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.

Association of Chlamydia trachomatis burden with the vaginal microbiota, bacterial vaginosis, and metronidazole treatment

“…In this study, while the use of relative abundances was sufficient to detect significant reductions of many BVAB and increases in Lactobacillus following successful MTZ treatment, the magnitude of the effects that these bacteria have on the genital environment are directly linked to their absolute abundances or burden, the quantification of which may be required to understand the relationship between the vaginal microbiota, genital environment, and chlamydial clearance. Future studies would benefit from the quantification of absolute abundances of dominant vaginal bacterial taxa or by inferring absolute abundances from total bacterial burden in combination with compositional data ( Tettamanti Boshier et al., 2020 ; Elnaggar et al., 2023a ; Elnaggar et al., 2023b ).…”

Microbial interactions amongGardnerella,PrevotellaandFannyhesseaprior to incident bacterial vaginosis: protocol for a prospective, observational study

“…Reactions will then undergo 40 cycles of amplification at 98°C for 15 s and 60°C for 15 seconds. Mixed vaginal plasmid standards made up of Gardnerella , Prevotella and Lactobacillus 55 will be run for each reaction ranging from 10 7 to 100 gene copies; values will be reported as 16S rRNA gene copies/specimen to estimate the total bacterial load. Measurements for total bacterial load will be used to adjust relative abundance measures acquired from vaginal microbiome sequencing data.…”

Molecular Characterization of Vaginal Microbiota Using a New 22-Species qRT-PCR Test to Achieve a Relative-abundance and Species-based Diagnosis of Bacterial Vaginosis

“…Molecular tests such as real-time polymerase chain reaction (RT-PCR), combined with the traditional clinical diagnostic criteria, have greatly improved the sensitivity and specificity of detecting BV pathogens [11,17,18]. They are also applicable in monitoring patient response to antibiotic therapy [1,19]. This approach has proven more useful for identifying patients at risk for recurrent BV [3,9,12,14,20,21].…”

“…Bacterial Vaginosis (BV), a condition in which the normal lactobacillus-rich vaginal microbiome becomes dominated by polymicrobial anaerobic bacterial species under non-acidic pH, remains the most common cause of abnormal vaginal discharge in reproductive-age women, with an estimated prevalence rate of 29% in North America [1,2]. The normal vaginal microbiome is dominated by three major Lactobacillus species, L. crispatus, L. jensenii, and L. gasseri, which protect the vagina by producing lactic acid, hydrogen peroxide, and bacteriocins that suppress bacterial growth [3].…”