Silent Reservoirs: Forensic Microbiology Uncovers Hidden Antimicrobial Resistance in Native Portuguese Laying Hens Raised Under Extensive Systems

Resumo

Introduction: Antimicrobial resistance (AMR) remains a major public health concern (Antunes et al., 2020), with antibiotic use in livestock farming recognized as a key contributor. Yet, the role of native Portuguese laying hens, raised under extensive systems with minimal antibiotic exposure, as potential reservoirs of resistant Escherichia coli remains largely overlooked (Miranda et al., 2023). Studying these birds offers a valuable model for understanding the natural ecology of AMR in the absence of direct selective pressure.

Objective: To assess the antibiotic resistance profiles of E. coli isolates from native Portuguese breeds within a One Health framework, and to explore the potential of Forensic Microbiology in tracking microbial sources through phenotypic and genetic profiling (Paruch & Paruch, 2024).

Materials and Methods: A total of 248 samples (124 cloacal swabs and 124 eggshells) were collected from birds with no recent antibiotic exposure. E. coli isolates were recovered using selective media, confirmed by PCR, and tested for susceptibility to 10 antibiotics following EUCAST/CLSI guidelines. Resistance genes were detected by PCR.

Results: Eighty-six E. coli strains were isolated (40 cloacal swabs, 46 from eggshells). Resistance to at least one antibiotic was observed in 98% of isolates. Cloacal isolates showed the highest resistance to gentamicin (97%), tetracycline (31%), and ampicillin (14%), while eggshell isolates showed resistance to gentamicin (96%), tetracycline (41%), and ampicillin (24%). Multidrug resistance was detected in 14% and 17% of cloacal and eggshell isolates, respectively. No ESBL or last-resort antibiotic resistance was found. The most frequently detected resistance genes were bla_TEM (45% cloacal, 36% eggshell) and sul2 (45% cloacal, 48% eggshell).

Discussion: The high prevalence of gentamicin resistance, despite limited antibiotic exposure, suggests environmental acquisition through contaminated water, soil, or wildlife, or co-selection pressures from other contaminants such as metals or disinfectants. The presence of resistance genes lacking phenotypic expression highlights the existence of silent reservoirs within extensive farming systems.

Conclusions: These findings reinforce the importance of continuous AMR monitoring and demonstrate the potential of forensic microbiology and microbial source tracking to pinpoint contamination origins, supporting more sustainable and biosecure poultry management practices.