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Single-cell tracking reveals antibiotic-induced changes in mycobacterial energy metabolism.

Maglica Ž, Özdemir E, McKinney JD - MBio (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), Lausanne, Switzerland zeljka.maglica@gmail.com.

No MeSH data available.


Related in: MedlinePlus

Antibiotic-induced ATP switching and PI staining as markers of live/dead status. ΔfbiC_MA cells were cultured in minimal-acetate flow medium, exposed to antibiotic, and analyzed as described in the legend to Fig. 3. See Movies S3 to S7 in the supplemental material. (A) Phase-contrast (grey) and fluorescence (red) images of bedaquiline-treated cells stained with PI at the end of the drug exposure period (24 h) and washout (48 h). (B) Fraction of PI-positive (red) and PI-negative (grey) cells at 24 and 48 h. Cells were scored as PI positive and nongrowing (red), PI negative and nongrowing (grey), or PI negative and growing (grey with green stripes). Data represent 20 movies from two independent experiments for each antibiotic: bedaquiline (n = 483), isoniazid (n = 1,270), streptomycin (n = 1,178), rifampin (n = 1,021), and ciprofloxacin (n = 615). (C) Lowest FRET/YFP ratios measured during antibiotic exposure (0 to 24 h). Cells were PI stained at 24 h and scored as PI positive (top) or PI negative (bottom) and growing or nongrowing during drug washout. Each symbol represents one cell (n = 30 per group except as noted). Red lines indicate means ± SD. (D) Lowest FRET/YFP ratios measured during antibiotic exposure (0 to 24 h) for cells that were scored as PI negative at 24 h. Cells were PI stained again at 48 h and scored as PI positive and nongrowing (red), PI negative and nongrowing (grey), or PI negative and growing (green). Each symbol represents one cell. (E) Time traces of FRET/YFP ratios of representative cells (three per antibiotic) that resumed growth after drug washout.
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fig4: Antibiotic-induced ATP switching and PI staining as markers of live/dead status. ΔfbiC_MA cells were cultured in minimal-acetate flow medium, exposed to antibiotic, and analyzed as described in the legend to Fig. 3. See Movies S3 to S7 in the supplemental material. (A) Phase-contrast (grey) and fluorescence (red) images of bedaquiline-treated cells stained with PI at the end of the drug exposure period (24 h) and washout (48 h). (B) Fraction of PI-positive (red) and PI-negative (grey) cells at 24 and 48 h. Cells were scored as PI positive and nongrowing (red), PI negative and nongrowing (grey), or PI negative and growing (grey with green stripes). Data represent 20 movies from two independent experiments for each antibiotic: bedaquiline (n = 483), isoniazid (n = 1,270), streptomycin (n = 1,178), rifampin (n = 1,021), and ciprofloxacin (n = 615). (C) Lowest FRET/YFP ratios measured during antibiotic exposure (0 to 24 h). Cells were PI stained at 24 h and scored as PI positive (top) or PI negative (bottom) and growing or nongrowing during drug washout. Each symbol represents one cell (n = 30 per group except as noted). Red lines indicate means ± SD. (D) Lowest FRET/YFP ratios measured during antibiotic exposure (0 to 24 h) for cells that were scored as PI negative at 24 h. Cells were PI stained again at 48 h and scored as PI positive and nongrowing (red), PI negative and nongrowing (grey), or PI negative and growing (green). Each symbol represents one cell. (E) Time traces of FRET/YFP ratios of representative cells (three per antibiotic) that resumed growth after drug washout.

Mentions: Propidium iodide (PI) is a membrane-impermeant DNA-intercalating agent that is commonly used as a selective stain to detect “dead” cells with damaged membrane permeability barriers. Due to its ability to accumulate gradually within live cells during prolonged incubation (33), which can have undesirable effects on cell physiology, PI staining is not well suited for continuous time-lapse studies, and it is typically used as an endpoint assay (4, 33, 34). In time-lapse experiments, we assessed the viability of antibiotic-treated cells by pulse-labeling cells with PI for 30 min after 24 h of antibiotic exposure and again for 30 min following an additional 24 h of postantibiotic recovery (Fig. 4A and B; also, see Fig. S2E in the supplemental material). We routinely observed that the fraction of PI-positive cells is higher at 48 h than at 24 h, indicating that many cells lose their membrane integrity during the postantibiotic recovery period. As with ATP switching, we observe substantial differences between different classes of antibiotics with respect to the timing and magnitude of PI staining. Again, bedaquiline and isoniazid represent opposite ends of the spectrum, generating 97% and 4% PI-positive cells, respectively, at the 48-h time point, while the other antibiotics that we tested generated intermediate numbers of PI-positive cells (Fig. 4B).


Single-cell tracking reveals antibiotic-induced changes in mycobacterial energy metabolism.

Maglica Ž, Özdemir E, McKinney JD - MBio (2015)

Antibiotic-induced ATP switching and PI staining as markers of live/dead status. ΔfbiC_MA cells were cultured in minimal-acetate flow medium, exposed to antibiotic, and analyzed as described in the legend to Fig. 3. See Movies S3 to S7 in the supplemental material. (A) Phase-contrast (grey) and fluorescence (red) images of bedaquiline-treated cells stained with PI at the end of the drug exposure period (24 h) and washout (48 h). (B) Fraction of PI-positive (red) and PI-negative (grey) cells at 24 and 48 h. Cells were scored as PI positive and nongrowing (red), PI negative and nongrowing (grey), or PI negative and growing (grey with green stripes). Data represent 20 movies from two independent experiments for each antibiotic: bedaquiline (n = 483), isoniazid (n = 1,270), streptomycin (n = 1,178), rifampin (n = 1,021), and ciprofloxacin (n = 615). (C) Lowest FRET/YFP ratios measured during antibiotic exposure (0 to 24 h). Cells were PI stained at 24 h and scored as PI positive (top) or PI negative (bottom) and growing or nongrowing during drug washout. Each symbol represents one cell (n = 30 per group except as noted). Red lines indicate means ± SD. (D) Lowest FRET/YFP ratios measured during antibiotic exposure (0 to 24 h) for cells that were scored as PI negative at 24 h. Cells were PI stained again at 48 h and scored as PI positive and nongrowing (red), PI negative and nongrowing (grey), or PI negative and growing (green). Each symbol represents one cell. (E) Time traces of FRET/YFP ratios of representative cells (three per antibiotic) that resumed growth after drug washout.
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fig4: Antibiotic-induced ATP switching and PI staining as markers of live/dead status. ΔfbiC_MA cells were cultured in minimal-acetate flow medium, exposed to antibiotic, and analyzed as described in the legend to Fig. 3. See Movies S3 to S7 in the supplemental material. (A) Phase-contrast (grey) and fluorescence (red) images of bedaquiline-treated cells stained with PI at the end of the drug exposure period (24 h) and washout (48 h). (B) Fraction of PI-positive (red) and PI-negative (grey) cells at 24 and 48 h. Cells were scored as PI positive and nongrowing (red), PI negative and nongrowing (grey), or PI negative and growing (grey with green stripes). Data represent 20 movies from two independent experiments for each antibiotic: bedaquiline (n = 483), isoniazid (n = 1,270), streptomycin (n = 1,178), rifampin (n = 1,021), and ciprofloxacin (n = 615). (C) Lowest FRET/YFP ratios measured during antibiotic exposure (0 to 24 h). Cells were PI stained at 24 h and scored as PI positive (top) or PI negative (bottom) and growing or nongrowing during drug washout. Each symbol represents one cell (n = 30 per group except as noted). Red lines indicate means ± SD. (D) Lowest FRET/YFP ratios measured during antibiotic exposure (0 to 24 h) for cells that were scored as PI negative at 24 h. Cells were PI stained again at 48 h and scored as PI positive and nongrowing (red), PI negative and nongrowing (grey), or PI negative and growing (green). Each symbol represents one cell. (E) Time traces of FRET/YFP ratios of representative cells (three per antibiotic) that resumed growth after drug washout.
Mentions: Propidium iodide (PI) is a membrane-impermeant DNA-intercalating agent that is commonly used as a selective stain to detect “dead” cells with damaged membrane permeability barriers. Due to its ability to accumulate gradually within live cells during prolonged incubation (33), which can have undesirable effects on cell physiology, PI staining is not well suited for continuous time-lapse studies, and it is typically used as an endpoint assay (4, 33, 34). In time-lapse experiments, we assessed the viability of antibiotic-treated cells by pulse-labeling cells with PI for 30 min after 24 h of antibiotic exposure and again for 30 min following an additional 24 h of postantibiotic recovery (Fig. 4A and B; also, see Fig. S2E in the supplemental material). We routinely observed that the fraction of PI-positive cells is higher at 48 h than at 24 h, indicating that many cells lose their membrane integrity during the postantibiotic recovery period. As with ATP switching, we observe substantial differences between different classes of antibiotics with respect to the timing and magnitude of PI staining. Again, bedaquiline and isoniazid represent opposite ends of the spectrum, generating 97% and 4% PI-positive cells, respectively, at the 48-h time point, while the other antibiotics that we tested generated intermediate numbers of PI-positive cells (Fig. 4B).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), Lausanne, Switzerland zeljka.maglica@gmail.com.

No MeSH data available.


Related in: MedlinePlus