Sentinel or Disperser? The Role of White Storks (Ciconia ciconia) in the Spread of Antibiotic-Resistant Bacteria

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Main Research Question

This study aimed to investigate the role of Ciconia ciconia in the spread of antibiotic-resistant bacteria (AMR), focusing on whether Ciconia ciconia acts as a "sentinel" (reflecting only the presence of local AMR) or a "transmitter" (facilitating the spread of AMR between different locations) during their seasonal movements between landfills (foraging grounds) and wetlands (habitats) in Spain.

2. Originality and Relevance of the Research Topic

Originality: While there are several studies on the carriage of AMR in wild birds, few have combined paired sampling of the same bird population across multiple seasons and relevant habitats with movement data. This study, combining GPS tracking data with microbiological testing, is innovative. Relevance: Whether migratory birds play a role in the spread of AMR has direct implications for AMR surveillance, public health risk assessment, and ecological management strategies. Research Gap: This study fills a gap in research that integrates the movement patterns of individual birds with comparative AMR microbiology across different habitats.

3. Comparison and Contributions to Published Studies:

This study provides spatially and temporally paired sampling data (the same white stork population was sampled at paired landfills and wetlands). This study achieved year-round sampling, covering both the autumn migration and wintering periods, a rarity in previous studies. The prevalence of Enterobacteriaceae and ESBL-producing bacteria was compared across different locations and sample types. Generalized linear models were used to assess the impact of location, sample type, and seasonal phase on AMR/MDR carriage rates.

4. Suggested Methodological Improvements:

Standardize the separation methods for water and fecal samples. Current differences in screening media  may affect the accuracy of prevalence comparisons. In addition to collecting stork feces, collect environmental samples from the landfill  to distinguish transient carriage from truly acquired resistance. Molecular typing (e.g., whole-genome sequencing, PCR) of isolates from different habitats to confirm whether the same strains are present in landfills and wetlands is key to verifying transmission.

5. Consistency of Conclusions with Evidence

The authors cautiously note in their conclusions that the evidence is insufficient to confirm that white storks are active transmitters of resistant bacteria, but indirect evidence of transmission exists in certain time periods and locations .

6. References

The references are generally relevant, but could be supplemented with a review of global wildlife AMR research from the past 3–5 years and additional research on landfill-foraging birds to enhance the comparative nature of the Discussion.

7. Additional Comments on Tables and Figures

It is recommended to include 95% confidence intervals in the resistance prevalence graphs.

Ensure that the image resolution meets journal publication requirements.

Author Response

As a note: All manuscript has been revised in order to improve English to more clearly express the research. All changes are marked in word document, while pdf is a clear version of the manuscript.

1) suggested Methodological Improvements:

Standardize the separation methods for water and fecal samples. Current differences in screening media  may affect the accuracy of prevalence comparisons. In addition to collecting stork feces, collect environmental samples from the landfill  to distinguish transient carriage from truly acquired resistance. Molecular typing (e.g., whole-genome sequencing, PCR) of isolates from different habitats to confirm whether the same strains are present in landfills and wetlands is key to verifying transmission.

Answer: Thank you very much for Your appreciation. New information has been included in the discussion section to address limitations and future studies. Lines: 491ff; 505ff; 519ff; 602ff; 630ff; 643ff

2) References

The references are generally relevant, but could be supplemented with a review of global wildlife AMR research from the past 3–5 years and additional research on landfill-foraging birds to enhance the comparative nature of the Discussion.

Answer: New information has been included in discussion section about global AMR in wildlife, with comparison of our results with other studies conducted with landfill-foraging wild birds. Lines: 495ff; 539ff; 549ff.

3) Additional Comments on Tables and Figures

It is recommended to include 95% confidence intervals in the resistance prevalence graphs.

Ensure that the image resolution meets journal publication requirements.

Answer: Thank You for Your suggestion. 95% CI has been added to most graphics. Besides, we have improved the quality and resolution of most graphics.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Cardona-Cabrera is a well-conceived and methodically executed study addressing an important One Health topic: the potential role of white storks in dispersing antimicrobial resistant bacteria (ARB) between anthropized and natural habitats. The authors integrate ecological data with microbiological and statistical analyses, and the work is valuable in its attempt to move beyond single-time-point surveillance toward longitudinal, paired-site sampling. The manuscript is well-structured, and the discussion is appropriately cautious in interpreting results. However, please read below some area for improvements:

1. Clarity in objectives vs. conclusions: while the introduction states a hypothesis about storks acting as dispersers, the conclusion centers on a lack of evidence for dispersal. It would help to explicitly restate in the Discussion that the methodology primarily tests for co-occurrence patterns, which is only an indirect indicator of dispersal.
2. Sample size imbalance: the notable disparity between landfill and wetland sample numbers (especially fecal samples) should be more prominently addressed in the limitations, as it likely reduced statistical power for wetland comparisons.
3. Water vs. fecal methodological differences: although the authors acknowledge different primary culture methods for water and feces, more discussion is needed on how this could bias species recovery and resistance prevalence.
4. Environmental context: the study could benefit from concurrent environmental sampling (landfill soils, leachate, wetland sediment) to strengthen interpretations of potential transmission pathways.
5. Figures and supplementary material: some key prevalence patterns (e.g., ESBL detection peaks) are mainly in text; clearer visual representation in main figures could improve accessibility for readers.
6. Terminology precision: occasional use of “dispersion” instead of “dispersal” may cause minor ambiguity; consistent terminology is recommended.
7. Genotypic data: while the focus is phenotypic resistance, even limited genotyping (e.g., for ESBL-positive isolates) could provide stronger evidence regarding strain similarity between sites.

Overall, in the reviewer opinion, the manuscript makes a meaningful contribution to the understanding of wild birds’ role in ARB ecology. The reviewer recommendation is moderate revisions to improve clarity on methodological constraints, to balance data presentation between landfill and wetland results, and to enhance discussion on future study design that could more directly test dispersal hypotheses.

Author Response

As a note: All manuscript has been revised in order to improve English to more clearly express the research. All changes are marked in word document, while pdf is a clear version of the manuscript.

1. Clarity in objectives vs. conclusions: while the introduction states a hypothesis about storks acting as dispersers, the conclusion centers on a lack of evidence for dispersal. It would help to explicitly restate in the Discussion that the methodology primarily tests for co-occurrence patterns, which is only an indirect indicator of dispersal.

Answer: Thank You for Your comment. We have tried to properly acknowledge and incorporate your suggestion. Line: 489ff

 

2. Sample size imbalance: the notable disparity between landfill and wetland sample numbers (especially fecal samples) should be more prominently addressed in the limitations, as it likely reduced statistical power for wetland comparisons.

Answer: Thank You for Your observation. The sections Study Limitations and further studies have been expanded to acomodate your suggestion Lines: 505ff

 

3. Water vs. fecal methodological differences: although the authors acknowledge different primary culture methods for water and feces, more discussion is needed on how this could bias species recovery and resistance prevalence.

Answer: Thank You for Your observation. The sections Study Limitations and further studies have been expanded to offer a deeper explanation about the limitation of applying two different screening Lines: 516ff; 528fff.

 

4. Environmental context: the study could benefit from concurrent environmental sampling (landfill soils, leachate, wetland sediment) to strengthen interpretations of potential transmission pathways.

Answer: Thank You for Your observation. The sections Study Limitations and further studies have been expanded to acomodate your suggestion Lines: 630ff; 638ff; 643ff.

 

5. Figures and supplementary material: some key prevalence patterns (e.g., ESBL detection peaks) are mainly in text; clearer visual representation in main figures could improve accessibility for readers.

Answer: Thank You for Your suggestion. Figures showing prevalence patterns have been modified to ease the reading of them. Besides, we have improved the quality and resolution of most graphics.

 

6. Terminology precision: occasional use of “dispersion” instead of “dispersal” may cause minor ambiguity; consistent terminology is recommended.

Answer: Thanks, this suggestion has been accepted

 

7. Genotypic data: while the focus is phenotypic resistance, even limited genotyping (e.g., for ESBL-positive isolates) could provide stronger evidence regarding strain similarity between sites.

Answer: Thank You, new paragraphs have been added to discuss the benefit of using a metagenomic approach Lines: 490ff; 602ff. We also acknowledge the need for further genotyping to try to come to any specific conclusions, although for the main objetive of this study this information could not be used for comparisons between paired locations as no ESBL-positive isolates were found in wetlands.

Author Response File: Author Response.pdf