# Evidence from a broad-range PNA probe links several Prevotella species to bacterial vaginosis

## TL;DR

A new probe shows that multiple Prevotella species, not just P. bivia, are linked to bacterial vaginosis, suggesting a broader role in the infection's development.

## Contribution

A new PNA probe with broader specificity for Prevotella species was developed and validated, revealing their wider involvement in bacterial vaginosis.

## Key findings

- The new Prevotella spp. PNA probe achieved 100% specificity and 96% sensitivity in vitro.
- The new probe detected significantly more Prevotella spp. in 66.6% of BV-positive clinical specimens compared to the P. bivia-specific probe.
- Prevotella spp. were also detected in 22.2% of healthy control specimens, suggesting broader presence.

## Abstract

Bacterial vaginosis (BV) is the most prevalent vaginal infection among reproductive-age women. It is associated with multiple adverse health outcomes in women including adverse pregnancy outcomes, an increased risk of pelvic inflammatory disease, infertility, and an increased risk of HIV and other sexually transmitted infections. BV is characterized by an imbalance in the vaginal microbiota, namely a decrease in protective Lactobacillus species and an overgrowth of facultative and strict 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).

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. Its performance was evaluated using a collection of 28 Prevotella strains representing 24 different species and 38 non-Prevotella spp. typically found in BV in order to assess its 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 vitro validation demonstrated that the new Prevotella spp. probe achieved a specificity of 100% and sensitivity of 96%. 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.

Our findings suggest that the involvement of Prevotella spp. in BV extends beyond P. bivia, implicating a wider range of species which could be present in the polymicrobial BV biofilm. The broader specificity of this new Prevotella spp. probe provides a valuable tool for future research on the vaginal microbiome and the pathogenesis of BV.