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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. RESULTS: A total of 150 vaginal swab samples from healthy women (115 pregnant and 35 non-pregnant) were categorized on the basis of Gram stain of direct smear as grade I (n = 112), grade II (n = 26), grade III (n = 9) or grade IV (n = 3). The composition of the vaginal microbial community of eight of these vaginal swabs (three grade I, two grade II and three grade III), all from non-pregnant women, were studied by culture and by cloning of the 16S rRNA genes obtained after direct amplification. Forty-six cultured isolates were identified by tDNA-PCR, 854 cloned 16S rRNA gene fragments were analysed of which 156 by sequencing, yielding a total of 38 species, including 9 presumptively novel species with at least five species that have not been isolated previously from vaginal samples. Interestingly, cloning revealed that Atopobium vaginae was abundant in four out of the five non-grade I specimens. Finally, species specific PCR for A. vaginae and Gardnerella vaginalis pointed to a statistically significant co-occurrence of both species in the bacterial vaginosis samples. CONCLUSIONS: Although historically the literature regarding bacterial vaginosis has largely focused on G. vaginalis in particular, several findings of this study--like the abundance of A. vaginae in disturbed vaginal microflora and the presence of several novel species--indicate that much is to be learned about the composition of the vaginal microflora and its relation to the etiology of BV.

SUBMITTER: Verhelst R

PROVIDER: S-EPMC419343 | biostudies-literature | 2004 Apr

REPOSITORIES: biostudies-literature

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

Verhelst Rita R Verstraelen Hans H Claeys Geert G Verschraegen Gerda G Delanghe Joris J Van Simaey Leen L De Ganck Catharine C Temmerman Marleen M Vaneechoutte Mario M

BMC microbiology 20040421

<h4>Background</h4>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. ...[more]

PMID: 15102329

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Project description:Bacterial vaginosis (BV) is a dysbiosis of the vaginal flora characterized by a shift from a Lactobacillus-dominant environment to a polymicrobial mixture including Actinobacteria and gram-negative bacilli. BV is a common vaginal condition in women and is associated with increased risk of sexually transmitted infection and adverse pregnancy outcomes such as preterm birth. Gardnerella vaginalis is one of the most frequently isolated bacterial species in BV. However, there has been much debate in the literature concerning the contribution of G. vaginalis to the etiology of BV, since it is also present in a significant proportion of healthy women. Here we present a new murine vaginal infection model with a clinical isolate of G. vaginalis. Our data demonstrate that this model displays key features used clinically to diagnose BV, including the presence of sialidase activity and exfoliated epithelial cells with adherent bacteria (reminiscent of clue cells). G. vaginalis was capable of ascending uterine infection, which correlated with the degree of vaginal infection and level of vaginal sialidase activity. The host response to G. vaginalis infection was characterized by robust vaginal epithelial cell exfoliation in the absence of histological inflammation. Our analyses of clinical specimens from women with BV revealed a measureable epithelial exfoliation response compared to women with normal flora, a phenotype that, to our knowledge, is measured here for the first time. The results of this study demonstrate that G. vaginalis is sufficient to cause BV phenotypes and suggest that this organism may contribute to BV etiology and associated complications. This is the first time vaginal infection by a BV associated bacterium in an animal has been shown to parallel the human disease with regard to clinical diagnostic features. Future studies with this model should facilitate investigation of important questions regarding BV etiology, pathogenesis and associated complications.

Project description:Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a change in vaginal flora away from predominantly Lactobacillus species. The cause of BV is unknown, but the condition has been implicated in diverse medical outcomes. The bacterium Atopobium vaginae has been recognized only recently. It is not readily identified by commercial diagnostic kits. Its clinical significance is unknown but it has recently been isolated from a tuboovarian abcess.Nucleotide sequencing of PCR amplified 16S rRNA gene segments, that were separated into bands within lanes on polyacrylamide gels by denaturing gradient gel electrophoresis (DGGE), was used to examine bacterial vaginal flora in 46 patients clinically described as having normal (Lactobacillus spp. predominant; Nugent score < or = 3) and abnormal flora (Nugent score > or = 4). These women ranged in age from 14 to 48 and 82% were African American.The DGGE banding patterns of normal and BV-positive patients were recognizably distinct. Those of normal patients contained 1 to 4 bands that were focused in the centre region of the gel lane, while those of BV positive patients contained bands that were not all focused in the center region of the gel lane. More detailed analysis of patterns revealed that bands identified as Atopobium vaginae were present in a majority (12/22) of BV positive patients, while corresponding bands were rare (2/24) in normal patients. (P < 0.001) Two A. vaginae isolates were cultivated from two patients whose DGGE analyses indicated the presence of this organism. Two A. vaginae 16S rRNA gene sequences were identified among the clinical isolates. The same two sequences were obtained from DGGE bands of the corresponding vaginal flora. The sequences differed by one nucleotide over the short (approximately 300 bp) segment used for DGGE analysis and migrated to slightly different points in denaturing gradient gels. Both isolates were strict anaerobes and highly metronidazole resistant.The results suggest that A. vaginae may be an important component of the complex bacterial ecology that constitutes abnormal vaginal flora. This organism could play a role in treatment failure if further studies confirm it is consistently metronidozole resistant.

Dataset Information

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

Publications

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

Similar Datasets

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