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Composition and function of the human-associated microbiota

Elisabeth M Bik
DOI: http://dx.doi.org/10.1111/j.1753-4887.2009.00237.x S164-S171 First published online: 1 November 2009

Abstract

The human body is an ecosystem harboring complex site-specific microbial communities. The majority of these human-associated microbes are found in the intestinal tract, where they play important roles in energy uptake, vitamin synthesis, and epithelial and immunity development. Recent molecular studies have characterized the human-associated microbiotas in more detail than conventional culture-dependent techniques, showing a large degree of microbial diversity and differences between anatomical sites and individuals. Investigating the composition and function of microbial symbionts will facilitate better understanding of their roles in human health and disease.

  • human-associated microbiota
  • intestinal microbiota
  • ribosomal genes

INTRODUCTION

The human body is home to complex communities of microorganisms. Their total number is estimated to be 1014; 10 times the number of human cells per individual.1 These microbial communities are found on our skin, in the mouth, nose, ears, vagina, and in the intestinal tract. Similar to environmental sources in which microbes are found, such as sea water and soil, the human body could be considered an ecosystem consisting of different niches, or a metacommunity consisting of many local communities. Each anatomical site has its own physiochemical characteristics, and each location is occupied with a specialized set of microbes.2 The majority of the human-associated microbes and the largest diversity are found in the intestinal tract, where microbial abundance increases from the stomach to the colon, with the highest number of microbes found in stools (1011 per mL).1 This complex ecosystem consists of bacteria, archaea, yeasts, and other eukaryotes.14

FUNCTIONS OF THE GUT MICROBIOTA

Over the past decades, the traditional view that all microorganisms cause disease has been replaced by the insight that most microbe-host interactions are non-pathogenic, commensal (with the microbes benefiting, while the host is unharmed), or even mutualistic (in which both partners benefit).2 Although not much is known yet about the possible beneficial roles of microbes in human-associated communities outside the intestinal tract, it becomes increasingly clear that the gut microbiota benefits the host in many different ways. Our intestinal symbionts play important roles in nutrient digestion and synthesis, energy metabolism, vitamin synthesis, epithelial development, and immune responses.5,6 In return, the intestinal microorganisms are provided with steady growth conditions and a constant stream of nutrients.1

The presence of an intestinal microbiota is not essential for survival of the host, but germ-free (GF) mice (born under sterile conditions and raised in a sterile environment) require 30% more energy in their diet,7 showing the significance of the indigenous microbiota in salvaging energy from food. This energy utilization by the gut microbiota works on different levels. First, specialized intestinal bacteria synthesize enzymes that human cells lack for the digestion of plant polysaccharides. Mammals can hydrolyze certain disaccharides and absorb monosaccharides, such as glucose and galactose, but they cannot break down most polysaccharides, such as those present in plant cell walls (fiber). The intestinal microbial community, however, is well equipped to degrade these biomolecules.8,9 The genome of the recently sequenced gut symbiont Bacteroides thetaiotaomicron encodes for 400 enzymes encoding the transport, binding, and digestion of complex sugars,10 including a well-understood starch utilization system.8,11 This microbial fermentation generates butyrate and other short-chain fatty acids that the host can use as energy sources and that help maintain the integrity of the intestinal epithelium.512

In addition to its direct role in increasing caloric uptake from diet by the fermentation of polysaccharides, the presence of a gut microbiota regulates fat storage in the host. GF mice were found to have less body fat than conventionally colonized mice, even though their chow consumption was higher.13 The presence of gut microbes promoted the absorption of monosaccharides from the gut lumen and the induction of hepatic lipogenesis, while suppressing the induction of fasting-induced adipocyte factor in the intestinal epithelium.13

The significance of the intestinal microbiota in vitamin synthesis has been recognized for many years. GF animals need to be supplemented with vitamin K and certain B vitamins.7 In conventionally colonized animals, these vitamins are produced by several intestinal genera, including Bacteroides and Eubacterium.14

In addition to the beneficial effects of the gut microbiota due to its metabolic activity, host-microbe interactions are essential for the host's defense against pathogenic infections, gut development, and epithelial homeostasis. First, as shown by the high susceptibility of GF animals to infections, the intestinal symbionts provide an important barrier to colonization by potential pathogens, called “colonization resistance,” by competing for the same nutrients and attachment sites as intruding microorganisms.216 In addition, the presence of a microbiota stimulates the development of the mucosal immune system. GF animals have hypoplastic Peyer's patches, reduced number of plasma cells, abnormal spleen and lymph nodes, and underdeveloped intestinal villus capillaries. Colonization with a single Bacteroides species can restore these deficiencies.1719

Both pathogenic and commensal bacteria secrete certain compounds with conserved patterns, such as lipopolysaccharide and lipoteicholic acid, that are recognized by Toll-like receptors (TLRs) of the host's innate immune system, triggering inflammatory and immune defense responses. It is not yet understood how the host can distinguish between pathogenic and commensal bacteria, and it was commonly believed that commensals could somehow evade recognition by the host's immune system. Surprisingly, a recent study showed that commensal bacteria interact with the intestinal surface and trigger a low degree of TLR-signaling (“tickling”).20 Without this interaction, the intestinal surface is more sensitive to injury, and less capable of inducing repair of the damaged surface. This indicates that the host epithelium and its immune cells do not simply tolerate commensal bacteria but depend on them to maintain the architectural integrity.20

THE GREAT ANOMALY

Given the importance of the indigenous microbiota for host physiology, determining the microbial composition of the microbial consortia associated with our bodies might provide novel insights into their role in human health and disease. Traditionally, the composition of a complex microbial community has been determined by microscopy and culture. While conventional microscopy can only describe differences based on morphology, culturing allows the researcher to differentiate bacterial species and strains based on a myriad of biochemical and immunological tests, such as ability to ferment certain sugars, growth on certain media, and agglutination with antibodies. Unfortunately, not all microorganisms can be cultured. In 1985, Staley and Konopka concluded that the biodiversity of a complex microbial community observed by microscope was higher than that by culturing on agar plates.21 They named this phenomenon “The Great Anomaly,” a term still used today. Many microbial species cannot be cultured because we do not know or cannot reproduce their preferred growth conditions. Depending on the composition of a microbial community, the percentage of unculturable bacteria can range from 10 to 99%.21 Therefore, research on microbial diversity is increasingly making use of culture-independent, molecular techniques.22,23

MOLECULAR TOOLS TO DETERMINE MICROBIAL DIVERSITY

To characterize the members of a complex microbial community, most modern molecular methods rely on the gene encoding the structural RNA of the small ribosomal subunit. The ribosome functions in the translation of RNA into protein, by assembling amino acids into polypeptide chains. It consists of two subunits, both of which are conglomerates of proteins and structural ribosomal RNA molecules (rRNAs). The gene encoding the rRNA of the small ribosomal subunit, called 16S rDNA in prokaryotes and 18S rDNA in eukaryotes, has certain properties that make it extremely suitable for phylogenetic analysis.24 First, because of its essential function in protein synthesis, ribosomes and thus rRNA genes are found in all living organisms. In addition, the ribosomal genes consist of both conserved and variable regions. Some of these regions are so conserved that a single set of “universal” primers can be used to amplify portions of the 16S rDNA of almost all bacteria. Finally, the sequences of the variable regions within the 16S rDNA gene can be used to determine phylogenetic relationships between organisms. Ribosomal RNA sequences of closely related bacterial species (e.g., of Escherichia coli and Enterobacter cloacae) are more similar than those of more distant bacterial groups (e.g., Escherichia coli and Staphylococcus aureus). Woese et al.25 were the first to realize that ribosomal RNA sequences could be used as a phylogenetic tool to describe the evolutionary relationships among organisms without the need to culture them. Using 16S rRNA sequences, Woese et al.25 discovered that the prokaryotes could be divided into two distinct groups, eubacteria (now called Bacteria) and archaebacteria (Archaea). Olsen et al.24 applied the amplification of ribosomal sequences to analyze complex microbial mixtures by constructing recombinant clone libraries; this was soon followed by the first culture-independent identification of a previously unidentified pathogen by Relman et al.26

To analyze the 16S rRNA gene of a sample containing a single bacterial species, the DNA is extracted and (part of) the 16S rDNA amplified using broad-range primers, with subsequent direct sequencing of the amplified product and comparison to published sequences. To determine the microbial diversity in a mixed community, the amplified products will need to be analyzed by constructing a clone library and sequencing an appropriate number of individual clones,24 or by labeling and hybridizing them to a microarray with specific probes (Figure 1).27 Alternative molecular tools to determine the composition of a microbial community include fluorescent in situ hybridization and gel-based profiling techniques such as denaturing gradient gel electrophoresis.28,29

Figure 1

Molecular analysis of complex microbial consortia based on the 16S rRNA gene. First, DNA is extracted from a sample containing an unknown community of microbial species. The ribosomal DNA is amplified using broad-range primers based on conserved regions and spanning variable regions, generating a mixture of PCR fragments. Several methods are available to determine the richness and relative abundance of the microbial amplicons, two of which are shown here. On the left, PCR products are cloned and transformed into Escherichia coli, with subsequent sequencing of the inserts, comparison to published sequences, and phylogenetic analyses. The species richness of the original sample will determine how many clones need to be sequenced to reach sufficient coverage. Alternatively (shown on the right), PCR products can be labeled and hybridized onto a microarray containing ten thousands of oligonucleotide probes. The amount of label bound to a specific spot is a measure for the relative abundance of the corresponding sequence.

Although the rDNA-based methods cannot discriminate between live and dead organisms, these techniques have discovered many previously unseen microbial groups and have rapidly enhanced our knowledge of phylogenetic relationships between microbial taxa.22,30 Publicly available databases have shown a marked increase of ribosomal DNA sequences; over 600,000 16S rDNA sequences have been published to date (August 2008). Using 16S rDNA-based techniques, studies have determined microbial diversity in environments ranging from sea water,31 soil,32 hot water springs,33 shower curtains,34 and recently, the human body.2 At least 50 bacterial phyla have been found in total, half of which consist entirely of uncultivated bacteria.23,30 Despite this enormous biodiversity, the majority of the human-associated bacteria are members of only four phyla (the “big four”, Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria).2 Most human-associated microbial consortia have been analyzed by at least one study; some key findings are presented in Figure 2, and reviewed below.

Figure 2

Phylum distribution in human-associated microbial communities characterized by sequencing of 16S-rDNA clone libraries. The lower portion lists the numbers of subjects, sites per subject, clone numbers, and numbers of phylotypes for each of the studies. A phylotype is defined by a group of sequences that are almost identical to each other, e.g., displaying 98% or more sequence identity. The sequence identification cut-off for each study is listed between parentheses.

SKIN MICROBIOTA

The human skin is an important organ for protecting the body against the external environment. It is exposed to a huge range of variations in temperature, humidity, and light exposure. The skin harbors its own community of microorganisms. A first study of the human skin bacterial biota by Gao et al. found 182 different species. Most clones were assigned to the Actinobacteria, Firmicutes, and Proteobacteria phyla. The composition of the skin microbiota was found to be similar between samples from the left and right forearm of most subjects, but highly variable among different timepoints and different individuals.35

ORAL MICROBIOTA

Studies of the microbial communities within the human mouth could provide more insight into the role of the indigenous microbiota in oral health and disease. Half of the adults in the United States have some form of periodontal disease, which, in its turn, can lead to systemic diseases such as bacterial endocarditis and preterm labor, signifying the important role of a seemingly local disease.36 In a molecular study of the oral microbiota from five human subjects without signs of periodontal disease, 141 bacterial phylotypes (“molecular species”) were found.37 The composition of the oral microbiota varied per site within each individual, as well as between individuals. The most dominant genera found in this study were Streptococcus, Gemella, Granulicatella, and Veillonella.37

MICROBIOTA OF THE ESOPHAGUS AND STOMACH

A 16S rDNA-based study of the bacteria associated with the human esophagus in four healthy individuals showed the presence of almost 100 different bacterial species. The composition of the esophageal microbiota was similar to that found in the upstream oral cavity, with Streptococcus, Prevotella, and Veillonella as the most prevalent groups.38

Downstream of the esophagus, the stomach with its harsh acid environment was traditionally believed to harbor few bacteria. The role of Helicobacter pylori in the development of human gastric disease is well studied, but little was known about the presence of other bacterial groups in the stomach. When we analyzed bacterial 16S rDNA sequences from the gastric biopsies of 23 subjects, we were surprised to identify a diverse community of 128 phylotypes, including H. pylori.39 The majority of the sequences were assigned to the Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria phyla. The presence of H. pylori did not seem to affect the relative abundance of the other members of the gastric community. Statistical analysis showed a large degree of intersubject variability. Furthermore, the gastric bacterial community was significantly different from the communities in the human mouth and esophagus, suggesting that the human stomach harbors a distinct microbiota.39

MICROBIOTA OF THE LARGE INTESTINE

In a large study on the microbiota in the human large intestine, Eckburg et al.40 analyzed multiple colonic sites and feces of three healthy individuals. Among 11,831 bacterial 16S rDNA sequences, 395 phylotypes were identified. In contrast, only a single phylotype, Methanobrevibacter smithii was observed among the 1524 sequences amplified with archaeal primers. Most sequences were assigned to the Firmicutes and Bacteroidetes phyla; minor phyla were Proteobacteria, Actinobacteria, Fusobacteria, and Verrucomicrobia. Intersubject differences were greater than the between-site differences within an individual.

By combining 16S rDNA sequences from this and other studies on the human gut microbiota in silico, on a total of 50 subjects, it was concluded that the human gastrointestinal tract harbors at least 1200 distinct microorganisms.41 Richness indices on the combined dataset estimated the total number of phylotypes, if sequencing efforts would be continued, to be between 2500 and 3000. That suggests a complete coverage of the microbial diversity of the human intestinal microbiota is still far away.

DEVELOPMENT OF THE INFANT INTESTINAL MICROBIOTA

It has long been assumed that each ecosystem is inhabited by a stable climax community that is best suited for the conditions found in that niche. However, studies on the human intestinal microbiota have shown a great level of interindividual differences in gut microbiotas among healthy individuals with a standard Western diet.40,41 Dethlefsen et al.29 have argued that both deterministic (diet, host genotype) as well as stochastic (random) effects might shape the composition of the intestinal microbial communities of an individual. To better understand how the human gut microbiota is assembled, we collected stool samples from 14 newborns, including a set of fraternal twins, in their first year of life. In addition, samples of feces were obtained from both parents, siblings if present, as well as breast milk and vaginal swabs from the mothers. The combined set included over 400 samples. Using broad-range primers, the 16S rDNA was amplified, labeled, and hybridized on an oligonucleotide microarray with over 10,000 spots.27,42

In the first weeks after birth, the baby stool samples were characterized by a limited number of bacterial species and highly unstable microarray profiles, but the diversity and stability of the microbial communities increased with age. Occasionally in the first months, some dramatic shifts in the bacterial composition were observed, sometimes associated with a period of antibiotic use. In the first 6 months, the baby stool samples clustered mainly per baby, suggesting that each newborn gut is colonized in a different pattern. Despite the instability and the differences between the babies in this study, the microbial communities became progressively more similar to one another. At the one-year timepoint, the baby stool profiles converged to an adult-like profile, with Bacteroides and Firmicutes as the most abundant taxa.42

Some very early baby stool timepoints (days after birth) were found to cluster with profiles from stool, vaginal swabs, or breast milk of the mother. In all cases, the profiles quickly changed over the next days. This suggests that initially the newborn gut is colonized with bacteria to which a baby happens to be exposed such as bacteria from the mother, but that those are quickly replaced by other bacteria that might be a better fit to that individual's intestinal niche. Other than the mother/early baby timepoints mentioned above, no clustering within families was observed, with the exception of fraternal twins, which displayed almost identical stool patterns and concurrent shifts.42

For each baby stool sample, we also determined the nearest-neighbor sample by Pearson correlation. For most samples, the nearest-neighbor sample was another sample from the same baby. Using this measure, the most similar pair of babies by far was the set of fraternal twins, babies 13 and 14 (Figure 3). These results, and the fact that we could not find comparable similarity in the communities of babies to an older sibling, suggest that the environment rather than genetic relationships plays a large role in the colonization events that shape the infant gut.42

Figure 3

Similarity of stool microbiota between babies. For all baby stool samples, Pearson correlation was used to calculate the nearest-neighbor sample based on the relative bacterial abundance microarray profiles. Subsequently, for each baby, the percent of nearest-neighbor samples from each baby was computed. The gray scale indicates the percent of samples from baby Y (in columns) that were nearest neighbors of the samples from baby X (in rows). Each row adds up to 100%.

Adapted from Palmer et al.42

INTESTINAL MICROBIOTA AND DISEASE

The increasing knowledge on the composition and function of the human-associated microbiota will help us better understand its role in human health and disease. Not surprisingly, the delicate balance between the host and its symbionts is sometimes disturbed. New insights into the role of the indigenous microbiota in systemic diseases, such as inflammatory bowel disease, are beginning to emerge.16

Recent studies have also suggested a link between intestinal biota and obesity. The feces of a wild-type mouse has a higher energetic value than that of an obese mouse model, indicating that the obese mouse is capable of extracting more energy out of the diet.43 GF mice inoculated with the gut community of an obese mouse increased more in weight than GF mice inoculated with the biota of a lean, wild-type mouse.43 In addition, the intestinal microbiota of the obese mice had less Bacteroidetes and more Firmicutes,44 while a similar difference has been found between lean and obese humans.45

In contrast to the putative role of the gut microbiota in certain human diseases, specific intestinal bacteria have therapeutic or prophylactic effects against infections, and can be used as probiotics.46 Given the large interest of the public in new health products, the use of these probiotic strains is likely to increase.

CONCLUSION

Although over 50 bacterial phyla have been described,23 the human microbiota is dominated by only four of these (Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria).2 In contrast to the limited number of phyla found in the human host, the number of different bacterial species and strains found in or on the human body is extremely high. In addition, the composition of the indigenous microbiota varies between anatomical sites (Figure 1), while each individual harbors a unique set of different bacterial strains.41

Despite the current knowledge on the population structure of the human-associated microbial communities, many questions remain. It is still unclear why these microbial consortia are so complex, and why each individual is associated with a different set of bacterial strains. What is the importance of the rare bacterial species within these ecosystems? How stable are these communities over time, and how do they respond to disturbances such as antibiotic use or infection with a pathogen? What are the roles of other members of the microbiota, such as archaea and fungi? How do all these different microbial species communicate with each other and with their host?

Fortunately, advances in molecular techniques will facilitate the performance of large-scale microbial community surveys. Ribosomal RNA-based microarrays27 and metagenomic analysis47 have already provided more insight into complex microbial consortia, while pyrosequencing will bring down the cost of sequencing analysis, enabling researchers to carry out large-scale ribosomal sequence surveys with tens of thousands of reads per sample.48,49 These and other powerful molecular ecology tools will provide us with improved insights into the complex relationships between humans and their symbionts.

Acknowledgments

Declaration of interest.  The author has no relevant interests to declare.

REFERENCES

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