Silent Reservoirs: Antibiotic-Resistant Escherichia coli in Autochtonous Portuguese Laying Hens.

Antimicrobial Resistance (AMR) is a critical public health challenge requiring a coordinated One Health approach. Escherichia coli is a key indicator of AMR and fecal contamination, as well as a zoonotic pathogen transmissible from animals to humans, often through contaminated products like meat and eggs. This study assessed the presence of antibiotic-resistant E. coli and associated resistance genes in 248 cloacal/eggshell samples collected from four autochthonous Portuguese laying hen breeds (Preta Lusitânica, Amarela, Branca, and Pedrês Portuguesa) raised under low antibiotic exposure. A total of 81 E. coli isolates were analyzed for phenotypic antibiotic susceptibility (EUCAST/CLSI) and genotypic resistance, using PCR. Resistance to at least one antibiotic was observed in 98.0% of the isolates. Gentamicin resistance was particularly high (97.1% cloacal; 95.7% eggshell isolates), followed by tetracycline (31.0% cloacal; 41.0% eggshell) and ampicillin (14.0% cloacal; 24.0% eggshell). Multidrug resistance (MDR) was observed in 14.3% of cloacal and 17.4% of eggshell isolates. Notably, no resistance was found against critically important antibiotics. The most prevalent resistance genes were sul2 (45.0% cloacal; 48.0% eggshell) and blaTEM (45.0% cloacal; 36.0% eggshell). Detection of resistant and MDR E. coli in low input systems suggests environmental acquisition, with chickens as reservoirs, highlighting the need for One Health surveillance.