component analysis (PCA) plot is a tool for identifying patterns in data by highlighting their similarities and differences. The PCA plot is generated by a statistical program that uses orthogonal (perpendicular) transformation to convert the observed data (which fall within the area shown) into an ordinate plot. The first principal component (PC1) accounts for the largest variation in the data, which is attributed to factor 1, and the second principal component (PC2) accounts for the second-largest variation in the data, which is attributed to factor 2. Shown is a PCA plot (A) and the corresponding radial phylogenetic tree (a phylogenetic relationship tree that does not make assumptions about ancestry, in contrast to the trees shown in Figure 5-2) (B) based on 16S rRNA sequence profiles depicting the relationship among the vaginal microbiotas of humans and nonhuman primates. Vaginal samples are color-coded to match their host species. Data from Yildirim S, Yeoman CJ, Janga SC, Thomas SM, Ho M, Leigh SR; Primate Microbiome Consortium, White BA, Wilson BA, Stumpf RM. 2014. ISME J 8:2431–2444.
In this way, 16S rRNA gene-based profiling can be used to characterize the content of complex microbial populations, such as those found in the colon or vagina, and enables researchers to compare the relative compositions of microbial populations within an individual and among multiple individuals. Microbiota profiles from individuals who are healthy should resemble one another (i.e., be more closely related in terms of composition), whereas profiles from individuals suffering from a particular type of infection or disease should likewise resemble each other, but be different from the profiles of healthy individuals. Consequently, comparative analysis can serve to distinguish microbiota profiles among individuals and provide some diagnostic assessment of an individual’s health status.
Cultivation-based studies took many years to complete a census for the human vaginal tract and came to the conclusion that Gram-positive Lactobacillus species were the dominant bacteria (present at over 90%) in most healthy women. It was believed that this Lactobacillus-dominant composition only changed during overt infection or symptomatic disease. The cultivation-independent studies using 16S rRNA gene-based microbial content analysis of the human vaginal tract showed no big surprises (Figure 5-2). Lactobacillus species were still the numerically dominant bacteria present in the population (at 80–90%), with the minor bacterial components being mostly other Gram-positive Firmicutes, such as Staphylococcus and Streptococcus species, Gardnerella vaginalis, and a few Gram-negative Proteobacteria, such as E. coli and Pseudomonas species. However, the phylogenetic DNA-based analysis did reveal a number of species-level differences in Lactobacilli content from the outcomes of cultivation-based studies, as well as considerable person-to-person, species-level variation in the non-Lactobacilli bacteria present. One finding that challenged some previous assumptions about what constitutes vaginal health was the finding that G. vaginalis, which previously was thought to be only associated with overgrowth during symptomatic bacterial vaginosis (BV), is actually a common constituent of the normal microbiota of asymptomatic, apparently healthy women.
One question that a researcher might ask in developing an animal model of disease that manifests with a shift in the normal microbiota composition is if animals, such as laboratory rodents that are often used as models for human disease, have a microbiota that is similar to that of humans. And, if not, would another animal such as a nonhuman primate serve as a more appropriate animal model? To address this issue, a researcher might compare the 16S rRNA gene profiles of the microbiota at particular body sites in potential animal models. An example that may help you appreciate this decision process is provided by a study to determine whether baboons could serve as an appropriate animal model for studying the microbiota of the human vaginal tract.
Why baboons? The reason for choosing baboons is that anatomically, hormonally, and reproductively, the baboon and human uteruses and vaginas resemble each other closely, and both are clearly different from that of rodents, such that mice or rats might be considered to serve as poor models for human vaginal disease. Baboons have been widely used as an animal model in gynecological and reproductive studies of the female genital tract. Topics of these studies have ranged from endometriosis to the efficacy of birth control methods to menopause. Baboons are easier to house and more accessible to researchers than chimpanzees, another candidate for an animal model. Since the captive animals have the same diet and the same environment, this study also provided a chance to ask whether the considerable individual-to-individual differences seen in the human subjects were due to differences in environment, genetics, or other factors. However, in contrast to the microbiota of the human and rodent vaginal tracts, no culture-based analysis of the baboon microbiota had ever been done previously.
The 16S rRNA gene analysis of the baboon microbiota yielded a totally unexpected finding: the microbiota of the baboon vaginal tract was quite different from that of the human vaginal tract. Whereas Lactobacillus species dominate the human vaginal tract with minor components of other bacteria, species of Gram-positive bacteria (Firmicutes) other than Lactobacillus dominated the microbiota of the baboon vagina: mostly Clostridia, but also Gram-negative Fusobacteria and members of the phylum Bacteroidetes. This difference is illustrated in the clustering analysis shown in Figure 5-4. The difference between the composition of the human and baboon microbiota is striking, particularly in view of the fact that baboons seem to be so closely related to humans. Most of the bacterial species found in the baboon vaginal tract have representatives related to those isolated from humans, but largely those isolated from the human mouth and colon rather than the vaginal tract. Even within these groups, however, the human sequences clustered independently from the baboon sequences, indicating that, in many cases, the baboon sequences were not closely related to the human sequences and might represent new genera.
Figure 5-4. A radial phylogenetic tree showing the 16S rRNA gene sequence profiles of human and baboon vaginal microbes clustering to six major taxonomic groups. This radial tree shows relationships based on near-complete 16S rRNA gene sequences from the vaginal microbial community of captive baboons (red) and published human sequences (blue). Results show that the vaginal microbiota were clustered to six major taxonomic groups. Although Gram-positive bacteria dominate the vaginal microbiota of baboons, as they do in humans, there are major differences between the vaginal microbiotas of humans and baboons. Unlike humans, Lactobacillus species did not dominate the baboon microbiota. Even within the same taxa of bacteria, the baboon sequences clustered separately from the human sequences. Reprinted from Rivera AJ, Frank JA, Stumpf R, Salyers AA, Wilson BA, Olsen GJ, Leigh S. 2011. Am J Primatol 73:119–129, with permission.
One question that immediately comes to mind considering these results is if the vaginal microbiotas of other nonhuman primates besides baboons are also different from that of humans. Indeed, a comparative 16S rRNA gene-based analysis of vaginal bacteria clearly demonstrated host species-specific vaginal microbial communities among humans and nonhuman primates (see Figure 5-3). Humans were distinct from other primates not only in microbial composition and diversity, but also because only humans possessed Lactobacillus-dominant vaginal microbiota. The reason for this interesting difference is still under investigation.
While phylogenetic relationship analysis based on 16S rRNA gene sequences has some clear advantages and can provide valuable insights regarding the microbiota composition, there are important limitations. In practice, given the complexity and diversity of the populations found in most parts of the body, a 16S rRNA gene analysis provides at best a representation of the most abundant genera and species in a particular site. This information, nevertheless, is extremely valuable because it narrows down the possible groups of bacteria in the population and can guide cultivation efforts. For example, the possible presence of anaerobic bacteria such as Prevotella species means that anaerobic conditions should be included in any attempt to cultivate members of the dominant groups.
A more serious limitation
