Lactobacillus crispatus dominant vaginal microbiome is associated with inhibitory activity of female genital tract secretions against Escherichia coli

Abstract

Objective: Female genital tract secretions inhibit E. coli ex vivo and the activity may prevent colonization and provide a biomarker of a healthy microbiome. We hypothesized that high E. coli inhibitory activity would be associated with a Lactobacillus crispatus and/or jensenii dominant microbiome and differ from that of women with low inhibitory activity.

Study design: Vaginal swab cell pellets from 20 samples previously obtained in a cross-sectional study of near-term pregnant and non-pregnant healthy women were selected based on having high (>90% inhibition) or low (<20% inhibition) anti-E. coli activity. The V6 region of the 16S ribosomal RNA gene was amplified and sequenced using the Illumina HiSeq 2000 platform. Filtered culture supernatants from Lactobacillus crispatus, Lactobacillus iners, and Gardnerella vaginalis were also assayed for E. coli inhibitory activity.

Results: Sixteen samples (10 with high and 6 with low activity) yielded evaluable microbiome data. There was no difference in the predominant microbiome species in pregnant compared to non-pregnant women (n = 8 each). However, there were significant differences between women with high compared to low E. coli inhibitory activity. High activity was associated with a predominance of L. crispatus (p<0.007) and culture supernatants from L. crispatus exhibited greater E. coli inhibitory activity compared to supernatants obtained from L. iners or G. vaginalis. Notably, the E. coli inhibitory activity varied among different strains of L. crispatus.

Conclusion: Microbiome communities with abundant L. crispatus likely contribute to the E. coli inhibitory activity of vaginal secretions and efforts to promote this environment may prevent E. coli colonization and related sequelae including preterm birth.

Figure 1. High E. coli inhibitory activity is associated with L. crispatus predominant microbiome.

Heat Map of community compositions and proportional abundances of bacteria. The first row of colored tiles indicates the level of E. coli inhibitory activity (claret = >90% inhibition and blue <20% inhibition). Each subsequent row represents the bacterial taxon and its proportional abundance (on a base 10 logarithmic scale). A taxon is only shown if ≥217 reads were assigned there for any sample (corresponding to retaining all reads above the 90^th percentile). The Roman numerals at the bottom of the figure correspond to vaginal microbiome groups as reported by Ravel et al. The last 3 samples have not been classified do to limited sample size in this region of the dendogram.

Figure 2. The microbiome associated with high E. coli inhibitory activity is significantly different than the microbiome in women with low activity (p = 0.042).

The pairwise Kantorovich-Rubinstein (KR) distances between sample microbiomes were calculated and a PERMANOVA principal component analysis was performed with E. coli inhibitory activity (A) and pregnancy (B) as the factors. The two axes represent the first two principal components of the pairwise KR distance matrix. The red point demarks the group centroid, while the black open points represent the sample coordinates in the first two principal components. The p-value test statistic is displayed at the top of each plot area, indicating the statistical significance of the difference in variances when samples were grouped according to the factor (i.e., E. coli inhibitory activity or pregnancy). The Eigen values, or the amount of variation in the data accounted for by each principal component, are found in parentheses adjacent to PC1 and PC2.

Figure 3. L. crispatus culture supernatants inhibit E. coli.

(a) Bar graphs depicting the E. coli cfu/ml after overnight incubation with 1∶10 dilution in normal saline of culture supernatants obtained from the indicated bacterial species or respective control media. The results were adjusted for differences in colony counts of the bacteria from which SCS were obtained and are presented as mean ± SD obtained from 3 independent experiments. (b) The spent culture supernatants obtained from three additional strains of L. crispatus (M35, SJ-3C, and 60) were normalized by diluting in culture media to that of the lowest growth (2×10⁷ cfu/ml) and then serial dilutions (neat, 1∶5, 1∶10 and 1∶25) were mixed with E. coli and tested for inhibitory activity. Results are mean ± SD from duplicate plates. The number symbol indicates that no bacterial colonies were observed on plates after incubation with undiluted L. crispatus 60 SCS. The asterisks represent a significant reduction in E. coli cfu relative to its control growth media (p<0.01).