Conventional antibiotic susceptibility testing (AST) assays such as broth microdilution and Kirby–Bauer disk diffusion are time-consuming (e.g., 24–72 h) and labor-intensive. Here, we present a microfluidic platform to perform AST assays with a broad range of antibiotic concentrations and controls. A culture medium stream was serially enriched with antibiotics along the length of the platform via diffusion and flow-directing mass convection mechanisms, generating a concentration gradient captured in a series of microchamber duplicates. We observed an agreement between the simulated and experimental concentration gradients and applicability to a variety of different molecules by changing the loading time according to a simple linear equation. The AST assay in our platform is based on bacterial metabolism, indicated by resazurin fluorescence. The small reaction volume enabled a minimum inhibitory concentration (MIC) to be determined in 4–5 h. Proof-of-concept functionality testing, using human isolates and clinically important antibiotics from different classes, indicated a high rate of agreement (94%: MIC within ±1 two-fold dilution of the reference method) of on-chip MICs and conventional broth microdilution. Overall, our results showed that this microfluidic platform is capable of determining antibiotic susceptibility in a rapid and reliable manner.

Antimicrobial resistance is a growing problem, necessitating rapid antimicrobial susceptibility testing (AST) to enable effective in-clinic diagnostic testing and treatment. Conventional AST using broth microdilution or the Kirby−Bauer disk diffusion are time-consuming (e.g., 24−72 h), labor-intensive, and costly and consume reagents. Here, we propose a novel gradientbased microchamber microfluidic (GM 2 ) platform to perform AST assay for a wide range of antibiotic concentrations plus zero (positive control) and maximum (negative control) concentrations all in a single test. Antibiotic lateral diffusion within enriched to depleted (C max and zero, respectively) cocurrent flowing fluids, moving alongside a micron-sized main channel, is led to form an antibiotic concentration profile in microchambers, connected to the depleted side of the main channel. We examined the tunability of the GM 2 platform, in terms of producing a wide range of antibiotic concentrations in a gradient mode between two consecutive microchambers with changing either the loading fluids' flow rates or their initial concentrations. We also tested the GM 2 platform for profiling bacteria associated with human Crohn's disease and bovine mastitis. Time to result for performing a complete AST assay was ∼ 3−4 h in the GM 2 platform. Lastly, the GM 2 platform tracked the bacterial growth independent of an antibiotic mechanism of action or bacterial species in a robust and easy-to-implement fashion.

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