PROJECT SUMMARY The continuing emergence and rapid dissemination of antibiotic-resistant pathogens are becoming a major threat to public health. Infections caused by carbapenem-resistant Enterobacterales and carbapenem-resistant Acinetobacter, which are difficult to treat, were recognized as one of the urgent threats among patients in medical facilities. Serious infections caused by antibiotic-resistant pathogens are associated with higher rates of morbidity and mortality and contribute to significant economic costs. Cefiderocol, which received FDA approval in 2019, is a novel siderophore-conjugated cephalosporin with expanded activity against carbapenem-resistant Gram- negative pathogens including strains that produce serine carbapenemases and metallo-β-lactamases. Cefiderocol-nonsusceptible isolates were reported in surveillance studies but the resistance mechanisms are largely unknown on many occasions. Antimicrobial heteroresistance describes a phenomenon where subpopulations of genetically homogeneous bacteria exhibit a range of susceptibilities to a particular antibiotic. Heteroresistance has considerable clinical relevance because antibiotic treatment may select more resistant populations. It is considered an important factor contributing to unexplained antibiotic treatment failure. Heteroresistance has been reported in various bacterial species to many classes of antibiotics. However, there is a lack of thorough research studying cefiderocol heteroresistance, the underlying mechanisms, and its clinical relevance. The overall goal of the proposed project is to study preexisting cefiderocol resistance and heteroresistance without prior cefiderocol exposure and the evolution of cefiderocol resistance after cefiderocol exposure in vitro and in patients. In Aim 1, we will investigate preexisting cefiderocol resistance and heteroresistance mechanisms in clinically isolated carbapenem-resistant Gram-negative pathogens. In Aim 2, we will study in vitro and clinical evolution of cefiderocol resistance following cefiderocol exposure, and characterize isolates with reduced cefiderocol susceptibility.