Multipad Agarose Plate (MAP): A Rapid and High-Throughput Approach for Antibiotic Susceptibility Testing

We describe a phenotypic antibiotic susceptibility testing (AST) method that can provide an eightfold speedup in turnaround time compared to the current clinical standard by leveraging advances in microscopy and single-cell imaging. A newly developed growth plate containing 96 agarose pads, termed the Multipad Agarose Plate (MAP), can be assembled at low costs. Pads can be prepared with dilution series of antibiotics. Bacteria are seeded on the pads and automatically imaged using brightfield microscopy, with a fully automated segmentation pipeline quantifying microcolony formation and growth rate. Using a test set of nine antibiotics with very different targets, we demonstrate that accurate minimum inhibitory concentration (MIC) measurements can be performed based on the growth rate of microcolonies within three hours of incubation with the antibiotic. Faster, reliable and high throughput methods for AST, such as MAP, could improve patient care by expediting treatment initiation and alleviating the burden of antimicrobial resistance. ### Competing Interest Statement AD declares to work for a company that operates in the application areas described in this work, but has no direct conflict of interest.