A novel Gardnerella, Prevotella, and Lactobacillus standard that improves accuracy in quantifying bacterial burden in vaginal microbial communities

Abstract

Bacterial vaginosis (BV) is the most common vaginal dysbiosis. In this condition, a polymicrobial biofilm develops on vaginal epithelial cells. Accurately quantifying the bacterial burden of the BV biofilm is necessary to further our understanding of BV pathogenesis. Historically, the standard for calculating total bacterial burden of the BV biofilm has been based on quantifying Escherichia coli 16S rRNA gene copy number. However, E. coli is improper for measuring the bacterial burden of this unique micro-environment. Here, we propose a novel qPCR standard to quantify bacterial burden in vaginal microbial communities, from an optimal state to a mature BV biofilm. These standards consist of different combinations of vaginal bacteria including three common BV-associated bacteria (BVAB) Gardnerella spp. (G), Prevotella spp. (P), and Fannyhessea spp. (F) and commensal Lactobacillus spp. (L) using the 16S rRNA gene (G:P:F:L, G:P:F, G:P:L and 1G:9L). We compared these standards to the traditional E. coli (E) reference standard using known quantities of mock vaginal communities and 16 vaginal samples from women. The E standard significantly underestimated the copy numbers of the mock communities, and this underestimation was significantly greater at lower copy numbers of these communities. The G:P:L standard was the most accurate across all mock communities and when compared to other mixed vaginal standards. Mixed vaginal standards were further validated with vaginal samples. This new G:P:L standard can be used in BV pathogenesis research to enhance reproducibility and reliability in quantitative measurements of BVAB, spanning from the optimal to non-optimal (including BV) vaginal microbiota.

Keywords: Gardnerella; Lactobacillus; Prevotella; bacterial burden; bacterial vaginosis; biofilm; qPCR standard; vaginal microbiome.

Figure 1

(A) Diagram of universal primer (UP) and specific primer (SP) locations in the V6 region of the 16S rRNA gene. (B–E) qPCR standards using the SP and UP for each vaginal bacteria of interest, Gardnerella spp. (G), Prevotella spp. (P), and Fannyhessea spp. (F), and Lactobacillus spp. (L). Data points are generated from three-fold dilutions of the purified plasmids starting at 10⁷ copies to 167 copies per reaction. Each point represents an average of 3 replicate qPCR reactions with corresponding error bars. Efficiency (E) is calculated based on the slope of the linear regression. Axes are labeled by the quantification cycle (Cq) and the number of copies per reaction (log10).

Figure 2

Mock vaginal communities using E and G:P:L mix standard. The equal parts G:P:L community is meant to represent a BV-like vaginal microbiome (top), G:P:L is representative of an intermediate microbiome (middle), and the 1G:9L is meant to represent an optimal vaginal microbiome (bottom). Known copy numbers are used as input, listed on the x-axis. The output is calculated using the corresponding standard and divided by the input to generate a percentage. A 100% output/input indicates the same copy number that was input was read as output from the standard. The resulting output/input was averaged and compared to 100% using two-way ANOVA, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 3

(A–D) Paired comparisons between the E and the four vaginal mixed standards across the three categories of Nugent score, normal, intermediate, and BV. The line between two points represents the same vaginal sample with each standard. The variability between these points was compared. All experiments were performed in triplicate and comparisons were made using two-way ANOVA, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.