Miniaturized Antimicrobial Susceptibility Test by Combining Concentration Gradient Generation and Rapid Cell Culturing

Abstract

Effective treatment of bacterial infection relies on timely diagnosis and proper prescription of antibiotic drugs. The antimicrobial susceptibility test (AST) is one of the most crucial experimental procedures, providing the baseline information for choosing effective antibiotic agents and their dosages. Conventional methods, however, require long incubation times or significant instrumentation costs to obtain test results. We propose a lab-on-a-chip approach to perform AST in a simple, economic, and rapid manner. Our assay platform miniaturizes the standard broth microdilution method on a microfluidic device (20 × 20 mm) that generates an antibiotic concentration gradient and delivers antibiotic-containing culture media to eight 30-nL chambers for cell culture. When tested with 20 μL samples of a model bacterial strain (E. coli ATCC 25922) treated with ampicillin or streptomycin, our method allows for the determination of minimum inhibitory concentrations consistent with the microdilution test in three hours, which is almost a factor of ten more rapid than the standard method.

Microfluidic advances in phenotypic antibiotic susceptibility testing

“…have developed a concentration gradient-forming microfluidic AST platform that tracks the number of bacteria within eight growth chambers (Kim et al 2015). Bacteria (5×10 5 CFU/mL) mixed with an antibiotic of interest and with buffer are injected into the two input ports, and a concentration gradient is generated via automatic or manual pressure (Fig.…”

“…use varying channel lengths to generate a concentration gradient on-chip; Bacteria are included in both the reagent and diluent; (b) Derzsi et al . use metering traps to deliver a nanoliter-sized bacterial sample to the storage well, followed by diluted reagent; The sequential merging of the diluent and the reagent in the metering traps forms a reagent plug that is approximately half as concentrated as the previous one; Panel a modified from (Kim et al 2015); Panel b modified and reprinted with permission (Derzsi et al 2016)…”

Rapid antibiotic sensitivity testing in microwell arrays

“…Multiple tests could be simultaneously performed on multiple samples 5,6,10,11 . Imaging to quantify microbial growth in the presence and absence of antibiotics was implemented using several techniques, including the monitoring of optical density 6,8,9,11 , bright field/phase contrast 11 and fluorescence measurements 6,8–10,13,14,18 . Several unconventional methods for monitoring AST have also been proposed, including RNA specific electrochemical biosensors 34 , pH changes of culture media during cell growth 10,35 , or asynchronous magnetic beads 36 .…”

“…Microscale systems usually require long and elaborated bacteria pre-loading and preparation steps 6 and thus remain time-consuming 6 . Some of the devices require complex infrastructure, such as use of pumps and syringes 9,11,13,14,16–18 . Many require the immobilization of bacteria with agarose within microchannels 11,15 or on agarose microparticles 37 .…”

Fast Antibiotic Susceptibility Testing based on single cell growth rate measurements

“…In particular, microfluidics (7,8) have made it possible to increase the signal to background ratio in the phenotypic assays by miniaturizing the bacterial incubation chambers (9). Using microfluidic approaches, it has been possible to push the time requirement for AST to 1-3h (10)(11)(12)(13)(14)(15).…”