Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes

Abstract

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.

A fruitful alliance: the synergy betweenAtopobium vaginaeandGardnerella vaginalisin bacterial vaginosis-associated biofilm

“…This is in accordance with prior reports on the association of A. vaginae with G. vaginalis. 9 11 15 16 24 We showed that the presence of both bacteria in the samples, regardless of their existence in a biofilm, was associated with an elevated or high Nugent score indicative for vaginal dysbiosis and BV. The highest probability of having a Nugent score higher than 3 was seen when both A. vaginae and G. vaginalis were part of a biofilm attaching to the vaginal epithelial cells.…”

“…11 Adherent A. vaginae and G. vaginalis were visualised in, respectively, 54.1% and 82.0% of samples with bacterial biofilm (detected using the universal BacUni-1 probe), suggesting an important role for both bacteria in this polymicrobial biofilm. Using FISH, we only found two samples containing A. vaginae (dispersed in both, adherent in one) in the absence of G. vaginalis , while more than one-third of the G. vaginalis -positive samples was negative for A. vaginae .…”

“…PNA FISH was performed as described earlier11 using species-specific probes for A. vaginae (AtoITM1) and G. vaginalis (Gard162) and the broad-range BacUni-1 probe. The hybridised samples were stored in the dark at room temperature for a maximum of 1 week before microscopic observation, using CLSM (LSM700, Zeiss, Oberkochen, Germany).…”

“…To study the potential role of A. vaginae and the synergy between A. vaginae and G. vaginalis in the biofilm, we used our newly developed peptide nucleic acid (PNA) A. vaginae probe11 together with an existing probe for G. vaginalis 21 and a universal bacterial probe22 to investigate the composition of vaginal biofilm and its importance in BV.…”

Multiplex PCR detection of problematic pathogens of clinically heterogeneous bacterial vaginosis in Bulgarian women

“…Bacteria such as G. vaginalis, Atopobium vaginae, and Mobiluncus spp. are recognized as predominant vaginal pathogens and as sensitive indicators in the diagnosis of BV, although many other BV-related pathogens have been found with variable frequency (7)(8)(9)(10)(11). A. vaginae and Mobiluncus spp.…”