Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

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

Version 1 : Received: 1 June 2021 / Approved: 2 June 2021 / Online: 2 June 2021 (08:32:03 CEST)

How to cite: Elitas, M.; Dhar, N.; McKinney, J. Revealing Antibiotic-Tolerance of the Mycobacterium smegmatis Xanthine/Uracil Permease Mutant Using Microfluidics and Single-Cell Analysis. Preprints 2021, 2021060052 (doi: 10.20944/preprints202106.0052.v1). Elitas, M.; Dhar, N.; McKinney, J. Revealing Antibiotic-Tolerance of the Mycobacterium smegmatis Xanthine/Uracil Permease Mutant Using Microfluidics and Single-Cell Analysis. Preprints 2021, 2021060052 (doi: 10.20944/preprints202106.0052.v1).

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 antibiotic-tolerance mechanisms of wild type and the msm2570::Tnmutant 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 with wild-type cells. This is the first study that characterized the antibiotic-tolerance phenotype of the msm2570::Tn mutant that has a transposon insertion in the msm2570 gene encoding a putative xanthine/uracil permease, which enrolls in uptake of nitrogen compound during nitrogen limitation. The experimental results indicate that the msm2570::Tn mutant can be further interrogated to reveal antibiotic killing mechanisms, in particularly, antibiotics those targets cell wall integrity.

Subject Areas

antibiotics; conventional; microbiology; microfluidics; microscopy; mycobacterium smegmatis; population; single cell

Comments (1)

Comment 1
Received: 10 June 2021
Commenter: Thomas K. Wood
The commenter has declared there is no conflict of interests.
Comment: I would like to note that our single-cell work with E. coli persisters is more “pioneering” than that of Van Melderen’s (your ref 14) in that ours was published 1.5 years before hers, and we were the first to show heterogeneity of waking at the single cell level (with all waking cells growing at exponentially at the same rate, once awaken) and the first to suggest the ribosome mechanism (2018, doi:10.1111/1462-2920.14093). Moreover, we showed by treating exponentially-growing cells with rifampicin, that persisters are formed from any cell type, so the reason you cite Van Melderen’s work was actually documented prior to her work. Also, her sentence, “the fate of the resulting persister cells have never been described up to this day” is patently false given our prior publication. Our work also shows 8 different ways that persisters are dormant, prior to Van Melderen. Many in the field have wondered how the Van Melderen work was published given that it contained very little that was new.

Furthermore, I do not believe your statement, “Still, very little is known about the single-cell kinetics and the underlying molecular mechanisms of these phenomena” given our subsequent work showing how persisters are formed and revive (attached Fig 1 summary, 2020).

Good luck with your review and hope your Intro will be updated prior to final publication.
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