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Resolution and characterization of distinct cpn60-based subgroups of Gardnerella vaginalis in the vaginal microbiota.

ABSTRACT: Bacterial vaginosis (BV), characterized by a shift of the vaginal microbiota from a Lactobacillus-dominated community to a dense biofilm containing a complex mixture of organisms, is an important risk factor in poor reproductive health outcomes. The Nugent score, based on Gram stain, is used to diagnose BV and Gardnerella vaginalis abundance in the sample is one factor determining Nugent score. A high Nugent score is indicative of BV but does not always correspond to the presence of clinical symptoms. G. vaginalis is recognized as a heterogeneous group of organisms, which can also be part of the normal, healthy vaginal microbiome. In addition, asymptomatic BV and non-Gardnerella types of BV are being recognized. In an attempt to resolve the heterogeneous group of G. vaginalis, a phylogenetic tree of cpn60 universal target sequences from G. vaginalis isolates was constructed that indicates the existence of four subgroups of G. vaginalis. This subdivision, supported by whole genome similarity calculation of representative strains using JSpecies, demonstrates that these subgroups may represent different species. The cpn60 subgroupings did not correspond with the Piot biotyping scheme, but did show consistency with ARDRA genotyping and sialidase gene presence. Isolates from all four subgroups produced biofilm in vitro. We also investigated the distribution of G. vaginalis subgroups in vaginal samples from Kenyan women with Nugent scores consistent with BV, Intermediate and Normal microbiota (n?=?44). All subgroups of G. vaginalis were detected in these women, with a significant difference (z?=?-3.372, n?=?39, p?=?0.001) in frequency of G. vaginalis subgroup B between BV and Normal groups. Establishment of a quantifiable relationship between G. vaginalis subgroup distribution and clinical status could have significant diagnostic implications.

SUBMITTER: Paramel Jayaprakash T

PROVIDER: S-EPMC3416817 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Resolution and characterization of distinct cpn60-based subgroups of Gardnerella vaginalis in the vaginal microbiota.

Paramel Jayaprakash Teenus T Schellenberg John J JJ Hill Janet E JE

PloS one 20120810 8

Bacterial vaginosis (BV), characterized by a shift of the vaginal microbiota from a Lactobacillus-dominated community to a dense biofilm containing a complex mixture of organisms, is an important risk factor in poor reproductive health outcomes. The Nugent score, based on Gram stain, is used to diagnose BV and Gardnerella vaginalis abundance in the sample is one factor determining Nugent score. A high Nugent score is indicative of BV but does not always correspond to the presence of clinical sym ...[more]

PMID: 22900080

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Project description:Increased abundance of Gardnerella vaginalis and sialidase activity in vaginal fluid is associated with bacterial vaginosis (BV), a common but poorly understood clinical entity associated with poor reproductive health outcomes. Since most women are colonized with G. vaginalis, its status as a normal member of the vaginal microbiota or pathogen causing BV remains controversial, and numerous classification schemes have been described. Since 2005, sequencing of the chaperonin-60 universal target (cpn60 UT) has distinguished four subgroups in isolate collections, clone libraries and deep sequencing datasets. To clarify potential clinical and diagnostic significance of cpn60 subgroups, we undertook phenotypic and molecular characterization of 112 G. vaginalis isolates from three continents. A total of 36 subgroup A, 33 B, 35 C and 8 D isolates were identified through phylogenetic analysis of cpn60 sequences as corresponding to four "clades" identified in a recently published study, based on sequencing 473 genes across 17 isolates. cpn60 subgroups were compared with other previously described molecular methods for classification of Gardnerella subgroups, including amplified ribosomal DNA restriction analysis (ARDRA) and real-time PCR assays designed to quantify subgroups in vaginal samples. Although two ARDRA patterns were observed in isolates, each was observed in three cpn60 subgroups (A/B/D and B/C/D). Real-time PCR assays corroborated cpn60 subgroups overall, but 13 isolates from subgroups A, B and D were negative in all assays. A putative sialidase gene was detected in all subgroup B, C and D isolates, but only in a single subgroup A isolate. In contrast, sialidase activity was observed in all subgroup B isolates, 3 (9%) subgroup C isolates and no subgroup A or D isolates. These observations suggest distinct roles for G. vaginalis subgroups in BV pathogenesis. We conclude that cpn60 UT sequencing is a robust approach for defining G. vaginalis subgroups within the vaginal microbiome.

Project description:ObjectivesTo examine the influence of sexual activity on the composition and consistency of the vaginal microbiota over time, and distribution of Gardnerella vaginalis clades in young women.MethodsFifty-two participants from a university cohort were selected. Vaginal swabs were self-collected every 3-months for up to 12 months with 184 specimens analysed. The vaginal microbiota was characterised using Roche 454 V3/4 region 16S rRNA sequencing, and G.vaginalis clade typing by qPCR.ResultsA Lactobacillus crispatus dominated vaginal microbiota was associated with Caucasian ethnicity (adjusted relative risk ratio[ARRR] = 7.28, 95%CI:1.37,38.57,p = 0.020). An L.iners (ARRR = 17.51, 95%CI:2.18,140.33,p = 0.007) or G.vaginalis (ARRR = 14.03, 95%CI:1.22,160.69, p = 0.034) dominated microbiota was associated with engaging in penile-vaginal sex. Microbiota dominated by L.crispatus, L.iners or other lactobacilli exhibited greater longitudinal consistency of the bacterial communities present compared to ones dominated by heterogeneous non-lactobacilli (p<0.030); sexual activity did not influence consistency. Women who developed BV were more likely to have clade GV4 compared to those reporting no sex/practiced non-coital activities (OR = 11.82, 95%CI:1.87,74.82,p = 0.009). Specimens were more likely to contain multiple G.vaginalis clades rather than a single clade if women engaged in penile-vaginal sex (RRR = 9.55, 95%CI:1.33,68.38,p = 0.025) or were diagnosed with BV (RRR = 31.5, 95%CI:1.69,586.87,p = 0.021).ConclusionsSexual activity and ethnicity influenced the composition of the vaginal microbiota of these young, relatively sexually inexperienced women. Women had consistent vaginal microbiota over time if lactobacilli were the dominant spp. present. Penile-vaginal sex did not alter the consistency of microbial communities but increased G.vaginalis clade diversity in young women with and without BV, suggesting sexual transmission of commensal and potentially pathogenic clades.

Project description:BackgroundBacterial vaginosis (BV) is one of the leading causes of vaginal complaints among women of childbearing age. The role of Gardnerella vaginalis remains controversial due to its presence in healthy and BV-type vaginal microflora. The phenotypic and genotypic heterogeneity of G. vaginalis suggested the existence of strain variants linked with different health conditions. We sought to analyze prevalence and distribution of G. vaginalis subgroups (clades) in BV-positive (n = 29), partial BV (n = 27), and BV-negative (n = 53) vaginal samples from Lithuanian women.MethodsVaginal samples were characterized by Amsel criteria and the Nugent method. Bacterial signatures characteristic of BV and concomitant infections were identified by culture and PCR. Using singleplex PCR assays, G. vaginalis subgroups were identified in 109 noncultured vaginal specimens by targeting clade-specific genes. Isolated G. vaginalis clinical strains were subtyped and the presence of the sialidase coding gene was detected by PCR. Data analysis was performed using GraphPad Prism statistical software.ResultsG. vaginalis was found in 87% of women without BV. Clade 4 was most frequently detected (79.4%), followed by clade 1 (63.7%), clade 2 (42.2%), and clade 3 (15.7%). Multi-clade G. vaginalis communities showed a positive association with Nugent score (NS) ? 4 (OR 3.64; 95% CI 1.48-8.91; p = 0.005). Clade 1 and clade 2 were statistically significantly more common in samples with NS 7-10 (OR 4.69; 95% CI 1.38-15.88; p = 0.01 and OR 6.26; 95% CI 2.20-17.81; p ? 0.001, respectively). Clade 3 and clade 4 showed no association with high NS (OR 0.88; 95% CI 0.26-3.04; p = 1.00 and OR 1.31; 95% CI 0.39-4.41; p = 0.767, respectively). The gene coding for sialidase was detected in all isolates of clade 1 and clade 2, but not in clade 4 isolates.ConclusionsWe showed an association between the microbial state of vaginal microflora and specific subgroups of G. vaginalis, the distribution of which may determine the clinical manifestation of BV. The frequent detection of clade 4 in the BV-negative samples might be due its lack of the gene coding for sialidase.

Dataset Information

Resolution and characterization of distinct cpn60-based subgroups of Gardnerella vaginalis in the vaginal microbiota.

Publications

Resolution and characterization of distinct cpn60-based subgroups of Gardnerella vaginalis in the vaginal microbiota.

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