Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA, United States.
Laboratory of Research in Biofilms Rosário Oliveira (LIBRO), Centre of Biological Engineering (CEB), University of Minho, Braga, Portugal.
Front Cell Infect Microbiol. 2023 Jun 19;13:1198113. doi: 10.3389/fcimb.2023.1198113. eCollection 2023.
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 16S rRNA gene copy number. However, 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) spp. (G), spp. (P), and spp. (F) and commensal 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) 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.
细菌性阴道病（BV）是最常见的阴道微生态失调。在这种情况下，多微生物生物膜会在阴道上皮细胞上发育。准确量化 BV 生物膜的细菌负担对于进一步了解 BV 发病机制是必要的。从历史上看，计算 BV 生物膜总细菌负担的标准一直基于量化 16S rRNA 基因拷贝数。然而，这种方法并不适合测量这种独特微环境中的细菌负担。在这里，我们提出了一种新的 qPCR 标准，用于量化阴道微生物群落中的细菌负担，从最佳状态到成熟的 BV 生物膜。这些标准包括三种常见的 BV 相关细菌（G）、（P）和（F）和共生菌（L）的不同组合，使用 16S rRNA 基因（G：P：F：L、G：P：F、G：P：L 和 1G：9L）。我们将这些标准与使用已知数量的模拟阴道群落和 16 名女性阴道样本的传统（E）参考标准进行了比较。E 标准显著低估了模拟群落的拷贝数，并且在这些群落的拷贝数较低时，这种低估更为显著。在所有模拟群落中，G：P：L 标准最为准确，与其他混合阴道标准相比也是如此。混合阴道标准进一步用阴道样本进行了验证。这种新的 G：P：L 标准可用于 BV 发病机制研究，以提高对从最佳到非最佳（包括 BV）阴道微生物群的 BVAB 定量测量的可重复性和可靠性。
