High-throughput, multiplex microfluidic test strip for the determination of antibiotic susceptibility in uropathogenic E. coli with smartphone detection

Abstract

Antibiotic resistance in urinary tract infections is a major global challenge and improved cost-effective and high throughput antibiotic susceptibility tests (AST) are urgently needed to inform correct antibiotic selection. We evaluated a high throughput microfluidic test strip for AST and minimum inhibitory concentration (MIC) determination in 20 urinary pathogenic E. coli (UPEC) isolates using six commonly prescribed or therapeutically beneficial antibiotics. The microfluidic MIC performs broth microdilution in 1 microliter volume capillaries, 100 X smaller than standard broth microdilution. Each test strip contains 10 parallel capillaries which are dipped into a single well of a 96 well plate, significantly increasing throughput over a microtitre plate. When tested with clinical UPEC isolates at standardised inoculum density, these devices gave 100% essential agreement (+/- 1 doubling dilution of antibiotic) to the gold standard microplate broth microdilution method described by CLSI. Although for some antibiotic/isolate combinations an earlier endpoint readout reduced accuracy, MIC test strips read at a 6h endpoint still gave 69 to 100 % essential agreement depending on the antibiotic. Growth could be detected significantly earlier than 6h, but with a trade-off between speed vs accuracy. These high-throughput, multiplexed test strips could be used to increase throughput and give faster results than microplates while retaining the core broth microdilution methodology of gold standard techniques for AST and MIC determination.

Direct microfluidic antibiotic resistance testing in urine with smartphone capture: significant variation in sample matrix interference between individual human urine samples

“…Microfluidic MIC test strips were prepared as described. 13 Briefly, microcapillary film with 10 parallel capillaries of 270 μm capillary diameter were hydrophilic coated by incubating 5 mg mL −1 polyvinyl alcohol (PVOH) ( M w 146, 000–186, 000, >99% hydrolysed, Sigma Aldrich, UK) for 2 h at room temperature. The PVOH was removed by washing the capillaries with sterile Ultrapure Milli-Q water.…”

“…The results of the AST are readable by both smartphone and robotic timelapse imaging, ideal for systematic analysis of matrix interference in microuidic microbiology assays. Having previously established proof-of-concept of microbial identication, viable cell counting, and monitoring bacteriophage lysis [12][13][14][15] in this system, we have not previously used the system to interrogate the impact of urine sample matrix on micro-uidic antibiotic resistance assays. Here we assessed in detail the impact of urine matrix on microuidic AST, by systematically quantifying minimum inhibitory concentration (MIC) in the presence or absence of a wide range of human urine samples.…”