole manuscript the numbers with two decimals.

Answer: As suggested, all numbers related to percentages along the manuscript were changed and put with two decimals.

Regarding the antibiotics concentrations, we cannot change the numbers (by placing two decimals) since the values are in agreement with the manufacturers, as you can see, for example below.

 

 

3. The Abstract:

• 21 should be Antibiotic resistance rates
• 24 multiple resistance - please include numbers

Answer: Accordingly, the corrections were done.

4. Keywords - please include multidrug resistance

Answer: The keyword was added.

5. The introduction section: l. 47-50 needs a reference

Answer: References were added like suggested.

6. Material and Methods: l. 133-136 needs a reference.

Answer: References were added like suggested.

please specify the definition of MDR used in this manuscript

Answer: Like asked we added the definition of MDR in L: 167-169 “Multidrug-resistant (MDR) is defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories.”

7. Section 3 Results and Discussion should be transformed into 2 different sections: 3. Results, 4. Discussion.

Answer: According to the Instructions for Authors of the Water Journal we can combine the Discussion with Results (see the sentence below). In view of the above, we place the presentation in this form for the reviewer's and the editor's consideration, since for this same journal we have previously published articles in this format (ex: Fonseca A.R, Santos J.A., Varandas S.G.P., Monteiro S.M., Martinho J.L., Cortes R.M.V., Cabecinha E. 2020. Current and Future Ecological Status Assessment: A New Holistic Approach for Watershed Management. Water 12, 2839; doi:10.3390/w12102839).

In the specific case, it seemed more adjusted since in view of the results we immediately present its interpretation and comparison with other studies.

“Discussion: Authors should discuss the results and how they can be interpreted in perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible and limitations of the work highlighted. Future research directions may also be mentioned. This section may be combined with Results.”

1. 191-192, can you please explain how it was effective in reducing resistance?

Answer: We introduced an explanation in the text.

1. 194-195 needs a number

Answer: Done.

1. 208-209, please removed the references; it is data from this study

Answer: We decided to keep the references by clarifying the sentence.

1. 255-258 needs a reference

Answer: Done.

Please try to compare your data with data from other studies regarding AMR resistance of Aeromonas spp.

Please include the limitations of your study.

Answer: As requested, we have not found studies supporting or contradicting our results since environmental conditions and human impact are very diverse. In this sense we included a sentence supporting the need of additional studies: “Thus, in view of the results obtained here, it would be important to carry out further studies to assess the variation in antibiotic resistance with seasons.”

8. The conclusion sections need to be revised. The conclusion should be more definitive rather than an extension of the discussion section.

 Answer: Done

9. Please include a list of abbreviations in alphabetical order.

 Answer: Done and the list of abbreviations are after the conclusions.

 

 

 

 

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

The main finding of this article is the description of Aeromonads in terms of antimicrobial resistance.

The study and results are clear but seem incomplet. Moreover, I noted some major and minor remarks.

 

Major remark:

 

L123-131: identifications of Aeromonas isolates have to be done up to species level. For that, genetic analyses are needed because these data are really missing in the further analysis.

L177: can the author explain how endogenous B-lactamases can represent a public health risk? Description at the phenotypic and genetic level of natural resistance on aeromonads is missing to evaluate the real public health risk.

L182: data on identification of Aeromonad species and of the resistance phenotypic profil of each of them are missing

L191: B-lactamase inhibitors restore amoxicillin or ticarcillin sensitivity of 6 and 8 strains, respectively that seems very low but coherent with literature. Can the authors discuss this point?

L195: This point is not shown on figure 2. Total phenotypic resistance profile is needed for all the 30 strains.

L199: Do the authors identify ESBL carried by these strains?

L205: What does it mean? What about the prevalence of the cphA gene or related carbapenemase-encoding genes?

L236: maybe research of mobile genetic elements are missing in the study; please discuss.

L318: what specific microbial parameter(s) do the author have identified as bioindicator(s)?

 

 

Minor remarks:

 

L35: more than use of water, it is anthropogenic activities that cause water pollutions

L37: ARGs are now considered as pollutants so, they may be list and presented here

L38: “posing” has to be replace by “pose”

L42-43: the relevance of this sentence is not obvious

L43: authors need to mention ARGs

L48: “types of sources”: can the authors be more precise?

L49: I think that "emergence" or "selection" are more appropriate than" arise"

L58-60: data are from a publication of 2000. Can the author verify these data? I think that more than 2 Aeromonas species can be considered as opportunistic pathogens for humans

L62: please replace closed by closely

L67: please clarify: is it really the source of AMR infections you are talking about, or of environmental AMR?

L71: can the authors precise the way of acquisition?

L73: it is not clear what gaps are the authors talking about?

L87: can the main activities mentioned below be represented on the map? Moreover, in the legend, there is a typography mistake: symbol is missing (  )

L97: can the authors give values? or mention these data on results section?

L107: please replace “replicas” by “replicates”

L110: It is not clear if authors mean three volumes of each replicate

L119: please replace “two-five colonies” by “up two to five”

L128: please clarify

L130: manufacturer's name is missing for API 20NE systems

L149: delete majuscule to “Isolates”

L208-209: the sentence has to be rewritten

L213-216: Does Standard deviation indicate that for some antibiogram, some strains were resistant or sensitive depending on the replicate? It is surprising. Can the authors explain this point? Please rewrite the sentence with “within the same antimicrobial”

L218-219: it has already been shown in fig. 2; this sentence has to be deleted

L220: Does it mean that the difference is not significative?

L226: it is difficult to understand what parameters are under consideration to conclude to the significant difference

L243: letters indicating significant difference are missing in the figure 4.

L246: Please replace low by lower

L287: the legend of the figure is not complete (red and grey histograms) and what is representing green line (MDR) is not clear.

 

 

Author Response

Response to Reviewer #2

 

Dear reviewer:

Thank you for your comments concerning our manuscript entitled “The role of aquatic ecosystems (River Tua, Portugal) as reservoirs of multidrug-resistant Aeromonas spp.)” (Ref: water-1094035). Those comments are all valuable and helpful for revising and improving our paper, as well as important for guiding significance to our studies.

 

We carefully revised the manuscript and all changes were marked by the red font (with the track changes tool), and addressed each comment in the point-by-point reply. We appreciate for your warm work earnestly, and hope that the correction will meet with approval. Once again, thank you for the careful review of our manuscript and the opportunity to respond to your comments.

 

Open Review

(x) I would not like to sign my review report
( ) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required
(x) Moderate English changes required
( ) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style

 

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	(x)	( )	( )	( )
Is the research design appropriate?	( )	( )	(x)	( )
Are the methods adequately described?	( )	( )	(x)	( )
Are the results clearly presented?	( )	( )	(x)	( )
Are the conclusions supported by the results?	( )	( )	(x)	( )

 

 

Comments and Suggestions for Authors

The main finding of this article is the description of Aeromonads in terms of antimicrobial resistance.

The study and results are clear but seem incomplet. Moreover, I noted some major and minor remarks.

 

Major remark:

 L123-131: identifications of Aeromonas isolates have to be done up to species level. For that, genetic analyses are needed because these data are really missing in the further analysis.

Answer: Thank you for the comment. To our understanding, this work shows the resistance patterns of Aeromonas spp. isolated from Tua River-Portugal. The ability to follow antimicrobial susceptibility trend over a period of time is key to monitor antimicrobial resistance dissemination in the aquatic environments. In fact, and over the last two decades, genetic approaches have been used for identification at the species level. Although, the main aim of the present work was not a genetic distinction between strains of genus Aeromonas. We are aware of the importance of this fact, mainly due to the phylogenetic discrepancies reported by Martínez-Murcia et al. (2005) to differentiate, for instance, A. salmonicida from A. bestiarum species with 16S rDNA. Reports, based on housekeeping genes have been provided coherent phylogenies of this genus (Martinez-Murcia et al., 2011).

However, it should be noted that the authors recognize the importance of this type of studies and as such are now starting to work on this.

L177: can the author explain how endogenous B-lactamases can represent a public health risk? Description at the phenotypic and genetic level of natural resistance on aeromonads is missing to evaluate the real public health risk.

Answer: Accordingly, the authors introduced a statement in view to clarify this issue.

L182: data on identification of Aeromonad species and of the resistance phenotypic profil of each of them are missing

Answer: Phenotypic characterization of Aeromonas isolates were performed according classical biochemical assays (lines 123-131). The genetic identification by 16S rDNA analysis is not the purpose of this study. As previously mentioned, research work concerning the identification of Aeromonas species, is ongoing.

L191: B-lactamase inhibitors restore amoxicillin or ticarcillin sensitivity of 6 and 8 strains, respectively that seems very low but coherent with literature. Can the authors discuss this point?

Answer: We introduced an explanation in the text.

L195: This point is not shown on figure 2. Total phenotypic resistance profile is needed for all the 30 strains.

Answer: Thank you for your comments and we agree that this point is not directly shown on Fig 2. Therefore, we insert a number as suggested by reviewer 1.

L199: Do the authors identify ESBL carried by these strains?

Answer: Thank you for your comment and after reading the text, we found that it was not really relevant, as well as suggested by another reviewer. So, we deleted the sentence.

L205: What does it mean? What about the prevalence of the cphA gene or related carbapenemase-encoding genes?

Answer: In fact, the environmental incidence of resistance to β-lactam antibiotics seems to be increasing. Our data shown a rate of Aeromonas resistance to imipenem around 43% (13 out 30 isolates). Report have been shown the relation of cphA gene and the resistance of Aeromonas to carbapenems, however in this work, this phylogenetic analysis is out of our main aim.

L236: maybe research of mobile genetic elements are missing in the study; please discuss.

Answer:  We thanks the valuable comments; however, the main objective of the present work was not focus on mechanisms of antibiotic resistance. Nonetheless, it should be noted that the authors recognize the importance of this type of studies that could be done in a next step.

L318: what specific microbial parameter(s) do the author have identified as bioindicator(s)?

Answer:  Aeromonas spp. are ubiquitous and with wide world distribution in aquatic ecosystems and has been studied as an indicator of the dissemination of antimicrobial resistance. This is reinforced before in the text at the end of the introduction section (last paragraph).

 

Minor remarks:

 L35: more than use of water, it is anthropogenic activities that cause water pollutions

Answer:  Done.

L37: ARGs are now considered as pollutants so, they may be list and presented here

Answer:  Done.

L38: “posing” has to be replace by “pose”

Answer:  Done.

L42-43: the relevance of this sentence is not obvious

L43: authors need to mention ARGs

Answer:  Done.

L48: “types of sources”: can the authors be more precise?

Answer:  Done.

L49: I think that "emergence" or "selection" are more appropriate than" arise"

Answer:  Done.

L58-60: data are from a publication of 2000. Can the author verify these data? I think that more than 2 Aeromonas species can be considered as opportunistic pathogens for humans.

Answer:  The article cited was published in 2010 and present an update review of the genus aeromonas since then. We also included in this sentence the reference [19] to support the 36 species described until now.

L62: please replace closed by closely

Answer:  Done.

L67: please clarify: is it really the source of AMR infections you are talking about, or of environmental AMR?

Answer:  Yes we talk about the AMR in the environment.

L71: can the authors precise the way of acquisition?

Answer:  We improved the sentence.

L73: it is not clear what gaps are the authors talking about?

Answer:  We improved the sentence for better understanding.

L87: can the main activities mentioned below be represented on the map? Moreover, in the legend, there is a typography mistake: symbol is missing (  )

Answer:  Done.

L97: can the authors give values? or mention these data on results section?

Answer:  No, we cannot, because we only could have accessed to qualitative data from Environment Portuguese Agency – APA.

L107: please replace “replicas” by “replicates”

Answer:  Done.

L110: It is not clear if authors mean three volumes of each replicate

Answer:  In text we clarified this issue.

L119: please replace “two-five colonies” by “up two to five”

Answer:  Done.

L128: please clarify

Answer:  Done.

L130: manufacturer's name is missing for API 20NE systems

Answer:  Done.

L149: delete majuscule to “Isolates”

Answer:  Done.

L208-209: the sentence has to be rewritten

Answer:  Done.

 

L213-216: Does Standard deviation indicate that for some antibiogram, some strains were resistant or sensitive depending on the replicate? It is surprising. Can the authors explain this point? Please rewrite the sentence with “within the same antimicrobial”

Answer:  We made a mistake, and now, with your helpful comment, the legend was better explained.

L218-219: it has already been shown in fig. 2; this sentence has to be deleted

Answer:  Done.

L220: Does it mean that the difference is not significative?

Answer:  Yes, it means that there are no statistically significant differences.

L226: it is difficult to understand what parameters are under consideration to conclude to the significant difference

Answer:  Thanks again for your observation. In fact, the statement was deleted, once it doesn’t bring any new information.

L243: letters indicating significant difference are missing in the figure 4.

Answer:  As no differences were detected, no letters were placed. In the figure caption, the text explains this lack of letters “No lettering indicates non-significant differences at a 95% confidence level”.

L246: Please replace low by lower

Answer:  Done.

L287: the legend of the figure is not complete (red and grey histograms) and what is representing green line (MDR) is not clear.

 Answer:  The clarification of the figure 5 legend was done.

 

 

 

 

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

Reviewer comments

 

General comments

The article titled “The role of aquatic ecosystems as reservoirs of multidrug-resistant Aeromonas spp.” Is well written. It described the presence of Aeromonas in two placements in Portugal. The novelty of the research has to do with the placements but Aeromonas has been previuosly uses as multidrug resistant indicators. 

It would be increse the relevance of the paper if  the authors could specify which specie is each of the 30 isolates of Aeromonas spp. Some Spatial analysis taking onto account the location of the cities and enterprises would be also valuable. Additionally, if the authors should explain at least one pathway for this multidrug resistance. See for example Biyela et al 2004 and Harnisz&Korzeniewska, 2018.

The authors should go deeper in the seasonal differences and explain the resistance. 

 

Specific comments

 

M&M section

Figure 1. Complete location sites in brackets. It is hard to see the location reference like name of town or even the course of the river.

Lines 89-103. Is there any study regarding the comment water pollution?

Line 108: What is the volume of the bottles? How are the bottles?

Line 160-169. This is part of the M&M.

Results and discussion section

The authors should discuss the presence or absence of resistance to some antibiotics in summer like (PRL, TZP, ATM) and (K,C).

Figure 4. It is hard to believe there is no significance between summer and autumn in in FOX site A. How it is explained?

 

Author Response

Response to Reviewer #3

 

Dear reviewer:

Thank you for your comments concerning our manuscript entitled “The role of aquatic ecosystems (River Tua, Portugal) as reservoirs of multidrug-resistant Aeromonas spp.)” (Ref: water-1094035). Those comments are all valuable and helpful for revising and improving our paper, as well as important for guiding significance to our studies.

 

We carefully revised the manuscript and all changes were marked by the red font (with the track changes tool), and addressed each comment in the point-by-point reply. We appreciate for your warm work earnestly, and hope that the correction will meet with approval. Once again, thank you for the careful review of our manuscript and the opportunity to respond to your comments.

 

 

(x) I would not like to sign my review report
( ) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required
( ) Moderate English changes required
( ) English language and style are fine/minor spell check required
(x) I don't feel qualified to judge about the English language and style

 

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	( )	(x)	( )	( )
Is the research design appropriate?	( )	(x)	( )	( )
Are the methods adequately described?	( )	( )	(x)	( )
Are the results clearly presented?	( )	( )	(x)	( )
Are the conclusions supported by the results?	( )	( )	(x)	( )

Comments and Suggestions for Authors

Reviewer comments

General comments

The article titled “The role of aquatic ecosystems as reservoirs of multidrug-resistant Aeromonas spp.” Is well written. It described the presence of Aeromonas in two placements in Portugal. The novelty of the research has to do with the placements but Aeromonas has been previuosly uses as multidrug resistant indicators. 

It would be increse the relevance of the paper if the authors could specify which specie is each of the 30 isolates of Aeromonas spp.

Answer: Thank you for the comment. The phenotypic characterization of Aeromonas isolates was carried out according to classic biochemical assays that are not robust to differentiate species. In fact, and over the last two decades, genetic approaches have been used for identification at the species level. We are aware of the importance of this fact, mainly due to the phylogenetic discrepancies reported by Martínez-Murcia et al. (2005) to differentiate, for instance, A. salmonicida from A. bestiarum species with 16S rDNA. Reports, based on housekeeping genes have been provided coherent phylogenies of this genus (Martinez-Murcia et al., 2011).

However, it should be noted that the authors recognize the importance of this type of studies and as such are now starting to work on this.

Some Spatial analysis taking onto account the location of the cities and enterprises would be also valuable.

Answer: We agree with the reviewer and as such this spatial analysis was made in the RD section. For a better understanding the pressures in the study area, the main activities which generate anthropic pressure in spatial terms, were represented in Figure 1.

Additionally, if the authors should explain at least one pathway for this multidrug resistance. See for example Biyela et al 2004 and Harnisz & Korzeniewska, 2018.

Answer: We agree with the comments and the introduction was improved.

The authors should go deeper in the seasonal differences and explain the resistance. 

 Answer: Done.

 

Specific comments

 M&M section

Figure 1. Complete location sites in brackets. It is hard to see the location reference like name of town or even the course of the river.

Answer: We totally agree and as suggested the Figure 1 was significantly improved. So, the main pressures in the study area, the main course and the main cities were now visible.

Lines 89-103. Is there any study regarding the comment water pollution?

Answer:  Your question is really pertinent but the available data are in reports from the Portuguese Environment Agency - APA, only qualitative data.

Line 108: What is the volume of the bottles? How are the bottles?

Answer:  Water samples were collected in 1 L sterile glass bottles. The correction was made in the text.

 

 

Line 160-169. This is part of the M&M.

Answer:  We partially agree with the reviewer and as such part of the text has moved on to section 2.4 of the MM.

Results and discussion section

The authors should discuss the presence or absence of resistance to some antibiotics in summer like (PRL, TZP, ATM) and (K,C).

Answer:  Done

Figure 4. It is hard to believe there is no significance between summer and autumn in in FOX site A. How it is explained?

Answer:  Done

 

 

 

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors,

First of all, I want to congratulate you on your works. The manuscript is more improved. Here are my comments:

1. The authors should use a point (not a comma) when presenting a number with two decimals l. 21 should be 83.33 .... 60 without 00 (when is 00 after a point), l. 22 26.67. Please check the whole manuscript carefully.
2. l. 185-186 needs a reference
3. The authors still have to  check the whole manuscript for English mistakes:

l. 11 for the high

l. 17 for the aquatic

l. 27-28 should be "Aquatic environments may provide an ideal setting for the acquisition and dissemination of antibiotic resistance because anthropogenic activities frequently impact them. "

l. 43-44 should be "ARGs are found in the clinical and natural environments and are linked to antibiotic-resistant bacteria (ARB) [4-9]. "

l. 67 please replace despite with although or despite the fact that

l. 69 please delete an - should be ``is insufficient knowledge``

l. 72 for the high

l.74 please replace are able to with can

l. 76 an indicator

l. 76 In this sense,

l. 80 please delete in order

l. 87 should be kilometers in, not kilometres of

l. 122-123 should be "The Tua River basin's hydrological regime presents significant interannual variability with an average annual runoff of 988.1 hm³. "

l. 134 please replace In addition with Also

l. 135 should be inflow

l. 141 in a cold

l. 155 Once confirmed,

l. 248 the susceptibility

l. 264 locations

l. 271-274 should be "It is noteworthy that it is precisely this site (T2) that presents the greatest anthropogenic pressure, namely, related to the production and refining of food oils and Agroindustrial Complex and all with the urban effluent discharged by the Mirandela city."

l. 281 the acquisition

l. 295 In the summer

l. 300 please replace was with were

l. 312 Therefore, a high

l. 331-332 should be "Thus, it would be important to carry out further studies to assess antibiotic resistance variation with seasons because of the results obtained here."

The authors should read the whole article carefully and correct the English mistakes. The list above contains some examples.

 

4. Please include the limitations of your study before the conclusions section.

 

Author Response

Dear Reviewer,

In reply to the review performed to the paper entitled "The role of aquatic ecosystems (River Tua, Portugal) as reservoirs of multidrug-resistant Aeromonas spp.”(ID water-1094035), we would like to thank you for your insightful suggestions. In this version (Round 2), we have attended all the comments and suggestions to improve the quality of the manuscript.

Thank you for detecting some mistakes (we corrected all the numbers along the whole manuscript carefully). We hope the modifications introduced in the manuscript are clear and concise enough as required in order to enable the publication of the manuscript.

Sincerely Yours,

Maria José Saavedra

Author Response File: Author Response.docx

Reviewer 2 Report

I thank the authors because the manuscript has been improved, clarifications done and the point to point reply is convincing even if I regret the absence of genetic data: identification of Aeromonas species; identification of resistance genes; identification of mobile elements underlying the potential impact on public health, with the potential transfer of resistance to allochthonous bacteria.

I still have minor requests:

L210: there is a mistake with the percentage of resistance to chloramphenicol

Fig: some words are underlined in red like they are bad-written; please correct

L349: authors have to precise “antibiotic classes of resistance”

Globally, I find it strange to give minutes when speaking about duration of incubation (18:00……).

I don’t understand why the authors changed all the points to commas in numeric data.

 

Author Response

Dear Reviewer,

In reply to the review performed to the paper entitled "The role of aquatic ecosystems (River Tua, Portugal) as reservoirs of multidrug-resistant Aeromonas spp.”(ID water-1094035), we would like to thank you for your insightful suggestions. In this version (Round 2), we have attended all the comments and suggestions to improve the quality of the manuscript. Thank you also for detecting some mistakes that were corrected

We hope the modifications introduced in the manuscript are clear and concise enough as required in order to enable the publication of the manuscript.

Sincerely Yours,

Maria José Saavedra

 

L210: there is a mistake with the percentage of resistance to chloramphenicol _Done

Fig: some words are underlined in red like they are bad-written; please correct _Done

L349: authors have to precise “antibiotic classes of resistance” _Done

Globally, I find it strange to give minutes when speaking about duration of incubation (18:00……)._ there was a mistake, already corrected.

I don’t understand why the authors changed all the points to commas in numeric data_ already corrected.

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

Reviewer comments v.2

 

Dear authors,

You introduce the following terms in your MS:

• acid-resistant strains
• antibiotic resistance genes (ARGs)

Please, note that from a geneticist point of view these concepts suppose a huge and extend part of the work. I would appreciate if you are more respectful using them if you really do not study this terms in the present work. This would lead to misunderstandings among readers, which reduces the reliability of the results. When it comes to strains, they must be previously characterized. In this sense, a table (as a corollary) with the classification of the strains according to the classical biochemical criteria would be appreciated. On the other hand, if the genes involved in resistance are not studied, it is better to discuss the valuable data from classical analyses that can elucidate general pathways of regulation. This work would improve the value of your results from a classical point of view.

 

 

Author Response

Dear Reviewer,

In reply to the review performed to the paper entitled "The role of aquatic ecosystems (River Tua, Portugal) as reservoirs of multidrug-resistant Aeromonas spp.”(ID water-1094035), we would like to thank you for your comments (in this version -Round 2- we have attended all the comments to improve the quality of the manuscript).

We hope the modifications introduced in the manuscript are clear and concise enough as required in order to enable the publication of the manuscript.

Sincerely Yours,

Maria José Saavedra

 

Answers:

You introduce the following terms in your MS:

• acid-resistant strains

Concerning the term “acid-resistant strains”, this is associated to “nalidixic acid-resistant strains” and we agree and corrected them.

 

• antibiotic resistance genes (ARGs)

Please, note that from a geneticist point of view these concepts suppose a huge and extend part of the work. I would appreciate if you are more respectful using them if you really do not study this terms in the present work. This would lead to misunderstandings among readers, which reduces the reliability of the results. When it comes to strains, they must be previously characterized. In this sense, a table (as a corollary) with the classification of the strains according to the classical biochemical criteria would be appreciated. On the other hand, if the genes involved in resistance are not studied, it is better to discuss the valuable data from classical analyses that can elucidate general pathways of regulation. This work would improve the value of your results from a classical point of view.

Thanks for the comments and we agree that these concepts are important work.

The approach to the concept of antibiotic resistance genes (ARGs), although not the objective of this study, is important to be mentioned it in the introductory section, since antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are reported in several studies on the aquatic ecosystem.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Dear Authors,

The manuscript looks much better and is more improved.

There are two aspects from my previous review, which you have not addressed:

1. l. 192-193 "Multidrug-resistant (MDR) is defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories" needs a reference
2. Please include clearly the limitations of your study before the conclusions section.

Author Response

Dear Reviewer,

In reply to the review performed to the paper entitled "The role of aquatic ecosystems (River Tua, Portugal) as reservoirs of multidrug-resistant Aeromonas spp.” (ID water-1094035), we would like to thank you for your insightful suggestions. In this version (Round 3), we have attended all the comments and suggestions to improve the quality of the manuscript.

We hope the modifications introduced in the manuscript are now clear and concise enough as required in order to enable the publication of the manuscript.

Sincerely Yours,

Maria José Saavedra

 

There are two aspects from my previous review, which you have not addressed:

1. 192-193 "Multidrug-resistant (MDR) is defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories" needs a reference.

Answer: We agree and a reference was put in the text:

34. Magiorakos A.P.; Srinivasan, A.; Carey, R.B., Carmeli, Y.; Falagas, M.E.; Giske, C.G.; Harbarth, S.; Hindler, J.F.; Kahlmeter, G.; Olsson-Liljequist, B.; Paterson, D.L.; Rice, L.B.; Stelling, J.; Struelens, M.J.; Vatopoulos, A.; Weber, J.T.; Monnet, D.L. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2011, 268-282. doi: 10.1111/j.1469-0691.2011.03570.x.

 

Please include clearly the limitations of your study before the conclusions section.

Answer: We agree about the importance to indicate the limitations of the study and so inserted them in the conclusions section.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript has been considerably improved but information about how an intrinsic resistance can represent potential risk to public health is missing and exploring what genes are involved in resistances of clinical interest could be helpfull.

Legend Fig. 4, please can the authors indicate the number or strains from T1 and T2 for each season

Author Response

Review 2

Dear Reviewer,

In reply to the review performed to the paper entitled "The role of aquatic ecosystems (River Tua, Portugal) as reservoirs of multidrug-resistant Aeromonas spp.” (ID water-1094035), we would like to thank you for your insightful suggestions. In this version (Round 3), we have attended all the comments and suggestions to improve the quality of the manuscript.

We hope the modifications introduced in the manuscript are now clear and concise enough as required in order to enable the publication of the manuscript.

Sincerely Yours,

Maria José Saavedra

 

Comments and Suggestions for Authors

The manuscript has been considerably improved but information about how an intrinsic resistance can represent potential risk to public health is missing and exploring what genes are involved in resistances of clinical interest could be helpfull.

Answer: a better explanation was put in the text about the potential risk of Aeromonas

(lines 352-358) The genus Aeromonas is regarded not only as an important disease-causing pathogen of cold-blooded species but also as the etiologic agent responsible for a variety of infectious complications in both immunocompetent and immunocompromised persons [23]. To highlight that carbapenems are often used as “last-line agents” or “antibiotics of last resort” for the treatment of severe infections due to multi-drug-resistant hospital-acquired bacteria [50]. However, the appearance of MDR seriously threatens this class of lifesaving antimicrobials.

 

 

Legend Fig. 4, please can the authors indicate the number or strains from T1 and T2 for each season

 

Answer: We clarify these issues and so, we improved the text

In the place T1, there was 13 strains and in the place T2 they found 17 strains.

T1  summer – 4 grown in GSP;  and 2 grown in GSP-IMP

T1 outumn – 5 grown in GSP; and 2 grown in GSP-IMP

T2  summer – 4 grown in GSP; and 3 grown in GSP-IMP

T2 autumn – 7 grown in GSP; and 3 grown in GSP-IMP

… Aeromonas spp. isolated from T1 (n = 13; 9 grown in GSP and 4 in GSP-IMP) and T2 (n = 17; 11 grown in GSP and 6 in GSP-IMP) river locations…. (lines 267-268)

… Aeromonas isolates at summer (4 strains grown in GSP and 2 grown in GSP-IMP) and autumn (5 grown in GSP; and 2 grown in GSP-IMP) seasons … (lines 301-303)

… in T2 (in summer, 4 strains grown in GSP and 3 grown in GSP-IMP versus in autumn 7 strains grown in GSP and 3 grown in GSP-IMP) … (lines 307-308)

Author Response File: Author Response.docx

Reviewer 3 Report

Reviewer comments v.3

 

Dear authors,

 

The MS has been considerably improved but some changes should be included.

In the study area section it should be mentioned all the cities in the course of the river and their population since this study emphasized the resistance to antibiotics.

The results from the section 2.3. Identification of isolates it is not specifically commented.

The authors used classical biochemical methods “(indole, Voges–Proskauer, methyl red, citrate reactions, gelatin liquefaction, nitrate reduction, urease test, glucose oxidation and carbohydrate fermentations were determined), Gram-negative staining, for the presence of normally positive cytochrome oxidase, catalase reaction, and growth in nutritive broth at 0% to grow in the presence of vibriostatic factor O/129 [23,31]. Additionally, commercial identification systems API 20NE (bioMérieux, https://www.biomerieux.com/)”. With these methods the authors identified several strains. In the place T1, there was 13 strains and in the place T2 they found 17 strains. They said that 20 strains were grown from agar plates (GSP) and 10 in GSP-IMP.

Please, specify which strains from each site are grown in GSP or GSP-IMP. Additionally, it is important to specify which strains are from autum or summer. Are they all found in both seasons? And places?

Have the K and C resistant strains any biochemical identification model?

Yours sincerely,

 

Author Response

Dear Reviewer,

In reply to the review performed to the paper entitled "The role of aquatic ecosystems (River Tua, Portugal) as reservoirs of multidrug-resistant Aeromonas spp.” (ID water-1094035), we would like to thank you for your insightful suggestions. In this version (Round 3), we have attended all the comments and suggestions to improve the quality of the manuscript.

We hope the modifications introduced in the manuscript are now clear and concise enough as required in order to enable the publication of the manuscript.

Sincerely Yours,

Maria José Saavedra

 

Comments and Suggestions for Authors

Dear authors,

The MS has been considerably improved but some changes should be included.

In the study area section it should be mentioned all the cities in the course of the river and their population since this study emphasized the resistance to antibiotics.

Answer: We agree with the reviewer and thus we improved the sub-chapter 2.1. Study Area with 2 sentences about the main population clusters affecting sampling sites (lines 131-134).

 

The results from the section 2.3. Identification of isolates it is not specifically commented.

The authors used classical biochemical methods “(indole, Voges–Proskauer, methyl red, citrate reactions, gelatin liquefaction, nitrate reduction, urease test, glucose oxidation and carbohydrate fermentations were determined), Gram-negative staining, for the presence of normally positive cytochrome oxidase, catalase reaction, and growth in nutritive broth at 0% to grow in the presence of vibriostatic factor O/129 [23,31]. Additionally, commercial identification systems API 20NE (bioMérieux, https://www.biomerieux.com/)”. With these methods the authors identified several strains. In the place T1, there was 13 strains and in the place T2 they found 17 strains. They said that 20 strains were grown from agar plates (GSP) and 10 in GSP-IMP. Please, specify which strains from each site are grown in GSP or GSP-IMP. Additionally, it is important to specify which strains are from autum or summer. Are they all found in both seasons? And places?

Answer: We clarify these issues and so we improved the text.

T1  summer – 4 grown in GSP;  and 2 grown in GSP-IMP

T1 outumn – 5 grown in GSP; and 2 grown in GSP-IMP

T2  summer – 4 grown in GSP; and 3 grown in GSP-IMP

T2 autumn – 7 grown in GSP; and 3 grown in GSP-IMP

… Aeromonas spp. isolated from T1 (n = 13; 9 grown in GSP and 4 in GSP-IMP) and T2 (n = 17; 11 grown in GSP and 6 in GSP-IMP) river locations…. (lines 267-268)

… Aeromonas isolates at summer (4 strains grown in GSP and 2 grown in GSP-IMP) and autumn (5 grown in GSP; and 2 grown in GSP-IMP) seasons … (lines 301-303)

… in T2 (in summer, 4 strains grown in GSP and 3 grown in GSP-IMP versus in autumn 7 strains grown in GSP and 3 grown in GSP-IMP) … (lines 307-308)

 

Have the K and C resistant strains any biochemical identification model?

Answer: There were no differences for the biochemical identification tests performed for isolates with resistance to K and C.

Author Response File: Author Response.docx