Discovery of novel antimicrobial resistance genes: integrons as a high-throughput gene capture and functional screening platform

Integrons are genetic elements that drive bacterial adaptation by capturing and expressing mobile gene cassettes. They play a key role in dissemination of antimicrobial resistance (AMR) genes, particularly in Gram-negative bacteria. In addition to known AMR determinants, integron gene cassettes carry a vast reservoir of novel genes whose functions are largely uncharacterised, making it difficult to assess their full contribution to the resistome. Contributing to this are limitations in current sequence-based prediction methods which often lack the ability to identify unknown AMR or other adaptive genes with novel mechanisms. To address this, we developed a high-throughput gene cassette capture system that utilises site-specific recombination activity of integrons and a counter selection strategy to capture and express gene cassettes from metagenomes. Coupling this platform with a functional screening approach allowed us to rapidly assay large libraries of environmental gene cassettes. Using this system, we recovered previously unknown AMR determinants while also providing insights into the prevalence of known clinical AMR genes in a range of environmental samples, including food and fertiliser. Here we provide experimental data on multiple novel bleomycin resistance genes and a stress response gene conferring gentamicin and tobramycin resistance. Our sequence analysis of the captured library also highlighted the diversity of the environmental cassette pool, with 656 unique cassettes recovered, the majority of which encoded proteins with unknown functions. The cassette capture system is a powerful tool for accessing hidden elements of the resistome and discovering novel adaptive genes that may go undetected using current sequence-based approaches.