Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities.

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.

The effect of vaginal microbial communities on colonization by Staphylococcus aureus with the gene for toxic shock syndrome toxin 1 (TSST-1): a case-control study.

Menstrual toxic shock syndrome is associated with vaginal colonization by Staphylococcus aureus strains that encode toxic shock syndrome toxin 1 (tst+). Interestingly, a small proportion of women are colonized by S. aureus tst+ but do not have symptoms of toxic shock syndrome. Here we sought to determine if differences in the species composition of vaginal bacterial communities reflect a differential risk of colonization by S. aureus capable of producing toxic shock syndrome toxin 1 (TSST-1). The composition of vaginal communities of women that were or were not colonized with S. aureus tst+ were compared based on terminal restriction fragment length polymorphism (T-RFLP) profiles and sequences of cloned 16S rRNA genes. There were no detectable differences in community composition or species rank abundance between communities of women vaginally colonized with S. aureus tst+ as compared to those that were not. Phylogenetic analysis of cloned 16S rRNA gene sequences showed that the predominant members of communities of women colonized with S. aureus tst+ were indistinguishable from those of other healthy women. The data suggest that the numerically dominant members of vaginal communities do not preclude colonization and proliferation of S. aureus tst+ within indigenous microbial communities of the vagina.

Understanding vaginal microbiome complexity from an ecological perspective.

The various microbiota normally associated with the human body have an important influence on human development, physiology, immunity, and nutrition. This is certainly true for the vagina wherein communities of mutualistic bacteria constitute the first line of defense for the host by excluding invasive, nonindigenous organisms that may cause disease. In recent years much has been learned about the bacterial species composition of these communities and how they differ between individuals of different ages and ethnicities. A deeper understanding of their origins and the interrelationships of constituent species is needed to understand how and why they change over time or in response to changes in the host environment. Moreover, there are few unifying theories to explain the ecological dynamics of vaginal ecosystems as they respond to disturbances caused by menses and human activities such as intercourse, douching, and other habits and practices. This fundamental knowledge is needed to diagnose and assess risk to disease. Here we summarize what is known about the species composition, structure, and function of bacterial communities in the human vagina and the applicability of ecological models of community structure and function to understanding the dynamics of this and other ecosystems that comprise the human microbiome.

The vaginal bacterial communities of Japanese women resemble those of women in other racial groups.

To determine whether different racial groups shared common types of vaginal microbiota, we characterized the composition and structure of vaginal bacterial communities in asymptomatic and apparently healthy Japanese women in Tokyo, Japan, and compared them with those of White and Black women from North America. The composition of vaginal communities was compared based on community profiles of terminal restriction fragments of 16S rRNA genes and phylogenetic analysis of cloned 16S rRNA gene sequences of the numerically dominant bacterial populations. The types of vaginal communities found in Japanese women were similar to those of Black and White women. As with White and Black women, most vaginal communities were dominated by lactobacilli, and only four species of Lactobacillus (Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri) were commonly found. Communities dominated by multiple species of lactobacilli were common in Japanese and White women, but rare in Black women. The incidence, in Japanese women, of vaginal communities with several non-Lactobacillus species at moderately high frequencies was intermediate between Black women and White women. The limited number of community types found among women in different ethnic groups suggests that host genetic factors, including the innate and adaptive immune systems, may be more important in determining the species composition of vaginal bacterial communities than are cultural and behavioral differences.