Detection of ESBL-producing Escherichia coli carrying blaCTXM-15 in magnificent frigatebird (Fregata magnificens) from Brazil: a one health perspective

Background: Wild birds are increasingly recognised as sentinels for antimicrobial resistance (AMR) in environments impacted by human activity, yet the role of seabirds in the dissemination and maintenance of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in Brazil remains unclear.

Methods: Cloacal swabs were collected from fifteen magnificent frigatebirds (Fregata magnificens) from the Cagarras Islands, a coastal archipelago. Bacterial isolation was performed using MacConkey agar supplemented with ceftriaxone, followed by identification using MALDI-TOF MS. Antimicrobial susceptibility testing was conducted using the disc diffusion method, and PCR screening was performed for ESBL genes. WGS and bioinformatics analysis were employed to characterise the isolate.

Results: One ceftriaxone-resistant E. coli isolate was recovered from an adult female bird. The isolate was identified as sequence type ST5614 and serotype O27:H14, carrying the bla_CTX-M-15 gene on an IncB/O/K/Z plasmid closely related to those described in human isolates. The strain showed resistance to multiple antimicrobials and harboured additional resistance genes including tet(A), sul1, sul2, mph(A), qnrS1, mrx(A), aph(3”)-Ib, aph(6)-Id, and ant(3”)-Ia.

Conclusion: Detection of bla_CTX-M-15 in F. magnificens may reflect the movement of clinically significant resistance genes at the human–wildlife interface, underscoring the value of seabirds as sentinels for environmental AMR surveillance. The findings highlight the interconnectedness of environmental, animal, and human health and reinforce the importance of wildlife surveillance in One Health AMR strategies.

1 Introduction

Multidrug-resistant (MDR) bacteria, defined as non-susceptibility to at least one agent in three or more antimicrobial categories (Magiorakos et al., 2012), have been detected in environmental sources such as rivers, sewage, and coastal waters. Wild birds are recognized as reservoirs and potential vectors of these pathogens, yet their role in the dissemination of antimicrobial resistance (AMR) remains poorly studied worldwide, including in Brazil (Sanganyado and Gwenzi, 2019; Paschoal et al., 2020). AMR is a major global health threat, exacerbated by improper disposal of antimicrobial-laden waste. Wild birds may acquire resistant strains from contaminated environments and contribute to their spread. The presence of MDR bacteria in birds without direct antimicrobial exposure highlights their potential as sentinels in AMR surveillance (Wang et al., 2017; Athanasakopoulou et al., 2022).

Extended spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) are a growing concern due to their resistance to critical antibiotics. While widely reported in hospitals, livestock, and companion animals, ESBL-EC have also been increasingly identified in wildlife and natural environments, suggesting broader ecological dissemination. In wild birds, especially those not exposed to antimicrobials, the detection of such resistant strains points to environmental acquisition and highlights the possible role of these animals as sentinels of antimicrobial resistance (Stedt et al., 2015; Fernandes et al., 2018; Silva et al., 2018; Salgado-Caxito et al., 2021).

The magnificent frigatebird (Fregata magnificens), a seabird widely distributed along tropical and subtropical coasts, nests on islands and disperses over large distances (Saviolli et al., 2016). Despite this, little is known about the occurrence of antimicrobial-resistant bacteria in its populations. Given its ecological role and exposure to anthropogenically influenced habitats, F. magnificens may help reveal resistance dynamics in coastal ecosystems (Zaluski et al., 2019). Expanding such investigations is crucial to support One Health strategies and address environmental dimensions of AMR.

This study investigated the presence of ESBL-producing E. coli in cloacal samples from 15 F. magnificens individuals inhabiting the Cagarras Islands, a coastal archipelago near Rio de Janeiro, Brazil. Microbiological analyses and whole genome sequencing were employed to characterize phenotypic and genotypic resistance profiles, providing new insights into the environmental dissemination of clinically relevant resistant bacteria and reinforcing the role of wildlife in One Health AMR surveillance strategies.

2 Materials and methods

2.1 Study area and sample collection

The study was conducted in the Cagarras Islands Natural Monument (MONA Cagarras), located off the coast of Rio de Janeiro State, Brazil (23°01′S, 43°12′W), approximately 5 km from Ipanema Beach. The archipelago comprises six main landforms: the Cagarras, Palmas, Comprida, and Redonda islands, as well as the islets Filhote da Cagarras and Filhote da Redonda (Figure 1). Samples were collected in October 2023 on Redonda Island—the largest and highest landmass in the region, covering approximately 395,500 m² and located 8.5 km from the mainland. This island is the main breeding and roosting site for F. magnificens and other marine bird species. Cloacal swabs were collected from each individual, placed in Cary Blair transport medium, kept refrigerated at 4 °C, and processed within 48 h. All sampling procedures were conducted under SISBIO permit no. 73163-6 and approved by the Animal Ethics Committee of the Oswaldo Cruz Institute (IOC), FIOCRUZ (Protocol L-019/2021).

Figure 1

Figure 1. Map showing the location of the Cagarras Islands Natural Monument (MONA Cagarras) off the coast of Rio de Janeiro, Brazil, highlighting Redonda Island, where samples were collected. The inset indicates the position of MONA Cagarras along the southeastern Brazilian coast. The scale bar is provided, and Redonda Island is located approximately 5 km from the mainland at Ipanema Beach. The image also features a Fregata magnificens, a common seabird in the region (source: Generated by authors using QGIS 3.32.2 based on public governmental shapefiles).

2.2 Bacterial isolation and identification

Cloacal samples were streaked onto MacConkey agar plates supplemented with ceftriaxone (2 μg/mL) and incubated overnight at 35 ± 2 °C for 18–20 h to select for ESBL-EC strains. After incubation, a limited bacterial growth was observed. Colonies displaying growth under these selective conditions were subcultured in BHI broth containing ceftriaxone (2 μg/mL) for confirmatory screening. Following this selective enrichment process, only one cloacal sample yielded growth of a single bacterial species in pure culture. This isolate was identified by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS, Bruker Daltonik, Germany).

2.3 Antimicrobial susceptibility testing

Acquired antimicrobial resistance genes were detected with ResFinder (Bortolaia et al., 2020), and insertion sequences and transposable elements were detected with ISfinder (Siguier et al., 2006). Comparative plasmid analysis was performed using BLAST Ring Image Generator (BRIG) v0.95 (Alikhan et al., 2011). Antimicrobial susceptibility profiles were determined by the disc diffusion method using the following antimicrobial agents: sulbactam/ampicillin [SAM, 20 (10/10 μg)], tetracycline (TET, 30 μg), imipenem (IPM, 10 μg), meropenem (MEM, 10 μg), ciprofloxacin (CIP, 5 μg), amikacin (AK, 30 μg), gentamicin (CN, 10 μg), sulphamethoxazole/trimethoprim (SXT, 25 μg), clindamycin (DS, 2 μg), levofloxacin (LEV, 5 μg), nalidixic acid (NAL, 30 μg), tigecycline (TGC, 15 μg), ceftriaxone (CRO, 30 μg) and ceftazidime (CAZ, 30 μg). Phylogenetic tree was constructed using Parsnp v2.1.4 (Kille et al., 2024) based on the whole-genome alignment of E. coli 99RCEF. Publicly available genomes of E. coli isolates belonging to the same ST were retrieved from EnteroBase5 and included in the analysis. The resulting core genome alignment was used to infer a maximum likelihood phylogeny with IQ-TREE (Minh et al., 2020). employing the best-fit substitution model selected by ModelFinder (Kalyaanamoorthy et al., 2017) and performing 1,000 ultrafast bootstrap (Hoang et al., 2018) replicates to assess branch support. The final tree was visualized, edited, and annotated using Interactive Tree Of Life (iTOL) v76 (Letunic et al., 2024). Polymyxin B minimum inhibitory concentration (MIC) was determined by the broth microdilution method, in accordance with BrCast/EUCAST guidelines.¹

2.4 PCR screening

Screening for ESBL genes was performed by PCR using specific primers for each target gene. The bla_CTX-M gene was amplified with primers Ctx-m-FW (5′-AAAAATCACTGCGCCAGTTC-3′) and Ctx-m-RV (5′-CCGTCGGTGACGATTTTAGCC-3′) (Saladin et al., 2002). For bla_TEM, primers Tem-F (5′-ATGAGTATTCAACATT TCCGTG-3′) and Tem-R (5′-TTACCAATGCTTAATCAGTGAG-3′) were used, and for bla_SHV, primers Shv-FW (5′-TTTATCGGCCCTCAC TCAAGG-3′) and Shv-RV (5′-GCTGCGGGCCGGATAACG-3′) were employed (Yuan et al., 2021). PCR cycling parameters (applied to all three assays): initial denaturation 94 °C for 3 min; 35 cycles of 94 °C for 45 s, 56 °C for 45 s, 72 °C for 45 s; final extension 72 °C for 10 min. As a positive control, the Klebsiella pneumoniae strain CCBH6556, harboring all three ESBL genes (GenBank: NZ_JBHFPY000000000.1), obtained from the Culture Collection of Hospital-Acquired Bacteria (CCBH), registered with the World Federation for Culture Collections (WFCC, WDCM 947), was used.

2.5 Whole genome sequencing and bioinformatics analysis

Genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN), according to the manufacturer’s protocol. Illumina libraries were prepared using the Illumina DNA Prep Kit and sequenced on a MiSeq platform (MiSeq Reagent Kit v2, 500 cycles). Raw Illumina reads were quality-filtered and adapter-trimmed using the CABGen web application (Duré et al., 2022). Oxford Nanopore Technologies (ONT) libraries were constructed with the Rapid Barcoding Kit 24 V14 (SQK-RBK114.24, ONT) and sequenced on a MinION Mk1B with an R10.4.1 flow cell, operated via MinKNOW software. Basecalling of ONT signal data was performed using Dorado.²

Hybrid de novo assembly was performed with Unicycler v0.4.9 (conservative mode) (Wick et al., 2017). Assembly completeness and contamination were evaluated using CheckM (Parks et al., 2015), and taxonomic classification and contaminant screening were performed with Kraken2 (Wood et al., 2019). Genome annotation was carried out using Prokka (Seemann, 2014). Plasmid replicon typing was performed with PlasmidFinder (Carattoli et al., 2014), serotyping with SerotypeFinder (Joensen et al., 2015), and multilocus sequence typing (MLST) using the Achtman scheme.³ Acquired antimicrobial resistance genes were detected with ResFinder (Bortolaia et al., 2020), and insertion sequences and transposable elements were detected with ISfinder.⁴ Comparative plasmid analysis was performed using BLAST Ring Image Generator (BRIG) v0.95.

Phylogenetic tree was constructed using Parsnp v2.1.4 (10.1093/bioinformatics/btae311) based on the whole-genome alignment of E. coli 99RCEF. Publicly available genomes of E. coli isolates belonging to the same ST were retrieved from EnteroBase⁵ and included in the analysis. The resulting core genome alignment was used to infer a maximum likelihood phylogeny with IQ-TREE (v2.2.010.1093/molbev/msaa015), employing the best-fit substitution model selected by ModelFinder and performing 1,000 ultrafast bootstrap replicates to assess branch support. The final tree was visualized, edited, and annotated using Interactive Tree Of Life (iTOL) v7.⁶

3 Results

A total of 15 F. magnificens individuals (10 adults and five juveniles; comprising two males, eight females, and five juveniles of undetermined sex) were captured on Redonda Island, Cagarras Archipelago. Cloacal swabs were obtained from all apparently healthy birds. Among the 15 samples, one ceftriaxone-resistant E. coli isolate was recovered from an adult female bird (6.6%; 95% CI: 0.16%–31.95%). Identification by MALDI-TOF MS confirmed the isolate as E. coli, and PCR detected the presence of the bla_CTXM gene.

The antimicrobial susceptibility profile revealed that the isolate was resistant to tetracycline, nalidixic acid, trimethoprim-sulfamethoxazole, clindamycin, ciprofloxacin, ceftriaxone and ceftazidime.

Whole genome sequencing identified the strain as sequence type ST5614 and serotype O27:H14. The analysis also revealed the presence of a 126,204 bp plasmid, named p99RCEF_blaCTX-M-15 (Figure 2), classified within the IncB/O/K/Z incompatibility group. This plasmid harbored the bla_CTXM-15 gene, as well as tet(A), sul1, sul2, mph(A), qnrS1, mrx(A), aph(3″)-Ib, aph(6)-Id, and ant(3″)-Ia.

Figure 2

Figure 2. Genomic comparison between IncB/O/K/Z plasmids in relation to p99RCEF_blaCTX-M-15 from E. coli 99RCEF. Inside out, the first ring is plasmid p99RCEF_blaCTX-M-15 from E. coli 99RCEF. The second ring is the plasmid GC content. The outer rings represent the nucleotide sequence of the corresponding DNA region of the following IncB/O/K/Z plasmids in different colors against the reference genome (p99RCEF_blaCTX-M-15): p1-S1-KEN-04-A from E. coli (blue), p1_A24359 from E. coli (green), and pJNQH940-1 from Salmonella enterica (purple). The last one is the gene annotations (black).

The bla_CTXM-15 gene was located within a Tn2-like element flanked by intact inverted repeats, although the element lacked a complete transposition module. The plasmid encoded a full set of conjugal transfer genes (traA to traY), toxin-antitoxin systems, pilus assembly proteins, colicin operons, recombinases, and components of a type IV secretion system.

Comparative genomic analysis showed that p99RCEF_blaCTX-M-15 shares high nucleotide sequence identity and structural similarity with three previously characterized IncB/O/K/Z plasmids carrying bla_CTXM-15: p1-S1-KEN-04-A (126,176 bp; GenBank NZ_CP145691.1) and p1_A24359 (131,757 bp; GenBank CP183674.1), both isolated from E. coli strains recovered from human faecal material in Germany and Switzerland, respectively, and pJNQH940-1 (127,061 bp; GenBank NZ_CP136142.1) from Salmonella enterica isolated from a renal transplant patient in China. These plasmids share a conserved genetic backbone, including mobile genetic elements and regions conferring antimicrobial resistance.

The isolate 99RCEF was identified as a phylogenetically distinct and basal lineage within the maximum likelihood phylogenetic tree compared to all other E. coli isolates of the same sequence type included in this study (Figure 3). While most isolates, predominantly derived from human, livestock or environmental sources, formed multiple well supported and genetically diverse clades, 99RCEF branched independently from the rest of the dataset. This separation was marked by a considerably long branch length and a strongly supported internal node with a bootstrap value of 100, providing robust statistical evidence for the phylogenetic distinctiveness and divergence of 99RCEF within the sequence type examined.

Figure 3

Figure 3. Maximum-likelihood phylogenetic tree based on core-genome single nucleotide polymorphisms (SNPs) from complete genomes of E. coli ST 5614, using 99RCEF as reference. The analysis includes all isolates of ST 5614 deposited in EnteroBase. Bootstrap values >40 are shown at the nodes. The colored circles indicate source of origin: red for human, orange for wild bird, green from environment, purple for livestock and gray for isolates with no available source information. Country and year of isolation are shown to the right of each isolate ID, when available in EnteroBase.

4 Discussion

The detection of an ESBL-EC strain carrying bla_CTXM-15 in F. magnificens from the Cagarras Islands represents a significant finding in the context of environmental AMR surveillance in Brazil. Wild birds, particularly those inhabiting environments impacted by human activities, have emerged as important sentinels for monitoring the spread of AMR, yet their role in the dissemination and maintenance of resistant bacteria remains underexplored in the country. The MDR bacteria in environmental matrices such as rivers, sewage, and coastal waters underscores the growing ecological dimension of AMR and the need for integrated One Health approaches.

Our study revealed the presence of an ESBL-EC strain resistant to multiple antimicrobials critically important in both animal production and human medicine, in a seabird species with no direct exposure to antibiotics. Furthermore, findings from protected or uninhabited islands show clinically relevant AMR in seabirds without clinical treatment, consistent with indirect anthropogenic contamination and environmental selection rather than direct medicinal use (Ewbank et al., 2021c). This finding aligns with previous reports from Brazil and other countries, where wild birds have been shown to harbor MDR bacteria, often with resistance profiles similar to those found in clinical and livestock settings (Costa et al., 2006; Pinto et al., 2015; Silva et al., 2020; Martín-Maldonado et al., 2022).

The acquisition of such bacteria by wild birds is likely linked to environmental contamination, particularly in areas receiving untreated domestic and hospital sewage, as is the case for Guanabara Bay adjacent to the Cagarras Islands. Recent environmental studies in Guanabara Bay and recreational beaches near the Cagarras Archipelago have already reported the presence of multidrug-resistant bacteria and resistance genes, including bla_CTXM variants in water and sediment samples (Montezzi et al., 2015; de Araujo et al., 2016; Costa et al., 2023). These findings highlight the chronic impact of untreated domestic and hospital sewage on the region, creating hotspots for the selection and persistence of antimicrobial resistance determinants. The proximity of the Cagarras Islands to these impacted coastal areas, together with the opportunistic foraging behavior of F. magnificens, which includes scavenging on fishing discards, kleptoparasitism, and feeding near coastal human-impacted habitats, strongly suggests that these seabirds may acquire resistant bacteria from such contaminated environments (Schunck et al., 2025). This environmental context supports our findings and emphasizes the role of anthropogenic pollution in shaping the resistome of wildlife in the region.

The detection of a bla_CTXM-15-positive IncB/O/K/Z plasmid in E. coli from F. magnificens aligns with recent evidence of the same plasmid type mediating multidrug resistance in clinical S. enterica isolate from blood of a hospitalised renal transplant patient in China (Ma et al., 2024). The strong genetic similarity between the p99RCEF_blaCTX-M-15 and clinical plasmids underscores the potential for interspecies and inter-environmental spread of critical resistance determinants, reinforcing the importance of One Health surveillance strategies.

The detection of an IncB/O/K/Z plasmid in F. magnificens contrasts with the predominance of IncF and IncI1 plasmids typically reported in seabirds (Wang et al., 2017; Athanasakopoulou et al., 2022). Furthermore, the identification of serogroup O27:H14 and ST5614 diverges from the more frequently observed serogroups in seabirds, such as O25b (ST131), O78 (ST117), and those associated with ST1159 and ST602 (Ewbank et al., 2022a; Dalazen et al., 2023). Notably, while serogroup O27 has been identified as the predominant serotype (75% of isolates) among Shiga toxin-producing E. coli strains from wild mammals including wild boar, red deer, otter, and fox in Portugal (Dias et al., 2022), its detection in marine birds appears to be previously unreported in the literature. The absence of documented O27 detection in Brazilian coastal waters or clinical surveillance studies further emphasizes the novelty of this finding and may suggest potential gaps in current epidemiological monitoring of E. coli serogroup diversity in marine environments. The detection of O27 E. coli in wild mammals coupled with its absence in current marine bird surveillance suggests that F. magnificens may serve as a carrier for this serogroup in this coastal environment.

The phylogenetic divergence of the 99RCEF isolate from other E. coli strains of the same ST may reflects a unique evolutionary history shaped by its host’s specific ecological context. Originating from a magnificent frigatebird inhabiting relatively isolated and anthropogenically impacted Cagarras islands, the founder population from which 99RCCEF belonged may have been subject to evolutionary forces such as genetic drift, positive selection and horizontal gene transfer (HGT) (Zdziarski et al., 2010; Martinez, 2012). These evolutionary processes may have influenced the development of its distinctive core phylogenetic markers, likely as an adaptive response to local antimicrobial selective pressures and interspecific competition within the microbial community. The ecological mobility and foraging behavior of F. magnificens likely facilitated contact with diverse microbial populations across different environments. This exposure to varied microbial communities, combined with local selective pressures, may collectively explain the genetic distinctiveness observed in isolate 99RCEF when compared to other strains of the same sequence type.

The epidemiological significance of detecting a plasmid with high structural similarity to those circulating in human and animal populations is considerable. Plasmids of the IncB/O/K/Z group are recognized as important vehicles for the global dissemination of bla_CTX-M-15 and other resistance genes (Rozwandowicz et al., 2018; Shirakawa et al., 2020; Ma et al., 2024). The occurrence of such plasmids in a wild seabird from a region heavily impacted by sewage discharge suggests that environmental interfaces play a crucial role in the maintenance and spread of AMR determinants (Coutinho et al., 2013).

Wildlife, including seabirds such as F. magnificens, may act as both carriers and vectors of clinically relevant resistance genes, facilitating their movement between environmental, animal, and human populations. The foraging behavior of F. magnificens, which includes feeding on fish and fishery waste in areas such as Guanabara Bay, increases their exposure to anthropogenically derived contaminants, including MDR bacteria. Our findings represent the first identification of the bla_CTXM-15 gene variant in F. magnificens from the Cagarras Islands. Previous research in the Alcatrazes Archipelago identified E. coli isolates harboring bla_CTX-M-2 and bla_CMY-2 genes in F. magnificens but did not detect bla_CTXM-15 (Ewbank et al., 2022b).

The detection of bla_CTX-M variants in birds from both archipelagos, despite their geographic separation, reinforces the idea that wild seabirds in Brazil are consistently exposed to antimicrobial-resistant bacteria of clinical relevance. Considering the strong flight capacity and foraging range of F. magnificens, it is plausible that individuals may transit between these and other coastal areas, either directly or through overlapping feeding grounds. Fregata magnificens breeds on islands across the Caribbean and tropical coasts of Central and South America (Nuss et al., 2016). While some individuals remain near colonies due to the prolonged breeding season, others, especially non-breeders, disperse widely. GPS data have recorded post-breeding movements up to 1,400 km (Weimerskirch et al., 2006), such mobility could contribute to the regional circulation of resistant bacteria and genetic elements across marine ecosystems influenced by anthropogenic pollution.

The presence of bla_CTXM-15 in wild birds has been reported worldwide, including Brazil (Guenther et al., 2012; Ben Yahia et al., 2018; Batalha de Jesus et al., 2019; Zurfluh et al., 2019; Beleza et al., 2024). These findings collectively may indicate the widespread environmental circulation of this clinically relevant resistance determinant. Our results provides important new insights into the genetic diversity and geographic spread of ESBL genes among marine avifauna along the Brazilian coastline by confirming the occurrence of bla_CTX-M-15 in the F. magnificens population, and the identification of the IncB/O/K/Z plasmid p99RCEF_blaCTX-M-15 adds to the understanding of the diversity and distribution of resistance elements in wildlife.

In summary, the first detection of ESBL-EC carrying bla_CTX-M-15 and a multidrug-resistant IncB/O/K/Z plasmid in F. magnificens from the Cagarras Islands underscores the interconnectedness of environmental, animal, and human health. While our sample size was constrained by the logistical challenges of accessing this protected marine environment, the study’s principal contribution lies in documenting an ecological bridge for a clinically important resistance gene rather than estimating prevalence. The genomic evidence demonstrates mechanistic connectivity that transcends sample size limitations, with the plasmid’s close structural relationship to clinical isolates providing clear evidence of cross-environmental exchange. These findings reinforce the importance of wildlife surveillance in One Health AMR strategies and highlight the need for continued monitoring of resistance dynamics in coastal ecosystems impacted by human activities.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, PRJNA1281631, https://www.ncbi.nlm.nih.gov/, SAMN49569905, https://www.ncbi.nlm.nih.gov/genbank/, NZ_CP196680.1.

Ethics statement

The animal study was approved by all sampling procedures were conducted under SISBIO (Sistema de Autorização e Informação em Biodiversidade) permit no. 73163-6 and approved by the Animal Ethics Committee of the Oswaldo Cruz Institute (IOC), FIOCRUZ (Protocol L-019/2021). The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

BP: Writing – review & editing. LS: Writing – review & editing. CM: Writing – review & editing. MS: Writing – review & editing. DR: Writing – review & editing. BS-K: Writing – review & editing. DS: Writing – review & editing. MO: Writing – review & editing. MB: Writing – review & editing. AD’A: Writing – review & editing. MS: Writing – review & editing. CR-d-S: Writing – original draft, Writing – review & editing.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This research was supported by the Fundação Oswaldo Cruz (Fiocruz), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number: 408882/2022-0) and the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, grant number: E-26/210.085/2020). MB received financial support from FAPERJ, grant number: JCNE/200.244/2023 and PROEP (CNPQ/PROEP/IOC/441653/2024-3).

Acknowledgments

We thank the Institute Chico Mendes for Biodiversity Conservation (ICMBio)—Cagarras Archipelago Natural Monument (MONA Cagarras; https://www.gov.br/icmbio/pt-br/assuntos/biodiversidade/unidade-de-conservacao/unidades-de-biomas/marinho/lista-de-ucs/mona-do-arquipelago-das-ilhas-cagarras), as well as Dra. Tatiane Teixeira Leite Ribeiro and Sr. Sergio Pires Jordão for their logistical support with the boat during the expedition.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The authors declare that no Gen AI was used in the creation of this manuscript.

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Publisher’s note

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Footnotes

1. ^http://www.eucast.org

2. ^https://github.com/nanoporetech/dorado

3. ^https://pubmlst.org/organisms/escherichia-spp

4. ^https://isfinder.biotoul.fr/blast.php

5. ^https://enterobase.warwick.ac.uk/species/index/ecoli

6. ^https://itol.embl.de/

References

Alikhan, N. F., Petty, N. K., Ben Zakour, N. L., and Beatson, S. A. (2011). BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 12:402. doi: 10.1186/1471-2164-12-402

Crossref Full Text | Google Scholar

Athanasakopoulou, Z., Diezel, C., Braun, S. D., Sofia, M., Giannakopoulos, A., Monecke, S., et al. (2022). Occurrence and characteristics of ESBL- and carbapenemase- producing Escherichia coli from wild and feral birds in Greece. Microorganisms 10:1217. doi: 10.3390/microorganisms10061217

PubMed Abstract | Crossref Full Text | Google Scholar

Batalha de Jesus, A. A., Freitas, A. A. R., de Souza, J. C., Martins, N., Botelho, L. A. B., Girão, V. B. C., et al. (2019). High-level multidrug-resistant Escherichia coli isolates from wild birds in a large urban environment. Microb. Drug Resist. 25, 167–172. doi: 10.1089/mdr.2018.0180

PubMed Abstract | Crossref Full Text | Google Scholar

Beleza, A. J. F., Maciel, W. C., Carreira, A. S., Marques, A. R., Lima, B. P., Nogueira, C. H. G., et al. (2024). Wild birds as reservoirs of multidrug-resistant Enterobacteria in Mulungu, Brazil. Braz. J. Poult. Sci. 26:eRBCA. doi: 10.1590/1806-9061-2022-1791

PubMed Abstract | Crossref Full Text | Google Scholar

Ben Yahia, H., Ben Sallem, R., Tayh, G., Klibi, N., Ben Amor, I., Gharsa, H., et al. (2018). Detection of CTX-M-15 harboring Escherichia coli isolated from wild birds in Tunisia. BMC Microbiol. 18:26. doi: 10.1186/s12866-018-1163-2

PubMed Abstract | Crossref Full Text | Google Scholar

Bortolaia, V., Kaas, R. S., Ruppe, E., Roberts, M. C., Schwarz, S., Cattoir, V., et al. (2020). ResFinder 4.0 for predictions of phenotypes from genotypes. J. Antimicrob. Chemother. 75, 3491–3500. doi: 10.1093/jac/dkaa345

PubMed Abstract | Crossref Full Text | Google Scholar

Carattoli, A., Zankari, E., García-Fernández, A., Voldby Larsen, M., Lund, O., Villa, L., et al. (2014). In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob. Agents Chemother. 58, 3895–3903. doi: 10.1128/aac.02412-14

PubMed Abstract | Crossref Full Text | Google Scholar

Costa, D., Poeta, P., Sáenz, Y., Vinué, L., Rojo-Bezares, B., Jouini, A., et al. (2006). Detection of Escherichia coli harbouring extended-spectrum beta-lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in Portugal. J. Antimicrob. Chemother. 58, 1311–1312. doi: 10.1093/jac/dkl415

Crossref Full Text | Google Scholar

Costa, W. F., Paranhos, R., Mello, M. P., Picão, R. C., and Laport, M. S. (2023). Occurrence of extended-spectrum β-lactamases-producing Escherichia coli isolates over gradient pollution in an urban tropical estuary. Environ. Microbiol. 25, 2041–2048. doi: 10.1111/1462-2920.16435

PubMed Abstract | Crossref Full Text | Google Scholar

Coutinho, F. H., Pinto, L. H., Vieira, R. P., Martins, O. B., Salloto, G. R. B., and Santoro, D. d. O. (2013). “Antibiotic resistance in aquatic environments of Rio de Janeiro, Brazil” in Perspectives in water pollution (IntechOpen). doi: 10.5772/54638

Crossref Full Text | Google Scholar

Dalazen, G., Fuentes-Castillo, D., Pedroso, L. G., Fontana, H., Sano, E., Cardoso, B., et al. (2023). CTX-M-producing Escherichia coli ST602 carrying a wide resistome in south American wild birds: another pandemic clone of one health concern. One Health 17:100586. doi: 10.1016/j.onehlt.2023.100586

PubMed Abstract | Crossref Full Text | Google Scholar

de Araujo, C. F. M., Silva, D. M., Carneiro, M. T., Ribeiro, S., Fontana-Maurell, M., Alvarez, P., et al. (2016). Detection of carbapenemase genes in aquatic environments in Rio de Janeiro, Brazil. Antimicrob. Agents Chemother. 60, 4380–4383. doi: 10.1128/AAC.02753-15

PubMed Abstract | Crossref Full Text | Google Scholar

Dias, D., Costa, S., Fonseca, C., Baraúna, R., Caetano, T., and Mendo, S. (2022). Pathogenicity of Shiga toxin-producing Escherichia coli (STEC) from wildlife: should we care? Sci. Total Environ. 812:152324. doi: 10.1016/j.scitotenv.2021.152324

PubMed Abstract | Crossref Full Text | Google Scholar

Duré, F. M., Silveira, M. C., Rocha-de-Souza, C. M., Leão, R. S., de Oliveira Santos, I. C., Albano, R. M., et al. (2022). CABGen: a web application for the Bioinformatic analysis of bacterial genomes. Front. Microbiol. 13:893474. doi: 10.3389/fmicb.2022.893474

PubMed Abstract | Crossref Full Text | Google Scholar

Ewbank, A. C., Esperón, F., Sacristán, C., Sacristán, I., Krul, R., Cavalcante de Macedo, E., et al. (2021c). Seabirds as anthropization indicators in two different tropical biotopes: a one health approach to the issue of antimicrobial resistance genes pollution in oceanic islands. Sci. Total Environ. 754:142141. doi: 10.1016/j.scitotenv.2020.142141

PubMed Abstract | Crossref Full Text | Google Scholar

Ewbank, A. C., Fuentes-Castillo, D., Sacristán, C., Cardoso, B., Esposito, F., and Fuga, B., Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli survey in wild seabirds at a pristine atoll in the southern Atlantic Ocean, Brazil: first report of the O25b-ST131 clone harboring blaCTX-M-8. Sci. Total Environ. 806:150539 (2022a). Available online at: (https://agris.fao.org/search/en/providers/122535/records/65dfa7e663b8185d9caddf41?utm_source=chatgpt.com)

Google Scholar

Ewbank, A. C., Fuentes-Castillo, D., Sacristán, C., Esposito, F., Fuga, B., Cardoso, B., et al. (2022b). World Health Organization critical priority Escherichia coli clone ST648 in magnificent frigatebird (Fregata magnificens) of an uninhabited insular environment. Front. Microbiol. 13:940600. doi: 10.3389/fmicb.2022.940600

PubMed Abstract | Crossref Full Text | Google Scholar

Fernandes, M. R., Sellera, F. P., Moura, Q., Souza, T. A., and Lincopan, N. (2018). Draft genome sequence of a CTX-M-8, CTX-M-55 and FosA3 co-producing Escherichia coli ST117/B2 isolated from an asymptomatic carrier. J. Glob. Antimicrob. Resist. 12, 183–184. doi: 10.1016/j.jgar.2018.01.015

PubMed Abstract | Crossref Full Text | Google Scholar

Guenther, S., Aschenbrenner, K., Stamm, I., Bethe, A., Semmler, T., Stubbe, A., et al. (2012). Comparable high rates of extended-Spectrum-Beta-lactamase-producing Escherichia coli in birds of prey from Germany and Mongolia. PLoS One 7:e53039. doi: 10.1371/journal.pone.0053039

PubMed Abstract | Crossref Full Text | Google Scholar

Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q., and Sy Vinh, L. (2018). UFBoot2: Improving the ultrafast bootstrap approximation. Mol. Biol. Evol., 35, 518–522. doi: 10.1093/molbev/msx281

Crossref Full Text | Google Scholar

Joensen, K. G., Tetzschner, A. M. M., Iguchi, A., Aarestrup, F. M., and Scheutz, F. (2015). Rapid and easy in silico serotyping of Escherichia coli isolates by use of whole-genome sequencing data. J. Clin. Microbiol. 53, 2410–2426. doi: 10.1128/jcm.00008-15

PubMed Abstract | Crossref Full Text | Google Scholar

Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A., and Jermiin, L. S. (2017). ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods, 14, 587–589. doi: 10.1038/nmeth.4285

Crossref Full Text | Google Scholar

Kille, B., Nute, M. G., Huang, V., Kim, E., Phillippy, A. M., and Treangen, T. J. (2024). Parsnp 2.0: scalable core-genome alignment for massive microbial datasets, Bioinformatics, 40:btae311. doi: 10.1093/bioinformatics/btae311

Crossref Full Text | Google Scholar

Letunic, I., and Bork, P. (2024). Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool, Nucleic Acids Research, 52, W78–W82. doi: 10.1093/nar/gkae268

Crossref Full Text | Google Scholar

Ma, W., Cui, X., Dong, X., Li, X., Liu, K., Wang, Y., et al. (2024). Characterization of nontyphoidal Salmonella strains from a tertiary hospital in China: serotype diversity, multidrug resistance, and genetic insights. Front. Cell. Infect. Microbiol. 13:1327092. doi: 10.3389/fcimb.2023.1327092

PubMed Abstract | Crossref Full Text | Google Scholar

Magiorakos, A.-P., Srinivasan, A., Carey, R. B., Carmeli, Y., Falagas, M. E., Giske, C. G., et al. (2012). Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 18, 268–281. doi: 10.1111/j.1469-0691.2011.03570.x

PubMed Abstract | Crossref Full Text | Google Scholar

Martinez, J. L. (2012). Bottlenecks in the transferability of antibiotic resistance from natural ecosystems to human bacterial pathogens. Front. Microbiol. 2:265. doi: 10.3389/fmicb.2011.00265

PubMed Abstract | Crossref Full Text | Google Scholar

Martín-Maldonado, B., Rodríguez-Alcázar, P., Fernández-Novo, A., González, F., Pastor, N., López, I., et al. (2022). Urban birds as antimicrobial resistance sentinels: white storks showed higher multidrug-resistant Escherichia coli levels than seagulls in Central Spain. Animals 12:2714. doi: 10.3390/ani12192714

PubMed Abstract | Crossref Full Text | Google Scholar

Minh, B. Q., Schmidt, H. A., Chernomor, O., Schrempf, D., Woodhams, M. D., von Haeseler, A., et al. (2020). IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era, Molecular Biology and Evolution, 37, 1530–1534. doi: 10.1093/molbev/msaa015

Crossref Full Text | Google Scholar

Montezzi, L. F., Campana, E. H., Corrêa, L. L., Justo, L. H., Paschoal, R. P., da Silva, I. L. V. D., et al. (2015). Occurrence of carbapenemase-producing bacteria in coastal recreational waters. Int. J. Antimicrob. Agents 45, 174–177. doi: 10.1016/j.ijantimicag.2014.10.016

PubMed Abstract | Crossref Full Text | Google Scholar

Nuss, A., Carlos, C. J., Moreno, I. B., and Fagundes, N. J. R. (2016). Population genetic structure of the magnificent Frigatebird Fregata magnificens (Aves, Suliformes) breeding colonies in the Western Atlantic Ocean. PLoS One 11:e0149834. doi: 10.1371/journal.pone.0149834

PubMed Abstract | Crossref Full Text | Google Scholar

Parks, D. H., Imelfort, M., Skennerton, C. T., Hugenholtz, P., and Tyson, G. W. (2015). CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 25, 1043–1055. doi: 10.1101/gr.186072.114

PubMed Abstract | Crossref Full Text | Google Scholar

Paschoal, R. P., Campana, E. H., de S. Castro, L., and Picão, R. C. (2020). Predictors of carbapenemase-producing bacteria occurrence in polluted coastal waters. Environmental Pollution 264:114776. doi: 10.1016/j.envpol.2020.114776

Crossref Full Text | Google Scholar

Pinto, A., Simões, R., Oliveira, M., Vaz-Pires, P., Brandão, R., and da Costa, P. M. (2015). Multidrug resistance in wild BIRD populations: importance of the food chain. J. Zoo Wildl. Med. 46, 723–731. doi: 10.1638/2012-0212.1

PubMed Abstract | Crossref Full Text | Google Scholar

Rozwandowicz, M., Brouwer, M. S. M., Fischer, J., Wagenaar, J. A., Gonzalez-Zorn, B., Guerra, B., et al. (2018). Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae. J. Antimicrob. Chemother. 73, 1121–1137. doi: 10.1093/jac/dkx488

PubMed Abstract | Crossref Full Text | Google Scholar

Saladin, M., Cao, V. T. B., Lambert, T., Donay, J.-L., Herrmann, J.-L., and Ould-Hocine, Z. (2002). Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol. Lett. 209, 161–168. doi: 10.1111/j.1574-6968.2002.tb11126.x

PubMed Abstract | Crossref Full Text | Google Scholar

Salgado-Caxito, M., Benavides, J. A., Adell, A. D., Paes, A. C., and Moreno-Switt, A. I. (2021). Global prevalence and molecular characterization of extended-spectrum β-lactamase producing-Escherichia coli in dogs and cats – a scoping review and meta-analysis. One Health 12:100236. doi: 10.1016/j.onehlt.2021.100236

PubMed Abstract | Crossref Full Text | Google Scholar

Sanganyado, E., and Gwenzi, W. (2019). Antibiotic resistance in drinking water systems: Occurrence, removal, and human health risks. Science of The Total Environment 669, 785–797. doi: 10.1016/j.scitotenv.2019.03.162

Crossref Full Text | Google Scholar

Saviolli, J. Y., Cunha, M. P. V., Guerra, M. F. L., Irino, K., Catão-Dias, J. L., and de Carvalho, V. M. (2016). Free-ranging frigates (Fregata magnificens) of the southeast coast of Brazil harbor extraintestinal pathogenic Escherichia coli resistant to antimicrobials. PLoS One 11:e0148624. doi: 10.1371/journal.pone.0148624

Crossref Full Text | Google Scholar

Schunck, F., Dores, F., Paes, N., Melo, M., Matinata, B., Bokermann, M., et al. (2025). Extra-marine occurrence of the magnificent frigatebird Fregata magnificens in the metropolitan region of São Paulo, Southeast Brazil. Mar. Ornithol. 51, 55–60. doi: 10.5038/2074-1235.51.1.1511

Crossref Full Text | Google Scholar

Seemann, T. (2014). Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068–2069. doi: 10.1093/bioinformatics/btu153

PubMed Abstract | Crossref Full Text | Google Scholar

Shirakawa, T., Sekizuka, T., Kuroda, M., Suzuki, S., Ozawa, M., Abo, H., et al. (2020). Comparative genomic analysis of third-generation-cephalosporin-resistant Escherichia coli harboring the blaCMY-2-positive IncI1 group, IncB/O/K/Z, and IncC plasmids isolated from healthy broilers in Japan. Antimicrob. Agents Chemother. 64:10.1128/aac.02385-19. doi: 10.1128/aac.02385-19

Crossref Full Text | Google Scholar

Siguier, P., Perochon, J., Lestrade, L., Mahillon, J., and Chandler, M. (2006). ISfinder: the reference centre for bacterial insertion sequences, Nucleic Acids Research, 34, D32–D36. doi: 10.1093/nar/gkj014

Crossref Full Text | Google Scholar

Silva, G. G. da C., Campana, E. H., Vasconcelos, P. C., Silva, N. M. V.da, Santos Filho, L., Leite, E. L., et al. (2020). Occurrence of KPC-producing Escherichia coli in Psittaciformes rescued from trafficking in Paraíba, Brazil. Int. J. Environ. Res. Public Health 18:95 doi: 10.3390/ijerph18010095

Crossref Full Text | Google Scholar

Silva, M. M., Sellera, F. P., Fernandes, M. R., Moura, Q., Garino, F., Azevedo, S. S., et al. (2018). Genomic features of a highly virulent, ceftiofur-resistant, CTX-M-8-producing Escherichia coli ST224 causing fatal infection in a domestic cat. J. Glob. Antimicrob. Resist. 15, 252–253. doi: 10.1016/j.jgar.2018.10.023

PubMed Abstract | Crossref Full Text | Google Scholar

Stedt, J., Bonnedahl, J., Hernandez, J., Waldenström, J., McMahon, B. J., Tolf, C., et al. (2015). Carriage of CTX-M type extended spectrum β-lactamases (ESBLs) in gulls across Europe. Acta Vet. Scand. 57:74. doi: 10.1186/s13028-015-0166-3

PubMed Abstract | Crossref Full Text | Google Scholar

Wang, J., Ma, Z.-B., Zeng, Z.-L., Yang, X.-W., Huang, Y., and Liu, J.-H. (2017). The role of wildlife (wild birds) in the global transmission of antimicrobial resistance genes. Zool. Res. 38, 55–80. doi: 10.24272/j.issn.2095-8137.2017.003

PubMed Abstract | Crossref Full Text | Google Scholar

Weimerskirch, H., Le Corre, M., Marsac, F., Barbraud, C., Tostain, O., and Chastel, O. (2006). Postbreeding movements of frigatebirds tracked with satellite telemetry. Condor 108, 220–225. doi: 10.1093/condor/108.1.220

Crossref Full Text | Google Scholar

Wick, R. R., Judd, L. M., Gorrie, C. L., and Holt, K. E. (2017). Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol. 13:e1005595. doi: 10.1371/journal.pcbi.1005595

PubMed Abstract | Crossref Full Text | Google Scholar

Wood, D. E., Lu, J., and Langmead, B. (2019). Improved metagenomic analysis with kraken 2. Genome Biol. 20:257. doi: 10.1186/s13059-019-1891-0

PubMed Abstract | Crossref Full Text | Google Scholar

Yuan, Y., Liang, B., Jiang, B.-W., Zhu, L.-W., Wang, T.-C., Li, Y.-G., et al. (2021). Migratory wild birds carrying multidrug-resistant Escherichia coli as potential transmitters of antimicrobial resistance in China. PLoS One 16:e0261444. doi: 10.1371/journal.pone.0261444

PubMed Abstract | Crossref Full Text | Google Scholar

Zaluski, S., Soanes, L. M., Bright, J. A., George, A., Jodice, P. G. R., Meyer, K., et al. (2019). Potential threats facing a globally important population of the magnificent frigatebird Fregata magnificens. Trop. Zool. 32, 188–201. doi: 10.1080/03946975.2019.1682352

Crossref Full Text | Google Scholar

Zdziarski, J., Brzuszkiewicz, E., Wullt, B., Liesegang, H., Biran, D., Voigt, B., et al. (2010). Host imprints on bacterial genomes—rapid, divergent evolution in individual patients. PLoS Pathog. 6:e1001078. doi: 10.1371/journal.ppat.1001078

PubMed Abstract | Crossref Full Text | Google Scholar

Zurfluh, K., Albini, S., Mattmann, P., Kindle, P., Nüesch-Inderbinen, M., Stephan, R., et al. (2019). Antimicrobial resistant and extended-spectrum β-lactamase producing Escherichia coli in common wild bird species in Switzerland. Microbiology 8:e845. doi: 10.1002/mbo3.845

PubMed Abstract | Crossref Full Text | Google Scholar