Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for the opportunity to evaluate the work entitled

“Environmental Dissemination of Antimicrobial-Resistant Escherichia coli and Salmonella spp. in Moroccan Broiler Farms”

After reading the manuscript, I leave below some suggestions for improvements so that the work can be considered for publication

 

1. Both in the title and in other points of the text, the scientific names of the species are not spelled correctly. Therefore, it is recommended to review and correct these points.

 

2. In fact, from the One Health perspective, animal husbandry has a great impact on selection, persistence, and spread of resistance, which directly influences the increase in antimicrobial resistance. But, in the introduction, the authors should emphasize this point a little more, such as the indiscriminate use of these chemotherapy drugs as promoters of animal growth.

 

3. The authors do not clearly present how the samples were collected. Some points should be better explained, such as: Were the collections carried out monthly, with regular spacing on days? How many samples were taken at each collection point (broiler farm)? In all, six environmental matrices (poultry manure, drinking troughs, farm soil, soil around 200 m from the farm, soil around 500 m from the farm, and well water) how many samples (1 or more?) were collected. In the months following the first collections, were the other collections carried out in the same location in each matrices? Were they always the same birds during the period? Or were there a harvest and reintroduction of new crops during the collection period? What was the basic diet of these birds? Were these birds treated with antibiotics for growth promotion?

 

4. Authors should verify whether an ethics committee involved in animal studies is necessary.

 

5. In the methodology, the authors describe different numbers of samples between collections. These variations should be better clarified.

 

6. In antibiogram assays, the authors use coli ATCC 25922. It would be important to have a standard strain for Salmonella as well.

 

7. In lines 495-497 the authors write: Multidrug resistance was defined as resistance to at least one antimicrobial agent in three or more antimicrobial classes. Intermediate isolates were considered non-resistant for statistical analysis, while resistant isolates were coded as resistant. Especially the isolated "intermediates" were not clear how they were classified in this category. It is recommended that authors present bibliographic references that support this classification criterion.

 

8. Table 1 of the results is not referenced in the text. Authors should reference it.

 

9. Figure 4 should be improved in terms of resolution and legend, in addition to explaining why in some months there no presentation of data is, such as in January and December 2024.

 

10. Because Table 3 represents 14 months for some antibiotics while for others 13 months are represented. What factor reflected in this time difference?

 

11. The authors rightly point out the limitations of the work, a very important point. However, it would be of great importance to apply some genotyping method of these isolates, such as ERIC PCR, for example. This data would greatly enrich the work, especially in the discussion, and, consequently, the conclusions.

Author Response

Point-by-Point Response to Reviewer 1

Manuscript: Phenotypic Antimicrobial Resistance in Escherichia coli and Salmonella spp. Recovered from Broiler-Farm Environmental Matrices in Morocco

Dear Reviewer,

We sincerely thank you for your careful evaluation and constructive recommendations. Your comments helped us improve the scientific nomenclature, One Health context, sampling description, quality-control explanation, MDR methodology, figures, tables, ethics statement, and limitations of the manuscript. Each reviewer comment is reproduced verbatim below, followed by our response and the exact location of the revision.

Revision package

Document	Purpose
Revised manuscript with Track Changes	Includes all revisions made in response to Reviewers 1 and 2, including nomenclature, sampling clarification, Table 1 citation, quality-control wording, MDR reference, figures, and limitations.
Supplementary Table S1	Provides row-level sampling metadata, farm-by-matrix and month-by-matrix distributions, sampling dates, and monthly sample/isolate denominators.
Supplementary Table S2	Provides isolate-level AST results, colistin MIC patterns, categorical interpretations, MDR classifications, class profiles, definitions, and breakpoint notes.

Reviewer comments

Comment 1

Reviewer comment:

Both in the title and in other points of the text, the scientific names of the species are not spelled correctly. Therefore, it is recommended to review and correct these points.

Response:

We thank the reviewer for identifying this issue. Scientific nomenclature was reviewed throughout the manuscript. The title and text now consistently use Escherichia coli at first mention, E. coli thereafter where appropriate, and Salmonella spp. when referring to multiple or unidentified members of the genus. Genus and species names are italicized, whereas the abbreviation “spp.” remains in roman type. Capitalization and abbreviated forms were also standardized in headings, tables, and figure captions.

Changes in the manuscript:

Title, lines 2–3; scientific nomenclature checked throughout the Abstract, Introduction, Results, Discussion, Methods, tables, and figure captions, lines 15–432.

Comment 2

Reviewer comment:

In fact, from the One Health perspective, animal husbandry has a great impact on selection, persistence, and spread of resistance, which directly influences the increase in antimicrobial resistance. But, in the introduction, the authors should emphasize this point a little more, such as the indiscriminate use of these chemotherapy drugs as promoters of animal growth.

Response:

We thank the reviewer and agree that the livestock contribution to antimicrobial selection pressure should be stated more clearly. The Introduction now explains that antibiotic growth promoters were historically used in intensive broiler production to improve feed conversion, growth performance, and disease control, and it connects veterinary antimicrobial use with the broader One Health resistance-selection landscape. We used the term “antimicrobials” rather than “chemotherapy drugs” to maintain standard veterinary and AMR terminology and avoided implying that antimicrobial use was measured on the participating farms.

Changes in the manuscript:

Introduction, lines 51–58.

Comment 3

Reviewer comment:

The authors do not clearly present how the samples were collected. Some points should be better explained, such as: Were the collections carried out monthly, with regular spacing on days? How many samples were taken at each collection point (broiler farm)? In all, six environmental matrices (poultry manure, drinking troughs, farm soil, soil around 200 m from the farm, soil around 500 m from the farm, and well water) how many samples (1 or more?) were collected. In the months following the first collections, were the other collections carried out in the same location in each matrices? Were they always the same birds during the period? Or were there a harvest and reintroduction of new crops during the collection period? What was the basic diet of these birds? Were these birds treated with antibiotics for growth promotion?

Response:

We thank the reviewer for this detailed and important comment. The sampling framework has been substantially expanded. Sampling was conducted on 35 separate dates between 24 November 2023 and 28 April 2025, with intervals of 1–63 days; therefore, collections were not monthly or regularly spaced. All 13 farms were visited repeatedly, with 39–46 samples collected per farm on 10–17 distinct dates. A total of 543 samples were collected: poultry manure (n = 78), drinking troughs (n = 79), farm soil (n = 98), soil approximately 200 m from the poultry houses (n = 79), soil approximately 500 m from the poultry houses (n = 79), and well water (n = 130). One or more matrices were collected at each visit according to availability, so sampling was not balanced across farms, dates, or matrices. The study followed environmental matrices rather than individual birds or identified flocks; repeated visits could occur during successive commercial broiler-production cycles. Flock diet and farm-level antimicrobial-use histories were not recorded and therefore cannot be added retrospectively. This absence is stated transparently as a limitation. Supplementary Table S1 provides the complete row-level metadata and farm/month/matrix distributions.

Changes in the manuscript:

Methods Sections 4.1–4.2, lines 290–316; Discussion limitations, lines 278–286; Supplementary Table S1.

Comment 4

Reviewer comment:

Authors should verify whether an ethics committee involved in animal studies is necessary.

Response:

We thank the reviewer. We verified the nature of the study and clarified the ethics statement. The work involved environmental samples only. No animals were handled, experimentally treated, sampled directly, restrained, or subjected to an intervention, and no human participants were included. Consequently, animal-study ethics approval was not applicable. The Institutional Review Board Statement and Informed Consent Statement were reformatted accordingly.

Changes in the manuscript:

Institutional Review Board and Informed Consent statements, lines 427–430.

Comment 5

Reviewer comment:

In the methodology, the authors describe different numbers of samples between collections. These variations should be better clarified.

Response:

We agree and have clarified the reason for the unequal numbers. Sampling intensity varied because visits depended on farm accessibility, active broiler-production cycles, biosecurity requirements, field logistics, and matrix availability. The design was purposive and unbalanced rather than a fixed monthly panel. The manuscript now reports the total number for each matrix, the range of samples and dates per farm, and explicitly directs readers to Supplementary Table S1 for farm-by-matrix, month-by-matrix, and monthly denominator summaries.

Changes in the manuscript:

Methods Sections 4.1–4.2, lines 290–316; Supplementary Table S1.

Comment 6

Reviewer comment:

In antibiogram assays, the authors use coli ATCC 25922. It would be important to have a standard strain for Salmonella as well.

Response:

We thank the reviewer for raising this quality-control point. We respectfully clarify that CLSI M100 Table 2A designates E. coli ATCC 25922 as the routine quality-control strain for Enterobacterales disk-diffusion testing; this group includes both E. coli and Salmonella spp. A separate Salmonella reference strain is therefore not required for routine verification of the disks, medium, and test performance used in this study. To remove ambiguity, the Methods now explicitly state that E. coli ATCC 25922 was used to monitor disk-diffusion performance for both bacterial groups. In addition, positive and negative controls were included in each Salmonella real-time PCR confirmation run.

Changes in the manuscript:

Molecular confirmation and AST quality control, lines 342–360; CLSI M100 is cited as Reference 48.

Comment 7

Reviewer comment:

In lines 495-497 the authors write: Multidrug resistance was defined as resistance to at least one antimicrobial agent in three or more antimicrobial classes. Intermediate isolates were considered non-resistant for statistical analysis, while resistant isolates were coded as resistant. Especially the isolated "intermediates" were not clear how they were classified in this category. It is recommended that authors present bibliographic references that support this classification criterion.

Response:

We thank the reviewer and have clarified the terminology and coding. Intermediate and susceptible-dose-dependent isolates were not reclassified as susceptible; their original categories remain visible in the isolate-level Supplementary Table S2. For binary resistance analyses, they were coded 0 because only the CLSI resistant category was treated as resistance. They were likewise not counted toward MDR, which required resistance to at least one agent in three or more antimicrobial classes. The exact S/I/SDD/R criteria are provided in Supplementary Table S2. CLSI M100 supports the categorical interpretations, and the internationally accepted MDR definition is now cited to Magiorakos et al.

Changes in the manuscript:

AST interpretation and MDR definition, lines 368–384; statistical coding, lines 391–395; References 48–49; Supplementary Table S2.

Comment 8

Reviewer comment:

Table 1 of the results is not referenced in the text. Authors should reference it.

Response:

We thank the reviewer. Table 1 is now explicitly cited in the Results immediately after the matrix-specific detection findings.

Changes in the manuscript:

Results Section 2.1, lines 82–89.

Comment 9

Reviewer comment:

Figure 4 should be improved in terms of resolution and legend, in addition to explaining why in some months there no presentation of data is, such as in January and December 2024.

Response:

We agree. Figure 4 was regenerated at publication quality with larger labels, separate panels for E. coli and Salmonella spp., a clearer legend, and a caption defining all antimicrobial abbreviations and denominators. The text now explains that monthly percentages are descriptive and that a percentage cannot be calculated when no isolate of the corresponding bacterial group was recovered and tested in a month. Months without an isolate denominator are therefore not represented as zero resistance. The full monthly sample and isolate denominators are supplied in Supplementary Table S1.

Changes in the manuscript:

Results Section 2.5 and revised Figure 4, lines 154–180; Supplementary Table S1.

Comment 10

Reviewer comment:

Because Table 3 represents 14 months for some antibiotics while for others 13 months are represented. What factor reflected in this time difference?

Response:

We thank the reviewer. The earlier inconsistency reflected months in which no isolate was available for a bacterial group or in which an organism–agent result could not be categorically interpreted, so a monthly resistance percentage or stable model estimate could not be produced. To avoid misleading comparisons, the former month-count presentation was replaced. Revised Table 3 now reports one mixed-effects model summary per eligible antimicrobial agent, while raw screening-only agents are clearly marked and excluded from modeling. Monthly denominators are reported separately in Supplementary Table S1, and models with sparse outcomes or unstable estimates were not interpreted.

Changes in the manuscript:

Results Section 2.6 and revised Table 3, lines 183–200; Statistical analysis, lines 391–405; Supplementary Table S1.

Comment 11

Reviewer comment:

The authors rightly point out the limitations of the work, a very important point. However, it would be of great importance to apply some genotyping method of these isolates, such as ERIC PCR, for example. This data would greatly enrich the work, especially in the discussion, and, consequently, the conclusions.

Response:

We agree that genotyping would substantially strengthen source attribution and the assessment of relatedness. However, ERIC-PCR, multilocus sequence typing, or whole-genome sequencing data were not available for the complete isolate collection and could not be generated retrospectively during the revision. We therefore did not infer clonality, persistence, transmission, or dissemination pathways. The manuscript was reframed as a phenotypic environmental baseline study, mechanistic statements were reduced, and the absence of genotyping was emphasized as a major limitation. Future work is specifically directed toward serotyping, whole-genome sequencing, plasmid analysis, hydrological assessment, and environmental source tracking, which would provide higher-resolution evidence of relatedness and transmission.

Changes in the manuscript:

Revised title, lines 2–3; Introduction, lines 64–72; Discussion and limitations, lines 263–286; Conclusions, lines 407–413.

Closing statement

We again thank Reviewer 1 for the constructive recommendations. We believe that the revised manuscript, together with the expanded supplementary tables, now presents the study design, analytical choices, phenotypic scope, and limitations more transparently and accurately.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

I have read the manuscript entitled “Environmental Dissemination of Antimicrobial-Resistant Escherichia coli and Salmonella spp. in Moroccan Broiler Farms”. The topic is relevant for the scope of the journal, as antimicrobial-resistant Enterobacterales in poultry-farm environments are important from a One Health perspective. The study includes a useful set of environmental matrices, including poultry manure, drinking troughs, farm soil, surrounding soils and well water, and the sample size is potentially valuable. However, in its present form, the manuscript requires major revision before it can be considered for publication.

The main strength of the study is that it attempts to assess both E. coli and Salmonella spp. across several environmental compartments of broiler farms in Morocco. The detection of multidrug-resistant isolates in manure, drinking troughs, farm soil and well water is relevant and could provide useful baseline data for environmental AMR surveillance. Nevertheless, the current manuscript has substantial methodological, analytical, interpretative and language-related weaknesses.

First, the sampling design needs to be described in much greater detail. The authors state that 543 environmental samples were collected from 13 broiler farms, but the manuscript does not clearly explain how farms were selected, how many samples were collected per farm, whether sampling was balanced across matrices and months, whether the same farms were repeatedly sampled, or whether multiple samples from the same farm and month were treated as independent observations. This is essential because the analysis currently treats isolates and detections as independent units, although clustering by farm, sampling date and matrix is likely. The lack of a clear sampling framework limits the interpretation of prevalence, geographical distribution, monthly variation and temporal trends.

Second, bacterial identification is insufficiently robust for the level of inference made. E. coli and Salmonella spp. were identified using culture methods and API 20E, but the manuscript does not report molecular confirmation, serotyping or WGS. This is particularly important for Salmonella, where species/subspecies confirmation and serovar identification would substantially improve the value of the study. Without serovar data, it is difficult to interpret the epidemiological and public-health relevance of the Salmonella isolates. The authors should at least acknowledge this as a major limitation and, ideally, provide serotyping or molecular confirmation.

Third, the antimicrobial susceptibility testing section requires important clarification and correction. The authors used the Kirby–Bauer disk diffusion method and CLSI 2021 interpretation. However, the manuscript should specify the exact CLSI document used, the relevant breakpoint tables, and whether all tested agents have appropriate interpretive criteria for E. coli and Salmonella. This is especially important for azithromycin, colistin and some β-lactams. The use of colistin disk diffusion is problematic because disk diffusion is not reliable for colistin susceptibility testing. Although the authors state that reduced-susceptibility isolates were further tested by MIC using MicroScan, it is unclear how screening was performed, how many isolates were retested, what MIC breakpoints were used, and whether broth microdilution was compliant with accepted standards. The colistin results should be interpreted very cautiously or removed unless the confirmatory method is fully documented.

Fourth, the analysis of monthly resistance trends is not sufficiently convincing in its current form. Logistic regression using month as a continuous predictor may be inappropriate if sampling was uneven across months, farms, matrices and bacterial counts. The manuscript itself notes that monthly sample numbers were not necessarily equal, but then proceeds to infer increasing and decreasing temporal trends. These models should account for farm, matrix and sampling intensity, or the authors should present the monthly data descriptively only. At minimum, the number of isolates tested per month and per matrix should be shown, and the limitations of the trend analysis should be discussed more explicitly.

Fifth, the manuscript repeatedly implies environmental dissemination beyond the poultry-house area, but the study design does not prove dissemination pathways. The detection of resistant bacteria in soil at 200 m or 500 m and in well water is important, but it does not demonstrate directionality, source attribution or spread from the farms. These findings should be described as “consistent with possible environmental dissemination” or as “detection in external environmental matrices”, rather than as evidence of dissemination. Without strain typing, genomic comparison, hydrological data, manure-management data or environmental-source tracking, transmission pathways cannot be inferred.

Sixth, the MDR analysis should be strengthened. The manuscript defines MDR as resistance to at least one agent in three or more antimicrobial classes, which is appropriate. However, the actual class-level MDR profiles are not shown. The authors should provide a table or supplementary table listing the resistance profiles by isolate or at least the most frequent MDR class combinations for E. coli and Salmonella. Reporting only the proportion of MDR isolates by matrix is not sufficient to understand the resistance burden.

Seventh, the manuscript lacks genotype-level information. For a paper submitted to Antibiotics and framed around environmental dissemination of AMR, phenotypic AST alone provides only a partial picture. The absence of resistance-gene detection, integron detection, plasmid analysis or WGS substantially limits mechanistic interpretation. The authors should either add molecular resistance data or clearly reframe the manuscript as a phenotypic baseline surveillance study. Statements about resistance genes, mobile genetic elements, plasmids, co-selection and horizontal gene transfer should be kept as background context unless directly investigated.

Eighth, the Discussion is too broad and often speculative. Several paragraphs discuss antimicrobial residues, plasmids, ARG transfer, groundwater contamination, food-chain entry and public-health risk, but these were not directly measured. The discussion should be shortened and focused more tightly on the actual findings: culture-based detection and phenotypic resistance in environmental samples. Claims about pathways such as runoff, dust, insects, footwear, manure handling, water systems and groundwater exposure should be framed as hypotheses requiring further study.

Ninth, the manuscript requires substantial language editing. There are many grammatical errors, awkward phrases, punctuation problems and formatting inconsistencies throughout the Abstract, Results, Discussion and Methods. Examples include “may circulate between animals”, “as result those sources”, “among Salmonella isolates, 84/127 ,66.1%”, “resistance mainly observed against 73.2% nalidixic acid”, “water systems that contains antibiotics as growth promoters”, “For Salmonella, MDR was also detected in 66.10% of isolates and in Lebanese 89.7% MDR found in poultry products”, and several incomplete or poorly structured sentences. The manuscript should undergo professional English editing before reconsideration.

Specific points requiring revision:

1. Clarify whether the study period ended in April or May 2025. The Abstract states sampling was conducted until 5 May 2025, whereas several Results sections describe detections from November 2023 to April 2025.
2. Clarify how the 13 farms were selected and whether they are representative of the region.
3. Provide farm-level and month-level sample counts for each matrix.
4. Clarify whether one isolate per positive sample was retained or whether multiple colonies/isolates could be obtained from a single sample.
5. Provide more details on API 20E confirmation and quality-control procedures.
6. For Salmonella, add serovar identification if possible. If not possible, explicitly state this as a limitation.
7. Correct the figure numbering. The manuscript refers to Figure 3 for geographical distribution, but the displayed figure is labelled Figure 2. Similar inconsistencies appear later.
8. Avoid using “prevalence” when the denominator is not population-based or when the sampling design was not representative. “Detection rate” may be more appropriate in several places.
9. Provide exact AST breakpoints and clarify how intermediate isolates were handled. The Methods state that intermediate isolates were considered non-resistant, which is acceptable if consistently applied.
10. Reconsider the inclusion and interpretation of colistin disk diffusion results. Colistin requires MIC-based methods.
11. Provide the number of isolates tested by month for the temporal resistance analysis.
12. Avoid overinterpreting logistic regression trends if sampling was uneven and clustered.
13. Provide class-level MDR profiles and not only MDR proportions.
14. Rephrase “broiler-farm environments can act as reservoirs” to “may act as reservoirs” unless persistence or transmission was directly demonstrated.
15. Shorten and focus the Discussion; several literature comparisons are inserted awkwardly and do not integrate well with the authors’ own data.
16. Correct the Funding statement. It currently begins with “This review is a part of…”, although the manuscript is an original research article.
17. Correct the institutional and informed consent statements; the formatting is currently incorrect.
18. Ensure consistency in region naming: the Abstract mentions Rabat–Sale–Zemmour–Zaer, whereas the Methods mention Rabat–Salé–Kénitra.
19. Check all percentages and punctuation in the Abstract and Results.
20. The manuscript should be carefully edited by a fluent scientific English speaker.

Overall, the topic is relevant and the dataset could be useful, but the current manuscript is not yet suitable for publication. The authors should substantially revise the sampling description, bacterial confirmation, AST interpretation, temporal analysis, MDR reporting, and discussion of dissemination pathways. The manuscript should also be reframed as a phenotypic environmental baseline study unless molecular AMR data are added.

Author Response

Point-by-Point Response to Reviewer 2

Manuscript: Phenotypic Antimicrobial Resistance in Escherichia coli and Salmonella spp. Recovered from Broiler-Farm Environmental Matrices in Morocco

Dear Reviewer,

We sincerely thank you for the careful evaluation and constructive recommendations. Your comments helped us improve the methodological transparency, analytical rigor, interpretation, supplementary reporting, and language of the manuscript. Reviewer comments are reproduced verbatim below, followed by our response and the location of each revision.

Line-number note: Line numbers refer to the Final/No Markup view of the revised manuscript. The submitted manuscript contains true Word Track Changes; selecting “Final” or “No Markup” displays the line numbering cited in this response.

Revision package

Document	Purpose
Revised manuscript with Track Changes	All textual revisions are recorded as true Word insertions/deletions. Figures 1, 3, and 4 and the numerical tables were updated.
Supplementary Table S1	Row-level sampling metadata plus farm-by-matrix, month-by-matrix, and monthly denominator summaries.
Supplementary Table S2	Raw and isolate-level AST, colistin MIC, revised MDR and class profiles, definitions, and exact breakpoint notes.

General comments

Opening assessment

Reviewer comment:

I have read the manuscript entitled “Environmental Dissemination of Antimicrobial-Resistant Escherichia coli and Salmonella spp. in Moroccan Broiler Farms”. The topic is relevant for the scope of the journal, as antimicrobial-resistant Enterobacterales in poultry-farm environments are important from a One Health perspective. The study includes a useful set of environmental matrices, including poultry manure, drinking troughs, farm soil, surrounding soils and well water, and the sample size is potentially valuable. However, in its present form, the manuscript requires major revision before it can be considered for publication.

The main strength of the study is that it attempts to assess both E. coli and Salmonella spp. across several environmental compartments of broiler farms in Morocco. The detection of multidrug-resistant isolates in manure, drinking troughs, farm soil and well water is relevant and could provide useful baseline data for environmental AMR surveillance. Nevertheless, the current manuscript has substantial methodological, analytical, interpretative and language-related weaknesses.

Response:

We thank Reviewer 2 for recognizing the relevance of the One Health topic, the range of environmental matrices, and the potential value of the dataset. In response to the major-revision recommendation, we substantially revised the sampling description, bacterial confirmation, AST interpretation, temporal analysis, MDR reporting, interpretation of external environmental detections, Discussion, and English language. We also added two detailed supplementary tables: Supplementary Table S1 provides the complete sampling framework and monthly denominators, and Supplementary Table S2 provides isolate-level AST, colistin MIC, MDR, class-profile, and breakpoint information.

Changes in the manuscript:

Title and Abstract, lines 2–40; Introduction, lines 46–72; Results, lines 74–233; Discussion, lines 237–285; Materials and Methods, lines 287–401; Supplementary Materials statement, lines 410–413.

Major comments

Major comment 1

Reviewer comment:

First, the sampling design needs to be described in much greater detail. The authors state that 543 environmental samples were collected from 13 broiler farms, but the manuscript does not clearly explain how farms were selected, how many samples were collected per farm, whether sampling was balanced across matrices and months, whether the same farms were repeatedly sampled, or whether multiple samples from the same farm and month were treated as independent observations. This is essential because the analysis currently treats isolates and detections as independent units, although clustering by farm, sampling date and matrix is likely. The lack of a clear sampling framework limits the interpretation of prevalence, geographical distribution, monthly variation and temporal trends.

Response:

We thank the reviewer for this important comment. Sections 4.1 and 4.2 now describe the study as purposive, non-probability sampling of farms listed in official registries, selected according to access and availability during active production cycles. We explicitly state that the 13 farms are not statistically representative of all farms in the region. All farms were sampled repeatedly; farm totals ranged from 39 to 46 samples collected on 10 to 17 distinct dates. Sampling was irregular and unbalanced across farms, months, and matrices. Supplementary Table S1 now contains row-level metadata, farm-by-matrix counts, month-by-matrix counts, and monthly sample and isolate denominators. Detection counts and rates are treated descriptively. For resistance-trend models, matrix was included as a fixed effect and farm as a random intercept; a farm–date visit effect was evaluated in sensitivity analyses.

Changes in the manuscript:

Sections 2.2 and 4.1–4.2, lines 89–104 and 288–313; Section 4.8, lines 381–401; Supplementary Materials, lines 410–413.

Major comment 2

Reviewer comment:

Second, bacterial identification is insufficiently robust for the level of inference made. E. coli and Salmonella spp. were identified using culture methods and API 20E, but the manuscript does not report molecular confirmation, serotyping or WGS. This is particularly important for Salmonella, where species/subspecies confirmation and serovar identification would substantially improve the value of the study. Without serovar data, it is difficult to interpret the epidemiological and public-health relevance of the Salmonella isolates. The authors should at least acknowledge this as a major limitation and, ideally, provide serotyping or molecular confirmation.

Response:

We thank the reviewer. The identification procedures have been expanded. API 20E incubation, APIweb interpretation, repeat testing of doubtful or low-discrimination profiles, and isolate storage are now specified. Importantly, all biochemically identified Salmonella spp. isolates were additionally confirmed using the innuDETECT Salmonella spp. real-time PCR assay, with DNA extracted from pure cultures and positive and negative controls included in each run. Serovar identification and WGS were not available for this phenotypic study. This limitation is now stated explicitly in both Methods and Discussion, and epidemiological and transmission claims have been reduced accordingly.

Changes in the manuscript:

Sections 4.3–4.5, lines 314–345; Discussion limitations, lines 277–285.

Major comment 3

Reviewer comment:

Third, the antimicrobial susceptibility testing section requires important clarification and correction. The authors used the Kirby–Bauer disk diffusion method and CLSI 2021 interpretation. However, the manuscript should specify the exact CLSI document used, the relevant breakpoint tables, and whether all tested agents have appropriate interpretive criteria for E. coli and Salmonella. This is especially important for azithromycin, colistin and some β-lactams. The use of colistin disk diffusion is problematic because disk diffusion is not reliable for colistin susceptibility testing. Although the authors state that reduced-susceptibility isolates were further tested by MIC using MicroScan, it is unclear how screening was performed, how many isolates were retested, what MIC breakpoints were used, and whether broth microdilution was compliant with accepted standards. The colistin results should be interpreted very cautiously or removed unless the confirmatory method is fully documented.

Response:

We thank the reviewer for identifying this critical issue. The revised manuscript now specifies CLSI M100, 31st edition (2021), Table 2A and the associated Salmonella footnotes. Exact interpretive criteria and agent-specific notes are provided in Supplementary Table S2. Intermediate and susceptible-dose-dependent categories were consistently treated as non-resistant. Direct amoxicillin and azithromycin disk results are retained only as raw screening observations and are excluded from categorical summaries, temporal models, and MDR classification because applicable general CLSI criteria were unavailable. Salmonella levofloxacin disk results are likewise not categorically interpreted. Colistin was not interpreted by disk diffusion. All 317 isolates were tested by the CLSI colistin broth disk elution MIC method. The revised Methods specify concentrations, inoculum, incubation, MIC reading, and Enterobacterales criteria (intermediate at MIC ≤2 µg/mL; resistant at MIC ≥4 µg/mL; no susceptible category). The Results, figures, MDR calculations, and Supplementary Table S2 were updated accordingly.

Changes in the manuscript:

Section 2.4, lines 121–151; Sections 4.6–4.7, lines 346–380; Supplementary Materials, lines 410–413.

Major comment 4

Reviewer comment:

Fourth, the analysis of monthly resistance trends is not sufficiently convincing in its current form. Logistic regression using month as a continuous predictor may be inappropriate if sampling was uneven across months, farms, matrices and bacterial counts. The manuscript itself notes that monthly sample numbers were not necessarily equal, but then proceeds to infer increasing and decreasing temporal trends. These models should account for farm, matrix and sampling intensity, or the authors should present the monthly data descriptively only. At minimum, the number of isolates tested per month and per matrix should be shown, and the limitations of the trend analysis should be discussed more explicitly.

Response:

We agree. Monthly detections and monthly resistance percentages are now presented as descriptive summaries, with explicit warnings that sampling totals and matrix composition varied substantially among months. The number of samples and isolates tested per month and matrix is supplied in Supplementary Table S1. The inferential analysis was revised to mixed-effects logistic regression with sampling matrix as a fixed effect and farm as a random intercept. A visit-level random intercept was assessed in sensitivity analyses. Models were restricted to outcomes with sufficient resistant and non-resistant observations, and Benjamini–Hochberg correction was applied by species. The text now refers to adjusted monthly associations rather than definitive temporal trends or seasonality.

Changes in the manuscript:

Sections 2.2 and 2.5–2.6, lines 89–104 and 153–199; Section 4.8, lines 381–401; Supplementary Materials, lines 410–413.

Major comment 5

Reviewer comment:

Fifth, the manuscript repeatedly implies environmental dissemination beyond the poultry-house area, but the study design does not prove dissemination pathways. The detection of resistant bacteria in soil at 200 m or 500 m and in well water is important, but it does not demonstrate directionality, source attribution or spread from the farms. These findings should be described as “consistent with possible environmental dissemination” or as “detection in external environmental matrices”, rather than as evidence of dissemination. Without strain typing, genomic comparison, hydrological data, manure-management data or environmental-source tracking, transmission pathways cannot be inferred.

Response:

We fully agree. The manuscript no longer presents detection in surrounding soils or well water as proof of dissemination from poultry houses. The title was reframed around phenotypic resistance, and the Abstract, Results, Discussion, and Conclusion now use cautious terms such as “detection in external environmental matrices” and explicitly state that source attribution, directionality, persistence, and transmission cannot be determined. Runoff, dust, insects, equipment, footwear, and manure transport are mentioned only as untested hypotheses for future work.

Changes in the manuscript:

Abstract, lines 27–40; Introduction, lines 59–72; Discussion, lines 264–276; Conclusion, lines 403–409.

Major comment 6

Reviewer comment:

Sixth, the MDR analysis should be strengthened. The manuscript defines MDR as resistance to at least one agent in three or more antimicrobial classes, which is appropriate. However, the actual class-level MDR profiles are not shown. The authors should provide a table or supplementary table listing the resistance profiles by isolate or at least the most frequent MDR class combinations for E. coli and Salmonella. Reporting only the proportion of MDR isolates by matrix is not sufficient to understand the resistance burden.

Response:

We thank the reviewer. MDR reporting has been expanded at both matrix and class levels. Table 4 now gives the number and percentage of MDR isolates for each species and environmental matrix. Table 5 reports the most frequent non-mutually exclusive three-class combinations. Supplementary Table S2 additionally provides the complete isolate-level drug profile, class profile, number of resistant classes, and MDR status. MDR was recalculated after excluding agents without applicable categorical criteria, resulting in 115/190 (60.5%) MDR E. coli and 82/127 (64.6%) MDR Salmonella spp.

Changes in the manuscript:

Section 2.7 and Tables 4–5, lines 202–233; Section 4.7, lines 375–380; Supplementary Materials, lines 410–413.

Major comment 7

Reviewer comment:

Seventh, the manuscript lacks genotype-level information. For a paper submitted to Antibiotics and framed around environmental dissemination of AMR, phenotypic AST alone provides only a partial picture. The absence of resistance-gene detection, integron detection, plasmid analysis or WGS substantially limits mechanistic interpretation. The authors should either add molecular resistance data or clearly reframe the manuscript as a phenotypic baseline surveillance study. Statements about resistance genes, mobile genetic elements, plasmids, co-selection and horizontal gene transfer should be kept as background context unless directly investigated.

Response:

We agree that phenotypic AST alone does not support mechanistic or genomic conclusions. Because resistance-gene, integron, plasmid, and WGS data were not available for this dataset, the manuscript has been explicitly reframed as a phenotypic environmental baseline study. Statements concerning mobile genetic elements and horizontal transfer are limited to background context, and the revised text clearly states that these mechanisms were not investigated. The Discussion and Conclusion identify serotyping, WGS, plasmid analysis, and source tracking as future work rather than interpreting them from the current data.

Changes in the manuscript:

Title, lines 2–3; Abstract, lines 15–18; Introduction, lines 64–72; Discussion, lines 259–285; Conclusion, lines 403–409.

Major comment 8

Reviewer comment:

Eighth, the Discussion is too broad and often speculative. Several paragraphs discuss antimicrobial residues, plasmids, ARG transfer, groundwater contamination, food-chain entry and public-health risk, but these were not directly measured. The discussion should be shortened and focused more tightly on the actual findings: culture-based detection and phenotypic resistance in environmental samples. Claims about pathways such as runoff, dust, insects, footwear, manure handling, water systems and groundwater exposure should be framed as hypotheses requiring further study.

Response:

We thank the reviewer and agree. The Discussion was substantially shortened and reorganized around the actual results: principal findings, detection by matrix, phenotypic resistance, MDR and class-level profiles, One Health implications, cautious interpretation of external matrices, and strengths/limitations. Claims about antimicrobial residues, ARG transfer, groundwater contamination, food-chain entry, and specific movement pathways were removed or explicitly labeled as untested hypotheses.

Changes in the manuscript:

Revised Discussion, lines 237–285.

Major comment 9

Reviewer comment:

Ninth, the manuscript requires substantial language editing. There are many grammatical errors, awkward phrases, punctuation problems and formatting inconsistencies throughout the Abstract, Results, Discussion and Methods. Examples include “may circulate between animals”, “as result those sources”, “among Salmonella isolates, 84/127 ,66.1%”, “resistance mainly observed against 73.2% nalidixic acid”, “water systems that contains antibiotics as growth promoters”, “For Salmonella, MDR was also detected in 66.10% of isolates and in Lebanese 89.7% MDR found in poultry products”, and several incomplete or poorly structured sentences. The manuscript should undergo professional English editing before reconsideration.

Response:

We thank the reviewer. The entire manuscript was comprehensively edited for grammar, syntax, punctuation, terminology, scientific tone, and consistency. Awkward or incomplete constructions were rewritten; bacterial names, percentages, spacing, capitalization, figure captions, and section headings were standardized. The examples identified by the reviewer are no longer present.

Changes in the manuscript:

Language and formatting revised throughout the manuscript, particularly Abstract, lines 15–40; Results, lines 74–233; Discussion, lines 237–285; and Methods, lines 287–401.

Specific points

Specific comment 1

Reviewer comment:

Clarify whether the study period ended in April or May 2025. The Abstract states sampling was conducted until 5 May 2025, whereas several Results sections describe detections from November 2023 to April 2025.

Response:

We thank the reviewer. We thank the reviewer. The end date has been checked against the sampling metadata and standardized as 28 April 2025 throughout the manuscript. The study period is now consistently reported as 24 November 2023 to 28 April 2025.

Changes in the manuscript:

Abstract Methods, lines 19–26; Section 4.1, lines 289–294.

Specific comment 2

Reviewer comment:

Clarify how the 13 farms were selected and whether they are representative of the region.

Response:

We thank the reviewer. The farm-selection procedure is now described as purposive, non-probability selection from official registries based on access and availability. We explicitly state that the farms should not be regarded as statistically representative of the entire region.

Changes in the manuscript:

Section 4.1, lines 295–301.

Specific comment 3

Reviewer comment:

Provide farm-level and month-level sample counts for each matrix.

Response:

We thank the reviewer. Supplementary Table S1 now provides row-level sampling metadata, farm-by-matrix counts, month-by-matrix counts, monthly sample totals, positive detections, and isolate denominators. The manuscript summarizes the imbalance and repeated sampling.

Changes in the manuscript:

Sections 2.2 and 4.1–4.2, lines 89–104 and 295–310; Supplementary Materials, lines 410–413.

Specific comment 4

Reviewer comment:

Clarify whether one isolate per positive sample was retained or whether multiple colonies/isolates could be obtained from a single sample.

Response:

We thank the reviewer. The Methods now state that one confirmed isolate per bacterial species and per positive environmental sample was retained. When several morphologically similar colonies were present, one purified representative colony was selected. The limitation of not assessing within-sample strain diversity is acknowledged.

Changes in the manuscript:

Section 4.6, lines 347–350; Discussion limitations, lines 277–283.

Specific comment 5

Reviewer comment:

Provide more details on API 20E confirmation and quality-control procedures.

Response:

We thank the reviewer. API 20E procedures now include incubation conditions, APIweb interpretation, repeat testing of doubtful or low-discrimination profiles, and storage. Salmonella identification was additionally confirmed by real-time PCR with positive and negative controls.

Changes in the manuscript:

Sections 4.3–4.5, lines 314–345.

Specific comment 6

Reviewer comment:

For Salmonella, add serovar identification if possible. If not possible, explicitly state this as a limitation.

Response:

We thank the reviewer. Serotyping could not be added because serovar data were not available. We have explicitly stated that serovar identification was not performed and identified this as a major limitation and priority for future studies.

Changes in the manuscript:

Section 4.5, lines 338–345; Discussion limitations, lines 277–285.

Specific comment 7

Reviewer comment:

Correct the figure numbering. The manuscript refers to Figure 3 for geographical distribution, but the displayed figure is labelled Figure 2. Similar inconsistencies appear later.

Response:

We thank the reviewer. Figure references and captions were checked and standardized. The geographical distribution is consistently Figure 2; phenotypic resistance is Figure 3; and monthly resistance is Figure 4.

Changes in the manuscript:

Figure captions, lines 91–92, 115–118, 148–151, and 175–179.

Specific comment 8

Reviewer comment:

Avoid using “prevalence” when the denominator is not population-based or when the sampling design was not representative. “Detection rate” may be more appropriate in several places.

Response:

We thank the reviewer. We agree. “Detection rate” is now used for positive samples, and the manuscript explicitly states that these values are not population prevalence because the sampling design was purposive and unbalanced.

Changes in the manuscript:

Results Section 2.1, lines 76–86; monthly interpretation, lines 93–104; farm representativeness, lines 295–301.

Specific comment 9

Reviewer comment:

Provide exact AST breakpoints and clarify how intermediate isolates were handled. The Methods state that intermediate isolates were considered non-resistant, which is acceptable if consistently applied.

Response:

We thank the reviewer. The exact CLSI document, table, disk contents, organism-specific applicability, and interpretation rules are now specified. Exact breakpoints are supplied in Supplementary Table S2. Intermediate and susceptible-dose-dependent results were consistently coded as non-resistant.

Changes in the manuscript:

Sections 4.6–4.7, lines 351–380; Supplementary Materials, lines 410–413.

Specific comment 10

Reviewer comment:

Reconsider the inclusion and interpretation of colistin disk diffusion results. Colistin requires MIC-based methods.

Response:

We thank the reviewer. Colistin disk diffusion has not been used for interpretation. All isolates were assessed by the CLSI colistin broth disk elution MIC method, and the revised Methods fully document concentrations, inoculum, incubation, MIC criteria, and the absence of a susceptible category.

Changes in the manuscript:

Results, lines 126–141 and 148–151; Section 4.6, lines 366–374.

Specific comment 11

Reviewer comment:

Provide the number of isolates tested by month for the temporal resistance analysis.

Response:

We thank the reviewer. The monthly numbers of samples analyzed, positive samples, and isolates tested are now provided in Supplementary Table S1. Figure 4 and its caption explicitly identify the monthly isolate denominator.

Changes in the manuscript:

Sections 2.2 and 2.5, lines 93–104 and 153–179; Supplementary Materials, lines 410–413.

Specific comment 12

Reviewer comment:

Avoid overinterpreting logistic regression trends if sampling was uneven and clustered.

Response:

We thank the reviewer. The regression has been revised as a mixed-effects model adjusted for matrix and farm clustering. The manuscript emphasizes that estimates are adjusted associations in an irregularly sampled dataset and are not proof of seasonality, causal change, or population-level trends.

Changes in the manuscript:

Section 2.6, lines 182–199; Section 4.8, lines 387–401.

Specific comment 13

Reviewer comment:

Provide class-level MDR profiles and not only MDR proportions.

Response:

We thank the reviewer. Class-level MDR profiles are now reported in Table 5, and complete isolate-level antimicrobial and resistance-class profiles are included in Supplementary Table S2.

Changes in the manuscript:

Section 2.7 and Tables 4–5, lines 202–233; Supplementary Materials, lines 410–413.

Specific comment 14

Reviewer comment:

Rephrase “broiler-farm environments can act as reservoirs” to “may act as reservoirs” unless persistence or transmission was directly demonstrated.

Response:

We thank the reviewer. The wording has been changed to “may represent reservoirs” or “may act as environmental reservoirs.” The Conclusion also states that detection does not establish origin, persistence, or direction of spread.

Changes in the manuscript:

Abstract Conclusions, lines 36–40; Conclusion, lines 403–409.

Specific comment 15

Reviewer comment:

Shorten and focus the Discussion; several literature comparisons are inserted awkwardly and do not integrate well with the authors’ own data.

Response:

We thank the reviewer. The Discussion has been shortened, reorganized, and focused on the observed culture-based detection and phenotypic resistance results. Literature comparisons are integrated only where they directly contextualize the findings.

Changes in the manuscript:

Revised Discussion, lines 237–285.

Specific comment 16

Reviewer comment:

Correct the Funding statement. It currently begins with “This review is a part of…”, although the manuscript is an original research article.

Response:

We thank the reviewer. The Funding statement now correctly identifies the work as original research conducted within the named One Health project; the incorrect phrase “This review is a part of” has been removed.

Changes in the manuscript:

Funding, lines 414–417.

Specific comment 17

Reviewer comment:

Correct the institutional and informed consent statements; the formatting is currently incorrect.

Response:

We thank the reviewer. The declarations were reformatted according to the journal style. The Institutional Review Board Statement explains why approval was not applicable, and the Informed Consent Statement is clearly stated as not applicable.

Changes in the manuscript:

Declarations, lines 423–428.

Specific comment 18

Reviewer comment:

Ensure consistency in region naming: the Abstract mentions Rabat–Sale–Zemmour–Zaer, whereas the Methods mention Rabat–Salé–Kénitra.

Response:

We thank the reviewer. The regional name has been standardized as Rabat–Salé–Kénitra throughout the Abstract, Figure 2 caption, and Methods.

Changes in the manuscript:

Abstract, lines 15–18; Figure 2 caption, lines 115–118; Section 4.1, lines 288–301.

Specific comment 19

Reviewer comment:

Check all percentages and punctuation in the Abstract and Results.

Response:

We thank the reviewer. All percentages were recalculated and cross-checked against the revised supplementary datasets. Punctuation, spacing, denominators, and species-specific values were corrected in the Abstract and Results. MDR values were recalculated after excluding non-interpretable screening agents.

Changes in the manuscript:

Abstract Results, lines 27–35; Results Sections 2.1–2.7, lines 76–233.

Specific comment 20

Reviewer comment:

The manuscript should be carefully edited by a fluent scientific English speaker.

Response:

We thank the reviewer. The manuscript was extensively edited for scientific English, including grammar, sentence structure, punctuation, terminology, consistency, and readability. A final proofread should still be performed after any journal formatting or editorial changes.

Changes in the manuscript:

Language revised throughout, especially lines 15–428.

Overall recommendation

Overall recommendation

Reviewer comment:

Overall, the topic is relevant and the dataset could be useful, but the current manuscript is not yet suitable for publication. The authors should substantially revise the sampling description, bacterial confirmation, AST interpretation, temporal analysis, MDR reporting, and discussion of dissemination pathways. The manuscript should also be reframed as a phenotypic environmental baseline study unless molecular AMR data are added.

Response:

We thank the reviewer for the constructive overall assessment. The manuscript has been substantially revised and is now explicitly presented as a phenotypic environmental baseline study. The revised version incorporates a transparent sampling framework, molecular confirmation of Salmonella spp., corrected and fully documented AST interpretation, MIC-based colistin testing, adjusted and cautious temporal analysis, isolate- and class-level MDR reporting, focused interpretation, and comprehensive language editing.

Changes in the manuscript:

Revisions are distributed across the Abstract through Supplementary Materials, lines 15–413.

Closing statement

We appreciate the reviewer’s detailed guidance. We believe that the revised manuscript and expanded supplementary material now provide a clearer, more rigorous, and appropriately cautious phenotypic baseline assessment of antimicrobial resistance in broiler-farm environmental matrices.

Sincerely,
The Authors

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors, Thank you for considering my suggestions for improvements the manuscript. After reading the new version, I recommend it for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for the extensive revision and for the detailed point-by-point response. In my opinion, the revised manuscript has substantially improved and now addresses the main concerns raised during the previous review round.

The manuscript has been appropriately reframed as a phenotypic environmental baseline study rather than as a source-attribution or transmission study. This is an important improvement. The title, Abstract, Introduction, Discussion and Conclusion now use more cautious wording, and the authors clearly state that the study design does not allow inference of persistence, source attribution, or direction of transmission.

The sampling design is now much more transparent. The authors clarify that the farms were selected purposively, that the sample is not statistically representative of all farms in the region, that repeated sampling was performed, and that sampling was irregular and unbalanced across farms, months and matrices. The addition of Supplementary Table S1 with farm-, month- and matrix-level information is useful and improves reproducibility.

The bacterial identification section has also improved. The API 20E procedure is now described more clearly, and the additional real-time PCR confirmation of Salmonella spp. substantially strengthens the reliability of the Salmonella results. The absence of serotyping and WGS is now appropriately acknowledged as a limitation.

The antimicrobial susceptibility testing section is now much clearer and methodologically stronger. The authors specify the CLSI document and interpretive framework, use E. coli ATCC 25922 as the quality-control strain, distinguish interpretable categorical results from raw screening observations, exclude agents without applicable general CLSI criteria from categorical summaries and MDR classification, and use an MIC-based method for colistin rather than disk diffusion. This satisfactorily addresses a major concern from the previous review.

The temporal analysis has also been improved. Monthly detection and resistance patterns are now presented descriptively and interpreted cautiously. The revised mixed-effects logistic regression, with adjustment for sampling matrix and farm-level clustering, is more appropriate for the irregular sampling design. The authors also correctly avoid presenting these associations as proof of seasonality, causal change, or population-level trends.

The MDR reporting is now more informative. The addition of matrix-specific MDR results, class-level MDR combinations, and isolate-level supplementary data provides a much clearer view of the resistance burden.

Overall, I consider the revised manuscript scientifically much stronger and substantially more cautious in its interpretation. I have no further major concerns. Before publication, I recommend only a final editorial check of the reference list and formatting. For example, a few references appear duplicated or incompletely formatted and should be checked carefully.