Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism

ABSTRACT ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond t...

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Autores principales:	Željka Maglica, Emre Özdemir, John D. McKinney
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/194b69edd8684d238517e8af42d96fcc
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spelling	oai:doaj.org-article:194b69edd8684d238517e8af42d96fcc2021-11-15T15:41:19ZSingle-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism10.1128/mBio.02236-142150-7511https://doaj.org/article/194b69edd8684d238517e8af42d96fcc2015-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02236-14https://doaj.org/toc/2150-7511ABSTRACT ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening. IMPORTANCE New antimicrobials are urgently needed to stem the rising tide of antibiotic-resistant bacteria. All antibiotics are expected to affect bacterial energy metabolism, directly or indirectly, yet tools to assess the impact of antibiotics on the ATP content of individual bacterial cells are lacking. The method described here for single-cell tracking of intracellular ATP in live bacteria has many advantages compared to conventional ensemble-averaged assays. It provides a continuous real-time readout of bacterial ATP content, cell vitality, and antimicrobial mechanism of action with high temporal resolution at the single-cell level. In combination with high-throughput microfluidic devices and automated microscopy, this method also has the potential to serve as a novel screening tool in antimicrobial drug discovery.Željka MaglicaEmre ÖzdemirJohn D. McKinneyAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 1 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Željka Maglica Emre Özdemir John D. McKinney Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism
description	ABSTRACT ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening. IMPORTANCE New antimicrobials are urgently needed to stem the rising tide of antibiotic-resistant bacteria. All antibiotics are expected to affect bacterial energy metabolism, directly or indirectly, yet tools to assess the impact of antibiotics on the ATP content of individual bacterial cells are lacking. The method described here for single-cell tracking of intracellular ATP in live bacteria has many advantages compared to conventional ensemble-averaged assays. It provides a continuous real-time readout of bacterial ATP content, cell vitality, and antimicrobial mechanism of action with high temporal resolution at the single-cell level. In combination with high-throughput microfluidic devices and automated microscopy, this method also has the potential to serve as a novel screening tool in antimicrobial drug discovery.
format	article
author	Željka Maglica Emre Özdemir John D. McKinney
author_facet	Željka Maglica Emre Özdemir John D. McKinney
author_sort	Željka Maglica
title	Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism
title_short	Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism
title_full	Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism
title_fullStr	Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism
title_full_unstemmed	Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism
title_sort	single-cell tracking reveals antibiotic-induced changes in mycobacterial energy metabolism
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/194b69edd8684d238517e8af42d96fcc
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Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism

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