Metagenomic insights into circulation and maintenance of antibiotic resistome in intensive dairy farm environments

Antibiotic resistance is an urgent global public health threat, with livestock recognized as a potential reservoir for antibiotic resistance genes (ARGs). Despite the extensive antibiotic use in dairy farms, little is known about ARG dynamics in these environments. This study was conducted on five intensive dairy farms in Greece to explore the presence of ARGs across the production cycle and potential exposure sources. Feces from lactating cows, pre-weaned and weaned calves, bulk-tank milk and colostrum, and environmental samples, including dust from feeding alleys and beams and air samples taken during routine farm activities, were collected. Stored solid and liquid manure and material from corresponding biogas treatment plants were also sampled. Shotgun metagenomic short-read sequencing was conducted on extracted DNAs to identify the antibiotic resistomes (ARG loads) and characterize microbiomes. Sequencing reads were aligned to reference ARGs (CARD, MEGARes, and ResFinder) and microbiome (Kraken) databases. ARGs to aminoglycosides, macrolide–lincosamide–streptogramin, tetracycline, sulfonamide–trimethoprim and β-lactams were abundant across samples. Pre-weaned calves carried high ARG loads, contrary to colostrum, reflecting increased antibiotic selective pressure in this age. Intriguingly, beam-dust samples harbored high load and diverse ARGs, including aac(6), aadA, aph(6), ant(6), aac(3), erm(A/B/F/X/C/35), mef(A), lnu(C/G), tet(A/H/K/L/M/Q/X), dfrA/D, sul1/2, and mcr and the ESBLs bla_TEM, bla_OXA, and bla_CTX-M. Unexpectedly, bla_IMP conferring resistance to carbapenems was detected in both animal and environmental samples, despite these antibiotics are not authorized for use in livestock. ARG maintenance in dust, along with lower but similar loads in air, poses a potential exposure risk for humans. Finally, biogas treatment samples, regardless of their differing microbiomes, were not free of ARGs and thereby could create, as fertilizers, an important channel for ARG transmission to humans and animals. In conclusion, avoidance of antibiotic misuse, application of strict biosafety measures and establishment of systematic surveillance are imperative for intensive dairy farming.