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 – there are millions in our “extended genome”2 compared to the nearly 23,000 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 vitro 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.3 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.4
Bacteria are no less persistent or proliferative inside the human body.
One prominent researcher called human skin a “virtual zoo of bacteria.”5 Another compared the diversity in the human gut to a rain forest.6 The human gut alone contains on average: 40,000 bacterial species,7 9 million unique bacterial genes and 100 trillion microbial cells.8 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.”9
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.10 To this point, only approximately 1% of this microbiota has been characterized and identified.11 The Human Microbiome Project aims to catalog the balance using an array of molecular sequencing techniques over the coming years.12 The combined genetic contributions of these microbes — in excess of 1,000,000 protein-coding genes — provide traits not encoded in our own genomes.13
Since the inception of the Human Microbiome Project in 2007, dozens of research teams have gathered data which redefine what it means to be human. Some commentators have gone so far as to refer to the human body as a superorganism whose “whose metabolism represents an amalgamation of microbial and human attributes.”14
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.
Nobody can pretend to know the complete life cycle and all the varieties of even a single bacterial species. It would be an assumption to think so.
Ernst Almquist, a colleague of Louis Pasteur
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.36 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 biofilm. One form of bacteria that has been studied for decades and about which a lot is known is the L-form.
Bacteria regularly engage in “shape shifting” between forms. For example, Paenibacillus dendritiformis bacteria survive overcrowding by switching between two distinct vegetative phenotypes.37
As a part of their natural life cycle, bacteria can transform into a variety of forms. One of those phases is the L-form. L-form bacteria, also known as cell wall deficient bacteria, are a phase of bacteria that are very small and lack cell walls.
Though the subject of a great deal of research over the last 100 years and implicated in a variety of diseases, L-forms remain largely misunderstood - or at the very least, underappreciated - by the medical research community. According to the Marshall Pathogenesis, L-forms are part of a metagenomic microbiota responsible for chronic disease.
Biofilms are densely packed communities of microbial cells that grow on living or inert surfaces and surround themselves with secreted polymers. Many bacterial species form biofilms, and their study has revealed them to be complex and diverse. The structural and physiological complexity of biofilms has led to the idea that they are coordinated and cooperative groups, analogous to multicellular organisms.38
Researchers have estimated that 60-80 percent of microbial infections in the body are caused by bacteria growing as a biofilm – as opposed to planktonic (free-floating) bacteria.
There is a perception that single-celled organisms are asocial, but that is misguided. When bacteria are under stress—which is the story of their lives—they team up and form this collective called a biofilm. If you look at naturally occurring biofilms, they have very complicated architecture. They are like cities with channels for nutrients to go in and waste to go out.
Andre Levchenko, PhD, Johns Hopkins University
Some external biofilm, namely chronic wounds and dental plaque, can be manually removed. Because of their inaccessibility and heightened resistance to certain antibiotic combinations and dosages, internal biofilm are more difficult to eradicate.
Biofilm bacteria are a part of what is known as the Th1 bacterial pathogens, which according to the Marshall Pathogenesis, collectively cause chronic disease. The Marshall Protocol targets the Th1 pathogens, in part, through the use of pulsed low doses of antibiotics, because they limit the growth of “persister cells.”
The genomes and the respective proteomes of microbes in the body frequently interact with those expressed by their human hosts. This is a key part of what is know as the interactome. The “massive”41 co-occurrence of protein-coding genes between microbes and humans speaks to the survival advantage of such homology, and the extent to which sequence overlap may play a key role in disease. Indeed, manipulation of host cell fate and orchestrated choreography of inflammatory responses are recurrent themes in the strategies of microbial pathogens.42 Bacteria affect host-cell pathways and human gene expression through a number of increasingly well-documented ways.
It is what bacteria do rather than what they are that commands attention, since our interest centers in the host rather than in the parasite.
Theobald Smith, M.D., circa 1904 43
Chronic diseases manifest in patients and within patient populations with a high degree of variability. Some people have five chronic diseases, and others have one. Some patients experience symptoms of disease early in life while others not until they are very old. According to the Marshall Pathogenesis, this variability can be attributed to several factors.
Over the course of a lifetime, patients pick up the approximately 90 trillion bacteria to which they play host.44 While some researchers refer to each person's unique microbiota as an individual's “pathogen burden” and other terms,45 46 we have referred to it as a person's “pea soup.” In everyday language, the term pea soup is otherwise used to refer to a dense fog – an apt metaphor for the human microbiota. The promiscuity with which bacteria exchange DNA as well as the sheer number of bacteria to which any given person plays host are both factors which severely limit researchers' ability to accurately predict species-species and species-disease interactions.
The process by which a person accumulates the bacteria which drive disease is known as “successive infection.” In successive infection, an infectious cascade of pathogens slow the immune response and allow for subsequent infections to proliferate, resulting in dysbiosis (microbial imbalances). In patients sick with chronic inflammatory diseases, successive infection is ongoing and has additive properties: generally speaking, the more sick people are, the more sick they tend to become. Like a person's pea soup, the process by which a person accumulates additional bacteria via successive infection has an inherent variability to it.
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.49 Other processes such as homologous recombination further muddle any kind of genomic coherence.50 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.51 52 When it comes to bacteria, the very definition of “species” may have to be reconsidered.53
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
For example, Hanage of Imperial College of London concluded that the classification of certain isolates of Neisseria was inherently “fuzzy.”54
That said, there is some evidence that broad classifications of species appear more often in certain kinds of tissue:
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.57 One commentator suggests the relationship between bacteria is actually more like that of a web.58
Enter metagenomics - a field which transcends the search for individual genomes. Literally “beyond genomics”59, 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 inflammation, 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.
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.60
The Marshall Pathogenesis 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 intracellular. 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 receptor which controls the innate immune response. 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 hundreds of genes.61
The term acute infection is used to refer to microbe living inside a host for a limited period of time, typically less than six months. However, an abundance of research has emerged suggesting that acute infections have long-lasting effects, predisposing a person to later onset of chronic diseases.
The purpose of the Marshall Protocol is to stimulate the immune response and improve the mix of microbes in the human body. In theory, this would free the immune response to target acute infections. Anecdotal reports from physicians and patients suggest that the MP is effective in this manner. To date, there have been no reports of tuberculosis or AIDS among MP patients.
Update Fierer: Science. 2009 May 29;324(5931):1190-2. Topographical and temporal diversity of the human skin microbiome.
Grice EA, Kong HH, Conlan S, Deming CB, Davis J, Young AC; NISC Comparative Sequencing Program, Bouffard GG, Blakesley RW, Murray PR, Green ED, Turner ML, Segre JA.
Genetics and Molecular Biology Branch, National Human Genome Research Institute, Bethesda, MD 20892, USA.
Comment in:Science. 2009 Aug 21;325(5943):944-5.
Human skin is a large, heterogeneous organ that protects the body from pathogens while sustaining microorganisms that influence human health and disease. Our analysis of 16S ribosomal RNA gene sequences obtained from 20 distinct skin sites of healthy humans revealed that physiologically comparable sites harbor similar bacterial communities. The complexity and stability of the microbial community are dependent on the specific characteristics of the skin site. This topographical and temporal survey provides a baseline for studies that examine the role of bacterial communities in disease states and the microbial interdependencies required to maintain healthy skin.
“microbiologists have discovered new viral genes in faeces. They find that the composition of virus populations inhabiting the tail ends of healthy intestines (as represented in our stools) is unique to each individual and stable over time. Even identical twins who share many of the same intestinal bacteria differed in their gut's viral make-up”
BMC Genomics. 2010 Sep 7;11:488. Molecular analysis of the diversity of vaginal microbiota associated with bacterial vaginosis.65
Ling Z, Kong J, Liu F, Zhu H, Chen X, Wang Y, Li L, Nelson KE, Xia Y, Xiang C. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China. Abstract BACKGROUND: Bacterial vaginosis (BV) is an ecological disorder of the vaginal microbiota that affects millions of women annually, and is associated with numerous adverse health outcomes including pre-term birth and the acquisition of sexually transmitted infections. However, little is known about the overall structure and composition of vaginal microbial communities; most of the earlier studies focused on predominant vaginal bacteria in the process of BV. In the present study, the diversity and richness of vaginal microbiota in 50 BV positive and 50 healthy women from China were investigated using culture-independent PCR-denaturing gradient gel electrophoresis (DGGE) and barcoded 454 pyrosequencing methods, and validated by quantitative PCR. RESULTS: Our data demonstrated that there was a profound shift in the absolute and relative abundances of bacterial species present in the vagina when comparing populations associated with healthy and diseased conditions. In spite of significant interpersonal variations, the diversity of vaginal microbiota in the two groups could be clearly divided into two clusters. A total of 246,359 high quality pyrosequencing reads was obtained for evaluating bacterial diversity and 24,298 unique sequences represented all phylotypes. The most predominant phyla of bacteria identified in the vagina belonged to Firmicutes, Bacteroidetes, Actinobacteria and Fusobacteria. The higher number of phylotypes in BV positive women over healthy is consistent with the results of previous studies and a large number of low-abundance taxa which were missed in previous studies were revealed. Although no single bacterium could be identified as a specific marker for healthy over diseased conditions, three phyla - Bacteroidetes, Actinobacteria and Fusobacteria, and eight genera including Gardnerella, Atopobium, Megasphaera, Eggerthella, Aerococcus, Leptotrichia/Sneathia, Prevotella and Papillibacter were strongly associated with BV (p < 0.05). These genera are potentially excellent markers and could be used as targets for clinical BV diagnosis by molecular approaches. CONCLUSIONS: The data presented here have clearly profiled the overall structure of vaginal communities and clearly demonstrated that BV is associated with a dramatic increase in the taxonomic richness and diversity of vaginal microbiota. The study also provides the most comprehensive picture of the vaginal community structure and the bacterial ecosystem, and significantly contributes to the current understanding of the etiology of BV. PMID: 20819230
Mycobacteria inhibition of IFN-gamma induced HLA-DR gene expression by up-regulating histone deacetylation at the promoter region in human THP-1 monocytic cells66
Infection of macrophages with mycobacteria has been shown to inhibit the macrophage response to IFN-gamma. In the current study, we examined the effect of Mycobacteria avium, Mycobacteria tuberculosis, and TLR2 stimulation on IFN-gamma-induced gene expression in human PMA-differentiated THP-1 monocytic cells. Mycobacterial infection inhibited IFN-gamma-induced expression of HLA-DRalpha and HLA-DRbeta mRNA and partially inhibited CIITA expression but did not affect expression of IFN regulatory factor-1 mRNA. To determine whether inhibition of histone deacetylase (HDAC) activity could rescue HLA-DR gene expression, butyric acid and MS-275, inhibitors of HDAC activity, were added at the time of M. avium or M. tuberculosis infection or TLR2 stimulation. HDAC inhibition restored the ability of these cells to express HLA-DRalpha and HLA-DRbeta mRNA in response to IFN-gamma. Histone acetylation induced by IFN-gamma at the HLA-DRalpha promoter was repressed upon mycobacteria infection or TLR2 stimulation. HDAC gene expression was not affected by mycobacterial infection. However, mycobacterial infection or TLR2 stimulation up-regulated expression of mammalian Sin3A, a corepressor that is required for MHC class II repression by HDAC. Furthermore, we show that the mammalian Sin3A corepressor is associated with the HLA-DRalpha promoter in M. avium-infected THP-1 cells stimulated with IFN-gamma. Thus, mycobacterial infection of human THP-1 cells specifically inhibits HLA-DR gene expression by a novel pathway that involves HDAC complex formation at the HLA-DR promoter, resulting in histone deacetylation and gene silencing.
Trevor? - “Crafty little buggers indeed! So what this is saying is that the mycobacteria are able to silence genes associated with the immune systems ability to react to infection by increasing HDAC. The use of HDAC inhibitors in culture lead to the clearance of recalcitrant intracellular bacterial infections. However, the use of HDAC inhibitors can lead to viremia as the body also uses HDAC to silence cellular machinary associated with viral replication as the HDAC inhibitors are non-specific in their action. Its a Catch22.”
Joyce,Good point. Helicobacter pylori were recently found circulating in peripheral blood, showing that the GI tract is not so well isolated from the rest of the body as had previously been believed.
I think the gut flora is key to introduction of new species to our systemic intraphagocytic microbiota over time, and also to how much of the innate immune system is occupied keeping the 'nasties' at bay in the GI tract.
The interesting thing is that although the MP abx profoundly change the gut flora, they do not wipe it out - as can be seen when discontinuing abx, the flora repopulates fairly quickly. I did once hear a presentation where it was claimed that a course of Cipro wiped out gut flora for a year. I don't believe that for a moment - the L-forms (like Hp) are clever little beasties…
Science. 2009 Dec 18;326(5960):1694-7. Epub 2009 Nov 5.
Bacterial community variation in human body habitats across space and time.
Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R. Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA. Abstract Elucidating the biogeography of bacterial communities on the human body is critical for establishing healthy baselines from which to detect differences associated with diseases. To obtain an integrated view of the spatial and temporal distribution of the human microbiota, we surveyed bacteria from up to 27 sites in seven to nine healthy adults on four occasions. We found that community composition was determined primarily by body habitat. Within habitats, interpersonal variability was high, whereas individuals exhibited minimal temporal variability. Several skin locations harbored more diverse communities than the gut and mouth, and skin locations differed in their community assembly patterns. These results indicate that our microbiota, although personalized, varies systematically across body habitats and time; such trends may ultimately reveal how microbiome changes cause or prevent disease.
From genomics to proteomics.
Tyers M, Mann M. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada M5G 1×5. firstname.lastname@example.org Abstract Proteomics is the study of the function of all expressed proteins. Tremendous progress has been made in the past few years in generating large-scale data sets for protein-protein interactions, organelle composition, protein activity patterns and protein profiles in cancer patients. But further technological improvements, organization of international proteomics projects and open access to results are needed for proteomics to fulfil its potential.
A total of 14,000 physical interactions obtained from the GRID database were represented with the Osprey network visualization system (see http://biodata.mshri.on.ca/grid). Each edge in the graph represents an interaction between nodes, which are coloured according to Gene Ontology (GO) functional annotation. Highly connected complexes within the data set, shown at the perimeter of the central mass, are built from nodes that share at least three interactions within other complex members. The complete graph contains 4,543 nodes of 6,000 proteins encoded by the yeast genome, 12,843 interactions and an average connectivity of 2.82 per node. The 20 highly connected complexes contain 340 genes, 1,835 connections and an average connectivity of 5.39.
We still don't understand what a very large proportion of our DNA actually does. Sure, we understand how the genes work, but genes make up far less than half the total size of a the human genome. The non-gene, 'non-coding', regions are loosely termed “Junk DNA.”
Well, a group at Oxford has started to hone in on one likely function: to perpetuate components of the Human Microbiome. Here is a simplified version of their hypothesis:
And the more complex concepts are in two papers at PLOS. First, a commentary:
and then the actual paper:
This is an important concept, which I have touched upon a few times, but generally felt it too complex to explain in detail. Now this paper, and the two commentaries above, can help me communicate the concept
It’s time for animals - including humans - to admit that the bacteria, viruses and other microbes have won. Our bodies are home to many times more bacterial cells than animal cells and countless trillions of viruses. Ancient retroviruses make up a good size chunk of our genome. Now, scientists have discovered that most any virus can set up shop in an animal's genomes and lay dormant for millions of years.
Mucosal Immunology (2011) 4, 133–138; doi:10.1038/mi.2010.89; published online 19 January 2011
A complex relationship: the interaction among symbiotic microbes, invading pathogens, and their mammalian host
M M Curtis1 and V Sperandio1
1Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
Correspondence: V Sperandio, (Vanessa.Sperandio@utsouthwestern.edu)
Received 1 October 2010; Accepted 13 December 2010; Published online 19 January 2011.
Topof page Abstract Symbiosis between microbes and their mammalian host is vital to maintaining homeostasis. Symbiotic microbes within the gastrointestinal tract provide an array of benefits to the host, including promotion of host immunity. A coordinated effort of the host and symbiotic microbes deters the colonization and survival of many invading pathogens. However, pathogens have devised strategies to overcome these mechanisms. Furthermore, some pathogens can hijack host hormones and bacterial autoinducers to induce virulence traits. Intra- and inter-species (bacteria/bacteria) and interkingdom (bacteria/host) communication orchestrates the complex relationship among symbiotic microbes, invading pathogens, and their mammalian host. Insight into this communication will provide a foundation for the development of targeted antimicrobial therapies.
Most bacteria harbor toxin–antitoxin (TA) systems, in which a bacterial toxin is rendered inactive under resting conditions by its antitoxin counterpart. Under conditions of stress, however, the antitoxin is degraded, freeing the toxin to attack its host bacterium. One such TA system, PezAT, has been difficult to study in the past because the PezT toxin is so toxic without its antitoxin counterpart that bacteria die before any useful measurements can be made. Here, we use a truncated version of PezT that kills bacteria more slowly than normal, allowing us to examine the mechanisms of how this TA system operates. We find that zeta toxins convert an essential building block of bacterial cell walls (known as UNAG) into a form that prevents normal cell wall growth, causing distortions in bacterial shape that leave the bacteria vulnerable to the hydrostatic pressure of its contents. Consequently, the bacteria burst, similar to what happens when they are treated with penicillin. These results may serve useful for designing new antibiotics. Additionally, our results support the hypothesis that activation of PezT during bacterial infections may be a method by which rapidly growing bacteria can instigate a suicide program, which would promote the release of virulence factors that facilitate spread of infections.
Differential Attraction of Malaria Mosquitoes to Volatile Blends Produced by Human Skin Bacteria
Were we but able to explain
The fiefdom of the microbe—
Why one man is his serf,
Another is his lord
When all are his domain….
Viruses that Infect Parasites that Infect Us: The Matryoshka Dolls of Human Pathogens
submitted by Chris Condayan on July 11, 2011 Tags: parasites, STC, viruses Source: schaechter.asmblog.org “We’re all too familiar with the viruses that can infect us, from the common cold to yellow fever virus to the endogenous retroviruses that make up a chunk of our genome. Many of us are also acquainted with parasites, such as tape worms or Giardia, that like to set up camp in the human body. But the world of parasites and viruses does not end there. Many parasites or endosymbionts can be infected with viruses. A classic example is Paramecium, which can harbor an endosymbiotic bacterium, Caedibacter, which in turn carries phages involved in making a toxin. But from the human point of view, things start to get particularly interesting when we consider the viruses that infect parasites of humans and how those viral infections—inside of a parasite inside of a person, somewhat like a Matryoshka nesting doll—may modulate the parasite’s interaction with its human host.”