Endocervical and vaginal microbiota in South African adolescents with asymptomatic Chlamydia trachomatis infection

Abstract

Adolescent girls and young women represent a key risk group for sexually transmitted infections (STIs). The vaginal microbiota is thought to play an important role in susceptibility to STIs such as Chlamydia trachomatis. We compared the microbiota of the lateral vaginal wall and endocervix, and assessed associations with C. trachomatis infection in South African adolescents. The endocervical and vaginal lateral wall microbiota were characterized by amplifying and sequencing the V4 region of the 16S rRNA gene and C. trachomatis diagnosed using molecular methods. Of the 72 girls included, 30 had asymptomatic C. trachomatis infections. Three major vaginal community types were identified; one Lactobacillus crispatus, one L. iners and one diverse, Gardnerella vaginalis dominant. The microbiota of the endocervix was significantly different from that of the lateral wall in terms of diversity. There were many differentially abundant taxa between the endocervix and lateral vaginal wall, including Achromobacter spanius and Enterococcus faecium. Women with C. trachomatis had higher relative abundance of G. vaginalis and other anaerobes. In this African adolescent cohort, significant differences between the lateral vaginal wall and endocervical microbiota diversity and composition were evident, although neither were strongly associated with C. trachomatis infection.

Figure 1

Composition of vaginal lateral wall and endocervical microbiota. (a) Heat map of the 30 most abundant taxa (rows) identified by 16S rRNA microbiome profiling using unsupervised hierarchical clustering with weighted UniFrac distances in all samples (columns). Unsupervised hierarchical clustering of 144 matched endocervial and vaginal lateral wall samples from 72 participants. The dendrogram was generated using average linkage clustering with weighted UniFrac distance, based on the relative abundance of taxa (merged at lowest taxonomic level) in each sample. Log2-transformed standardized read counts are illustrated by the colour key. Annotation bars above the heatmap depict community type cluster (top bar), BV status based on Nugent scoring (upper middle bar), C. trachomatis (Ct) infection status (lower middle bar) and anatomical sampling site (bottom bar). Samples that did not meet the minimum probability of ≥60% of belonging to any of the three clusters were excluded from downstream analyses (n = 4, “no.cluster” in figure). (b) Barplot of the 30 top most abundant taxa identified by 16S rRNA microbiome profiling. Samples are grouped by microbial compositional subtype (C1, C2, C3) established using Fuzzy clustering with weighted UniFrac distances and ordered based on the abundance of the most dominant species in each community type (G. vaginalis, L. crispatus and L. iners, respectively). Samples that did not meet the minimum probability of ≥60% of belonging to any of tree clusters (n = 4) were excluded from the figure. Shannon diversity Index for each sample is depicted below the barplot. (c) Principal Coordinates Analysis (PCoA) of samples colored by compositional subtypes generated using Fuzzy clustering with weighted UniFrac distances. Samples are colored by compositional subtype (C1, C2, C3), with BV status displayed as shapes. Samples that did not meet the minimum probability of ≥60% of belonging to any of these clusters were excluded from downstream analyses (n = 4, “no.cluster” in figure).

Figure 2

Taxa significantly different between vaginal lateral wall versus endocervical microbiota. (a) Principal Coordinates Analysis (PCoA) of the vaginal and endocervical microbiota using weighted UniFrac distances. Individual samples are colored by anatomical sampling site (endocervical:EC and lateral vaginal wall:LW) with BV status displayed as shapes. (b) Boxplot depicting the alpha diversity of the vaginal lateral wall (LW) and endocervical (EC) microbiota. (c) Taxa significantly differentially abundant and/or frequent by anatomical site category by metagenomeSeq (FDR ≤0.05, coefficient ≥1.25, taxa present in ≥20% of samples in at least one of the two groups being compared). Unsupervised clustering of samples (columns) by Bray-Curtis distance; heat map scale: log2-transformed standardized counts. (d) The top ten most influential taxa by random forests analysis. The x-axis indicates the mean decrease in Gini Index (length of bar represents predictive ability of each taxon). (e) Barplot depicting within-subject pair-wise distances (weigthed UniFrac) between sampling sites (ECvsLW) and the mean between-subject pair-wise distances (weighted UniFrac) between either endocervical samples (ECvsEC) or lateral wall samples (LWvsLW).

Figure 3

Taxa significantly different in C. trachomatis infected versus uninfected individuals in the endocervical microbiota. (a) Taxa significantly differentially abundant and/or frequent by C. trachomatis category in the endocervical (EC) microbiota by metagenomeSeq (FDR ≤0.05, coefficient ≥1.25, taxa present in ≥20% of samples in at least one of the two groups being compared). Unsupervised clustering of samples (columns) by Bray-Curtis distance; heat map scale: log2-transformed standardized counts. The OTU assigned C. trachomatis taxonomy was excluded for this analysis. (b) The top 20 most influential taxa by random forests analysis. The x-axis indicates the mean decrease in Gini Index (length of bar represents predictive ability of each taxon), where a larger index indicates greater predictive power. Taxa that were significantly differentially abundant and/or frequent in C. trachomatis infected versus uninfected individuals (FDR ≤0.05, coefficient ≥1.25, taxa present in ≥20% of samples in at least one of the two groups being compared); hierarchical clustering (Bray-curtis distance); heat map scale: log2-transformed standardized counts. The OTU assigned C. trachomatis taxonomy was excluded for this analysis. (c) Principal Coordinates Analysis (PCoA) of the endocervical microbiota using weighted UniFrac distances. Individual samples are colored by C. trachomatis infection status with fuzzycluster displayed as shapes.