Prevalence of Antibiotic Resistant E. coli Strains Isolated from Farmed Broilers and Hens in Greece, Based on Phenotypic and Molecular Analyses
, Ioannis A. Giantsis 2,*, Konstantinos V. Papageorgiou 1, Georgios A. Delis 1, Vangelis Economou 1, Spyridon K. Kritas 1, Evangelia N. Sossidou 3 and Evanthia Petridou 1
Round 1
Reviewer 1 Report
Please find enclosed all my comments
Comments for author File: 
Comments.pdf
Author Response
Reviewer 1
I would thank the authors for this interesting manuscript. However I found that the manuscripts have presented multiple flaws that have substantially affected its quality.
Response: We are grateful to the reviewer for finding our study interesting as well as for the valuable detailed comments. The reviewer indeed helped us to upgrade the way we describe the results and the scientific sound of our study. We trust that after addressing all comments following her/his suggestions, the multiple flaws were corrected and the quality of the manuscript has been substantially increased.
- The main concern is related to the results presentation. The results are poorly presented and they did not define the total number of the strains and how did they obtain them from the total sample. Additionally, the second paragraph contains multiple redundant data that was described in the first paragraph. The results should also supported by a table (or tables) and replace the two first figures. They should also include what were the factors that could be associated with the high/low antibiotic resistance level.
Response: Based on the reviewer’s recommendation and suggestions, the Results were substantially revised, presenting the findings in a Table, modifying the Figures according to the specific suggestions and clarifying the total numbers.
- The second concern is related to the fact that your isolates were not biochemically identified. Tryp-141 tone Bile X-glucuronide agar is used as a presumptive medium for E coli not for its definitive identification (you should at least add Oxydase and indole tests).
Response: We apologize for missing this part. The isolates were molecularly identified using the PCRs developed and described Clermont et al. 2000. Details have been added in the revised manuscript (please see Sections 2.3, 3.3 and Table 1)
- The other concern is related to the introduction and the discussion. Both of them are too long with some „orphan” paragraphs without efforts to summarize (see below).
Response: In accordance to the below comment, both Introduction and Discussion were deeply revised (see detailed responses to below comments).
- At last, the number of reference is too high for a manuscript. Also, they need to be updated since the most recent was published in 2021 and about 4% only of the 71 references are published after the year 2020.
Response: In accordance to the reviewer’s comment, more than 10 recent (2020 and afterwards) references were added in several parts of the manuscript, mainly in the Introduction and Discussion.
Thus, I suggest to the authors to take more time and revise all the parts of the manuscript.
I have also other comments and suggestions:
Title:
Delete “genotypes in” from the title (Prevalence of antibiotic resistant E. coli strains isolated from farmed broilers and hens in Greece, based on phenotypic and molecular analyses)
Response: We agree with the reviewer’s point and thus the title was accordingly modified
Abstract:
- “The use of antimicrobials is beneficial for livestock health; however the overuse and misuse can increase the resistance towards β-lactams, quinolones and colistin.” Your statement here is false, since the misuse can increase resistance to all antibiotics not only to these 3 types of antibiotics.
Response: The sentence was corrected following the reviewer’s recommendation.
- “The aim of the present study was the phenotypic and molecular examination of the presence of β-lactams, quinolones and colistin”. This is not your objective since you have also examined the presence of resistance to tetracycline, chloramphenicol.... which are not from the mentioned families. Reformulate
Response: In accordance to the reviewer’s comment, this part of the abstract was substantially rephrased.
- The approach of writing in the abstract differs from the results. You did not mention the results of resistance in the different samples (broilers, layer hens) in any part of the results. For example we do not find the number of 317 samples in the main text and all of the percentages are different between the abstract and the results.
Response: Both the Abstract and the Results were carefully revised and are totally consistent in the revised manuscript. Also, separate results for broilers and laying hens are shown in the new Table 2.
- Resistance of broiler and laying hens samples to 3rd... (change samples to isolates or strains).
Response: Corrected according the reviewer’s comment
- In the same line: “found” may be “was found to be....” (or any other expression of your choice). The same remark for line 24 (and line 206).
Response: All corrected according the reviewer’s comment
- The conclusion of line 25-26 has no relation with your work.
Response: In agreement with the reviewer’s comment, this sentence was deleted and was replaced by a conclusion based on our results.
Introduction
Please revise and summarize the introduction. It should focus on the antibiotic resistance of E coli strains. You do not need to describe each antibiotic alone. Additionally you should report the mechanisms and the rate of resistance in the world and what is done in your country in this chapter (what are the published studies). Correct also your objectives.
Response: Taking into consideration the comment of the reviewer, several parts of the discussion were deleted. Also and most importantly, in accordance to this comment, the mechanisms of action as well as the rate of resistance both worldwide and in Greece are provided in a new paragraph in the Introduction. Also, the objectives of the study were revised in the scope paragraph.
Material and Methods:
- You should first add the period of the study;
Later, you should add how did you collect your samples, from which region, town..., The number of animals in the flocks, the number of samples in each flock, the frequency of sampling, How did the sample have been pooled?......
Response: Information regarding period and regions of the collections, number of animals in the flocks, total number of samples and pools and collection methodology were provided in two additional paragraphs in the revised manuscript, as recommended by the reviewer (please see Section 2.1 in the revised manuscript)
- The references used in line 148 and 155 are the same even “the Clinical and Laboratory Standard Institute (CLSI)” is not “the European Committee on Antimicrobial Susceptibility Testing (EUCAST)”.
Response: The additional reference was provided as the reviewer correctly mentioned (Reference number 55 in the revised manuscript).
- Line 165-168: add what will be the result and what is the reference used.
Response: Analytical details for the AmpC β-lactamases test as well as the corresponding reference were provided in the revised manuscript (please see Section 2.2)
- Molecular investigation of Antimicrobial Resistance Genes: You should add the sequence of the primers and the size of the band.
Response: In accordance to the reviewer’s comment, a Table was added in the revised manuscript providing the primer sequences and the size of the expected bands (please see Table 1 in the revised manuscript)
- Statistical analysis: we can not see where you used the logistic regression model in the results. Is this model really applicable in your case? What statistical tests you used before applying this model?
Response: We agree with the reviewer and we apologize for this mistake. Indeed, this model is not applicable in our case. Instead we used a simpler χ2 was performed to compare phenotypic with molecular findings, resulting in no correlation between these two datasets.
Results:
- I suggest revising and organizing all your results. Paragraphs 1 and 2 especially contain multiple repetitive data (i.e. nalidixic acid (73.4%) is repeated in line 203 and 207...).
One can not understand what the difference between % of paragraph 1 and 2 is. Try to reformulate and be clearer.
Response: The Results part was substantially reformulated, the repeated parts were deleted and also a Table was created to better present the findings (please Section 3.1 and Table 2 in the revised manuscript).
- Also, you should begin your results by defining the total number of isolated strains to be cleaner for the reader.
Response: Modified according to the reviewer’s comment
- Line 199: “All the strains showed resistance to at least more antimicrobial substance, except ampicillin”. What do you mean here exactly? your sentence is not clear.
Response: The sentence was rephrased according to the reviewer’s comment
- The same applies for the sentence in line 214: “Only one strain exhibited sensitivity to all antimicrobial agents except ampicillin.”
Response: This sentence was deleted according to the reviewer’s comment
- Line 202: The highest rates of resistance were shown to... (the verb shown is not adequate here).
Response: The verb was replaced with “observed” as recommended by the reviewer
- Line 206: “Regarding quinolones, resistance rates found significantly high”. the sentence is incorrect. revise please.
The same for the sentence: “Higher resistance percentages observed against...”
Response: The sentences were deleted in accordance to the general reformulation of the Section 3.1
- Line 211: “Application of the minimum inhibitory concentration (MIC) assay showed that.....” You do not repeat the sentence since you have mentioned this in materials and methods. You have just to describe the antibiotic resistance.
Response: This part was deleted in accordance to the general reformulation of the Section 3.1, as recommended by the reviewer.
- Figure 1 and 2 have no sense. Additionally, their quality is very poor and their big size is not adequate with your manuscript. I suggest replacing them with tables of high quality and if you insist to put them you should improve their quality and reduce their size to be in the same line.
Response: As suggested by the reviewer, the quality of the figures was increased; their sizes were reduced and are in the same line as one figure in the revised manuscript (Figure 1 in the revised manuscript).
- In figure 3 you should mention the size of the band, the positive and the negative control. (You should also explain what was the reference strain used in your study).
Response: Corrected according to the reviewer’s comment (please see Figure 2 in the revised manuscript)
Discussion:
- The discussion is too long, try to summarize it to be as concise and clear as possible. Beginning with an introduction of 20 lines is not adequate. Reduce and summarize this part to some lines only.
Response: As suggested by the reviewer, the first part of the discussion was largely reduced. Particularly the two first paragraphs were embedded in one summarized.
- Line 305: correct the style of referencing (24, 58, 59).
Response: Corrected according the reviewer’s comment
- Line 304-306: “However, the problem with the ESBL-production from E. coli stains in poultry has been poorly described”. Your statement here is false. Multiple studies exist regarding ESBL-producing E coli in poultry. Please update your references.
Response: The statement was modified according to the reviewer’s comment as follows “The problem with the ESBL-production from E. coli strains has been also recently described in poultry” and also some references were added.
- The statement of line 338-345 is not adequate for your study.
Response: This part was deleted in the revised manuscript, as correctly recommended by the reviewer.
- You should add the limitation of the study at the last of the discussion.
Response: The major limitation of our findings is the detection of only one resistance gene, which has been added in the last paragraph of the discussion as recommended by the reviewer.
- The conclusion has no relation with your work. It should contain the most important results of your study and eventually some recommendations.
Response: The conclusion paragraph was deleted and rewritten following the reviewer’s recommendations.
References:
You should add the number corresponding to each reference. We can not find which reference is 1 or 2...
Response: We apologize for this mistake; it was probably due to an error by a non-updated browser. It has been accordingly corrected in the revised manuscript
At last, I recommend to the authors to revise substantially all the manuscript and proofread it for English language.
Response: The manuscript has been proofread and errors in the written Engligh were corrected, as suggested by the reviewer.
Reviewer 2 Report
Dear authors,
The manuscript is well-written and organized. Minor English adjustments and additional citations must be included to justify some of the claims. Before I give in-depth revisions, I have high reservations about the methodology used to confirm the E. coli isolates. Currently, in the manuscript, you only conducted presumptive analysis and have presumptive positive results for E. coli. Have you confirmed the isolates to be E. coli by PCR or WGS? This is a critical step and must be conducted to claim resistance to isolates that are only presumptive positives. Without molecular confirmation of the isolates, I recommend rejecting the manuscript.
Author Response
The manuscript is well-written and organized. Minor English adjustments and additional citations must be included to justify some of the claims. Before I give in-depth revisions, I have high reservations about the methodology used to confirm the E. coli isolates. Currently, in themanuscript, you only conducted presumptive analysis and have presumptive positive results for E. coli. Have you confirmed the isolates to be E. coli by PCR or WGS? This is a critical step and must be conducted to claim resistance to isolates that are only presumptive positives. Without molecular confirmation of the isolates, I recommend rejecting the manuscript.
Response: We are grateful to the second reviewer for the well writing and organisation of our manuscript.
As suggested by the reviewer, the written English have been corrected in the entire manuscript and also more than 10 additional recent references have been added in the revised manuscript.
We apologise that by mistake we did not mention the molecular identification of E. coli isolates. The isolates were molecularly identified using the PCRs developed by Clermont et al. 2000. Details have been added in the revised manuscript (please see Sections 2.3, 3.3 and Table 1)
Reviewer 3 Report
R1
In this study, the researches investigated the prevalence of antibiotic resistant E. coli strains isolated from broiler and laying hen farms in Greece. The analyses included cultivation of bacteria, detection of specific antimicrobial genes through PCR and sequencing, and antibiotic susceptibility testing using disk diffusion and microdilution assays. Considering the constantly growing global problem of antimicrobial resistance, the topic of the manuscript is important and the results provide interesting information on the resistance rates in commensal E. coli strains in Greece. Although the manuscript is mostly well written and provides information about the material and methods in details, there are some sections which are a bit confusing and require rephrasing in order to be more clear. Therefore I would recommend this manuscript for publication after a revision.
line 32 have become a highly important public health issue worldwide
line 39 species, as well as the spread
line 55 Clermont et al. described one additional phylogenetic group (phylogroup G) in 2019 (doi:10.1111/1462-2920.14713). also, there are several cryptic clades which have been previously described. please add it when mentioning other phylogenetic groups.
line 78 cattle
line 97 in the past years, many
line 99 exhibiting
line 131 fom every flock
line 135 91 broiler and 15 laying hen flocks
line 137 if available, more than one flock per farm was sampled
line 138 on arrival to the laboatory
line 143 italicize E. coli
line 148 add full description of the agar (manufacturer, city, country)
line 163 inhibition zone
line 173 a molecular level
line 174 polymerase chain reaction (PCR) – write full name when mentioning for the first time
line 199 'All the strains showed resistance to at least more antimicrobial substance, except ampicillin.' – what do you mean by that?
lines 200-201, 215-216 I suggest writing numbers in the sentence and putting percentages in the brackets
line 224 low for ceftriaxone
lines 202-226 in this section, you mention results of the antibiotic susceptibility testing in two separate paragraphs. you also mention different numbers and percentages for same antimicrobials. is maybe each pragraph meant to be describing different poultry categories (one for broilers, one for laying hens)?
lines 228-231 is the first sentence describing the total results, and the second one only laying hens? I would suggest adding a figure or a table with the results (numbers and percentages) that you mention in the text.
line 228 phenotypes
line 245 statistical
line 250 Research regarding antimicrobial usage and the growth of antimicrobial resistance in food-producting animals, more specifically poultry industry, remains a relatively poorly investigated field in Greece.
line 253 antimicrobial resistance in broilers and laying hens.
lines 256-260 remove the description of methods. it's repetitive and redundant.
line 272 after the introduction of quinolones
line 273 both from
line 280 rates to nalidixic acid
line 281 trimethoprim (40.7%), while
line 283 are lower
line 284 pattern was observed
line 286 Furthermore, other studies on poultry showed the resistance levels
line 292 data on antimicrobial resistance in laying hens is less available
line 293 resistance rates to
line 296, 297 resistance to
line 319 was found
line 335-337 all relevant information on mcr gene at a global scale are of major importance in order to prevent public health threats and apply proper measures.
Author Response
In this study, the researches investigated the prevalence of antibiotic resistant E. coli strains isolated from broiler and laying hen farms in Greece. The analyses included cultivation of bacteria, detection of specific antimicrobial genes through PCR and sequencing, and antibiotic susceptibility testing using disk diffusion and microdilution assays. Considering the constantly growing global problem of antimicrobial resistance, the topic of the manuscript is important and the results provide interesting information on the resistance rates in commensal E. coli strains in Greece. Although the manuscript is mostly well written and provides information about the material and methods in details, there are some sections which are a bit confusing and require rephrasing in order to be more clear. Therefore I would recommend this manuscript for publication after a revision.
Response: We are grateful to the third reviewer for recognizing the importance of our work as well as the correct utilization of methodologies. We trust that after all reviewers’ suggestions and recommendations the confusing parts were corrected and the sections needing rephrasing are now clear to the reader in the revised manuscript.
line 32 have become a highly important public health issue worldwide
Response: corrected according to the reviewer’s comment
line 39 species, as well as the spread
Response: corrected according to the reviewer’s comment
line 55 Clermont et al. described one additional phylogenetic group (phylogroup G) in 2019(doi:10.1111/1462-2920.14713). also, there are several cryptic clades which have been previously described. please add it when mentioning other phylogenetic groups.
Response: The additional phylogenetic group was referred in the revised manuscript, together with the reference suggested by the reviewer. Moreover, the cryptic clades statement was added according to the reviewer’s comment.
line 78 cattle
Response: corrected according to the reviewer’s comment
line 97 in the past years, many
Response: corrected according to the reviewer’s comment
line 99 exhibiting
Response: corrected according to the reviewer’s comment
line 131 fom every flock
Response: corrected according to the reviewer’s comment
line 135 91 broiler and 15 laying hen flocks
Response: corrected according to the reviewer’s comment
line 137 if available, more than one flock per farm was sampled
Response: This part was rephrased according to the first reviewer’s comment
line 138 on arrival to the laboratory
Response: corrected according to the reviewer’s comment
line 143 italicize E. coli
Response: corrected according to the reviewer’s comment
line 148 add full description of the agar (manufacturer, city, country)
Response: added, as recomended by the reviewer
line 163 inhibition zone
Response: corrected according to the reviewer’s comment
line 173 a molecular level
Response: corrected according to the reviewer’s comment
line 174 polymerase chain reaction (PCR) – write full name when mentioning for the first time
Response: full name was provided as recomended by the reviewer
line 199 'All the strains showed resistance to at least more antimicrobial substance, exceptampicillin.' – what do you mean by that?
Response: This part was indeed confusing and has been deleted from the revised manuscript
lines 200-201, 215-216 I suggest writing numbers in the sentence and putting percentages in the brackets
Response: the results section was deeply revised and all numbers are now shown in the new Table 2, considering also this particular comment of the reviewer
line 224 low for ceftriaxone
Response: this part was deleted and rewritten together with the rest results
lines 202-226 in this section, you mention results of the antibiotic susceptibility testing in two separate paragraphs. you also mention different numbers and percentages for same antimicrobials. is maybe each pragraph meant to be describing different poultry categories (onefor broilers, one for laying hens)?
Response: The section was rewritten and the finding were better clarifie in the new Table 2.
lines 228-231 is the first sentence describing the total results, and the second one only layinghens? I would suggest adding a figure or a table with the results (numbers and percentages) that you mention in the text.
Response: This part was rewritten for clarification, whereas the two figures were embedded in a new one of better quality (please see section 3.2 in the revised manuscript).
line 228 phenotypes
Response: Deleted, in accordance with the rewriting of the section 3.2
line 245 statistical
Response: corrected according to the reviewer’s comment
line 250 Research regarding antimicrobial usage and the growth of antimicrobial resistance in food-producting animals, more specifically poultry industry, remains a relatively poorly investigated field in Greece.
Response: This part was deleted in accordance to the first reviewer’s suggestion for shortening the Discussion
line 253 antimicrobial resistance in broilers and laying hens.
Response: corrected according to the reviewer’s comment
lines 256-260 remove the description of methods. it's repetitive and redundant.
Response: Deleted, according to the reviewer’s comment
line 272 after the introduction of quinolones
Response: corrected according to the reviewer’s comment
line 273 both from
Response: corrected according to the reviewer’s comment
line 280 rates to nalidixic acid
line 281 trimethoprim (40.7%), while
line 283 are lower
Response: These parts were deleted and summarized for better flow of the text
line 284 pattern was observed
Response: corrected according to the reviewer’s comment
line 286 Furthermore, other studies on poultry showed the resistance levels
Response: corrected according to the reviewer’s comment
line 292 data on antimicrobial resistance in laying hens is less available
Response: corrected according to the reviewer’s comment
line 293 resistance rates to
Response: corrected according to the reviewer’s comment
line 296, 297 resistance to
Response: corrected according to the reviewer’s comment
line 319 was found
Response: corrected according to the reviewer’s comment
line 335-337 all relevant information on mcr gene at a global scale are of major importance in order to prevent public health threats and apply proper measures.
Response: corrected according to the reviewer’s comment
Round 2
Reviewer 1 Report
See attached file
Comments for author File: Comments.pdf
Author Response
Sustainability-2299461: Prevalence of antibiotic resistant E. coli strains isolated from farmed broilers and hens in Greece, based on phenotypic and molecular analyses
I would thank the authors for their efforts to improve the quality of this manuscript. However I found that some of my comments were not taken into consideration in the revised version (or at least a convincing answer in the rebuttal letter). Also, the manuscript still has multiple gaps that you will find below:
Response: We are grateful to the reviewer for recognizing our effort to improve the quality of the manuscript. We hope that after implementing the second round of her/his comments, the detected gaps have been eliminated and corrected
I will begin with my concerns that still be without responses:
- The main concern is related to the results presentation. The results are poorly presented and they did not define the total number of the strains and how did they obtain them from the total sample. Additionally, the second paragraph contains multiple redundant data that was described in the first paragraph. The results should also supported by a table (or tables) and replace the two first figures. They should also include what were the factors that could be associated with the high/low antibiotic resistance level.
Even efforts are made here some gaps still exists (see below).
Re: the results were further modified based on the reviewer’s below comments. Particularly, the total number of isolates analyzed were clarified in the beginning of the results, the percentages of microbial resistance separately for broilers and laying hens were added in Table 2, the Figure 1 was deleted and the text was rephrased and corrected in several parts.
- The second concern is related to the fact that your isolates were not biochemically identified. Tryp-141 tone Bile X-glucuronide agar is used as a presumptive medium for E coli not for its definitive identification (you should at least add Oxydase and indole tests).
The authors did not answer to this comment
Re: Although identification of E. coli was also molecularly identified as described in 2.3, Oxydase and indole biochemical tests were additionally performed following the reviewer’s comment (please see section 2.1 in the revised manuscript).
- The other concern is related to the introduction and the discussion. Both of them are too long with some ‘orphan” paragraphs without efforts to summarize (see below).
This is the main concern in the revised version; even more, the authors added a paragraph to the introduction making it longer.
Re: The additional paragraph provides data regarding the mechanisms and the rate of resistance isolated from different organisms in Greece, following the recommendation of the reviewer in a detailed comment below. Nevertheless, both introduction and discussion are intensively shortened in the revised manuscript, omitting unnecessary information. The introduction is now summarized and focused on the resistance of E. coli strains, whereas it was also modified and revised in several parts.
- At last, the number of reference is too high for a manuscript. Also, they need to be updated since the most recent was published in 2021 and about 4% only of the 71 references are published after the year 2020.
Even the references have been updated with some recent ones; their number has reached 89 references which is a high number for such a manuscript and such results
Re: As recommended by the reviewer, the number of references has been significantly reduced, and is now 63 references, in line with the deletion of unnecessary information.
Abstract:
Add the period of study
Re: The period of the study was added in the revised abstract in accordance to the reviewer’s recommendation.
Introduction
Please revise and summarize the introduction. It should focus on the antibiotic resistance of E coli strains. You do not need to describe each antibiotic alone. Additionally you should report the mechanisms and the rate of resistance in the world and what is done in your country in this chapter (what are the published studies). Correct also your objectives.
This point is still lacking in your introduction. Summarizing the introduction and deleting unnecessary details is necessary
Re: The introduction, in the current form, in the second revision of the revised manuscript, has been intensively shortened, deleting unnecessary details as recommended by the reviewer. Particularly, it is approximately 50 lines shorter.
Results:
- Also, you should begin your results by defining the total number of isolated strains to be cleaner for the reader.
I did not understand why did you delete the total number of strains (106) from the results.
Re: We apologize for misunderstanding the previous comment of the reviewer. The total number of strains (106) has been added again in the Results of the revised manuscript.
- Line 211: “Application of the minimum inhibitory concentration (MIC) assay showed that.....” You do not repeat the sentence since you have mentioned this in materials and methods. You have just to describe the antibiotic resistance.
You have to delete this sentence. You should write just your results. “they are resistant or sensitive to colistin.
Re: corrected according to the reviewer’s comment
- Figure 1 and 2 have no sense. Additionally, their quality is very poor and their big size is not adequate with your manuscript. I suggest replacing them with tables of high quality and if you insist to put them you should improve their quality and reduce their size to be in the same line.
The figures are still of poor quality and are not adapted for such a journal (Sustainablity)
Re: the figures have been deleted from the revised manuscript in accordance with the initial recommendation of the reviewer.
Discussion:
- The discussion is too long, try to summarize it to be as concise and clear as possible. Beginning with an introduction of 20 lines is not adequate. Reduce and summarize this part to some lines only.
I still have a concern about this
Re: In accordance to the reviewer’s comment, the discussion was further shortened; particularly the first part was summarized in no more than five lines. Also some other parts were further deleted and/or summarized.
The other remarks are cited below:
Line 13-14: correct as: “....however their overuse....
Re: corrected according to the reviewer’s comment
Line 14: “...increase resistance to antibiotics”. Reformulate since the term antibiotics was cited in the beginning of the sentence (antimicrobials).
Re: reformulated by replacing “antibiotics” with “these compounds”
Line 16-19: delete and reformulate in the association with the last sentence of the objectives. Add the study period.
Re: reformulated as follows: “Resistance of E. coli strains was examined against various antibiotics including several families of compounds such as…”
Line 20: you should begin with the total number of strains (106).
Re: corrected as follows: “In total 106 strains were investigated, sampled during the years 2016-2019 from 91 poultry farms,…”
Line 27: “rather not in laying hens”. delete please
Re: deleted, as recommended by the reviewer
Line 28: “based on our..” correct please
Re: corrected according to the reviewer’s comment
Line 35: “as the bacteria evolve resisting mechanisms”, delete please
Re: deleted, as recommended by the reviewer
Line 39-41: This sentence has no relation with the last one
The same remark in Line 45-46. Additionally 67% in animals or humans??? explain please.
Re: this part has been rephrased and summarized in accordance to the general comments of the reviewer above (please see the first paragraph of the Introduction in the revised manuscript)
Line 51: delete E coli (you can begin with “it”). Delete “facultative anaerobic” (it was cited in line 48).
Re: this part was partially deleted and summarized in accordance to the reviewer’s recommendation to shorten the Introduction (please see the second paragraph of the Introduction in the revised manuscript)
Line 76-91: The ideas this paragraph are insufficient (to define the hypothesis) and not organized. You talk about antibiotic resistance in line 73 than E coli infection, later you return to resistance. Reorganize please.
Re: The part of this paragraph concerning infestation was deleted, whereas the whole part has been divided in two new paragraphs in an effort to be better organized.
Line 76: replace infestation by infection
Re: this part was deleted in the revised manuscript
Line 86 and 91: E coli should be in italic
Re: corrected according to the reviewer’s comment
At last, I insist that the introduction should be substantially revised and summarized.
Re: following the reviewer’s recommendation, as already stated in the response of a previous comment, the introduction has been substantially reduced and summarized.
Line 161-164: delete “from the region” in line 162 and 163.
Re: corrected according to the reviewer’s comment
Line 164: “in Greece, in an attempt to cover the largest 164 part of poultry production in the country”. delete please
Re: corrected according to the reviewer’s comment
Line 169: replace hrs by h
Re: corrected according to the reviewer’s comment
Line 177: correct 37°C
Re: corrected according to the reviewer’s comment
Line 207: correct “ E. coli identity confirmation” as: phylogeny
The same in line 212
Re: both corrected according to the reviewer’s comment
I suggest also as an alternative to separate between the phylogeny and the antibiotic resistance.
Re: we agree with the reviewer’s suggestions and therefore the previous 2.3 was separated in two new sections, 2.3 and 2.4.
Line 243: define the total number
Re: the total number has been added as recommended by the reviewer
Line 249-254: why did you delete the %? add them please
Re: the % are now provided in the Table 2, this is the reason for deleting them from the text. However, if the reviewer insists, we can add them again in the manuscript body.
Line 251: Delete “Table 2” you should cite it one time in the manuscript.
Re: deleted, according to the reviewer’s comment.
Line 252: correct 3rd please.
Re: corrected according to the reviewer’s comment
Line 260: delete as suggested before
Re: deleted and rephrased according to the reviewer’s comment
Line 263-265: I think that there is a problem in the organization of your ideas. Try to revise please (from the beginning of the results).
Re: the sentence was deleted for clearance
Table 2: You should define what were Intermediate strains considered? Explain in the methods please.
Re: an explanation was added in the section 2.2 of the Materials and Methods in the revised manuscript
Add the percentage for each antibiotic (broilers, hens).
Re: the percentages were added as recommended by the reviewer
Line 294: “whereas it was not detected in laying hens (Figure 2)”. Delete please
Re: deleted according to the reviewer’s comment
Line 308: Systemic; delete please
Re: this part was deleted in the context of the general revision and shortening of the discussion
Line 329: Delete “quinolones” and reformulate please
Re: the sentence was reformulated as follows: “Increased rates of E. coli resistant strains to quinolones isolated from farm animals are often observed…”, in accordance to the reviewer’s comment
Line 333: delete table 2.
Re: deleted according to the reviewer’s comment
Line 339-340: the sentence repeat word by word what was written in line 332-334.
Re: the repeated sentence was deleted from the first mentioning and the whole paragraph was reformulated
Line 344: delete “the” before resistance levels
Re: deleted according to the reviewer’s comment
Line 347-349: What do you mean here?
Re: the sentence was rephrased for clearance as follows: “…resistant phenotypes are occasionally produced by non resistant genotypes”
Line 350: “ data on antimicrobial resistance in laying hens is less available” where ? I don’t think so. be careful please
Re: the sentence was deleted in accordance to the reviewer’s comment
Line 354: “stands for...” revise please
Re: replaced by “was”, in accordance to the reviewer’s comment
Line 356: replace “levels” by “a level”
Re: corrected according to the reviewer’s comment
Line 366: Although it’s not very clear if ESBL production from E. coli represents a major... It my be
Re: corrected according to the reviewer’s comment
Line 379: “a study to lying...” revise please
Re: replaced with “concerning”,in accordance to the reviewer’s comment
At last I insist that the discussion needs a substantial revision
Re: the discussion has been substantially revised. We hope that this version satisfies the reviewer’s expectations
Conclusion
What are the main results, Resistance? genes?
Revise please by adding the main results of your study
Re: according to our opinion, the most noteworthy finding or our study is the identification of the resistance gene, and therefore this statement was further highlighted in the revised Conclusions section
At last, I recommend to the authors to revise substantially all the manuscript.
Re: we trust that after implementing all the suggestions of the reviewer and substantially revising the entire manuscript, it has been deeply upgraded and will satisfy the reviewer’s expectations.
Reviewer 3 Report
Dear authors,
You have improved the manuscript immensely after the first revision. It is now much clearer and easier to follow the thought, although there are still a few sentences which require rephrasing. I suggest acceptance of this manuscript for publication after a minor revision.
154-155 the samples were received by a walk-through of the poultry chamber - what do you consider under the 'poultry chamber'?
190 not necessary to put the manufacturer twice (Oxoid Ltd., Basingstoke, UK)
202 inducer of what?
231 target
Author Response
Dear authors,
You have improved the manuscript immensely after the first revision. It is now much clearer and easier to follow the thought, although there are still a few sentences which require rephrasing. I suggest acceptance of this manuscript for publication after a minor revision.
Response: We are grateful to the reviewer for recognizing our effort to improve the manuscript. All her/his final recommended corrections have been embedded in the revised manuscript.
154-155 the samples were received by a walk-through of the poultry chamber - what do you consider under the 'poultry chamber'?
Re: the term “poultry chamber” was replaced by the term “broiler or the hen house unit” for clearance, as mentioned by the reviewer
190 not necessary to put the manufacturer twice (Oxoid Ltd., Basingstoke, UK)
Re: corrected according to the reviewer’s comment
202 inducer of what?
Re: “of antibiotic resistance”, added in the revised manuscript according to the reviewer’s comment
231 target
Re: corrected according to the reviewer’s comment
Round 3
Reviewer 1 Report
I would thank the authors for their efforts. the manscript is better like this.
I have just some "minor coments":
Line 21: correcte as: ...91 poultry farm including 75 broiler farms and 16 laying hen farms, originating.....
Line 23: sulfamethoxazole (s in lower case)
Line 51:" Also,....described." delete thissentence please
Line 54: "E. coli..." add as a new paragraph in a new line
Line 72-74: "whereas....daiey farm [18]"delete please. the ESBL are defined in Line 75 and you can not begin with he prevalence and return to the definition.
Line 75: replace however byanother adaptedadverd (furthemore, moreover...)
Line 225: "as previously characterized [58]." delete please (results not discussion)
Line 249: correct as: "According to the EFSA, data."
Line 280: correct as: "...Asian countries and Algeria with a percentage..."
Line 282: correct as: "...are in general in agreement..."
Author Response
We are grateful to the reviewer who really helped us to improve the quality of our manuscript. All minor comments have been implemented in the 3rd version, which now is certainly improved. The changes are shown with the "track changes" tool in the revised version
