Combinatorial Antimicrobial Susceptibility Testing Enabled by Non-Contact Printing

Abstract

We demonstrate the utility of non-contact printing to fabricate the mAST—an easy-to-operate, microwell-based microfluidic device for combinatorial antibiotic susceptibility testing (AST) in a point-of-care format. The wells are prefilled with antibiotics in any desired concentration and combination by non-contact printing (spotting). For the execution of the AST, the only requirements are the mAST device, the sample, and the incubation chamber. Bacteria proliferation can be continuously monitored by using an absorbance reader. We investigate the profile of resistance of two reference Escherichia coli strains, report the minimum inhibitory concentration (MIC) for single antibiotics, and assess drug–drug interactions in cocktails by using the Bliss independence model.

Microfluidics for antibiotic susceptibility testing

“…74 Further upscaling of chamber-based devices for AST resulted in systems containing over 1000 chambers. 75 Such an increase in throughput allowed for analysis of the efficacy of cocktails of antibiotics in search of synergistic, additive, or antagonistic relations. Pipetting over 1000 antibiotic samples into an array of microscopic wells is not feasible manually, so the authors used a droplet spotting machine.…”

“…From the approaches to AST described here, this ease-of-use is best represented by printing antibiotics to microfluidic chambers or lab-on-a-disc devices: such chambers with pre-printed drugs could be used as cartridges to which samples would be added with a pipette, and the AST readout would be performed in the physician's office quickly. Antibiotics are being printed and dried commercially to 96-well-plates, so new microfluidic AST assays should be compared to such well-plate-based solutions: although well-plates are compatible with robotic liquid handling systems, their throughput is limited to 96 reactions per plate (compare to a thousand reactions on a chip with pre-printed antibiotics 75 ) and single-cell analysis in not viable in bulk wells as bacteria can swim in a large reaction volume and individual cells are virtually impossible to follow on a large scale. Pheno-molecular assays were shown to be the fastest ASTs from clinical samples to readouts, 167 however it is questionable if such an assay would be run by a physician: rather, a lab technician would work on the assay in a centralized hospital laboratory.…”

“…However, the to-date presented chamber-based solutions require the end-user to perform only a single pipetting step or two steps at most to run AST. 75 4.1.2.2. Serial dilution in droplets.…”

Droplet Microfluidics for High-Throughput Analysis of Antibiotic Susceptibility in Bacterial Cells and Populations

“…In our solution, various numbers of antibiotic molecules and different antibiotics were printed into >1000 nanoliter-sized chambers to screen for resistance to cocktails of antibiotics (Figure 2). 14 When a bacterial solution is pipetted into the chip with printed antibiotics, the liquid is sucked into the chambers as the chip tries to refill its polymer structure with gas as the chip had been degassed before the experiment. As the channels branch fractally from the inlet, there is no pressure difference between chambers and the filling is accurate.…”

Synergistic interactions of ionic liquids and antimicrobials improve drug efficacy

“…One of the most widely used combinatorial formulations in clinical practice is amoxicillin with clavulanic acid, where amoxicillin is an inhibitor of peptidoglycan synthesis while clavulanic acid inhibits the β-lactamase enzyme which degrades amoxicillin ( Huttner et al., 2019 ). Similar approaches combine two molecules which each independently exhibit antimicrobial activity and combine the dosing for treatment ( Montelongo-Peralta et al., 2019 ; Opalski et al., 2020 ). Many of these combinations are designed such that the two antimicrobials act via different mechanisms, ideally each enhancing the activity of the other.…”