Multiplexed, rapid phenotypic antibiotic susceptibility testing based on angle-resolved light scattering imaging of microfluidic droplets

Introduction: There is an urgent need for rapid, high-throughput phenotypic antimicrobial susceptibility testing (AST) capable of assessing a microbial sample's susceptibility to multiple antibiotics. Objectives: In this study, we have established a multiplexed rapid AST platform that employs droplet microfluidics for high-throughput single-cell based analysis, 2D angle-resolved light scattering for growth detection, and fluorescence detection via optical fibers to identify the antibiotic condition within each droplet. Methods: For this, multiple antibiotic conditions are coded with fluorescence dyes and encapsulated with single cells to enable the testing of multiple antibiotics in a single experiment. We utilize convolutional neural networks (CNNs) and statistical models to assess the growth of various Staphylococcus aureus strains and determine the probability of susceptibility to different antibiotics. Results: Our platform achieved a 95 % categorical agreement with the disc diffusion reference method after just three hours of incubation, demonstrating the same level of accuracy as the established VITEK 2 system for the tested strains and antibiotics. Notably, our platform reduced the incubation time by 5–11 h compared to VITEK 2 and by 13–17 h compared to the gold standard disc diffusion method. Conclusions: With the presented innovations, our technology takes a big step towards realizing true phenotypic determination of antibiotic resistance profiles for timely antimicrobial treatment decisions.