Conserved protein sequence-structure signatures identify emerging antibiotic resistance genes from the human microbiome

Bacteria exhibiting antimicrobial resistance (AMR) is a problem that has grown to become a significant public health challenge worldwide. Antibiotic resistance genes (ARGs), determinants of AMR, mostly emerge from non-clinical settings. Identifying previously undetected ARGs in the human microbiome which confer resistance to clinical concentrations of antibiotics is a crucial component of addressing AMR, yet can be hindered by their low homology to existing ARGs. Here, we attempt to address this by focussing on functionally important protein regions. ARG-PASS (ARG-PAirwise Sequence vs Structure) represents a novel protein function prediction method which leverages a one-class support vector machine trained on pairwise primary and tertiary distributions of structurally conserved regions of proteins encoded by ARGs. ARG-PASS was applied to six reference strains of the Human Microbiome Project. Nine candidates were selected for experimental verification and all were functionally confirmed when expressed in E.coli, belonging to ARG classes: APH, dfr, class B and C {beta}-lactamases, and penicillin binding proteins. We also used ARG-PASS directly on protein structures within the AlphaFold database and predicted a phnP gene (metallo-{beta}-lactamase fold), which is highly divergent from existing {beta}-lactamases and had activity against ampicillin. In total, 80% of the tested genes confer resistance at CLSI resistant breakpoints and the remainder represent pre-resistance genes, with activity but not at clinically relevant minimum inhibitory concentrations. We suggest pre-resistance genes may preferentially evolve into clinically relevant resistance determinants. ARG-PASS represents a novel and precise method of identifying previously uncharacterized ARGs from DNA databases, contributing to resistance surveillance and antibiotic stewardship.