Hydrophobic Agent Permeability Assay (HAPA) for Rapid Antibiotic Susceptibility Testing

Timely and accurate antibiotic susceptibility testing (AST) is essential for effective infection management and combating antimicrobial resistance. Standard clinical methods, such as broth microdilution (BMD) and disk diffusion, depend on monitoring optical density or inhibition zone diameters and typically require 24 hours or longer to complete. Recent approaches that track single-cell division and morphological changes within the first four hours have shown promise for rapid AST but suffer from limited throughput. Here, we introduce a subcellular phenotyping strategy, termed the Hydrophobic Agent Permeability Assay (HAPA), that monitors bacterial outer membrane permeability dynamics, as a direct functional readout of antibiotic efficacy. In details, we find that susceptible Gram-negative bacteria will rapidly increase membrane permeability upon antibiotic exposure, while resistant cells are impermeable, which difference can be visualized by hydrophobic dyes. Remarkably, such a straightforward readout, as a unique indicator, at the cell envelope coincides with MIC-level inhibition, enabling discrimination of breakpoint responses, which only takes from minutes, to two hours, and four hours, for carbapenem, β-lactam, and aminoglycoside and quinolone drugs, respectively. From the tested 75 clinical isolates of the most representative Gram-negative members of the ESKAPE pathogens, including Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii , and Pseudomonas aeruginosa , HAPA achieved 97.1% categorical agreement with BMD, with low minor error (1.33%), major error (2.50%), and very major error (1.23%) rates, exceeding established regulatory performance benchmarks. By quantitatively correlating bacterial sensitivity to antibiotics to the dynamic membrane permeability responses, HAPA provides a high-throughput, accurate, and clinically translatable solution for rapid AST.