Heterogeneity of vaginal microbial communities within individuals

Abstract

Recent culture-independent studies have revealed that a healthy vaginal ecosystem harbors a surprisingly complex assemblage of microorganisms. However, the spatial distribution and composition of vaginal microbial populations have not been investigated using molecular methods. Here, we evaluated site-specific microbial composition within the vaginal ecosystem and examined the influence of sampling technique in detection of the vaginal microbiota. 16S rRNA gene clone libraries were prepared from samples obtained from different locations (cervix, fornix, outer vaginal canal) and by different methods (swabbing, scraping, lavaging) from the vaginal tracts of eight clinically healthy, asymptomatic women. The data reveal that the vaginal microbiota is not homogenous throughout the vaginal tract but differs significantly within an individual with regard to anatomical site and sampling method used. Thus, this study illuminates the complex structure of the vaginal ecosystem and calls for the consideration of microenvironments when sampling vaginal microbiota as a clinical predictor of vaginal health.

FIG. 1.

Schematic representation of vaginal sampling locations. Three swab samples were collected, from the fornix, cervix, and lower one-third (outer) region of the vaginal canal. Two vaginal scrapings were collected, from the upper one-third and the lower one-third of the vagina. An ectocervical lavage sample was obtained by washing the vaginal canal with sterile saline solution.

FIG. 2.

Phylogenetic distribution of vaginal bacteria from eight healthy, asymptomatic subjects. Numbers of clones for each phylotype are indicated in parentheses. The tree was produced using DNADIST and FITCH in PHYLIP. Bootstrap values of >50 from 100 resamplings (* indicates bootstrap value of <50, and − represents no clustering in the method) are indicated at each node (Fitch/parsimony/maximum likelihood). The scale bar represents 0.02 substitutions per site. Thermotoga maritima was used as the outgroup. The right side of the figure displays relative abundances of the phylotypes by subject and by group. The numbers above the abundance columns for the subjects correspond to the subject identification number. G1 to G4 correspond to the four groups into which the subjects were divided.

FIG. 3.

Grouping of subjects by bacterial composition at the phylum level. The histogram (lower panel) shows the phylum level bacterial composition of each subject, and the dendrogram (upper panel) displays the clustering of the eight subjects. Four groups were identified using a cutoff distance value of 20%. Over 500 clone sequences each were analyzed for groups 1 and 2, while about 1,000 clone sequences each were analyzed for groups 3 and 4.

FIG. 4.

Clustering of vaginal samples within individuals based on microbial composition. The histograms (lower portion of each panel) show the phylum level bacterial composition of each subject, and the dendrograms (upper portion of each panel) display the clustering of the sample types within each of the eight subjects. CX, swab cervix library; FX, swab fornix library; OT, swab outer library; SL, scrape lower library; SU, scrape upper library; LA, lavage library.

FIG. 5.

Distance ratios of between-sample versus within-sample distances for repeat libraries from subjects 403 and 409. (A) Bray-Curtis dissimilarity distances for the repeat libraries for the two most diverse subjects (no. 403 and 409) used to calculate distance ratios. The P values for single factor analysis of variance comparing between-sample distances versus within-sample distances are denoted as follows: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (B) Linear correlation of Bray-Curtis distances with the calculated distance ratios.