Revealing Antibiotic Tolerance of the Mycobacterium smegmatis Xanthine/Uracil Permease Mutant Using Microfluidics and Single-Cell Analysis

Abstract

To reveal rare phenotypes in bacterial populations, conventional microbiology tools should be advanced to generate rapid, quantitative, accurate, and high-throughput data. The main drawbacks of widely used traditional methods for antibiotic studies include low sampling rate and averaging data for population measurements. To overcome these limitations, microfluidic-microscopy systems have great promise to produce quantitative single-cell data with high sampling rates. Using Mycobacterium smegmatis cells, we applied both conventional assays and a microfluidic-microscopy method to reveal the antibiotic tolerance mechanisms of wild-type and msm2570::Tn mutant cells. Our results revealed that the enhanced antibiotic tolerance mechanism of the msm2570::Tn mutant was due to the low number of lysed cells during the antibiotic exposure compared to wild-type cells. This is the first study to characterize the antibiotic tolerance phenotype of the msm2570::Tn mutant, which has a transposon insertion in the msm2570 gene—encoding a putative xanthine/uracil permease, which functions in the uptake of nitrogen compounds during nitrogen limitation. The experimental results indicate that the msm2570::Tn mutant can be further interrogated to reveal antibiotic killing mechanisms, in particular, antibiotics that target cell wall integrity.

Microfluidics as a Novel Technique for Tuberculosis: From Diagnostics to Drug Discovery

“…Single cells were studied using microfluidics and time-lapse microscopy and provided evidence that the mutant strain was more tolerant to INH compared to the wild-type strain. This current example is proof that microfluidics can achieve an improved understanding of resistance mechanisms of mycobacteria to antibiotics and aid the discovery of new antimicrobials [161].…”

Microfluidics as a Novel Technique for Tuberculosis: From Di-agnostics to Drug Discovery

“…Single cells were studied using microfluidics and time-lapse microscopy and provided evidence that the mutant strain was more tolerant to INH compared to the wild-type strain. This current example is proof that microfluidics can achieve improved understanding of resistance mechanisms of mycobacteria to antibiotics and aid the discovery of new antimicrobials [169].…”

Microfluidic systems for infectious disease diagnostics

“…Cells were trapped by means of a semipermeable membrane in PDMS microfluidic channels, enabling stable flow conditions for on-chip culture and rapid medium exchange. 234 The response of live mycobacteria to antibiotic stress could also be monitored dynamically by wavelength modulated Raman spectroscopy. 235 For this purpose, M. smegmantis was acoustically trapped in a microfluidic glass chamber.…”