Exploring the diversity and burden of antimicrobial resistance genes carried by white stork (Ciconia ciconia) throughout the breeding season in Madrid, Spain

Abstract

Anthropogenic environments are critical hotspots for the emergence and persistence of antimicrobial resistance (AMR). Extensive livestock pastures and landfills are of particular concern due to their high bacterial diversity and frequent wildlife visitation, facilitating the transfer of antimicrobial resistance genes (ARGs). The white stork (Ciconia ciconia) heavily exploits landfills and extensive pastures in Spain, providing an ideal model to study AMR emergence. We evaluated the diversity and burden of ARGs in breeding white storks across three stages of the breeding season, reflecting different foraging strategies. Fecal samples from 31 nests were collected during two breeding seasons (2020−2021) and screened for 21 ARGs across eight antibiotic classes using real-time qPCR. ARGs were universally present, and over 70 % of nests contained fecal samples harboring genes conferring resistance to three or more antibiotic classes, indicating a high burden of potential multi-drug resistance at the community level. Generalized linear models identified breeder age and sampling period as strong predictors of total ARG burden (p < 0.001), with older breeders contributing more ARG burden early in the season, although this effect diminished later. Landfill use showed only a weak association with ARG burden, while nest success approached significance. These findings suggest that foraging dynamics, rather than landfill exposure alone, strongly influence ARG acquisition in white storks. Agricultural pastures and other anthropogenic environments may play a larger role in shaping resistomes than previously assumed. Understanding how wildlife foraging behavior drives AMR carriage is crucial to better characterize AMR transmission risk at the wildlife-livestock interface.