Humans

Community sequencing of total gut microbiota taken from obese and lean twins show substantial differences in their compositions. Total population sequences were analyzed to determine the levels of enzymes involved in carbohydrate, lipid, and amino acid metabolism. Obesity is associated with phylum-level differences in the microbiota, a significantly reduced bacterial diversity, and an increase in the population expression of enzymes which result in an increased efficiency of calorie harvest in the diets of the obese twins.

Type I diabetes is an autoimmune disease that is correlated with a multiplicity of predisposing factors, including an aberrant intestinal microbiota, a leaky intestinal mucosal barrier, and intrinsic differences in immune responsiveness. Various animal models for diabetes have shown a role for bacteria in the onset of the disease. Community DNA sequencing of intestinal flora comparing healthy and autoimmune children showed that autoimmune children had relatively unstable gut biomes with significantly decreased levels of species diversity, and the populations showed large scale replacement of Firmicutes species with Bacteroidetes species.

Human skin represents the most extensive organ of the human body, whose functions include protecting the body from pathogens, preventing loss of moisture, and participating in the regulation of body temperature. Considered as an ecosystem, the skin supports a range of microbial communities that live in distinct niches. Hair-covered scalp lies but a few inches from exposed neck, which in turn lies inches away from moist hairy underarms, but these niches are, at a microbial level, as distinct as a temperate forest would be compared with savanna and tropical rain forest. Studies characterizing the microbiota that inhabit these different niches are providing insights into the balance between skin health and disease.[23]

T cell differentiation to Th1, Th2, Th17 and Treg linages

Prevention of urogenital diseases in women depends on healthy vaginal microbiomes, but what is meant by "healthy" has not been understood. Community population studies using advanced sequencing methodologies (including pyrosequencing) are yielding insights into the range of microbial diversity in the human vagina. An unexpected finding was the prevalence of Prevotella species, which are known to positively affect the growth of Gardnerella vaginalis and Peptostreptococcus anaerobius, two species linked to bacterial vaginosis, by providing these disease-associated bacteria with key nutrients.

A proposal has been made to classify people by enterotype, based on the composition of the gut microbiome. By combining 22 newly sequenced fecal metagenomes of individuals from four countries with previously published data sets, three robust clusters were identified that are not nation or continent specific.[25][26]

The traditional view of the immune system is that it is a complex assembly of organs, tissues, cells and molecules that work together to eliminate pathogens. Modifications to this traditional view, that the immune system has evolved to control microbes, have come from the discovery that microbes coevolve with and exert control upon the immune system. It is known that germ-free animals possess an underdeveloped immune system. The biology of the T helper 17 cells (Th17) has generated interest due to their key role in inflammatory processes. Excessive amounts of the cell are thought to play a key role in autoimmune diseases such as multiple sclerosis, psoriasis, juvenile diabetes, rheumatoid arthritis, Crohn's disease, and autoimmune uveitis. It has been discovered that specific microbiota direct the differentiation of Th17 cells in the mucosa of the small intestine.

Immune system

The symbiotic relationship between animal host and microbiota has a significant impact on shaping the immune system. The immune system is able to recognize the types of bacteria that are harmful to the host and combats them, while allowing the helpful bacteria to carry out their functions. After an infant is born completely sterile, their gut is quickly populated by commensal bacteria that affect the immune response, resulting in future tolerance to that bacteria. This early colonization helps to establish the symbiotic microbiome inside the host early in its life. The bacteria are also able to stimulate lymphoid tissue associated with the gut mucosa. This enables the tissue to produce antibodies for pathogens that may enter the gut. It has been found that bacteria may also play a role in the activation of TLRs (toll-like receptors) in the intestines. TLRs are a type of PRR (pattern recognition receptor) used by host cells to help repair damage and recognize dangers to the host. This could be important in immune tolerance and autoimmune diseases. Pathogens could influence this symbiotic coexistence leading to immune dysregulation and susceptibility to diseases. This could provide new direction for managing immunological and metabolic diseases.

Human microbiome

Flowchart illustrating how the human microbiome is studied on the DNA level.

For the members of the human microbiome, see human microbiome

The estimate of the human microbiome is of about 39 trillion microbial cells, outnumbering human cells 1.3 to 1, with an uncertainty of 25% and a variation of 53% over the population of male subjects, and that each defecation event may change the ratio to favor human cells over bacteria to 1 to 1.1. In diseased individuals altered microbiota are associated with diseases such as neonatal necrotizing enterocolitis, inflammatory bowel disease[48] and vaginosis.[49]

Studying the human microbiome

For the study of human microbiome, see Human Microbiome Project.

The problem of elucidating the human microbiome is essentially identifying the members of a microbial community which includes bacteria, eukaryotes, and viruses. This is done primarily using DNA-based studies, though RNA, protein and metabolite based studies are also performed.[50] DNA-based microbiome studies typically can be categorized as either targeted amplicon studies or more recently shotgun metagenomic studies. The former focuses on specific known marker genes and is primarily informative taxonomically, while the latter is an entire metagenomic approach which can also be used to study the functional potential of the community. One of the challenges that is present in human microbiome studies but not in other metagenomic studies is to avoid including the host DNA in the study.

Presence of a core microbiome

Aside from simply elucidating the composition of the human microbiome, one of the major questions involving the human microbiome is whether there is a "core", that is, whether there is a subset of the community that is shared between most humans.[52][53] If there is a core, then it would be possible to associate certain community compositions with disease states, which is one of the goals of the Human Microbiome Project. It is known that the human microbiome is highly variable both within a single subject and between different individuals. For example, the gut microbiota of humans is markedly dissimilar between individuals, a phenomenon which is also observed in mice.[54] Hamady and Knight show that one can rule out the possibility that any species is shared among all humans at more than 0.9% abundance in the gut or at more than 2% abundance on hands.[53] Although there is very little species level conservation between individuals, it has been shown that this may be a result of functional redundancy as different communities tend to converge on the same functional state.

On 13 June 2012, a major milestone of the Human Microbiome Project (HMP) was announced by the NIH director Francis Collins. The announcement was accompanied with a series of coordinated articles published in Nature[56][57] and several journals in the Public Library of Science (PLoS) on the same day. By mapping the normal microbial make-up of healthy humans using genome sequencing techniques, the researchers of the HMP have created a reference database and the boundaries of normal microbial variation in humans. From 242 healthy U.S. volunteers, more than 5,000 samples were collected from tissues from 15 (men) to 18 (women) body sites such as mouth, nose, skin, lower intestine (stool), and vagina. All the DNA, human and microbial, were analyzed with DNA sequencing machines. The microbial genome data were extracted by identifying the bacterial specific ribosomal RNA, 16S rRNA. The researchers calculated that more than 10,000 microbial species occupy the human ecosystem and they have identified 81 – 99% of the genera.

Role in psychology

Depression

Microbes are also implicated in depression. The pathogenic bacterium Borrelia burgdorferi causes Lyme disease which causes depression in up to 2/3 of all cases.[58] Non-pathogenic bacteria are also implicated in depression in which bacterial populations are suppressed. Increasing serotonin levels through selective serotonin reuptake inhibitors is the primary treatment of depression in humans. Human patients with depression are less able to properly digest fructose,[59] which is also associated with a reduction in tryptophan production.[60] Eliminating fructose from their diet improved their depression.

Anxiety

Gut microbes are also implicated in anxiety disorders. In humans, anxiety disorders are common in patients with disturbed gut flora.

Autism

Autistic populations have a unique microbiome consisting of more clostridial species.[63] Half of all autistic children with gastrointestinal dysfunction were found to have the bacterium Sutterella which was completely absent in non-autistic children with gastrointestinal dysfunction.[64] There is evidence that for some children with late-onset autism antibiotics can alleviate symptoms temporarily.

Research methods

Targeted amplicon sequencing

Targeted amplicon sequencing relies on having some expectations about the composition of the community that is being studied. In target amplicon sequencing a phylogenetically informative marker is targeted for sequencing. Such a marker should be present in ideally all the expected organisms. It should also evolve in such a way that it is conserved enough that primers can target genes from a wide range of organisms while evolving quickly enough to allow for finer resolution at the taxonomic level. A common marker for human microbiome studies is the gene for bacterial 16S rRNA (i.e. "16S rDNA", the sequence of DNA which encodes the ribosomal RNA molecule).[50] Since ribosomes are present in all living organisms, using 16S rDNA allows for DNA to be amplified from many more organisms than if another marker were used. The 16S rDNA gene contains both slowly evolving regions and fast evolving regions; the former can be used to design broad primers while the latter allow for finer taxonomic distinction. However, species-level resolution is not typically possible using the 16S rDNA. Primer selection is an important step, as anything that cannot be targeted by the primer will not be amplified and thus will not be detected. Different sets of primers have been shown to amplify different taxonomic groups due to sequence variation.

Targeted studies of eukaryotic and viral communities are limited and subject to the challenge of excluding host DNA from amplification and the reduced eukaryotic and viral biomass in the human microbiome.

After the amplicons are sequenced, molecular phylogenetic methods are used to infer the composition of the microbial community. This is done by clustering the amplicons into operational taxonomic units (OTUs) and inferring phylogenetic relationships between the sequences. Due to the complexity of the data, distance measures such as UniFrac distances are usually defined between microbiome samples, and downstream multivariate methods are carried out on the distance matrices. An important point is that the scale of data is extensive, and further approaches must be taken to identify patterns from the available information. Tools used to analyze the data include VAMPS, QIIME and mothur.

Metagenomic sequencing

Main article: Metagenomics

Metagenomics is also used extensively for studying microbial communities. In metagenomic sequencing, DNA is recovered directly from environmental samples in an untargeted manner with the goal of obtaining an unbiased sample from all genes of all members of the community. Recent studies use shotgun Sanger sequencing or pyrosequencing to recover the sequences of the reads. The reads can then be assembled into contigs. To determine the phylogenetic identity of a sequence, it is compared to available full genome sequences using methods such as BLAST. One drawback of this approach is that many members of microbial communities do not have a representative sequenced genome.

Despite the fact that metagenomics is limited by the availability of reference sequences, one significant advantage of metagenomics over targeted amplicon sequencing is that metagenomics data can elucidate the functional potential of the community DNA. Targeted gene surveys cannot do this as they only reveal the phylogenetic relationship between the same gene from different organisms. Functional analysis is done by comparing the recovered sequences to databases of metagenomic annotations such as KEGG. The metabolic pathways that these genes are involved in can then be predicted with tools such as MG-RAST, CAMERA and IMG/M.

RNA and protein-based approaches

Metatranscriptomics studies have been performed to study the gene expression of microbial communities through methods such as the pyrosequencing of extracted RNA. Structure based studies have also identified non-coding RNAs (ncRNAs) such as ribozymes from microbiota. Metaproteomics is a new approach that studies the proteins expressed by microbiota, giving insight into its functional potential.