Vaginal Bacterial Microbiome Analysis Using 16S rRNA Nanopore Sequencing Confirms Inverse Association Between Gardnerella and Lactobacillus Genera

Abstract

Objective: The aim of this analysis was to explore vaginal bacterial microbiome using nanopore sequencing for the identification of species, dominance, and possible associations. Methods: Vaginal swabs were collected within one menstrual cycle, PCR was performed with modified primers 27F and 1429R. Nanopore sequencing of the 16S rRNA gene library was performed using the SQK-RBK114.96 kit (Oxford Nanopore) and a FLO-MIN114 flow cell on the MinKNOW platform (software version 24.02.8) with default parameters. Subsequent analysis was carried out using EPI2ME (version 5.2.2) with the wfmetagenomics workflow. Results: In this study, 135 vaginal microbiome samples were analyzed, collected in different menstrual phases from 35 women. The samples were divided into two groups based on the relative abundance of Gardnerella: in 46 samples it exceeded 20%. Among the samples with high Gardnerella levels, only 14 cases (30.4%) showed a Lactobacillus abundance greater than 20%, compared to 65 cases (73.0%) in the low Gardnerella group. The odds ratio calculation revealed that the likelihood of detecting Lactobacillus >20% was more than six times lower in the high Gardnerella group (odds ratio = 0.16; χ² = 28.89; p < 0.0001). Conclusions: Here we note a statistically significant inverse association between Gardnerella and Lactobacillus genera, which likely reflects competition for the same ecological niche. 16S rRNA sequencing is very useful for the quick analysis of the major species up to the genus level. Using this methodology the status of vaginal microbiota, physiological vs. pathological, can quickly be assessed without microorganism cultivation