Fig 1.
SYTO-13 labelling of P. carinii trophic and cystic forms.
All P. carinii life cycle stages were stained in red (panels A–D) with a home-made anti-Pneumocystis polyclonal antibody, recognized by an Alexa-647-conjugated secondary antibody. SYTO-13 nuclear staining of viable P. carinii is displayed for corresponding fields (panels E–H). Differential Interference Contrast (DIC) pictures of corresponding fields are also shown (I–L). Panels (B, F, J) and (D, H, L), show an isolated cystic form with one or several labelled nuclei. In the other panels, cystic forms (arrowheads) and trophic forms are well visible. The white arrows show non-viable Pneumocystis organisms stained in red but did not display any nuclear green fluorescence (Panels C, G, K). Bar = 5 μm.
Fig 2.
Viability assessment of P. carinii using SYTO-13 by flow cytometry analysis.
The data are displayed as histograms displaying 10,000 P. carinii collected events. The axes represent the relative number of cells (y axis) and the cell-associated fluorescence intensity on a logarithmic scale (x axis). Panel A shows the gated (R1) P. carinii population labelled with a rat specific polyclonal antibody and a goat anti-rat IgG antibody conjugated to Alexa-647 (FL4 channel). The intensity of SYTO-13 live-cell nucleic acid staining is analyzed within the gated P. carinii population (FL1 channel) for P. carinii organisms cultured during 4 days without (panel B), with 0.15 μM (panel C) or 90 μM (panel D) of pentamidine. The gated population R2 represents the viable Pneumocystis cells. The percentages of viability are indicated for each histogram. The presented histograms are representative of one experiment.
Table 1.
In vitro pharmacodynamic parameters of pentamidine, atovaquone and trimethoprim-sulfamethoxazole (TMP/SMX) calculated using the SYTO-13 assay.
Fig 3.
Relationships between drug concentrations and P. carinii viability inhibition of pentamidine, atovaquone and trimethoprim-sulfamethoxazole (TMP/SMX).
Values of P. carinii viability inhibition are calculated from the SYTO-13 live-cell staining assay. To calculate the percentage of viability inhibition in relation with drug concentrations, one drug-free control was included in each assay. All susceptibility assays were set up in triplicate.