antibiotic resistance and virulence genes in the sense that they are present in high numbers in an area in which other bacteria are transiently present for 24–48 hours, which is more than enough time for DNA transfers to occur. Only recently has it been possible to test this hypothesis by using molecular sequencing methods to follow the host-to-host transmission and movement of particular genes through and within the human colon. In fact, evidence is mounting that gene transfers via conjugative elements like plasmids and conjugative transposons occur frequently in the colon. These transfers occur between different species and genera, including between colonic bacteria and bacteria from different sites.
The reservoir hypothesis has loomed large in the debate over possible adverse consequences of the overuse of antibiotics, particularly on the farm. Antibiotic-resistant bacteria arise on the farm due to selection by the use of antibiotics as feed additives or to prevent infections in crowded populations of animals. The problem is that these antibiotic-resistant bacteria are already moving through the food supply and into the human intestinal tract, where the resistance genes could be transferred to bacteria permanently or temporarily residing in the human colon. These concerns regarding the increased spread of antibiotic resistance are compounded by the recent realization of the importance of a normal microbiota in maintaining health and by the fact that antibiotic treatment leads to loss of not just the disease-causing pathogen, but also the beneficial bacteria that maintain a healthy microbiota. Perturbation of the normal healthy microbiota (dysbiosis) has been correlated with inflammatory and autoimmune diseases, such as inflammatory bowel disease (IBD) and Crohn’s disease, and with increased susceptibility to other types of infections by opportunistic pathogens such as C. difficile.
Microbiota of the Vaginal Tract
The microbiota of the female vaginal tract has already been introduced in the example about the application of DNA-based analysis of complex microbial populations, but that description did not explore the special features of the site that presumably explain the composition of the microbiota of the vagina. The vagina is a complex site. The vaginal tract experiences considerable periodic hormonal changes associated with the menstrual cycle. Although mucin secretions are constantly bathing the vaginal mucosa, the flow of fluids seen is not nearly as fast as that seen in the intestinal tract, except during menstruation. Most of the time, the bacteria are loosely or strongly associated with the vaginal mucosa.
The microbiota of the vagina of most healthy humans consists mainly of Gram-positive Lactobacillus species. These lactobacilli are fermentative bacteria that produce mainly lactic acid and probably contribute to the normally low pH (less than 5) of the vaginal tract, which generally acts as a powerful protective barrier against colonization by many disease-causing bacteria. Unfortunately, many other pathogenic bacteria can survive and multiply at pH 5. Another possible contribution of lactobacilli to protecting the vaginal tract against disease-causing bacteria is that some of them produce hydrogen peroxide, which is toxic to many microbes, not just bacteria. There is no question that women who take antibiotics that kill or inhibit the growth of lactobacilli often develop yeast infections (vaginitis) or bacterial dysbiosis (vaginosis). The availability of new molecular techniques has now allowed in-depth analysis of the vaginal microbiota and interrogation of their roles in vaginal health and reproduction.
BV was once considered to be a minor disease, characterized by slight discomfort, mild inflammation, a fishy odor, and an unhealthy vaginal microbiota that lacked lactobacilli. More recently, BV has been shown to be associated with a number of disorders, including increased risk of premature labor and birth, although the mechanism of this connection is still unclear, as well as increased susceptibility to infection with sexually transmitted pathogens, such as HIV, Neisseria gonorrhoeae, or Chlamydia trachomatis.
In contrast to diseases caused by a single pathogen, BV arises from a shift in the microbiota from a predominantly lactobacilli population to a predominantly Gram-negative one, principally G. vaginalis. In fact, a woman with a significant concentration of Gram-negative bacteria like G. vaginalis had previously been assumed by physicians to be exhibiting disease. As mentioned before, DNA-based analyses of a large number of apparently healthy women revealed a surprising finding: many of them carried high concentrations of G. vaginalis (see Figure 5-18). And, interestingly, while about 70% of the healthy women had predominantly lactobacilli, about 30% had very few or no lactobacilli.
Figure 5-18. Temporal and interindividual dynamics of vaginal bacterial communities. Shown are the 16S rRNA gene-based bacterial community composition profiles of eight different healthy women sampled twice weekly over the course of 16 weeks. Colors indicate the relative abundance of each taxonomical group (phylotype) present in each sample. Red bars along the bottom of the plot indicate dates of menstruation. Reprinted from Gajer P, Brotman RM, Bai G, Sakamoto J, Schütte UM, Zhong X, Koenig SS, Fu L, Ma ZS, Zhou X, Abdo Z, Forney LJ, Ravel J. 2012. Sci Transl Med 4(132):132ra52, with permission.
More detailed metagenomic and multi-omic analyses have already demonstrated not just considerable person-to-person variation but variations within a person over the course of the menstrual cycle (Figure 5-18), as well as among different sites in the vaginal tract and with other factors such as hormonal status, sexual activity, age, and pregnancy. Clearly, the microbiota of the vaginal tract is turning out to have as complex a population as that of the other body sites. And, once again, an in-depth discussion of these fascinating topics is beyond the scope of this textbook, so we have included a few suggested readings at the end of this chapter.
The Other Microbiota: The Forgotten Eukaryotes
The content of this chapter has so far focused largely on bacteria (with a brief foray into the archaea of the gut), so it is appropriate to end with a brief description of microbes that have been largely ignored in most studies, but which nevertheless have an impact on the bacterial communities and our immune system: the eukaryotic microbes and viruses. This is particularly true of recent examinations of the microbiotas of the human colon, mouth, and vagina, but is also relevant to the microbiotas of other areas.
From recent metagenomic studies, fungi and bacteriophage play a significant role in the human health, but the DNA-based analyses of these components of the oral, vaginal, and colonic microbiota (the mycobiome and virome, respectively) have been relatively understudied. There are several challenges that have hampered progress in this area. Fungi are normally found as minor components of the healthy microbiota (overgrowth is usually associated with disease). Many fungi are difficult to cultivate or are uncultivable. It is also often difficult to lyse and extract genomic DNA from many fungi. To capture the mycobiome, recent metagenomic sequencing efforts have used the internal transcribed spacer (ITS) region of the rRNA genes as a basis for comparative sequencing analysis, analogous to the 16S rRNA sequencing analysis in bacteria.
Like the mycobiome, the virome (viruses and phage) has also been understudied. Again, there are a number of key challenges that need to be overcome for this field to move forward. Despite the fact that viruses are relatively abundant in numbers, they only make up a small portion of the total DNA or RNA in a sample. Virions are difficult to isolate, purify, and study, particularly in terms of finding suitable host cells for viral propagation. From those sequencing studies that have been undertaken, it appears that the bulk of the sequences are completely novel, with few, if any, homologs in the databases. This makes sequence annotation and comparative genomic analyses difficult to perform and, more significantly, it makes viral classification, evolution, and characterization within complex mixtures a daunting task. Nevertheless, many valiant efforts are currently underway.
In developing countries especially, there is another important eukaryotic component of the colonic microbiota that consists of protozoa and helminths (e.g., tapeworms). We tend to think of protozoa and helminths as pathogens, but this picture may not be entirely correct. Many people
