<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

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

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

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

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

The presence of the putative <i>Gardnerella vaginalis</i> sialidase A gene in vaginal specimens is associated with bacterial vaginosis biofilm

<div><p>Bacterial vaginosis (BV) is a difficult-to-treat recurrent condition in which health-associated lactobacilli are outnumbered by other anaerobic bacteria, such as <i>Gardnerella vaginalis</i>. Certain genotypes of <i>G</i>. <i>vaginalis</i> can produce sialidase, while others cannot. Sialidase is known to facilitate the destruction of the protective mucus layer on the vaginal epithelium by hydrolysis of sialic acid on the glycans of mucous membranes. This process possibly facilitates adhesion of bacterial cells on the epithelium since it has been linked with the development of biofilm in other pathogenic conditions. Although it has not been demonstrated yet, it is probable that <i>G</i>. <i>vaginalis</i> benefits from this mechanism by attaching to the vaginal epithelium to initiate biofilm development. In this study, using vaginal specimens of 120 women enrolled in the Ring Plus study, we assessed the association between the putative <i>G</i>. <i>vaginalis</i> sialidase A gene by quantitative polymerase chain reaction (qPCR), the diagnosis of BV according to Nugent score, and the occurrence of a BV-associated biofilm dominated by <i>G</i>. <i>vaginalis</i> by fluorescence in situ hybridisation (FISH). We detected the putative sialidase A gene in 75% of the <i>G</i>. <i>vaginalis</i>-positive vaginal specimens and found a strong association (p<0.001) between the presence of a <i>G</i>. <i>vaginalis</i> biofilm, the diagnosis of BV according to Nugent and the detection of high loads of the <i>G</i>. <i>vaginalis</i> sialidase A gene in the vaginal specimens. These results could redefine diagnosis of BV, and in addition might guide research for new treatment.</p></div

Overview of different subsets of samples analysed with fluorescence in situ hybridisation (n = 462), Nugent score (n = 527), <i>G</i>. <i>vaginalis</i> quantitative polymerase chain reaction (n = 528) and <i>G</i>. <i>vaginalis</i> sialidase quantitative polymerase chain reaction (n = 393).

<p>Overview of different subsets of samples analysed with fluorescence in situ hybridisation (n = 462), Nugent score (n = 527), <i>G</i>. <i>vaginalis</i> quantitative polymerase chain reaction (n = 528) and <i>G</i>. <i>vaginalis</i> sialidase quantitative polymerase chain reaction (n = 393).</p

The association between quantitative polymerase chain reaction results for <i>G</i>. <i>vaginalis</i> and <i>G</i>. <i>vaginalis</i> sialidase of vaginal samples and fluorescence in situ hybridisation and Nugent score results of vaginal slides.

<p>The association between quantitative polymerase chain reaction results for <i>G</i>. <i>vaginalis</i> and <i>G</i>. <i>vaginalis</i> sialidase of vaginal samples and fluorescence in situ hybridisation and Nugent score results of vaginal slides.</p

Vaginal microbiome of participants at time of removal of contraceptive ring: presence and absence of a vaginal biofilm with confocal laser scanning microscopy after fluorescence in situ hybridisation by species.

<p>(53 results unavailable due to inadequate quality of samples for confocal laser scanning microscopy).</p

Association of vaginal dysbiosis and biofilm with contraceptive vaginal ring biomass in African women - Fig 4

<p>Visualisation of biomass on intravaginal ring surface by scanning electron microscopy at A. 1000x and B. 4000x magnification: Phenotype 2—condense biofilm of bacilli on vaginal epithelial cells.</p