A microfluidics system for drug susceptibility testing in mycobacterium smegmatis.

Abstract

Tuberculosis remains a major health concern worldwide. The emergence of strains resistant to anti-tuberculosis drugs has further complicated management of this disease. In addition, the prolonged multidrug chemotherapy (6 to 9 months) required to effectively sterilize drug tolerant mycobacteria that persist in human tissues, impedes disease management efforts. Hence, drug susceptibility tests that can easily detect drug resistant strains in a minimal amount of time whilst detecting both genetic and phenotypic resistant mechanisms is one of the priorities of TB control programs. The aim of this project was to develop a culture-based drug susceptibility testing platform for tuberculosis using microfluidics technology. A microfluidic platform has many advantages such as large scale integration, high throughput screening, automation and requires minimal human involvement. Such qualities are ideal for a diagnostic test. We designed and built the microdialyser which is a physical cell culture system that has micro-sized cell culture chambers that are as small as 200 pL in volume, mimicking the volume of a membrane bound compartment of human macrophage—a cell that harbours TB bacilli. This system provides a unique advantage of performing drug susceptibility testing in a micro-sized environment which resembles an in vivo growth environment. With the miracle of miniaturization, the microdialyser can support 120 microdialyser units to operate independently. Since each microdialyser unit is very small it consume minimal reagents and therefore reduces costs. Each culture system is monitored in situ to provide automated, real time, non-invasive measurement of cell density. The microdialyser system was used to perform drug susceptibility tests using M. smegmatis cells. This system revealed an epigenetic drug-tolerant phenotype of M. smegmatis that appears when mycobacteria are cultured in a space-confined bioreactor and disappears in larger growth chambers with respect to rifampicin. We then investigated possible underlying mechanisms that caused the drug tolerance, which eluded detection in conventional culture systems. In view of these results we highly recommend that this system be further validated with M. tuberculosis isolates so that it can be employed for routine drug susceptibility testing in clinical settings.