Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis

Abstract

Background: The pathogenesis of bacterial vaginosis remains largely elusive, although some microorganisms, including Gardnerella vaginalis, are suspected of playing a role in the etiology of this disorder. Recently culture-independent analysis of microbial ecosystems has proven its efficacy in characterizing the diversity of bacterial populations. Here, we report on the results obtained by combining culture and PCR-based methods to characterize the normal and disturbed vaginal microflora.

Microbes on the human vaginal epithelium

“…We found 4 of 20 subjects with a significant number of Peptostreptococcus reads and another 3 subjects with a few reads of Peptostreptococcus. Atopobium was found in two of eight subjects by Verhelst et al (22) and was the only microbe in one of five subjects by Zhou et al (23). Four of our 20 subjects had a small number of Atopobium reads, subjects 03 (3 reads), 05 (41 reads), 09 (53 reads), and 43 (74 reads; Tables 9, 11, 15, and 23, respectively).…”

“…As a direct consequence of our deep sequencing, we found an additional four subjects with a significant number of Streptococcus reads as well as three subjects with a few reads of Streptococcus. Verhelst et al (22) found no subjects with Streptococcus but one of eight subjects with Peptostreptococcus. We found 4 of 20 subjects with a significant number of Peptostreptococcus reads and another 3 subjects with a few reads of Peptostreptococcus.…”

“…It is unlikely that rDNA polymorphisms would be detected by this procedure. Verhelst et al (22) did not report the number of sequence reads per subject. Of the eight subjects, four had essentially only Lactobacillus, one had Atopobium, two had Atopobium plus Prevotella, and one had Peptostreptococcus plus Peptoniphilus.…”

“…There are three recently published studies using 16S rDNA gene-based technology to identify microbes in the human female urogenital tract (21)(22)(23). Although the three published studies took samples from different sites within the vagina and used different primer pairs (yielding different sizes of amplified products), different amplification conditions, different women (from Canada, Belgium, and the U.S., respectively), and at unreported times during the menstrual cycle, these women comprise a reasonable comparison group for our experiments.…”

“…Verhelst et al (22) studied eight healthy subjects ''attending our out-patient clinic for a routine gynecological visit.'' Verhelst et al (22) amplified a 524-base fragment of 16S rDNA; ''the 16S rRNA gene was amplified and sequenced from selected clones and from cultured isolates that could not be identified by tDNA-PCR'' (tDNA-PCR is a technique that uses one tRNA-based primer for PCR amplification). It is unlikely that rDNA polymorphisms would be detected by this procedure.…”