Prevalence and Genomic Characterization of ESBL-Producing Escherichia coli in Livestock and Farmers in Catalonia, Spain

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 Prevalence and Genomic Characterization of ESBL-Producing Escherichia coli in Livestock and Farmers in a High-Density Pig Farming Area

In this study, ESBL-producing Escherichia coli were investigated in livestock and farmers using fecal samples collected from selected farms. The study relied on routine microbiological techniques and whole-genome sequencing. Although the study is field-based, the results appear unexpected, particularly the absence of ESBL-producing Escherichia coli in all 360 tested samples that collect from cattle. This finding requires further clarification, as it may reflect methodological limitations or sampling bias, potentially indicating an unintentional bias toward pigs rather than other animal species. The authors should address this issue and provide a more detailed explanation.

Following requests are necessary to be addressed to improve quality of the study.

 

Title:

• In my opinion, the title should be revised to accurately reflect the actual aim and findings of the study. The current title implies that the effect of high-density pig farming areas on the occurrence of Enterobacteriaceae in pigs, other livestock, and humans was investigated. However, the study does not directly assess this relationship, and the presented results do not provide evidence to support such a conclusion. To avoid misleading readers, any unsupported claim regarding the impact of high-density pig farming should be removed from the title or rephrased to better align with the study objectives and the evidence presented.

Abstract:

• Line 17: The first sentence of the abstract should provide a concise background that highlights the research problem and establishes the rationale for the study. It should clearly introduce the issue being addressed and support the study hypothesis before describing the study objectives or methods.
• Line 27 : The result stating, "whereas no ESBL-, AmpC-, or carbapenemase-producing isolates were identified in cattle (0/360) or poultry (0/171), and a low prevalence was observed in horses (7.0%, 15/214), is unexpected and appears inconsistent with the existing literature. In my opinion, this finding requires further explanation and discussion. The authors should clarify the possible reasons for the complete absence of resistant isolates in cattle and poultry, including considerations related to the sampling strategy, study population, geographical variation, detection methods, or other factors that may have influenced these results.
• Line 32 : The authors need to clearly describe how many samples were submitted for whole-genome sequencing (WGS), specify the animal groups from which these samples were selected, and define the criteria used for their selection. At present, this information is unclear and requires clarification to ensure transparency and reproducibility of the study.
• Line 42 : According to the results, despite the high-density pig farming environment, only a low number of ESBL-producing Escherichia coli were detected in livestock and humans. This finding suggests that pigs may not represent a significant source of ESBL-producing E. coli in this particular setting. However, this conclusion should be interpreted with caution, and the authors may need to reconsider or moderate their claims in the conclusion section, ensuring they are fully supported by the presented data.
• At the end of this section, the authors should clearly state the limitations of the study, along with recommendations for future research. This will help to properly contextualize the findings and guide further investigations in this area.

Introduction:

• The introduction should include a clearly written paragraph that supports the study hypothesis and provides a strong scientific rationale for the work. In particular, the authors should explain how pig farms may act as reservoirs for ESBL-producing Escherichia coli, including the potential role of virulence and antimicrobial resistance genes associated with mobile genetic elements. This background is essential to justify the study design and to better position the research within the existing literature.

Methos:

• Line 102: The authors need to clarify whether the samples were collected from healthy or clinically ill animals, particularly those with diarrheal symptoms, as such cases may represent a more important source of ESBL-producing Escherichia coli transmission to other animals, humans, and the environment compared to healthy animals. This information is essential for accurately interpreting the epidemiological relevance of the findings and their implications for transmission dynamics.
• Line 132: It is not clearly described from which animal groups the samples submitted for whole-genome sequencing (WGS) were selected. The authors should clearly specify the source populations and provide the criteria used for selecting the 100 samples for WGS analysis. This information is essential to ensure transparency, reproducibility, and proper interpretation of the genomic results.

 

Author Response

Comment 1: Title: In my opinion, the title should be revised to accurately reflect the actual aim and findings of the study. The current title implies that the effect of high-density pig farming areas on the occurrence of Enterobacteriaceae in pigs, other livestock, and humans was investigated. However, the study does not directly assess this relationship, and the presented results do not provide evidence to support such a conclusion. To avoid misleading readers, any unsupported claim regarding the impact of high-density pig farming should be removed from the title or rephrased to better align with the study objectives and the evidence presented.

Response 1: Thank you for pointing this out. This revised version accurately reflects the descriptive and genomic scope of the study without overstating causal relationships. We have revised the title to remove any implicit causal inference and ensure alignment with the study design:

“Prevalence and genomic characterization of ESBL-producing Escherichia coli in livestock and farmers in Catalonia, Spain”

 

Abstract:

Comment 2: Line 17: The first sentence of the abstract should provide a concise background that highlights the research problem and establishes the rationale for the study. It should clearly introduce the issue being addressed and support the study hypothesis before describing the study objectives or methods.

 

Response 2: Thank you for pointing this out. We agree that the original opening sentence did not sufficiently establish the scientific context. We have rewritten the first sentence to highlight antimicrobial resistance as a global One Health issue and clearly justify the study. A concise and focused background statement has been added to introduce the research problem before describing objectives.

We added these sentences:

Background/Objectives: Extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae represent an increasing One Health concern because food-producing animals may act as reservoirs of antimicrobial-resistant bacteria with potential transmission to humans.

 

Comment 3: Line 27: The result stating, "whereas no ESBL-, AmpC-, or carbapenemase-producing isolates were identified in cattle (0/360) or poultry (0/171), and a low prevalence was observed in horses (7.0%, 15/214), is unexpected and appears inconsistent with the existing literature. In my opinion, this finding requires further explanation and discussion. The authors should clarify the possible reasons for the complete absence of resistant isolates in cattle and poultry, including considerations related to the sampling strategy, study population, geographical variation, detection methods, or other factors that may have influenced these results.

Response 3: We thank the reviewer for this thoughtful observation. We agree that the absence of ESBL-, AmpC-, or carbapenemase-producing E. coli among cattle and poultry samples deserves further discussion. In this regard, the following sentences have been added to the discussion and the corresponding bibliography:

An unexpected finding of our study was the absence of ESBL-, AmpC-, or carbapenemase-producing E. coli among cattle and poultry samples. Although ESBL-producing Enterobacterales have been reported in both animal sectors in several countries, prevalence estimates vary considerably according to geographical location, farming systems, biosecurity practices, sampling strategies, and antimicrobial usage patterns. Therefore, these results should be interpreted within the specific epidemiological context of the study area and should not be extrapolated to broader cattle or poultry populations. Previous studies have shown substantial geographic and host-associated variability in the distribution of ESBL-producing E. coli, highlighting the importance of local ecological and management factors in shaping resistance patterns.

In addition, accumulating One Health evidence suggests that the epidemiology of ESBL-producing E. coli may differ markedly between regions and livestock production systems.

The cross-sectional design of the present study provides only a temporal snapshot of colonization and may not capture intermittent or low-frequency carriage events. Although selective culture methods were applied, low-prevalence colonization cannot be completely excluded. Further multicentre and longitudinal investigations are warranted to determine whether these findings reflect genuinely low circulation of resistant strains or temporal and ecological variability within livestock production systems.

 

• Bibliography:

Dohmen W, Dorado-García A, Bonten MJ, Wagenaar JA, Mevius D, Heederik DJ. Risk factors for ESBL-producing Escherichia coli on pig farms: A longitudinal study in the context of reduced use of antimicrobials. PLoS One. 2017 Mar 21;12(3):e0174094. doi: 10.1371/journal.pone.0174094.

Bergšpica I, Kaprou G, Alexa EA, Prieto M, Alvarez-Ordóñez A. Extended Spectrum β-Lactamase (ESBL) Producing Escherichia coliin Pigs and Pork Meat in the European Union. Antibiotics (Basel). 2020 Oct 7;9(10):678. doi: 10.3390/antibiotics9100678.

Ewers C, Bethe A, Semmler T, Guenther S, Wieler LH. Extended-spectrum β-lactamase-producing and AmpC-producing Escherichia coli from livestock and companion animals, and their putative impact on public health: a global perspective. Clin Microbiol Infect. 2012 Jul;18(7):646-55. doi: 10.1111/j.1469-0691.2012.03850

Miltgen G, Berti V, Milenkov M, Schmitt H, Wagenaar JA, Armand-Lefevre L. Circulation of extended-spectrum β-lactamase and plasmid-borne cephalosporinase-producing Escherichia coli from a One Health perspective: a narrative review. Clin Microbiol Infect. 2026 Apr;32(4):578-590. doi: 10.1016/j.cmi.2025.12.003

 

Comment 4: Line 32: The authors need to clearly describe how many samples were submitted for whole-genome sequencing (WGS), specify the animal groups from which these samples were selected, and define the criteria used for their selection. At present, this information is unclear and requires clarification to ensure transparency and reproducibility of the study.

 

Response 4: Thank you for pointing this out. We agree with this comment. We added and improved these sentences:

A total of 162 isolates were analyzed by WGS, showing high genetic diversity. Among the 127 Escherichia coli isolates, the most prevalent were ST10 (18/127, 14.2%), followed by ST453 (7/127, 5.5%), ST515 (7/127, 5.5%), and ST10562 (7/127, 5.5%).

 

Comment 5: Line 42: According to the results, despite the high-density pig farming environment, only a low number of ESBL-producing Escherichia coli were detected in livestock and humans. This finding suggests that pigs may not represent a significant source of ESBL-producing E. coli in this particular setting. However, this conclusion should be interpreted with caution, and the authors may need to reconsider or moderate their claims in the conclusion section, ensuring they are fully supported by the presented data.

Response 5: Thank you for pointing this out. We agree and have moderated the conclusions.

We added and improved these sentences:

Swine appear to constitute the primary reservoir of ESBL-producing E. coli. The genomic relatedness observed between animal and human isolates supports shared exposure to a common ecological pool of multidrug-resistant bacteria. However, the cross-sectional design precludes inference of transmission pathways or transmission directionality. Longitudinal studies are needed to elucidate the dynamics of interspecies transmission.

 

Comment 6: At the end of this section, the authors should clearly state the limitations of the study, along with recommendations for future research. This will help to properly contextualize the findings and guide further investigations in this area.

Response 6: We thank the reviewer for this important suggestion. We agree that a clearer and more structured presentation of the study limitations and future research directions strengthens the interpretation of our findings.

Accordingly, we have revised the final paragraph of the Discussion to explicitly outline the main limitations of our study. We added and improved these sentences:

This study has several limitations. First, its cross-sectional design provides only a snapshot of colonization and does not allow assessment of temporal dynamics or determination of the directionality of transmission between animals and humans. Second, the number of participating farmers was relatively limited, particularly among swine workers, which may reduce the precision of prevalence estimates and limit the ability to detect less frequent transmission events. Third, the study was conducted in a single high-density livestock region, which may affect the generalizability of the findings to other geographical settings with different farming practices.

In addition, the study focused primarily on colonization in apparently healthy animals and humans, and did not include a systematic evaluation of clinically ill animals, which may represent different epidemiological scenarios with potentially increased bacterial shedding and transmission.

Future research should address these limitations through longitudinal study designs that enable the assessment of transmission dynamics over time and across different ecological compartments. Expanding the sample size and including more diverse human and animal populations would improve the robustness and external validity of the findings. Furthermore, the application of long-read sequencing technologies would allow more detailed characterization of plasmid structures, facilitating a better understanding of horizontal gene transfer and the dissemination of antimicrobial resistance within and between species. Integrating environmental sampling (e.g., water, soil, and farm surfaces) would also provide a more comprehensive view of the transmission pathways within a One Health framework.

 

Introduction:

Comment 7: The introduction should include a clearly written paragraph that supports the study hypothesis and provides a strong scientific rationale for the work. In particular, the authors should explain how pig farms may act as reservoirs for ESBL-producing Escherichia coli, including the potential role of virulence and antimicrobial resistance genes associated with mobile genetic elements. This background is essential to justify the study design and to better position the research within the existing literature.

Response 7: We thank the reviewer for this insightful comment and fully agree that a clearer scientific rationale improves the framing of the study. In response, we have added a new paragraph in the Introduction and the corresponding bibliography :

Intensive pig production systems constitute a particularly relevant ecological niche for the emergence and maintenance of ESBL-producing Escherichia coli. High animal density, frequent antimicrobial exposure, and close contact between animals and farm workers create conditions that favour bacterial persistence, selection, and dissemination [8,11]. In this context, E. coli, as a commensal organism of the intestinal microbiota, plays a key role as a reservoir of antimicrobial resistance determinants [12].

Importantly, ESBL genes are frequently located on mobile genetic elements, especially conjugative plasmids, which facilitate horizontal gene transfer across bacterial populations and host species [12,13]. These plasmids often co-harbour additional antimicrobial resistance and virulence-associated genes, promoting both adaptation to the intestinal environment and survival under selective pressure [12,13]. Consequently, pig farms may act not only as reservoirs but also as amplification points for multidrug-resistant E. coli with the potential to spread through direct contact, environmental contamination, and the food chain [8–10].

 

Methods:

Comment 8: Line 102: The authors need to clarify whether the samples were collected from healthy or clinically ill animals, particularly those with diarrheal symptoms, as such cases may represent a more important source of ESBL-producing Escherichia coli transmission to other animals, humans, and the environment compared to healthy animals. This information is essential for accurately interpreting the epidemiological relevance of the findings and their implications for transmission dynamics.

Response 8: We thank the reviewer for highlighting this important point. We have clarified in the Methods section with these sentences:

Animals were sampled as part of routine veterinary surveillance programs, and the vast majority were clinically healthy at the time of sampling. No systematic inclusion of animals with clinical disease, such as diarrhoeal symptoms, was performed. Therefore, the findings of this study primarily reflect colonization patterns in apparently healthy livestock rather than infection-related dynamics.

 

Comment 9: Line 132: It is not clearly described from which animal groups the samples submitted for whole-genome sequencing (WGS) were selected. The authors should clearly specify the source populations and provide the criteria used for selecting the 100 samples for WGS analysis. This information is essential to ensure transparency, reproducibility, and proper interpretation of the genomic results.

Response 9: Thank you for pointing this out. The following sentences were incorporated:

A total of 162 Enterobacteriaceae isolates recovered from selective culture media (ESBL-, AmpC-, or carbapenemase-producing strains) were included in the WGS analysis. Isolates were derived mainly from swine, as well as from horses and human samples. No isolates from cattle or poultry were available for sequencing due to the absence of growth on selective media. Therefore, isolate selection was comprehensive and based on microbiological criteria rather than predefined sampling.

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for the opportunity to review the manuscript entitled "Prevalence and Genomic Characterization of ESBL-Producing Escherichia coli in Livestock and Farmers in a High-Density Pig Farming Area" by Marina Pujol et al. The manuscript addresses an interesting topic. However, several methodological and analytical issues should be addressed before the manuscript can be considered for publication. 

The abstract would benefit from the inclusion of a brief introductory sentence to better contextualize the study. In addition, line 26 should clearly report the number of carbapenem-resistant isolates detected in swine.

The Introduction is well structured, and I have no major comments. However, the Materials and Methods section requires substantial revision. The authors should clearly indicate the number of samples collected from each animal species on each farm. Without this information, it is difficult to evaluate whether the sampling strategy may have introduced bias into the prevalence estimates. I am also extremely concerned about the ethical aspects of the study. Although fecal sampling from farm animals is minimally invasive, ethical approval is required. My main concern, however, is the collection of human samples. The absence of approval from a Human Research Ethics Committee is a serious issue that may compromise the publication of the study. Obtaining informed consent alone is generally not sufficient and institutional ethical approval is mandatory for studies involving human participants.

Results: The authors state that the prevalence was higher in certain groups. How was this conclusion reached without a statistical comparison? Appropriate statistical analyses should be performed to support these statements.

The MLST results deserve a more in-depth analysis. In particular, the authors should discuss the clonal complexes represented by the identified sequence types. I also recommend performing an eBURST analysis to better illustrate the population structure of the isolates.

Figure 5 would be easier to interpret if percentages were presented in addition to the absolute number of isolates.

Table 1 contains mostly descriptive information and could be moved to the Supplementary Material.

Lines 223–226 refer to data that are "not shown." If these findings are important enough to be discussed, they should either be presented in a figure or supplementary table or described more thoroughly in the text. In its current form, this section is confusing.

Overall, the manuscript contains an excessive number of tables and figures. Several of them simply present raw data without highlighting the main findings or biological significance. As a result, the manuscript often reads as a compilation of results rather than a structured scientific analysis. I encourage the authors to be more selective and prioritize the figures and tables that best support the central conclusions of the study.

One major limitation is the lack of analyses exploring the relationship between isolates obtained from linked sources, particularly animals and humans from the same farm or at least from the same species (for example, swine and swine farms). At a minimum, the MLST data should be used to evaluate whether isolates from the same epidemiological setting share sequence types or clonal complexes. Beyond this, it would be highly informative to compare the genetic contexts of the ESBL and AmpC genes, including the associated mobile genetic elements, between human and animal isolates. Such analyses would substantially strengthen the One Health component of the study.

Gene names should be italicized consistently throughout the manuscript. 

Figure 6 contains too many panels, making it visually overwhelming and difficult to interpret. Some of these data could be summarized in the text or presented in a simple table instead. The focus should be on the most relevant findings rather than presenting every result in detail

Author Response

We would like to sincerely thank you for the time and effort devoted to the careful evaluation of our manuscript. We greatly appreciate the constructive comments and thoughtful suggestions provided by the reviewers, which have been invaluable in improving the quality, clarity, and scientific rigor of our work. We have carefully considered each comment and revised the manuscript accordingly. Below, we provide a detailed point-by-point response to each of the reviewers' comments, indicating the corresponding changes made in the revised manuscript.

Abstract

Comment 1: Inclusion of a brief introductory sentence to better contextualize the study.

Response 1: We sincerely thank the reviewer for this valuable suggestion. We agree that providing a brief contextual introduction improves the clarity of the abstract and better frames the objectives of the study. Accordingly, we have added the following sentence at the beginning of the abstract:

"Extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae represent an increasing One Health concern because food-producing animals may act as reservoirs of antimicrobial-resistant bacteria with potential transmission to humans."

In addition, to better reflect the content of this section, the heading has been changed from Objectives to Background and Objectives.

Comment 2: Line 26 should clearly report the number of carbapenem-resistant isolates detected in swine.

Response 2: We thank the reviewer for this helpful comment. We agree that the number of carbapenem-resistant isolates represents an important finding and should be explicitly reported in the abstract. We have therefore revised the Results section of the abstract as follows:

"ESBL-producing Enterobacteriaceae were predominantly detected in swine, with 63.5% (127/200) of animals testing positive, including four ESBL+AmpC-producing isolates and two carbapenem-resistant Enterobacteriaceae."

Materials and Methods

Comment 3: The authors should clearly indicate the number of samples collected from each animal species on each farm.

Response 3: We sincerely thank the reviewer for this valuable suggestion. We agree that providing a detailed breakdown of the sampling strategy improves the transparency of the study. Because this information is extensive, we considered that presenting it as supplementary material would improve readability while making the complete sampling information readily available. Accordingly, we have added a new supplementary table:

“Table S1. Sampling strategy. Number of livestock and farmers sampled on each farm according to livestock species.”

Comment 4: I am also extremely concerned about the ethical aspects of the study. Although fecal sampling from farm animals is minimally invasive, ethical approval is required. My main concern, however, is the collection of human samples. The absence of approval from a Human Research Ethics Committee is a serious issue that may compromise the publication of the study. Obtaining informed consent alone is generally not sufficient and institutional ethical approval is mandatory for studies involving human participants.

Response 4: We sincerely thank the reviewer for raising this important point. We apologize for not including this information in the original version of the manuscript. The study involving human participants was reviewed and approved by the Clinical Research Ethics Committee of the Consorci Hospitalari de Vic (approval No. 2021165), and all participants provided written informed consent prior to enrolment. We have now incorporated this information into the Materials and Methods section to clarify that all ethical requirements for research involving human participants were fulfilled.

Results

Comment 5: The authors state that the prevalence was higher in certain groups. How was this conclusion reached without a statistical comparison? Appropriate statistical analyses should be performed to support these statements.

Response 5:

We thank the reviewer for this important observation. In response, we performed statistical comparisons of ESBL prevalence among occupational groups using Fisher’s exact test, which was considered the most appropriate method given the limited sample size and the presence of zero-count cells. The Results section has been revised accordingly and now includes the statistical comparison between swine farmers and the remaining livestock workers. These analyses confirmed that ESBL-producing Enterobacteriaceae were significantly more frequent among swine farmers than among other occupational groups.

We added in results:

The prevalence of ESBL/AmpC- and carbapenemase-producing Enterobacteriaceae differed significantly across livestock species (p < 0.001). Swine showed the highest prevalence (127/200 [63.5%]), compared with horses (15/214 [7.0%]), cattle (0/360), and poultry (0/171). The prevalence of ESBL-producing Enterobacteriaceae was significantly higher among swine farmers than among all other livestock workers combined (7/15 [46.7%] vs. 2/49 [4.1%], p < 0.001).

We have now incorporated this information into the Materials and Methods section:

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics software version 30.0. Categorical variables were expressed as frequencies and percentages. The chi-square test (Fisher's exact test) and Student's t-test were used to compare the epidemiological characteristics between animal species, occupational groups, and positivity status. Statistical significance was set at p<0.05.

Comment 6: The MLST results deserve a more in-depth analysis. In particular, the authors should discuss the clonal complexes represented by the identified sequence types. I also recommend performing an eBURST analysis to better illustrate the population structure of the isolates.

Response 6: We sincerely thank the reviewer for this valuable suggestion. We agree that a more detailed analysis of the MLST data strengthens the interpretation of the population structure of the isolates. Accordingly, we have performed a goeBURST analysis and incorporated the results into the revised manuscript as a new figure (Figure 2). We have also modified the corresponding section in the Results as follows:

"A wide diversity of sequence types (STs) was identified among ESBL-producing E. coli isolates, with no single ST predominating. Figure 2 presents the goeBURST network generated from MLST-derived allelic profiles, showing the relationships among the isolates."

Comment 7: Figure 5 would be easier to interpret if percentages were presented in addition to the absolute number of isolates.

Response 7: We sincerely thank the reviewer for this helpful suggestion. We agree that including percentages improves the interpretation of the figure. Accordingly, we have revised Figure 5 to display the prevalence (%) for each virulence-associated gene.

Comment 8: Table 1 contains mostly descriptive information and could be moved to the Supplementary Material.

Response 8: We sincerely thank the reviewer for this helpful suggestion. We agree that Table 1 primarily contains descriptive information and is better suited for the Supplementary Material. Accordingly, it has been moved to the Supplementary Material as Table S2.

Comment 9: Lines 223–226 refer to data that are "not shown." If these findings are important enough to be discussed, they should either be presented in a figure or supplementary table or described more thoroughly in the text. In its current form, this section is confusing.

Response 9: We sincerely thank the reviewer for this valuable comment and apologize for the lack of clarity in the original version of the manuscript. The sentence referring to isolates with unknown sequence types was indeed misplaced. It has now been relocated within the section describing the sequence types of swine E. coli isolates, immediately before the reference to Figure 2, where these isolates are actually represented. We have also revised the wording to clarify this point and avoid any ambiguity. We hope that this modification improves the readability of the section and resolves the confusion.

Comment 10: Overall, the manuscript contains an excessive number of tables and figures. Several of them simply present raw data without highlighting the main findings or biological significance. As a result, the manuscript often reads as a compilation of results rather than a structured scientific analysis. I encourage the authors to be more selective and prioritize the figures and tables that best support the central conclusions of the study.

Response 10: We sincerely thank the reviewer for this thoughtful suggestion. We agree that reducing the number of figures and tables improves the readability of the manuscript and helps maintain the focus on the main findings. Accordingly, Tables 1, 2, and 3, which mainly contain descriptive or reference information, have been moved to the Supplementary Material (Tables S2–S4). In addition, Figure 1 has also been transferred to the Supplementary Material (Figure S1) and Figure 2 was removed. These changes have substantially reduced the number of figures and tables included in the main manuscript while ensuring that all relevant information remains available to the reader.

Comment 11: One major limitation is the lack of analyses exploring the relationship between isolates obtained from linked sources, particularly animals and humans from the same farm or at least from the same species (for example, swine and swine farms). At a minimum, the MLST data should be used to evaluate whether isolates from the same epidemiological setting share sequence types or clonal complexes. Beyond this, it would be highly informative to compare the genetic contexts of the ESBL and AmpC genes, including the associated mobile genetic elements, between human and animal isolates. Such analyses would substantially strengthen the One Health component of the study.

Response 11: We sincerely thank the reviewer for this thoughtful and constructive suggestion. We agree that farm-level comparisons between animal and human isolates would be of considerable interest and could further strengthen the One Health perspective of the study. We carefully evaluated this possibility; however, the distribution of positive human isolates across farms did not allow a robust epidemiological comparison. Although the prevalence of ESBL-producing E. coli among swine farmers was high, positive human isolates were unevenly distributed between farms, so farm-level comparisons could be misleading because any observed similarities or differences might simply reflect the limited and unbalanced sampling rather than the true epidemiological relationships between animal and human isolates. For this reason, we considered that the population-level analyses presented in the manuscript provide a more robust and scientifically sound interpretation of our findings. We have acknowledged this limitation in the Discussion.

Comment 12: Gene names should be italicized consistently throughout the manuscript. 

Response 12: We sincerely thank the reviewer for this careful observation. Gene nomenclature has been reviewed throughout the entire manuscript, and all gene names have now been consistently italicized in accordance with standard scientific conventions.

Comment 13: Figure 6 contains too many panels, making it visually overwhelming and difficult to interpret. Some of these data could be summarized in the text or presented in a simple table instead. The focus should be on the most relevant findings rather than presenting every result in detail

Response 13: We sincerely thank the reviewer for this valuable suggestion. We agree that the original version of Figure 6 contained a large amount of information, making it difficult to interpret. To improve clarity and readability, we have replaced Figure 6 with a table that presents the same data in a more concise and accessible format. We believe that this modification facilitates comparison of antimicrobial resistance genes across the different antibiotics while maintaining all the relevant information.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript provides a valuable "One Health" assessment of ESBL-producing Escherichia coli in livestock and farm workers, combining prevalence data with WGS analysis. Overall, the manuscript is well-written and addresses an important topic in antimicrobial resistance surveillance. I have only a few comments aimed at improving clarity and enhancing the interpretation of key findings.

Lines 155-157: The authors report basic demographic data for the farmers (age and sex), but these variables are not used subsequently in the analyses or discussed in the results. If stratified or multivariate analyses were not performed, the authors should briefly mention this limitation or consider removing these variables if they are not relevant to the study objectives.

Lines 252-253 and 310-400: The detection of carbapenem-resistant isolates carrying the blaOXA-48 gene in pigs, identified in both E. coli (ST58) and K. oxytoca (ST145) and associated with IncL plasmids, is one of the most clinically relevant findings of the study, yet it is only briefly mentioned in the Results and not sufficiently explored in the Discussion. Given the clinical importance of OXA-48 carbapenemases and the well-known role of IncL plasmids in their clinical spread, this finding deserves further discussion within the One Health framework. The authors should better contextualize its public health relevance and consider strengthening its interpretation in the Conclusions.

Author Response

This manuscript provides a valuable "One Health" assessment of ESBL-producing Escherichia coli in livestock and farm workers, combining prevalence data with WGS analysis. Overall, the manuscript is well-written and addresses an important topic in antimicrobial resistance surveillance. I have only a few comments aimed at improving clarity and enhancing the interpretation of key findings.

Comment 1: Lines 155-157: The authors report basic demographic data for the farmers (age and sex), but these variables are not used subsequently in the analyses or discussed in the results. If stratified or multivariate analyses were not performed, the authors should briefly mention this limitation or consider removing these variables if they are not relevant to the study objectives.

Response 1:

We thank the reviewer for this valuable comment.

We agree that age and sex were not subsequently used in the analyses and were not directly related to the primary objectives of the study, which focused on the prevalence and genomic characterization of ESBL-producing Enterobacteriaceae in livestock and farm workers. To improve the clarity and focus of the manuscript, we have removed the detailed demographic description and retained only the number of farmers included in the study population.

The Results section now states: “A total of 64 farmers representing four livestock sectors were enrolled.”

Comment 2: Lines 252-253 and 310-400: The detection of carbapenem-resistant isolates carrying the blaOXA-48 gene in pigs, identified in both E. coli (ST58) and K. oxytoca (ST145) and associated with IncL plasmids, is one of the most clinically relevant findings of the study, yet it is only briefly mentioned in the Results and not sufficiently explored in the Discussion. Given the clinical importance of OXA-48 carbapenemases and the well-known role of IncL plasmids in their clinical spread, this finding deserves further discussion within the One Health framework. The authors should better contextualize its public health relevance and consider strengthening its interpretation in the Conclusions.

 

Response 2:

Abstract results: Two carbapenem-resistant isolates carrying blaOXA-48 were identified in swine, including E. coli ST58 and K. oxytoca ST145, both associated with IncL plasmids.

 

 

Abstract conclusión: The identification of blaOXA-48-producing Enterobacterales associated with IncL plasmids further highlights the public health relevance of livestock-associated antimicrobial resistance

 

Discusion Although carbapenem-resistant isolates were rare, the detection of blaOXA-48 in both E. coli and K. oxytoca recovered from swine deserves particular attention. OXA-48 is one of the most clinically significant carbapenemases worldwide and is frequently associated with successful epidemic plasmids, particularly those belonging to the IncL family [27]. In our study, blaOXA-48 was identified in two phylogenetically distinct bacterial species and associated with IncL plasmids, suggesting the potential involvement of horizontal gene transfer mechanisms rather than clonal spread alone. While the low number of isolates precludes definitive epidemiological conclusions. The presence of OXA-48-producing Enterobacterales in livestock is concerning from a One Health perspective, as food-producing animals may contribute to the maintenance and environmental dissemination of clinically relevant resistance determinants. Continuous genomic surveillance is therefore warranted to monitor the occurrence and spread of carbapenemase-producing Enterobacterales at the human–animal interface.

 

Bibliography: Prasad S, Dratvova B, Gryndlerova A, Brajerova M, Kabelikova P, Tkadlec J, Drevinek P, Krutova M. IncL plasmid-mediated dissemination of OXA-48 β-lactamase and bla_CTX-M-15 gene amplification identified via long-read sequencing in carbapenem-resistant Enterobacterales. JAC Antimicrob Resist. 2026 Jan 7;8(1):dlaf254. doi: 10.1093/jacamr/dlaf254.

 

Conclusion: The detection of blaOXA-48-carrying Enterobacterales associated with IncL plasmids highlights the potential emergence of clinically relevant carbapenem resistance mechanisms in livestock.

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank most the requested correction  has been performed 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors made a clear effort to revise the manuscript, and its overall quality has improved substantially. I believe the study is now suitable for publication.