Exploring the diversity of Gardnerella vaginalis in the genitourinary tract microbiota of monogamous couples through subtle nucleotide variation

Abstract

Background: Bacterial vaginosis (BV) is an enigmatic disease of unknown origin that affects a large percentage of women. The vaginal microbiota of women with BV is associated with serious sequelae, including abnormal pregnancies. The etiology of BV is not fully understood, however, it has been suggested that it is transmissible, and that G. vaginalis may be an etiological agent. Studies using enzymatic assays to define G. vaginalis biotypes, as well as more recent genomic comparisons of G. vaginalis isolates from symptomatic and asymptomatic women, suggest that particular G. vaginalis strains may play a key role in the pathogenesis of BV.

Methodology/principal findings: To explore G. vaginalis diversity, distribution and sexual transmission, we developed a Shannon entropy-based method to analyze low-level sequence variation in 65,710 G. vaginalis 16S rRNA gene segments that were PCR-amplified from vaginal samples of 53 monogamous women and from urethral and penile skin samples of their male partners. We observed a high degree of low-level diversity among G. vaginalis sequences with a total of 46 unique sequence variants (oligotypes), and also found strong correlations of these oligotypes between sexual partners. Even though Gram stain-defined normal and some Gram stain-defined intermediate oligotype profiles clustered together in UniFrac analysis, no single G. vaginalis oligotype was found to be specific to BV or normal vaginal samples.

Conclusions: This study describes a novel method for investigating G. vaginalis diversity at a low level of taxonomic discrimination. The findings support cultivation-based studies that indicate sexual partners harbor the same strains of G. vaginalis. This study also highlights the fact that a few, reproducible nucleotide variations within the 16S rRNA gene can reveal clinical or epidemiological associations that would be missed by genus-level or species-level categorization of 16S rRNA data.

Figure 1. Shannon entropy analysis per column for 65,710 aligned G. vaginalis sequences.

Peaks in entropy indicate nucleotide variation at given locations. While the X-axis of the figure indicates the location of the given column in the full length G. vaginalis 16S rRNA gene sequence, bar at the top superimposes the approximate locations of hyper-variable regions V4 (557–662), V5 (800–861) and V6 (981–1027) on G. vaginalis sequences that were used in this study.

Figure 2. Hierarchical clustering results of vaginal swab samples.

Samples were clustered (clustering significance: p<0.001, UniFrac significance: p = 0.016) based on the UniFrac distance metric. Red, orange and green colors indicate samples from BV, intermediate and normal Nugent categories, respectively.

Figure 3. Oligotype profiles in various female patients and their sexual partners.

Different colors in the pie charts correspond to different oligotypes. In every set, the pie chart on the left represents the sample collected from the female patient.