Improved understanding of the bacterial vaginal microbiota of women before and after probiotic instillation

Abstract

The vaginal bacterial microbiota of 19 premenopausal women was examined by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing of the V2-V3 region of the 16S rRNA gene. Ten of the women were studied further to investigate the effect and persistence of vaginally inserted capsules containing viable lactobacilli. PCR-DGGE indicated that most subjects had a microbiota represented by one to three dominant DNA fragments. Analysis of these fragments revealed that 79% of the women possessed sequences with high levels of similarity to Lactobacillus species sequences. Sequences homologous to Lactobacillus iners sequences were the most common and were detected in 42% of the women tested. Alteration of the vaginal microbiota could be detected by PCR-DGGE in several women after the instillation of lactobacilli. Additionally, randomly amplified polymorphic DNA analysis of lactobacilli isolated from selective media demonstrated that the exogenous strains could be detected for up to 21 days in some subjects. This study demonstrates that non-culture-based techniques, such as PCR-DGGE, are useful adjuncts for studies of the vaginal microbiota.

FIG. 1.

DGGE of the V2-V3 16S rRNA gene amplicons from vaginal samples: profiles for 19 subjects (zero time, prestudy samples). The arrowheads indicate the DNA fragments sequenced from specific lanes, while in unmarked lanes only the dominant fragment was sequenced. BLAST sequence homologies are shown in Table 1.

FIG. 2.

DGGE profiles of the vaginal microbiota from five women during the study. Lanes L contained known isolates L. fermentum RC-14 (a) and L. rhamnosus GR-1 (b). Lanes 1 to 5 contained amplicons from samples taken at zero time (prestudy) and at 3, 7, 14, and 21 days after instillation of capsules containing lactobacilli, respectively. The arrowheads indicate DNA fragments that were sequenced. Presumptive identities based on closest BLAST homologies are as follows: for subject 263, lane 1, L. delbrueckii; for subject 265, lane 1, L. crispatus; for subject 265, lane 3, Pseudomonas sp.; for subject 260, lane 1, L. iners; for subject 260, lane 2, L. crispatus; for subject 261, lane 1 (from top to bottom), L. iners, Arthrobacter sp., and G. vaginalis; for subject 261, lane 2, Pseudomonas sp.; for subject 261, lane 3, S. agalactiae; for subject 261, lane 5, L. iners (all fragments); for subject 268, lane 1, B. fibrisolvens and G. vaginalis (fragments 1 and 2, respectively).