Table 1.
Probe specifications.
Table 2.
Specificity testing in duplicate of PNA probes using cultured bacteria.
The signal was considered positive if it had a positive counterpart in the DAPI stain and displayed a positive signal simultaneously with the broad-range probe. The signal was considered negative if no signal was seen with the species-specific probe.
Fig 1.
Dispersed bacteria versus biofilm.
Confocal laser scanning images with 400x magnification of G. vaginalis biofilm in 2 vaginal slides (A and B) in a superimposed image: vaginal epithelial cells DAPI in blue and G. vaginalis specific PNA-probe Gard162 with Alexa Fluor 647 in red. A: vaginal sample with dispersed bacteria; B: vaginal sample with bacteria in biofilm.
Fig 2.
Distribution of samples according to FISH.
Aggregates of bacterial cells attached to the vaginal epithelial cells, were defined as biofilm. Separate scattered bacterial cells, without the presence of biofilm, were defined as dispersed only bacteria.
Fig 3.
Montage of confocal laser scanning images with 400x magnification of G. vaginalis biofilm, negative for A. vaginae, in 4 vaginal samples (A-D) in a superimposed image: vaginal epithelial cells DAPI in blue and G. vaginalis specific PNA-probe Gard162 with Alexa Fluor 647 in red. For clarity we omitted the BacUni-1 plane; the bacteria that did not hybridize with Gard162 are visible in DAPI blue.
Fig 4.
Superimposed image of polymicrobial biofilm of A. vaginae and G. vaginalis.
Montage of confocal laser scanning images with 400x magnification of polymicrobial biofilm in 6 vaginal samples (A-F) in a superimposed image: vaginal epithelial cells DAPI in blue, G. vaginalis specific PNA-probe Gard162 with Alexa Fluor 647 in red and A. vaginae specific PNA-probe AtoITM1 with Alexa Fluor 488 in green. For clarity we omitted the BacUni-1 plane; the bacteria that are not bound by the specific probes are visible in DAPI blue.
Fig 5.
Polymicrobial biofilm of A. vaginae and G. vaginalis in different panes.
Confocal laser scanning image with 400 x magnification of polymicrobial biofilm in different panes, A: vaginal epithelial cells DAPI in blue, B: all bacteria, BacUni-1 PNA-probe with Alexa Fluor 555 in yellow, C: A. vaginae specific PNA-probe AtoITM1 with Alexa Fluor 488 in green, D: G. vaginalis specific PNA-probe Gard162 with Alexa Fluor 647 in red (superimposed image can be seen in Fig 3A).
Table 3.
Performance of A. vaginae (AtoITM1) and G. vaginalis (Gard162) PNA probes, compared to qPCR results, for 119 vaginal slides.
Table 4.
Presence of A. vaginae and G. vaginalis, as assessed by FISH, in relation to A. vaginae and G. vaginalis loads as determined by qPCR for 119 vaginal samples.