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. <i>Escherichia coli</i> 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 <i>E. coli</i> and associated resistance genes in 248 cloacal/eggshell samples collected from four autochthonous Portuguese laying hen breeds (<i>Preta Lusitânica</i>, <i>Amarela</i>, <i>Branca</i>, and <i>Pedrês Portuguesa</i>) raised under low antibiotic exposure. A total of 81 <i>E. coli</i> 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 <i>sul</i>2 (45.0% cloacal; 48.0% eggshell) and <i>bla</i>_TEM (45.0% cloacal; 36.0% eggshell). Detection of resistant and MDR <i>E. coli</i> in low input systems suggests environmental acquisition, with chickens as reservoirs, highlighting the need for One Health surveillance.