Characterization of Microbial Communities Found in the Human Vagina by Analysis of Terminal Restriction Fragment Length Polymorphisms of 16S rRNA Genes

Abstract

To define and monitor the structure of microbial communities found in the human vagina, a cultivationindependent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for the isolation of genomic DNA and an optimal strategy for T-RFLP analyses. The various genera in the model community could best be resolved by digesting amplicons made using bacterial primers 8f and 926r with HaeIII; fewer strains could be resolved using other primer-enzyme combinations, and no combination successfully distinguished certain species of the same genus. To demonstrate the utility of the approach, samples from five women that had been collected over a 2-month period were analyzed. Differences and similarities among the vaginal microbial communities of the women were readily apparent. The T-RFLP data suggest that the communities of three women were dominated by a single phylotype, most likely species of Lactobacillus. In contrast, the communities of two other women included numerically abundant populations that differed from Lactobacillus strains whose 16S rRNA genes had been previously determined. The T-RFLP profiles of samples from all the women were largely invariant over time, indicating that the kinds and abundances of the numerically dominant populations were relatively stable throughout two menstrual cycles. These findings show that T-RFLP of 16S rRNA genes can be used to compare vaginal microbial communities and gain information about the numerically dominant populations that are present.

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

“…This result suggests that the presence of lactobacilli may not exclude potential pathogens from the vagina; likewise, the presence of G. vaginalis is not strictly related to clinically manifested disturbances. In healthy vaginas, G. vaginalis could represent a "sentinel species," a term used to refer to an indigenous species that is particularly sensitive to changes in biological, physical, or chemical characteristics of the environment and responds through an increase in population size (12). The identification of potential sentinel species can help to predict whether there is an increased risk of an individual contracting an infection due to shifts in the composition of the microbial communities or changes in the abundance of specific populations.…”

“…1. According to the data reported in previous studies (9,12,24,41), a low level of bacterial diversity appeared to characterize the communities of healthy vaginas. Lactobacilli represented the dominant inhabitants of the normal vaginal tract, as all the women in the NI group had at least one sequence homologous to a sequence of a Lactobacillus species.…”

“…In recent years, molecular techniques based on the analysis of rRNA gene sequences have been developed, providing powerful tools to reveal the phylogenetic diversity of the microorganisms found within complex ecosystems and understand community dynamics (12,24,41). PCR-denaturing gradient gel electrophoresis (PCR-DGGE) represents a rapid and reliable technique that has been used successfully to identify the bacterial compositions of different ecological niches, including the vaginal ecosystem (9,10,13,14,35).…”

Assessment of vaginal microbiota in Brazilian women with and without bacterial vaginosis and comparison with Nugent score

“…In contrast, culture-independent molecular methods have been proven to be efficient tools for phylogenetic diversity studies and understanding the dynamics of these communities [15][16][17][18]. Molecular assays based on genetic fingerprinting and 16S rDNA sequence analysis have enabled bacterial diversity investigation in different microbial communities [17,19].…”

Uso da biblioteca genômica RNAr 16S como ferramenta para o estudo da microbiota fecal humana

“…A definição exata da microbiota intestinal ainda é muito difícil, uma vez que os procedimentos de coleta de tecidos são invasivos e a informação obtida a partir da análise da microbiota das fezes não fornece resultados precisos sobre a microbiota de mucosa intestinal. Porém alguns estudos demonstraram que a microbiota fecal reflete a microbiota do cólon intestinal (TANNOCK, 2005 Apesar de serem considerados iniciadores universais, os dados descritos na literatura indicam o uso destes iniciadores em comunidades específicas, como por exemplo, o iniciador universal 8F tem sido utilizado para avaliar a microbiota vaginal humana (COOLEN et al, 2005), além da microbiota intestinal humana (LIN et al, 2013;DURBÁN et al, 2011;SHEN et al, 2010;CARROLL et al, 2012).…”