On-Chip Isoniazid Exposure of Mycobacterium smegmatis Penicillin-Binding Protein (PBP) Mutant Using Time-Lapse Fluorescent Microscopy

Abstract

Antibiotic resistance has been one of the biggest threats to global health. Despite the available prevention and control strategies and efforts in developing new antibiotics, the need remains for effective approaches against antibiotic resistance. Efficient strategies to cope with antimicrobial resistance require a quantitative and deeper understanding of microbial behavior, which can be obtained using different techniques to provide the missing pieces of the current antibiotic-resistance puzzle. Microfluidic-microscopy techniques are among the most promising methods that contribute modernization of traditional assays in microbiology. They provide monitoring and manipulation of cells at micro-scale volumes. Here, we combined population-level, culture-based assays with single-cell resolution, microfluidic-microscopy systems to investigate isoniazid response of Mycobacterium smegmatis penicillin-binding protein (PBP) mutant. This mutant exhibited normal growth in plain medium and sensitivity to stress responses when treated with thermal stress (45 °C), detergent stress (0.1% sodium dodecyl sulfate), acid stress (pH 4.5), and nutrient starvation (1XPBS). The impact of msm0031 transposon insertion on drug-mediated killing was determined for isoniazid (INH, 50 µg/mL), rifampicin (RIF, 200 µg/mL), ethionamide (ETH, 200 µg/mL), and ethambutol (EMB, 5 µg/mL). The PBP mutant demonstrated remarkable isoniazid-killing phenotype in batch culture. Therefore, we hypothesized that single-cell analysis will show increased lysis kinetics and fewer intact cells after drug treatment. However, the single-cell analysis data showed that upon isoniazid exposure, the percentage of the intact PBP mutant cells was 24%, while the percentage of the intact wild-type cells was 4.6%. The PBP mutant cells exhibited decreased cell-lysis profile. Therefore, the traditional culture-based assays were not sufficient to provide insights about the subpopulation of viable but non-culture cells. Consequently, we need more adequate tools to be able to comprehend and fight the antibiotic resistance of bacteria.

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

“…Microfluidic devices and microscopy techniques have been widely used to reveal antibiotic tolerance mechanisms of bacteria, but only a few studies have reported their observations or findings with underlying genetic mechanisms [11][12][13][14][15][16][17][18][19][20][21][22][23]. We investigated the antibiotic tolerances of M. smegmatis wild-type and the msm2570::Tn mutant cells using microfluidic bacteria culture platform which is capable of providing stable flow and rapid medium change [13].…”

“…In the past decade, microfluidics and microscopy methods have arisen to overcome the limitations of traditional tools [11][12][13][14][15][16][17][18][19][20][21][22][23]. The advantages of microfluidic assays over macro-scale, traditional batch culture methods include increased limit of detection, higher sampling rate, higher specificity, and compatibility with several imaging techniques [11][12][13][14][15][16][17][18][19][20][21][22][23]. Among these pioneering studies, Goormaghtigh and Van Melderen observed Escherichia coli (E. coli) cells that generated ofloxacin persisters at single-cell level using a commercially available CellASIC ONIX B04A-03 Microfluidic Plate (Millipore, USA) and the CellASIC ONIX Microfluidic System [14].…”

“…On the other hand, McKinney group mostly focused on mycobacterial persistence and widely investigated behavior of Mycobacterium smegmatis (M. smegmatis), model organism of Mycobacterium tuberculosis (M. tuberculosis, Mtb) in the presence of first-line antituberculosis drugs [11,13,20,21]. Their ground-breaking studies showed that persistence was a dynamic behavior of antibiotic-stressed mycobacteria based on single-cell analysis using a microfluidic-microscopy system [13].…”

“…In this study, we used a microfluidic chip integrated with dialysis membrane, published in [13,20,21]. We elucidated an increased antibiotic-tolerance mechanism for M. smegmatis.…”

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

“…In the past decade, microfluidic and microscopy methods have arisen to overcome the limitations of traditional tools [11][12][13][14][15][16][17][18][19][20][21][22]. The advantages of microfluidic assays over macro-scale, traditional, batch culture methods include increased limit of detection, higher sampling rate, higher specificity, and compatibility with several imaging techniques [11][12][13][14][15][16][17][18][19][20][21][22]. Among these pioneering studies, Kim and co-workers presented that persister cells wake based on ribosome content [8].…”