Merging Metagenomics and Spatial Epidemiology To Understand the Distribution of Antimicrobial Resistance Genes from Enterobacteriaceae in Wild Owls

Antimicrobial resistance (AMR) is a well-documented phenomenon in bacteria from many natural ecosystems, including wild animals. However, the specific determinants and spatial distribution of resistant bacteria and antimicrobial resistance genes (ARGs) in the environment remain incompletely understood. In particular, information regarding the importance of anthropogenic sources of AMR relative to that of other biological and ecological influences is lacking. We conducted a cross-sectional study of AMR in great horned owls ( Bubo virginianus) and barred owls ( Strix varia) admitted to a rehabilitation center in the midwestern United States. A combination of selective culture enrichment and shotgun metagenomic sequencing was used to identify ARGs from Enterobacteriaceae Overall, the prevalence of AMR was comparable to that in past studies of resistant Enterobacteriaceae in raptors, with acquired ARGs being identified in 23% of samples. Multimodel regression analyses identified seasonality and owl age to be important predictors of the likelihood of the presence of ARGs, with birds sampled during warmer months being more likely to harbor ARGs than those sampled during cooler months and with birds in their hatch year being more likely to harbor β-lactam ARGs than adults. Beyond host-specific determinants, ARG-positive owls were also more likely to be recovered from areas of high agricultural land cover. Spatial clustering analyses identified a significant high-risk cluster of tetracycline resistance gene-positive owls in the southern sampling range, but this could not be explained by any predictor variables. Taken together, these results highlight the complex distribution of AMR in natural environments and suggest that both biological and anthropogenic factors play important roles in determining the emergence and persistence of AMR in wildlife. IMPORTANCE Antimicrobial resistance (AMR) is a multifaceted problem that poses a worldwide threat to human and animal health. Recent reports suggest that wildlife may play an important role in the emergence, dissemination, and persistence of AMR. As such, there have been calls for better integration of wildlife into current research on AMR, including the use of wild animals as biosentinels of AMR contamination in the environment. A One Health approach can be used to gain a better understanding of all AMR sources and pathways, particularly those at the human-animal-environment interface. Our study focuses on this interface in order to assess the effect of human-impacted landscapes on AMR in a wild animal. This work highlights the value of wildlife rehabilitation centers for environmental AMR surveillance and demonstrates how metagenomic sequencing within a spatial epidemiology framework can be used to address questions surrounding AMR complexity in natural ecosystems.