From ecology to evolution: plasmid- and colicin-mediated persistence of antibiotic resistant Escherichia coli in gulls

  1. Michaela Ruzickova
  2. Jana Palkovicova
  3. Kristina Nesporova
  4. Marketa Rysava
  5. Rene Pariza
  6. Simon Krejci
  7. Ivan Literak
  8. Monika Dolejska

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Abstract

Antimicrobial resistance in wildlife is an emerging concern within the One Health concept. Gulls, due to their synanthropic behaviour and long-distance migration, are recognised as vectors and secondary reservoirs of resistant bacteria. These birds can facilitate the environmental spread of resistant strains across ecosystem boundaries. Understanding their role in shaping microbial communities is essential for assessing the broader ecological impact. This study investigates the persistence and competitive dynamics of cephalosporin-resistant Escherichia coli in Caspian gulls captured at their breeding colony at a water reservoir and subsequently monitored in captivity for three months, representing the longest in vivo experiment of its kind conducted on wild birds.

We observed sustained colonization and long-term shedding of resistant E. coli throughout the entire study, marking the longest documented carriage of resistant bacteria in wild birds to date. Notably, rapid dissemination of various E. coli sequence types (STs) with CTX-M-1 was observed, with ST11138 rapidly outcompeting other strains, including the initially dominant ST11893. Genomic analyses revealed that ST11138 harboured F24:A-:B1 and IncI1/ST3/CTX-M-1 plasmids encoding colicins and corresponding immunity genes, likely conferring a competitive advantage.

Our findings underscore the role of bacteriocin-mediated interactions in shaping microbial communities and highlight the importance of plasmid-encoded traits in the persistence of resistant strains in wildlife. Importantly, our findings underscore the ecological novelty of longitudinal in vivo tracking of AMR persistence in natural hosts and highlight the need to consider ecological and microbiome-level interactions when assessing the environmental dimension of AMR under the One Health concept.

Importance

Antimicrobial resistance in wildlife is an emerging concern within the One Health framework, with gulls recognized as important vectors and secondary reservoirs of resistant bacteria. Due to their synanthropic behaviour and long-distance migration, these birds can facilitate the spread of resistant strains across ecosystems. However, the role of wildlife in resistance dynamics remains underexplored, especially in long-term, natural settings. Our study is unique in its scope and duration, representing the longest in vivo experiment of its kind conducted on wild birds. By capturing these processes in live hosts under naturalistic conditions and across an extended period, our study provides rare and ecologically grounded insights into how antimicrobial resistance is maintained outside clinical or laboratory settings. Our findings show sustained colonization and long-term shedding of resistant E. coli , with strain ST11138 outcompeting others. Genomic analyses reveal plasmid-encoded traits, highlighting the novel ecological and evolutionary mechanisms underlying resistance maintenance in wildlife.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17461982.

    Summary

    This manuscript presents the longest in vivo experimental study to date on the persistence and dynamics of cephalosporin-resistant Escherichia coli in wild Caspian gulls. It offers valuable insights into how antimicrobial resistance (AMR) is maintained and transmitted within wildlife reservoirs. Through whole-genome analyses, the authors demonstrate that strain ST11138, which carries plasmid-encoded colicin production and related immunity genes, quickly outcompetes other strains. This highlights that competitive mechanisms can allow resistant strains to persist even in the absence of antibiotics.

    Major Revisions

    The sample size in this study consists of five Caspian gulls. This limited number may affect the statistical power and the generalizability of the results to the broader population of Caspian gulls or to other wild bird species. It would be beneficial to acknowledge this limitation and discuss how the limited number of participants may influence the interpretation and generalizability of the results.

    The manuscript emphasizes in the importance section (around line 56) that this research is the "longest in vivo experimental study" of its kind. While its longer duration is clearly beneficial, it would be helpful for readers to have more explanation about why extending the duration makes such a significant difference.

    The authors noted a significant difference between AUC values between the ST. While the AUCs in rich media LB-broth is a measure of intrinsic growth potential or growth rate, it is widely acknowledged to be a poor predictor of competitive fitness in complex, nutrient-limited environments like the avian gut. The paper's primary claim is based on competitive advantage in the gut, which the separate competition assay addresses more directly. The results should be discussed as differences in intrinsic growth rate (a valuable finding for characterizing the STs), and the direct competition assay should be prioritized as the primary evidence for competitive fitness. The discussion should explicitly address the limitation of LB-based growth curves as a proxy for in vivo gut fitness.

    Minor Revisions

    In Table 1, the timing and pattern of strain dominance varied among individual gulls. Some birds showed a rapid transition to a single dominant strain within the first few weeks, while others changed more gradually or maintained multiple strains for a longer time. While you briefly mentioned this in the results and discussion section, clarifying the reason behind this would help readers better understand the diversity and natural variability in your results.

    The decision to house the gulls in captivity and use a standardized diet is certainly practical for experimental control. However, it would be beneficial if you could expand a bit more on how these artificial conditions might have influenced the patterns of microbial competition and persistence you observed. Readers may benefit from some comments on how limited diet variety, reduced environmental exposure, and a more controlled setting could have shaped the dynamics compared to what might occur in free-living gulls in more variable habitats.

    Out of the 94 E. coli isolates identified in your study, only 62 were selected for whole-genome sequencing, which might leave some readers wondering how this selection was made or if anything important might have been overlooked. Providing a brief explanation about this would be helpful.

    The isolation method relied on selecting only five to six colonies per selective plate. This raises a question about whether less abundant colonies were missed. Authors should justify this selection strategy in the methods or include a statement acknowledging that this approach primarily tracked the most dominant strains.

    Overall Impression

    I found the overall narrative both interesting and relevant, given increasing concerns about antimicrobial resistance in wildlife. The study does a good job connecting detailed, long-term field observations with molecular evidence, helping us better understand how resistance traits persist.

    Competing interests

    The authors declare that they have no competing interests.

    Use of Artificial Intelligence (AI)

    The authors declare that they used generative AI to come up with new ideas for their review.

  2. Version published to 10.1101/2025.09.30.679527 on bioRxiv