A Systems Biology Approach Investigating the Effect of Probiotics on the Vaginal Microbiome and Host Responses in a Double Blind, Placebo-Controlled Clinical Trial of Post-Menopausal Women

<div><p></p><p>A lactobacilli dominated microbiota in most pre and post-menopausal women is an indicator of vaginal health. The objective of this double blinded, placebo-controlled crossover study was to evaluate in 14 post-menopausal women with an intermediate Nugent score, the effect of 3 days of vaginal administration of probiotic <i>L. rhamnosus</i> GR-1 and <i>L. reuteri</i> RC-14 (2.5×10⁹ CFU each) on the microbiota and host response. The probiotic treatment did not result in an improved Nugent score when compared to when placebo. Analysis using 16S rRNA sequencing and metabolomics profiling revealed that the relative abundance of <i>Lactobacillus</i> was increased following probiotic administration as compared to placebo, which was weakly associated with an increase in lactate levels. A decrease in <i>Atopobium</i> was also observed. Analysis of host responses by microarray showed the probiotics had an immune-modulatory response including effects on pattern recognition receptors such as TLR2 while also affecting epithelial barrier function. This is the first study to use an interactomic approach for the study of vaginal probiotic administration in post-menopausal women. It shows that in some cases multifaceted approaches are required to detect the subtle molecular changes induced by the host to instillation of probiotic strains.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="http://clinicaltrials.gov/ct2/show/NCT02139839" target="_blank">NCT02139839</a></p></div

Summary of Nugent Score Change - Proportion of Improvement from Baseline.

1<p>Improvement in Nugent Score: indicated by shift of intermediate score (4–6) at baseline to normal score (0–3) at follow-up visits. Baseline Nugent score for study Phase-I was collected at visit 3, and baseline Nugent score for study Phase-II was collected at visit 6.</p>2<p><i>p</i>-value is based on McNemars test to determine results of cross-over treatment regimen.</p

Microbiota heat map.

<p>Each column represents the microbiota of a single sample with the 50 most abundant OTUs displayed with their taxonomies and the remainder pooled. Samples are clustered by the participant of origin and organized from first to last visit from left to right. The time points immediately following administration of the placebo or probiotic are indicated with a blue or green arrow (respectively). OTUs representing the putative OTUs for <i>L. rhamnosus</i> GR-1 and <i>L. reuteri</i> RC-14 have been bolded.</p

Selected genus relative abundances following probiotic and placebo interventions.

<p>(A) <i>Lactobacillus</i>, (B) <i>Gardnerella</i>, (C) <i>Atopobium</i>, (D) <i>Prevotella</i>, (E) <i>Streptococcus</i>, (F) <i>Staphylococcus</i>. Following probiotic administration for 3 days, the proportion of <i>Lactobacillus</i> is significantly increased while that of <i>Atopobium</i> is decreased and <i>Staphylococcus</i> is increased. Placebo interventions increased <i>Streptococcus</i> and <i>Staphylococcus</i> abundance. *FDR<0.1, **FDR<0.05, ***FDR<0.01.</p

Gene Ontology Enrichment in genes differentially expressed by probiotic intervention.

<p>Genes differentially expressed (≥2-fold change, p<0.05) were subjected to GO enrichment analysis. Functions heavily relate to innate immune system responses including responses to both Gram-positive and Gram-negative bacteria.</p

UPGMA clustering of all participants microbiota based upon weighted UniFrac distances.

<p>In general, participants cluster most closely with themselves. The sample tips are colored by the participant of origin.</p

CONSORT flow diagram.

<p>CONSORT flow diagram.</p

Time series of participant 17.

<p>During the administration of probiotic between visits 6 and 7, the abundance of lactobacilli significantly increases, decreasing the proportional abundance of both <i>Atopobium</i> and <i>Gardnerella</i>. This increase is not due solely to the probiotic strains as a significant increase in indigenous <i>L. gasseri</i>/<i>johnsonii</i> takes place.</p