High-resolution, high-throughput detection of hidden antibiotic resistance with the dilution-and-delay (DnD) susceptibility assay.

Rising rates of antibiotic treatment failure highlight the complexity of resistance mechanisms. While genetically encoded resistance is well established, recent clinical studies have uncovered noncanonical mechanisms driven by phenotypic heterogeneity, such as heteroresistance, persistence, or adaptive resistance. Yet, standard susceptibility tests lack the resolution or throughput to detect these diverse phenotypic mechanisms. To address these limitations, we develop a scalable, high-resolution assay-Dilution-and-Delay (DnD)-by implementing two basic principles of bacterial growth. DnD detects rare drug-insensitive cells at frequencies as low as 1 in 100 million, while also reporting bulk population inhibition as the conventional MIC. We demonstrate this capability across synthetic communities, heteroresistance, persistence, and evolutionary progression. These high-resolution data expose how traditional susceptibility assays are constrained by statistical detection limits. Scaling up DnD for high-throughput application to ~120 clinical isolates reveals that under-the-radar multidrug resistance or tolerance is common. By combining resolution with scalability, DnD provides an advanced platform for antibiotic susceptibility testing with broad impact on basic research, clinical practice, and epidemiological surveillance. It supports a new framework for measuring, defining, and understanding antibiotic resistance.