Wildlife rehabilitation centers serve a vital ecological function by offering care and support to injured, sick, or orphaned animals (Willette et al. 2023). Through their efforts, these centers facilitate the conservation and recovery of numerous species, playing a key role in safeguarding biodiversity (Willette et al. 2023). However, while these facilities offer significant benefits to wildlife, they may also inadvertently create conditions that favor the acquisition and spread of human-associated bacterial pathogens (Sellera 2019). Herein, we present the identification and genomic characterization of international clones of ESBL-positive E. coli isolated from wild raptors undergoing rehabilitation in Brazil.

In June 2024, during a local epidemiological investigation conducted to monitor the presence of WHO critical priority bacteria in wild hosts, rectal/cloacal samples from 49 animals undergoing rehabilitation at the "Orquidário Municipal de Santos" zoological park in Santos, Southeast Brazil were collected and transported to the research laboratory within 72 h at room temperature using charcoal-containing Amies medium. Sample collection was carried out during clinical examinations already required for animal care and followed the standard veterinary procedures routinely conducted at this rehabilitation center. The species studied include: black-eared opossum (Didelphis marsupialis, n = 26), pale baywing (Agelaioides fringillarius, n = 5), red-rumped agouti (Dasyprocta leporina, n = 4), crested caracara (Caracara plancus, n = 4), black vulture (Coragyps atratus, n = 2), striped owl (Asio clamator, n = 2), American barn owl (Tyto furcata, n = 2), burrowing owl (Athene cunicularia, n = 1), rusty-barred owl (Strix hylophila, n = 1), American kestrel (Falco sparverius, n = 1), and roadside hawk (Rupornis magnirostris, n = 1).

Bacterial isolation was performed by streaking swab samples onto plates of MacConkey agar (Kasvi, Spain) supplemented with 2 mg/L of ceftriaxone (Blau Farmacêutica, Brazil). The plates were incubated at 35 ± 2 °C for up to 24 h and colonies growing were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing was carried out using disk diffusion (Cefar, Brazil) and concentration gradient strip (Liofilchem, Italy) methods for the following antimicrobials: ceftriaxone, cefotaxime, aztreonam, ceftazidime, cefepime, amoxicillin/clavulanic acid, cefoxitin, ertapenem, imipenem, meropenem, amikacin, gentamicin, ciprofloxacin, levofloxacin, tetracycline, trimethoprim/sulfamethoxazole, and chloramphenicol. The results were interpreted by the Clinical and Laboratory Standards Institute (M100TM, 34th ed., 2024). The MDR profile (resistance to at least one antimicrobial agent in three or more antimicrobial categories listed in the Enterobacteriaceae table) of the strains was determined as previously defined by Magiorakos et al. (2012).

The phenotypic production of ESBL was evaluated using the double-disk synergy test (Jarlier et al. 1988), while the molecular confirmation was performed by conventional polymerase chain reactions targeting CTX-M group-encoding genes, including bla, bla, bla, and bla (Dropa et al. 2015).

Genomic DNA was extracted by PureLink™ Genomic DNA Mini Kit (Thermo Fisher Scientific, USA). DNA libraries were prepared using the Rapid Plus DNA Library Prep Kit for Illumina (RK20208, Biozym, Germany) and genomic sequencing was performed by Illumina HiSeq platform (Illumina Inc., USA). Read trimming, de novo assembly, quality check, and annotation were carried out using BBDuk (Geneious Prime v.2025), SPAdes v.3.15.2 (https://github.com/ablab/spades), CheckM (Parks et al. 2015) and RAST server (https://rast.nmpdr.org/), respectively. Phylogenetic groups were determined by ClermonTyping (http://clermontyping.iame-research.center/). Multi-locus sequence typing, CH typing, serotyping, antimicrobial resistance genes (ARGs), virulence genes, and plasmid replicons were determined using MLST v.2.0, CHTyper v.1.0, SerotypeFinder v.2.0, ResFinder v.4.7.2, VirulenceFinder v.2.0, and PlasmidFinder v.2.1, respectively, which are available at the Center for Genomic Epidemiology (https://www.genomicepidemiology.org/services/). The association between plasmid replicons and bla genes was investigated using MobileElementFinder v.0.1.3 (https://cge.food.dtu.dk/services/MobileElementFinder/). All beforementioned tools were used with default parameters. The genetic contexts of the bla and bla genes were analyzed by nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and Geneious Prime 2025.1.2 and compared using Easyfig (https://mjsull.github.io/Easyfig/).