Abstract

Background. Bacterial vaginosis (BV) is the most prevalent vaginal infection among reproductive-age women, with adverse pregnancy complications and an increased risk of HIV and sexually transmitted infection acquisition. BV is characterized by an imbalance in the vaginal microbiota, namely a decrease of Lactobacillus species and an overgrowth of anaerobic bacteria, leading to the development of a polymicrobial biofilm. Despite extensive research, the etiology of BV remains unclear, and its pathophysiology is not fully understood. It has been hypothesized that P. bivia, in combination with Gardnerella spp, plays an important role in the early development of the BV biofilm. We previously developed a peptide nucleic acid (PNA) probe specifically targeting P. bivia to investigate its role as a potential early colonizer. However, our recent findings have raised doubts about the specificity of this association, suggesting a broader involvement of other Prevotella species in incident BV (iBV).
Methods. A new PNA probe targeting Prevotella spp. 23S rRNA was developed compared to the existing P. bivia‑specific probe. This new probe was optimized in vitro through a variation of hybridization temperatures and times, and its performance was evaluated using a collection of 28 Prevotella strains representing 24 different species, and 37 non-Prevotella spp. typically found in BV, in order to assess sensitivity and specificity. Both probes were tested on vaginal swab specimens from women with and without BV to assess the bacterial count and detection of Prevotella species in iBV.
Results. In vitro validation demonstrated that the new Prevotella spp. probe achieved a specificity of 94% and sensitivity of 92%. As expected, its broader detection allowed identification of a wider range of Prevotella spp. compared to the P. bivia-specific probe, which was intentionally restricted to a single species. Application to clinical specimens revealed that the new probe identified a significantly higher count of Prevotella spp. in 6/9 (66.6%) BV-positive specimens compared to the P. bivia-specific probe. In 2/9 (22.2%) healthy control specimens, greater Prevotella spp. detection was also observed.
Conclusions. Our findings suggest that the involvement of Prevotella in BV extends beyond P. bivia, implicating a wider range of species which could be present in the BV polymicrobial biofilm. The broader specificity of the new Prevotella probe provides a valuable tool for future research on the vaginal microbiome.