Antimicrobial resistance (AMR) is a major threat to global human health. Improved AMR surveillance is one of several priorities identified by the World Health Organization to mitigate the burden of AMR and improve the use of existing antibiotics. Complicating surveillance is the fact that existing AMR genes do not explain all antibiotic-resistant infections. Using a combination of forced evolution and metabolic rescue experiments, we have identified a novel mechanism of resistance to sulfamethoxazole (a potent inhibitor of folate biosynthesis) in the major human pathogen Group A Streptococcus (GAS). Â Resistance is mediated by a substrate-binding protein (ThfT) that expands the substrate profile of an endogenous ABC transporter to include multiple end products of the folate biosynthesis pathway. As this mechanism of AMR involves acquisition of these metabolites directly from the host it remains undetectable by in vitro antibiotic susceptibility testing methods. We propose that ThfT-mediated sulfamethoxazole resistance constitutes a new paradigm of bacterial AMR that is only active during an infection, and highlights the need to understand the activity of antibiotics in the context of the infections they are designed to treat. In this presentation, I will outline our recent data that has characterised this new mechanism of antibiotic resistance, and how we are now using this research to improve the treatment and prevention of GAS infections in remote Aboriginal and Torres Strait Islander communities.