Microbes in the human body

According to a recent National Institutes of Health (NIH) estimate, 90% of cells in the human body are bacterial, fungal, or otherwise non-human.¹ Although many have concluded that bacteria surely enjoy a commensal relationship with their human hosts, only a fraction of the human microbiota has been characterized, much less identified. The sheer number of non-human genes represented by the human microbiota – 1,000,000+ compared to the nearly 32,000 in the human genome – implies we have just begun to fathom the full extent to which bacteria work to facilitate their own survival.

The NIH's ongoing initiative, the Human Microbiome Project, aspires to catalog the human microbiome, also referred to as the human metagenome. Emerging insights from environmental sampling studies have shown, for example, that in vitroA technique of performing a given procedure in a controlled environment outside of a living organism - usually a laboratory. based methods for culturing bacteria have drastically underrepresented the size and diversity of bacterial populations. One environmental sample of human hands found 100 times more species than had previously been detected using purely culture-based methods. Another study which also employed high throughput genomic sequencing discovered high numbers of hydrothermal vent eubacteria on prosthetic hip joints, a species once thought only to persist in the depths of the ocean.

Recent research has demonstrated that the diversity, prevalence and persistence of bacteria has been consistently underestimated. Microbes form most of the world's biomass: there are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a milliliter (gram) of fresh water.² Studies have found bacteria in areas previously thought to be completely sterile. A broad diversity of bacteria were found at all of the “clean rooms” where NASA spacecraft are assembled and in spite of the highly desiccated, nutrient-bare conditions within.³

Bacteria are no less persistent or proliferative inside the human body.

One prominent researcher called human skin a “virtual zoo of bacteria.”⁴ Another compared the diversity in the human gut to a rain forest.⁵ According to Asher Mullard, “Between them [the bacteria in our bodies], they harbor millions of genes, compared with the paltry 20,000 estimated in the human genome. To say that you are outnumbered is a massive understatement.”⁶

The global initiative known as the Human Microbiome Project currently estimates that the microorganisms that live inside or on Homo sapiens outnumber somatic (body) and germ cells [germ cells as in gametes, not bacteria] by a factor of ten.⁷ To this point, only approximately 1% of this microbiota has been characterized and identified.⁸ The Human Microbiome Project aims to catalog the balance using an array of molecular sequencing techniques over the coming years.⁹ The combined genetic contributions of these microbes — in excess of 1,000,000 protein-coding genes — provide traits not encoded in our own genomes.¹⁰

Genes in the human microbiota far outnumber those in the human genome.

Researchers have long known that traditional methods for identifying bacteria are effective at identifying only a fraction of the bacteria in a given sample. New genomic based methods such as polymerase chain reaction (PCR) detect bacterial forms based on the presence of bacterial DNA or RNA. These new techniques are leading to some unexpected insights about bacteria.

• Bacteria are everywhere including the world's most hostile environments – According to Penn State researcher Jennifer Loveland-Curtze, “Microbes comprise up to one-third or more of the Earth’s biomass, yet fewer than 8,000 microbes have been described out of the approximately 3,000,000 that are presumed to exist,”
  • NASA “clean rooms” – One would think that the the one place on Earth where bacteria do not exist is in the NASA “clean rooms” – the supposedly sterile places used to assemble aircraft. A 2007 research team compared the prevalence of bacteria found using traditional culture-based methods and ribosomal RNA gene sequence analysis. The four geographically diverse samples taken show a broad diversity in the types of bacteria able to grow in the most hostile environments including almost 100 types of bacteria, about 45 percent of which were previously unknown to science.¹¹ The findings were something of a shock for NASA, an agency now forced to wonder exactly how many unknown pathogens have been taken to the moon and Mars.
  • Two miles below the surface of a Greenland glacier – A Penn State team found viable “ultrasmall bacteria” in a glacial core¹² – a habitat which is low-temperature, high-pressure, reduced-oxygen, and nutrient-poor. The core was estimated to be 120,000 years old.
• Each person has a unique mix of pathogens – A study led by Dr. Noah Fierer used a high-throughput method for PCR testing to identify the number and species of bacteria present on the hands of 51 undergraduate students leaving an exam room. Each student whose bacterial “fingerprint” – that is, their unique combination of bacteria – was sequenced, carried on average 3,200 bacteria from 150 species on their hands. Only five species were found on all the students’ hands, while any two hands – even belonging to the same person – had only 13% of their bacterial species in common.¹³
• Many bacteria cannot be cultured using traditional cultivation techniques – Using PCR, Fierer's team found that the hands of students subjects contained 332,000 genetically distinct bacteria belonging to 4,742 different species. 45% of the species detected were considered rare. This marked a hundred-fold increase in the number of bacterial speciess detected in previous studies that had relied on purely culture-based methods (such as the Petri dish) to characterize the human hand microbiota.¹⁴ These conclusions are supported by the aformentioned study of NASA clean rooms, which found that only 0.1 to 55% of viable cells found via PCR were able to grow on defined culture medium.¹⁵
• A number of bacteria never though to exist in man, do, and in large numbers. – A 2007 study, for example, found hydrothermal vent eubacteria on a prosthetic hip joint, which represents fully 6% of the bacteria sequences and analyzed.¹⁶ Hydrothermal vent eubacteria otherwise grow best above 176°F (80°C).

Free-floating (planktonic) bacteria may be consistent with the popular conception of bacteria in the human body, but these types of bacteria are in the minority.¹⁷ Bacteria are distinguished by nothing if not their diversity – diversity in form, size, and habitat. Indeed, bacteria can float in the bloodstream, but they can also live inside human cells. They can exist in communities known as biofilmA structured community of microorganisms encapsulated within a self-developed protective matrix and living together.. One form of bacteria that has been studied for decades and about which a lot is known is the L-formDifficult-to-culture bacteria that lack a cell wall and are not detectable by traditional culturing processes. Sometimes referred to as cell wall deficient bacteria..

Bacterial pathogens operate by attacking crucial intracellular pathways in their hosts. These pathogens usually target more than one intracellular pathway and often interact at several points in each of these pathways to commandeer them fully.¹⁹ These well-documented strategies include:

• modulation of host-cell death to facilitate survival in the host²⁰
• modulation of vesicular traffic, a strategy which provides a protective niche within host cells, including in macrophages and neutrophils, which normally kill bacteria²¹
• secretion of proteins, which are similar in effect to substances known to be toxic to humans²²
• creation of ligands such as capnine which bind and block the Vitamin D ReceptorA nuclear receptor located throughout the body that plays a key role in the innate immune response. ²³

Using electron microscopy, Dr. William Wirostko et al identified cell wall deficient bacteria within immune cells of patients with juvenile rheumatoid arthritis and uveitis. This image suggests that pathogens have access to the human cell's transcriptional machinery.

According to one analysis, 463 human genes are changed during an infection with Mycobacterium tuberculosis.²⁴ Of the 463 genes whose expression were changed. 366 of them were known genes. The other genes were unknown – mutations. Of the 366 human genes affected, 25 were upregulated and 341 were downregulated. Two more significant effects were the downregulation of the CD14 receptor which was downregulated 2.3-fold, and the VDRThe Vitamin D Receptor. A nuclear receptor located throughout the body that plays a key role in the innate immune response. receptor which was downregulated 3.3-fold. The mutated genes function in various cellular processes including intracellular signalling, cytoskeletal rearrangement, apoptosis, transcriptional regulation, cell surface receptors, cell-mediated immunity and cellular metabolic pathways.

It is quite plausible that “autoimmunity,” in which it is believed that the body is attacking itself, is caused by bacterial-induced alteration of human genes. All a bacterium would need to do in order to generate an apparent “autoimmuneA condition or disease thought to arise from an overactive immune response of the body against substances and tissues normally present in the body” reaction would be to interfere with the genes necessary for the production of proteins against which autoantibodies are produced.

Pathogenic bacteria have a variety of ways of disrupting the activity of and causing damage to human genes.

• Horizontal gene transfer – Bacteria can insert their DNA into human DNA.
• Interruption of transcription and translation of DNA and RNA – Intracellular pathogens, which inhabit the cytoplasm, can interfere with the steps involved in the transcription and translation processes. Such interference results in genetic mutations, meaning that human DNA is almost certainly altered, over time. The more pathogens people accumulate, the more their genome is potentially altered.
• Disruption of DNA repair mechanisms – Since environmental factors such as exposure to ultraviolet light result in as many as one million individual molecular lesions per cell per day, the potential of intracellular bacteria to interfere with DNA repair mechanisms also greatly interferes with the integrity of the genome and its normal functioning. If the rate of DNA damage exceeds the capacity of the cell to repair it, the accumulation of errors can overwhelm the cell and result in early senescence, apoptosis or cancer. Problems associated with faulty DNA repair functioning result in premature aging, increased sensitivity to carcinogens, and correspondingly increased cancer risk.

Lifelong persistent symbiosis between the human genome and the microbiota [the large community of chronic pathogens that inhabit the human body] must necessarily result in modification of individual genomes. It must necessarily result in the accumulation of ‘junk’ in the cytosol, it must necessarily cause interactions between DNA repair and DNA transcription activity.

Trevor Marshall, PhD

The highly variable range of human genetic mutations induced by bacteria have been identified with some success by researchers with the Human Genome Project. Rather than serving as markers of particular diseases, such mutations generally mark the presence of those pathogens capable of affecting DNA transcription and translation in the nucleus.

Traditionally, bacteria have been understood to:^{26 27}

• reproduce asexually
• not recombine their genetic material with other bacterial species
• be members of a clearly defined (or definable) species
• for a single species, be largely clones of one another

Recent analyses of bacterial DNA have revealed that these assumptions are misplaced. To a much greater extent than ever anticipated, bacteria rapidly and frequently share their DNA with their fellow prokaryotes – even distantly related bacteria – through a process called horizontal gene transfer.²⁸ Other processes such as homologous recombination further muddle any kind of genomic coherence.²⁹ As a result the diversity and variability among bacteria are much greater than anticipated.

Given the rapid diversification in the microbial world, it has become increasingly difficult to classify bacteria with traditional approaches.^{30 31} When it comes to bacteria, the very definition of “species” may have to be reconsidered.³²

There's no single such thing as a microbial species. There's too much diversity in the range of biological collections that we might call species. Recognizing the variability between different groups, we'll probably abandon the notion of there being a single cutoff in terms of species definition…. The species concept is doomed to radical irrelevance because we don't actually need it any more. Metagenomics will come in and shift the paradigm for it…. More [novel] organisms are created through [genetic] recombination than through mutation.

W. Ford Doolittle, PhD speaking at Metagenomics 2006

Horizontal gene transfer can produce organisms effectively belonging to several species at once. Each circle represents an individual genome with the arrows representing the transfer of genetic material. The all-blue, all-gold and red/green circles represent genomes from three different bacterial groups that might be designated species. Source: Doolittle 2006

That said, there is some evidence that broad classifications of species appear more often in certain kinds of tissue:

• The human gut seems to consist of large numbers of Firmicutes and Bacteroidetes.³³
• Grice et al showed that there was greater diversity between different regions of the human skin in a single person than between similar skin regions of different people.³⁴

If species are defined by a shared gene pool, phylogenetic trees (such as the kind used to describe how Darwin's finches have common ancestors) do not satisfactorily model the relationships among bacteria – not when one organism could be a member of two or more otherwise quite distinct “species” simultaneously.³⁵ One commentator suggests the relationship between bacteria is actually more like that of a web.³⁶

Enter metagenomics - a field which transcends the search for individual genomes. Literally “beyond genomics”³⁷, metagenomics is an approach which looks at how whole communities of bacteria develop and interact including biofilm bacteria, intracellular bacteria, and L-form bacteria. Metagenomics provides a way of understanding the mysterious majority of microbes, which have been historically difficult to culture and classify. It is an approach, which involves taking a sample from the environment, pooling the DNA from all the different species present, fracturing it into a mixture of relatively short fragments and then sequencing the lot.

Metagenomics has begun to provide valuable insights into which communities of microbes cause disease. Given that each gene codes for a protein and that a number of proteins have harmful effects, the presence of a particular gene can and has signalled the presence of a pathogenic form of bacteria.

For more than a century, researchers have confined their thinking to Koch's Postulates, which erroneously dictated that a given infectious disease is always caused by a single microbial species. Indeed, a small minority of diseases such as leprosy are caused by a single pathogen.

However, over the years, researchers have cataloged ample evidence of why certain chronic diseases appear to be caused by pathogens: the inflammationThe complex biological response of vascular tissues to harmful stimuli such as pathogens or damaged cells. It is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue., the granuloma, the typical co-infections, the unique non-pathological microbial communities, etc. But, rarely have researchers found evidence of a single infectious agent, and that is because chronic diseases aren't caused by an individual species of microbe, but by ever-evolving, patient-specific whole communities of microbes. A fully realized understanding of metagenomics offers this key insight into chronic disease pathology.

Newer cultivation techniques have associated bacterial count in a pregnant woman's amniotic fluid to age at delivery. Early pregnancy is correlated with worse health outcomes for the newborn.

The genomic diversity and relative importance of distinct genotypes within natural bacterial populations have remained largely unknown and may remain so for years to come.³⁸

The Marshall PathogenesisA description for how chronic inflammatory diseases originate and develop. makes no claims about which individual microbial species, if there are such things, are to blame for chronic disease. Besides, such a consideration is ancillary. The unique and difficult to define mix of pathogens an individual has is known as his or her pea soup – one of the definitions of which is “a dense fog.”

At least some of the bacteria which cause disease are often intraphagocytic. That is, they persist inside phagocytes, the very immune cells meant to kill them. These microbes take hold progressively through a process called successive infection. Chronic forms of bacteria are able to survive and reproduce by generating substances which block and turn off the Vitamin D Receptor, a key nuclear receptorIntracellular receptor proteins that bind to hydrophobic signal molecules (such as steroid and thyroid hormones) or intracellular metabolites and are thus activated to bind to specific DNA sequences which affects transcription. which controls the innate immune responseThe body's first line of defense against intracellular and other pathogens. According to the Marshall Pathogenesis the innate immune system becomes disabled as patients develop chronic disease.. So logical and powerful is this survival mechanism that it seems very likely that this is the primary mode by which chronic pathogenic forms persist. It simply makes too much evolutionary sense for pathogens not to take full advantage of a receptor, which according to one recent study, transcribes over 27,000 genes.³⁹