Abstract Dental calculus, the calcified form of the mammalian oral microbial plaque biofilm, is a rich source of oral microbiome, host, and dietary biomolecules and is well preserved in museum and archaeological specimens. Despite its wide presence in mammals, to date, dental calculus has primarily been used to study primate microbiome evolution. We establish dental calculus as a valuable tool for the study of nonhuman host microbiome evolution, by using shotgun metagenomics to characterize the taxonomic and functional composition of the oral microbiome in species as diverse as gorillas, bears, and reindeer. We detect oral pathogens in individuals with evidence of oral disease, assemble near-complete bacterial genomes from historical specimens, characterize antibiotic resistance genes, reconstruct components of the host diet, and recover host genetic profiles. Our work demonstrates that metagenomic analyses of dental calculus can be performed on a diverse range of mammalian species, which will allow the study of oral microbiome and pathogen evolution from a comparative perspective. As dental calculus is readily preserved through time, it can also facilitate the quantification of the impact of anthropogenic changes on wildlife and the environment.

Highlights d Wild animals can be used as indicators of antibiotic contamination of environments d Bear oral microbiota from the pre-antibiotic era reveal a baseline of natural AMR d Bear AMR load tracked human antibiotic use over the last 80 years d Bear AMR load decreased after Swedish strategy to curb AMR was implemented

Animals and their associated microbiomes share a long evolutionary history, influenced by a complex interplay between extrinsic environmental and intrinsic host factors. However, we know little about microbiome responses to long-lasting environmental and host-centred processes, which require studying microbiome changes through time. Here, we apply a temporal metagenomics approach to dental calculus, the calcified oral microbial biofilm. We establish dental calculus as a valuable tool for the study of host microbiome evolution by characterising the taxonomic and functional composition of the oral microbiome in a variety of wild mammals. We detect oral pathogens in individuals with evidence of oral disease, assemble near-complete bacterial genomes from historical specimens, characterise antibiotic resistance genes even before the advent of industrial antibiotic production, reconstruct components of the host diet and recover host genetic profiles. Our work demonstrates how dental calculus can be used in the future to study the evolution of oral microbiomes and pathogens, and the impact of anthropogenic changes on wildlife and the environment.

Following the advent of industrial-scale antibiotics production in the 1940s, antimicrobial resistance (AMR) has been on the rise and now poses a major global health threat. Because AMR can be exchanged between humans, livestock and wildlife, evaluating the potential of wild animals to act as AMR reservoirs is essential. We used shotgun metagenomics sequencing of dental calculus, the calcified form of the oral microbial biofilm, to determine the abundance and repertoire of AMR genes in the oral microbiome of Swedish brown bears from museum specimens collected over the last 200 years. Our temporal metagenomics approach allowed us to establish a baseline of natural AMR in the pre-antibiotics era and to quantify a significant increase in total AMR load and diversity of AMR genes that is correlated with human antibiotics use. We also demonstrated that Swedish public health policies were effective in limiting AMR spillover into wildlife.