Characterization of the vaginal micro- and mycobiome in asymptomatic reproductive-age Estonian women

Abstract

The application of high-throughput sequencing methods has raised doubt in the concept of the uniform healthy vaginal microbiota consisting predominantly of lactobacilli by revealing the existence of more variable bacterial community composition. As this needs to be analyzed more extensively and there is little straightforward data regarding the vaginal mycobiome of asymptomatic women we aimed to define bacterial and fungal communities in vaginal samples from 494 asymptomatic, reproductive-age Estonian women. The composition of the vaginal microbiota was determined by amplifying bacterial 16S rRNA and fungal internal transcribed spacer-1 (ITS-1) regions and subsequently sequencing them using 454 Life Sciences pyrosequencing. We delineated five major bacterial community groups with distinctive diversity and species composition. Lactobacilli were among the most abundant bacteria in all groups, but also members of genus Gardnerella had high relative abundance in some of the groups. Microbial diversity increased with higher vaginal pH values, and was also higher when a malodorous discharge was present, indicating that some of the women who consider themselves healthy may potentially have asymptomatic bacterial vaginosis (BV). Our study is the first of its kind to analyze the mycobiome that colonizes the healthy vaginal environment using barcoded pyrosequencing technology. We observed 196 fungal operational taxonomic units (OTUs), including 16 OTUs of Candida spp., which is more diverse than previously recognized. However, assessing true fungal diversity was complicated because of the problems regarding the possible air-borne contamination and bioinformatics used for identification of fungal taxons as significant proportion of fungal sequences were assigned to unspecified OTUs.

Figure 1. Distribution of 432 samples according to number of sequences and number of OTUs.

The cutoff value is set to 400 sequences. Round and square brackets in the figure indicate to the exclusion and inclusion of adjacent value, respectively.

Figure 2. Relative abundance of bacterial OTUs found in the vaginal communities of healthy Estonian women (n = 432).

Bacterial taxonomic assignments are indicated on the right of the heatmap at the Genus and Species level. The relative abundance is color coded and indicated by the color key on the left top of the map. The tree on the top of the heatmap characterizes the similarity of analyzed samples based on OTU composition of vaginal microbiota.

Figure 3. Two-dimensional (2D) plots describing the principal component analysis (PCA) of OTU composition among analyzed samples.

The plots represent 2D projections of a multidimensional analysis where the relative abundance of each specific OTU defines a dimension. Both plots are the projection of the same analysis viewed at a different angle. The plots visualize the clustering and variability of studied vaginal bacterial communities. First (a) 2D plot of the first two PCA components describes the clustering of groups I–V. Second (b) 2D plot of third and fourth PCA component confirms that the samples belonging to non-classifiable group (0) are not clustering into separate or any other entity. The variance described by the respective PCA components (Axis-1 and Axis-2) is written in brackets.

Figure 4. The distribution of 10 most relatively abundant OTUs in determined vaginal bacterial community groups.

The distribution is presented based on mean relative abundance values (x-axis) of these OTU-s among bacterial community groups (y-axis).

Figure 5. Relative abundance of the most abundant bacterial and fungal OTUs found in the vaginal communities of 181 women.

Taxonomic assignments are indicated on the right of the heatmap at the Genus and Species level. The relative abundance is color coded and indicated by the color key on the left top of the map. The tree on the top of the heatmap characterizes the similarity of analyzed samples.