Abstract Background This study focuses on the AMR profiles in E. coli isolated from captive mammals at EcoZoo San Martín, Baños de Agua Santa, Ecuador, highlighting the role of wildlife as reservoirs of resistant bacteria. Aims The aim of this research is to investigate the antimicrobial resistance profiles of E. coli strains isolated from various species of captive mammals, emphasizing the potential zoonotic risks and the necessity for integrated AMR management strategies. Materials & Methods A total of 189 fecal samples were collected from 70 mammals across 27 species. These samples were screened for E. coli, resulting in 90 identified strains. The resistance profiles of these strains to 16 antibiotics, including 10 β‐lactams and 6 non‐β‐lactams, were determined using the disk diffusion method. Additionally, the presence of Extended‐Spectrum Beta‐Lactamase (ESBL) genes and other resistance genes was analyzed using PCR. Results Significant resistance was observed, with 52.22% of isolates resistant to ampicillin, 42.22% to ceftriaxone and cefuroxime, and 27.78% identified as ESBL‐producing E. coli. Multiresistance (resistance to more than three antibiotic groups) was found in 35.56% of isolates. Carnivorous and omnivorous animals, particularly those with prior antibiotic treatments, were more likely to harbor resistant strains. Discussion These findings underscore the role of captive mammals as indicators of environmental AMR. The high prevalence of resistant E. coli in these animals suggests that zoos could be significant reservoirs for the spread of antibiotic‐resistant bacteria. The results align with other studies showing that diet and antibiotic treatment history influence resistance profiles. Conclusion The study highlights the need for an integrated approach involving veterinary care, habitat management, and public awareness to prevent captive wildlife from becoming reservoirs of antibiotic‐resistant bacteria. Improved waste management practices and responsible antibiotic use are crucial to mitigate the risks of AMR in zoo environments and reduce zoonotic threats.
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