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

PLoS One. 2015 Aug 25;10(8):e0136658. doi: 10.1371/journal.pone.0136658. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actinobacteria / genetics
  • Actinobacteria / isolation & purification*
  • Actinobacteria / pathogenicity
  • Biofilms*
  • Female
  • Gardnerella vaginalis / genetics
  • Gardnerella vaginalis / isolation & purification*
  • Gardnerella vaginalis / pathogenicity
  • Humans
  • In Situ Hybridization, Fluorescence
  • Nucleic Acid Probes / genetics
  • Peptide Nucleic Acids / genetics
  • Vagina / microbiology
  • Vaginosis, Bacterial / genetics
  • Vaginosis, Bacterial / microbiology*
  • Vaginosis, Bacterial / pathology

Substances

  • Nucleic Acid Probes
  • Peptide Nucleic Acids

Grant support

Funding was received by TC from European and Developing Countries Clinical Trials Partnership: SP.2011.41304.043, http://www.edctp.org/; by VJ from Combined Highly Active Anti-Retroviral Microbicides under EU FP7: 242135, http://cordis.europa.eu/fp7/home_en.html; and by VJ from Dormeur Investment Service Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.