Use of flow cytometry to monitor Legionella viability

Abstract

Legionella viability was monitored during heat shock treatment at 70 degrees C by a flow cytometric assay (FCA). After 30 min of treatment, for 6 of the 12 strains tested, the FCA still detected 10 to 25% of cells that were viable but nonculturable (VBNC). These VBNC cells were able to produce ATP and to be resuscitated after culture on amoebae.

FIG. 1.

Kinetic analysis by FCA of Legionella extermination after heat shock treatment. A 3-day culture of L. pneumophila sg 1 on BCYE with 0.1% α-ketoglutarate was subjected to a heat shock at 70°C from 0 to 60 min. The bacteria were visualized on a dot plot of PI red fluorescence (FL3) versus Syto 9 green fluorescence (FL1). The P1 region corresponds to dead cells. Syto 9-stained bacteria with intact membranes were used to delineate the P2 region (viable, culturable cells). The P3 region (VBNC cells) corresponds to bacteria in an intermediate state with compromised membranes (see the text).

FIG. 2.

Evaluation of the characteristics of a suspension of L. pneumophila sg 1 PHB before (0 min) and 10 and 60 min after a heat shock treatment at 70°C. Cells were evaluated by an FCA (A), solid culture on BCYE with 0.1% α-ketoglutarate (B), ATP production (C), and solid culture on BCYE with 0.1% α-ketoglutarate after resuscitation on amoebae (D). Representative results (A, B, and D) or means ± standard errors (C) from four independent experiments are shown. RLU, relative light units; SE, standard errors.

FIG. 3.

Resistance patterns of various Legionella strains subjected to a heat shock treatment. After a 3-day culture on BCYE with 0.1% α-ketoglutarate, 12 Legionella strains were subjected to a heat shock treatment at 70°C from 0 to 60 min, and FCAs were performed. For each strain, the result at each time point shown is the percentage of bacteria detected in both the P2 (viable, culturable cells) and P3 (VBNC cells) regions and is expressed as the mean ± standard error from two to five independent experiments. Lp1, L. pneumophila sg 1.