Unravelling the bacterial vaginosis-associated biofilm : a multiplex Gardnerella vaginalis and Atopobium vaginae fluorescence in situ hybridization assay using peptide nucleic acid probes

Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis

Dispersed bacteria versus biofilm.

<p>Confocal laser scanning images with 400x magnification of <i>G</i>. <i>vaginalis</i> biofilm in 2 vaginal slides (A and B) in a superimposed image: vaginal epithelial cells DAPI in blue and <i>G</i>. <i>vaginalis</i> specific PNA-probe Gard162 with Alexa Fluor 647 in red. A: vaginal sample with dispersed bacteria; B: vaginal sample with bacteria in biofilm.</p

Superimposed image of polymicrobial biofilm of <i>A</i>. <i>vaginae</i> and <i>G</i>. <i>vaginalis</i>.

<p>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, <i>G</i>. <i>vaginalis</i> specific PNA-probe Gard162 with Alexa Fluor 647 in red and <i>A</i>. <i>vaginae</i> 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.</p

Polymicrobial biofilm of <i>A</i>. <i>vaginae</i> and <i>G</i>. <i>vaginalis</i> in different panes.

<p>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: <i>A</i>. <i>vaginae</i> specific PNA-probe AtoITM1 with Alexa Fluor 488 in green, D: <i>G</i>. <i>vaginalis</i> specific PNA-probe Gard162 with Alexa Fluor 647 in red (superimposed image can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136658#pone.0136658.g003" target="_blank">Fig 3A</a>).</p

Specificity testing in duplicate of PNA probes using cultured bacteria.

<p>¹(+) Presence of hybridization</p><p>²(-) Absence of hybridization.</p><p>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.</p

<i>G</i>. <i>vaginalis</i> biofilm.

<p>Montage of confocal laser scanning images with 400x magnification of <i>G</i>. <i>vaginalis</i> biofilm, negative for <i>A</i>. <i>vaginae</i>, in 4 vaginal samples (A-D) in a superimposed image: vaginal epithelial cells DAPI in blue and <i>G</i>. <i>vaginalis</i> 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.</p