The Gut Microbiome

Collectively, the human body's microorganisms contain an enormous amount of bacteria. It has been recognized since the late 1970s that the number of bacterial cells in and on the human body exceeds at least an astronomical amount the number of human cells.

More recently, in 2010, it was reported that our ' accessory genome ' estimated number of unique bacterial genes exceeds the number of our genes by a margin of 150.

While the bacteria are 1,000 times smaller than human cells, they nevertheless make up about 2 percent of the body mass (1.5 kg), making them collectively equal in size to the human brain (1.4 kg) or the liver (1.6 kg), leading some to draw attention to our inhabitant microbes as an entire human organ.

Conversely, the human-microbial bond was also equivalent to that of a superorganism, a colony of bees, or a holobiont, such as a coral reef.

A holobiont is an agglomeration of a host and the numerous other species that live in or near it, forming a distinct ecological unit. A holobiont's components are individual organisms or bionts, whereas the hologenome is the aggregate genome of all bionts.

Recognizing the need for a collective term referring to the large number of relatively unexplored and mostly unnamed microorganisms populating the human body, Joshua Lederberg coined the term microbiome in 2001 to "mean the ecological population of commensal, symbiotic and pathogenic microorganisms that share our body space and have all but been overlooked as determinants of health and disease.

The concept microbiome has already tended to be used more stringently, referring to the cumulative molecular (especially genomic) data obtained from a microorganism population, rather than the microorganisms themselves, resulting in some research misunderstanding.

For the specifics of this research, the word microbiome has been used as an ecological population of microorganisms following its original published description by Lederberg, the concept microbiota as a synonym shall also be employed.

Subsequently, the word hologenome is used to refer to the host's collective fenomas and its microbiome. Finally, to refer to combined genomic and proteomic data gathered from an environmental sample, the terms metagenome and metaproteome will be employed.

In tandem with significant technological developments in microbiology and molecular methods, these concepts have evolved to investigate complex microbial systems.

 Early studies of human-associated microbes focused on individual bacterial species and strains being isolated and cultured. This leads to a further explanation and naming of hundreds of human microbiome species using those strategies.

Nonetheless, culture-free molecular analyzes of prokaryotic 16S ribosomal RNA (rRNA) and the 16S rRNA gene using methods first introduced by Carl Woese and Norman Pace show that at least 60-80 percent of bacteria inhabiting the human body can not be cultivated in a laboratory.

New molecular methodologies have been developed to access these unculturable members, particularly terminal restriction fragment length polymorphism (T-RFLP) molecular fingerprinting, single target PCR, multiplex PCR, quantitative PCR (qPCR), fluorescent in situ hybridization (FISH), checkerboard hybridization, and 16S microarrays based on rRNA, inter alia.

The most effective of these methods merged PCR amplification of 16S rRNA genes from a mixed microbial sample using universal bacterial primers, followed by cloning PCR products into a plasmid vector, transformation of the plasmids into competent Escherichia coli cells, plating and colony selection, plasmid purification, and sequencing of up to several thousand clones by Sanger.

Such sequences could then be grouped into distinct phylotypes which correspond approximately to species of bacteria by proximity (usually 97.0 percent or 98.5 percent sequence similarity).

This approach was successfully used in 2010 to classify 600 predominant members of the human oral microbiome. However, again, it is not feasible to characterize uncommon or less prevalent oral morphological traits using this method given the high-cost, labor-intensive, and low-performance nature of the cloning and sequencing phases.

The advent of NGS has influenced microbiome investigations by removing the cloning requirement and allowing millions of 16S rRNA gene sequences to be obtained at a fraction of the cost per base compared to conventional techniques.

Notwithstanding shorter reading lengths and shifting focus to hypervariable regions of the individual 16S rRNA gene rather than the entire genome, the expanded sequencing depth has identified a wide range of low-abundance taxa within the human microbiome, increasing the established taxonomic diversity of the oral microbiome alone by more than one order of magnitude.

Latest generation sequencing has also enabled the combined analysis of all genes and genomes within a microbiome, microbiome metagenomics, feasible as well. This has allowed microbiome research to move further than taxonomic ' who's there ' questions to practical ' what they're doing. '

Besides, the amplification of the single-cell genome and bioinformatics advances in sequence aggregation from metagenomic datasets allow complete genome reconstruction of unculturable taxa, offering our first glimpse of the potential role of these mysterious organisms.

Concerning primitive microbiomes, NGS provides two more benefits compared to previous methods. Firstly, the technique can be applied to dead cells as a culture-free method, and secondly, NGS is designed for fragmented DNA inside the range of sizes typical of ancient DNA (< 300bp).

Advances in metagenomic techniques in the field of metaproteomics are also being compared. Previously, microbial protein detection relied primarily on antibody-based procedures, often tied to gel electrophoresis (e.g., Western Blotting). Proteomics-based approaches advanced through the use of 2D (pI versus MWt) comparative gels with specific locations eluted and examined by soft simultaneous mass spectroscopy (MS / MS) ionization.

Such ' top-down ' approaches are gradually being replaced by ' bottom-up ' solutions, also known as shotgun proteomics, which perform mass spectrometric analyses of peptides from the complete protein extract's enzymatic digestion.

These bottom-up methods allow for higher performance and are also more applicable to primitive protein research because they do not involve intact molecules of protein.

Such advanced molecular methods provide unparalleled insights into the activity and malfunction of human microbial populations and provide a framework for understanding the role of gut microbiota in human health and disease.

 Since the early 2000s, numerous studies have used a range of molecular methodologies to investigate the structure and function of the human microbiome.

A significant boost to these activities began in 2008, following the launch in Europe of the National Institutes of Health Human Microbiome Project (HMP) in the United States and the Human Intestinal Tract Metagenomics (MetaHIT) project.

Although human-associated microorganisms were historically seen at best as passive commensal tag-longs or irritations to be rinsed or flossed away, we now recognize that human oral, intestinal, skin and uritogenital microbiota play critical roles in sustaining host health by supporting essential functions in digestion and metabolism, vitamin development, and immune system education and maintenance.

Nonetheless, when threatened by poor diet, illness, stress, antimicrobial drugs, and other environmental disturbances, human microbiome ecology can migrate from a mutualistic to a dysbiotic state, leading to local and systemic diseases as diverse as obesity, type II diabetes, irritable intestinal disease and colon cancer, periodontal and dental decay, atherosclerosis and endocarditis.

The healthy human microbiome also contains several common, but highly severe, opportunistic pathogens, such as Streptococcus pneumonia, Haemophilus influenzae, Neisseria meningitidis, Clostridium difficile, Propionibacterium acnes and Staphylococcus aureus, some of which are implicated in hospital and community infections and present a particular risk to the elderly and the immune system.

Increasingly, multiple antibiotic-resistant compounds are overwhelmingly detected in normal oral and gastrointestinal microbiota in healthy individuals.

This appears to suggest that the use of antibiotic therapy may affect non-clinical goals, either through the direct clinical implementation or via indirect growth stimulation or prophylactic treatment in livestock, and may result in long-term intrinsic reservoirs of antibiotic resistance.

Furthermore, antibacterial remedies can perturb healthy bacterial communities themselves, resulting in further complications.

For example, wide-spectrum antibiotic therapeutic courses are known to disrupt the gut and uritogenital microbiota, where they can induce antibiotic-associated colitis and bacterial vaginosis, alike.

As a result, the interest in probiotic and prebiotic remedies for the treatment of disrupted microbiomas is increasing, but lack of basic knowledge about what defines a healthy microbiome, as well as a greater understanding of the propagation and development of healthy microbiota, are hindering factors in the development of these therapies.

While significant efforts have been made to classify healthy gut and oral microbiomas, recent studies on rural, non-Western populations have expressed concerns as to whether the microbiota we currently identify as natural has been influenced by recent effects of the modern Western diet, hygiene, antibiotic prominence, and lifestyle.

The industrial revolution has dramatically reduced our contact with natural environments and has fundamentally changed our relationship with food production.

The human oral and gut microbiomes, found at the entry point of our food and the locus of food digestion, have developed in circumstances of regular exposure to several environmental and zoonotic microbes which are no longer in play in today's globalized food chain.

In addition, the food itself has shifted from the wild natural products eaten by our hunter-gatherer ancestors to today's industrialized supermarkets packed with an excess of highly processed Western foods containing chemically refined levels of sugar, oil, and salt, not to include antimicrobial preservatives, petroleum-based dyes, and many other additives.

This dietary shift has changed the pressure of selection on our microbiomas. For example, disorders such as dental caries and gum disease are defined under the ' ecological plaque hypothesis ' as oral ecological disasters of cultural and eating habits.

Although it may be already evident that the human microbiome plays a crucial role in making us human, in maintaining us healthy, and in making us sick, we know remarkably few of the diversity, variation, and evolution of the human microbiome both recently and in the past.

Instead, many questions remain: 

When and how did our bacterial populations become distinctly human?

And what would that imply for today and the future of our microbiomes?

How do we develop and distribute microbiomas, and to what extent are our cultural traditions and installed environments affected by this?

How have contemporary western diets, hygiene standards, and antibiotic prominence affected the function of ' normal ' microbiomas?

Are we still in mutual symbiosis with our microbiomes or are the so-called ' civilizational diseases ' – heart disease, obesity, type II diabetes, asthma, allergies, osteoporosis – testimony that our microbiomes are not ecologically balanced and dysbiotic?

Who are the representatives of the human microbiome on an even more in-depth level, how did they happen to inhabit us, and how long would they have been there? Who is ' our microbial self '?

Research of rural and indigenous communities, and crowdfunding initiatives such as the American Gut, the Earth Microbiome Project, and uBiome aim to describe current microbiomes over a variety of modern settings.

Moreover, even the most thorough analysis of modern microbiota can give a limited perspective into pre-industrial microbiomas.

By comparison, direct study of ancient microbiomes from isolated places and historical periods in the past might provide a fascinating perspective of the coevolution of microbes and organisms, host-microbial ecology, and improvement of the state of human health over time.