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Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of Calcein Acetoxymethyl Ester.

Krämer CE, Singh A, Helfrich S, Grünberger A, Wiechert W, Nöh K, Kohlheyer D - PLoS ONE (2015)

Bottom Line: Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity.Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase.Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.

View Article: PubMed Central - PubMed

Affiliation: IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany.

ABSTRACT
Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single cell cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity. Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.

No MeSH data available.


Related in: MedlinePlus

Metabolic activity sensing under iron limitation at single cell level.(A) Mean single cell fluorescence traces of all descendants of a progenitor cell are shown under intermittent iron supply. A daughter cell with higher calcein fluorescence than its siblings (light blue arrow) generated growing descendants with increased mean single cell fluorescence traces (light blue lines) in comparison to other descendants of the initial progenitor cell (grey mean single cell fluorescence traces). A single spontaneously non-growing cell changed from a dividing state to a non-growing state loosing esterase activity and intracellular calcein due to lysis (indicated by black arrow, lysed cell shown in (C) and (D), respectively). (B) Mean single cell fluorescence traces of spontaneously non-growing cells of a microcolony are shown. Lysing cells (red lines) lost fluorescence spontaneously after lysis (red arrow). However, they were still detectable as apparently intact cells (end of recognition marked with red asterisks). The mean single cell fluorescence traces of a spontaneously non-growing but metabolically active cell (blue line) are shown in comparison. Mean single cell fluorescence increases shortly after cell birth (blue arrow). (C) A lysed but apparently intact cell (marked with black dashed line) and a cell directly before performing lysis (marked with red dashed line) are depicted. (D) Lysed cells still appear to be intact cells (red dashed line and black dashed line, respectively) after lysis. These non-growing cells showed no calcein fluorescence and were considered to be metabolically non-active. (E) A non-growing but metabolically active cell after re-supply of iron with elevated mean single cell fluorescence (marked by blue dashed line).
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pone.0141768.g008: Metabolic activity sensing under iron limitation at single cell level.(A) Mean single cell fluorescence traces of all descendants of a progenitor cell are shown under intermittent iron supply. A daughter cell with higher calcein fluorescence than its siblings (light blue arrow) generated growing descendants with increased mean single cell fluorescence traces (light blue lines) in comparison to other descendants of the initial progenitor cell (grey mean single cell fluorescence traces). A single spontaneously non-growing cell changed from a dividing state to a non-growing state loosing esterase activity and intracellular calcein due to lysis (indicated by black arrow, lysed cell shown in (C) and (D), respectively). (B) Mean single cell fluorescence traces of spontaneously non-growing cells of a microcolony are shown. Lysing cells (red lines) lost fluorescence spontaneously after lysis (red arrow). However, they were still detectable as apparently intact cells (end of recognition marked with red asterisks). The mean single cell fluorescence traces of a spontaneously non-growing but metabolically active cell (blue line) are shown in comparison. Mean single cell fluorescence increases shortly after cell birth (blue arrow). (C) A lysed but apparently intact cell (marked with black dashed line) and a cell directly before performing lysis (marked with red dashed line) are depicted. (D) Lysed cells still appear to be intact cells (red dashed line and black dashed line, respectively) after lysis. These non-growing cells showed no calcein fluorescence and were considered to be metabolically non-active. (E) A non-growing but metabolically active cell after re-supply of iron with elevated mean single cell fluorescence (marked by blue dashed line).

Mentions: On a single cell basis, iron starvation caused heterogeneous phenotypes at the end of iron limitation and during the first five hours after iron resupply as illustrated in Fig 8 and S7 Video. The mean single cell traces of all growing and metabolically active descendants of one progenitor cells are shown in comparison with a spontaneously non-viable cell that lost its metabolic activity after lysis (Fig 8A). Bacteria showed an increase in their mean single cell fluorescence initially after media switch to CGXII + 4% GLC—iron and to CGXII + 4% GLC, respectively. The bacterial cells that were growth inhibited due to iron limitation showed comparable mean single cell fluorescence as their descendants after recovery during re-supply of iron.


Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of Calcein Acetoxymethyl Ester.

Krämer CE, Singh A, Helfrich S, Grünberger A, Wiechert W, Nöh K, Kohlheyer D - PLoS ONE (2015)

Metabolic activity sensing under iron limitation at single cell level.(A) Mean single cell fluorescence traces of all descendants of a progenitor cell are shown under intermittent iron supply. A daughter cell with higher calcein fluorescence than its siblings (light blue arrow) generated growing descendants with increased mean single cell fluorescence traces (light blue lines) in comparison to other descendants of the initial progenitor cell (grey mean single cell fluorescence traces). A single spontaneously non-growing cell changed from a dividing state to a non-growing state loosing esterase activity and intracellular calcein due to lysis (indicated by black arrow, lysed cell shown in (C) and (D), respectively). (B) Mean single cell fluorescence traces of spontaneously non-growing cells of a microcolony are shown. Lysing cells (red lines) lost fluorescence spontaneously after lysis (red arrow). However, they were still detectable as apparently intact cells (end of recognition marked with red asterisks). The mean single cell fluorescence traces of a spontaneously non-growing but metabolically active cell (blue line) are shown in comparison. Mean single cell fluorescence increases shortly after cell birth (blue arrow). (C) A lysed but apparently intact cell (marked with black dashed line) and a cell directly before performing lysis (marked with red dashed line) are depicted. (D) Lysed cells still appear to be intact cells (red dashed line and black dashed line, respectively) after lysis. These non-growing cells showed no calcein fluorescence and were considered to be metabolically non-active. (E) A non-growing but metabolically active cell after re-supply of iron with elevated mean single cell fluorescence (marked by blue dashed line).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4625966&req=5

pone.0141768.g008: Metabolic activity sensing under iron limitation at single cell level.(A) Mean single cell fluorescence traces of all descendants of a progenitor cell are shown under intermittent iron supply. A daughter cell with higher calcein fluorescence than its siblings (light blue arrow) generated growing descendants with increased mean single cell fluorescence traces (light blue lines) in comparison to other descendants of the initial progenitor cell (grey mean single cell fluorescence traces). A single spontaneously non-growing cell changed from a dividing state to a non-growing state loosing esterase activity and intracellular calcein due to lysis (indicated by black arrow, lysed cell shown in (C) and (D), respectively). (B) Mean single cell fluorescence traces of spontaneously non-growing cells of a microcolony are shown. Lysing cells (red lines) lost fluorescence spontaneously after lysis (red arrow). However, they were still detectable as apparently intact cells (end of recognition marked with red asterisks). The mean single cell fluorescence traces of a spontaneously non-growing but metabolically active cell (blue line) are shown in comparison. Mean single cell fluorescence increases shortly after cell birth (blue arrow). (C) A lysed but apparently intact cell (marked with black dashed line) and a cell directly before performing lysis (marked with red dashed line) are depicted. (D) Lysed cells still appear to be intact cells (red dashed line and black dashed line, respectively) after lysis. These non-growing cells showed no calcein fluorescence and were considered to be metabolically non-active. (E) A non-growing but metabolically active cell after re-supply of iron with elevated mean single cell fluorescence (marked by blue dashed line).
Mentions: On a single cell basis, iron starvation caused heterogeneous phenotypes at the end of iron limitation and during the first five hours after iron resupply as illustrated in Fig 8 and S7 Video. The mean single cell traces of all growing and metabolically active descendants of one progenitor cells are shown in comparison with a spontaneously non-viable cell that lost its metabolic activity after lysis (Fig 8A). Bacteria showed an increase in their mean single cell fluorescence initially after media switch to CGXII + 4% GLC—iron and to CGXII + 4% GLC, respectively. The bacterial cells that were growth inhibited due to iron limitation showed comparable mean single cell fluorescence as their descendants after recovery during re-supply of iron.

Bottom Line: Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity.Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase.Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.

View Article: PubMed Central - PubMed

Affiliation: IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany.

ABSTRACT
Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single cell cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity. Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.

No MeSH data available.


Related in: MedlinePlus