Progress Uncovering the Mysteries of the Human Microbiome

Nigel R. A. Beeley, Ph.D.

Although there has been an interest in the diversity and impact of human-hosted bacteria for many years, the human microbiome project (HMP) began in earnest in 2008, focused on bacterial populations found in five body sites (oral, skin, vaginal, gut, and nasal/lung) (1). The objective was to examine how changes in the human microbiome are associated with human health or disease. The emphasis was on culture-independent methods of microbial community characterization, including metagenomics as well as extensive whole genome sequencing and deep sequencing of bacterial 16S rRNA sequences amplified by PCR from diverse harvested human samples. The use of sequences from the slowly evolving 16S rRNA (16S ribosomal RNA) portion of a bacterial genome was pioneered by Woese and Fox in their definitions of the three domains Archaea, Bacteria and Eucarya (2).

“One can think about whether we are predominantly feeding ourselves with our microbiome coming along for the ride versus if we are predominantly feeding our microbiome with ourselves just coming along for the ride.”

A brief history of events which led to the HMP are worthy of review. Scientific progress can sometimes be held back by theories which were valid upon their initial incarnation but have endured over the years way beyond their sell-by date. Such is the case in the world of microbiology, where Koch’s postulates, first formulated in 1884, had a lasting impact on parts of the microbial world until the 1950’s (3). Koch’s postulates were four criteria designed to establish a causative relationship between a microbe and a disease and it is the second of these that has hindered progress in the microbiology community. It states that “the microorganism must be isolated from a diseased organism and grown in pure culture.” Microbiologists knew for some time that 100,000’s of different strains of bacteria existed, but up until twenty years ago perhaps as few as 1% of these had actually been characterized through isolation and growth in culture. Finding suitable culture conditions for bacterial growth has been straightforward for some of the more common aerobic bacteria, but much more challenging for others, such as the anaerobic bacteria and any strains of bacteria that had a commensal component to their survival. The classic example is C. difficile, named not because of its modern day notoriety as the scourge of hospital based infections, but because it was difficult to grow in culture. C. difficile, like other members of the Clostridium family, is also a bacteria that has a highly resistant spore form (4). Eventually techniques of genomic analysis have made it possible to conclusively identify the presence of bacterial strains regardless of Koch’s second postulate requiring isolation and culture and those techniques have been refined and expanded to characterize entire biomes today.

Physiologists began to ask questions about the role of bacterial populations via in-vivo experiments. One approach involved the development of control animals that lack the population of bacteria that is conventional of the species, called “gnotobiotic” (5). The germ free status can be created through birthing and raising animals in an aseptic environment, or with a big dose of antibiotics (with some physiological differences). Some surprising microbiome effects were observed with different strains of mice, such as ob/ob (obese) and db/db (diabetic) mice as well as gnotobiotic mice, which resulted in a realization that the makeup of the microbiome played a key role in murine energy expenditure which in turn had a direct impact on both obesity and type II diabetes (6).

Enter stage East (and later stage West), J. Craig Venter and next generation gene sequencing technologies. The first genome to be sequenced using massively parallel shotgun gene sequencing methodology (1996) was from a thermophilic bacteria, Methanocaldococcus jannaschii, isolated from a hydrothermal vent near Woods Hole and of interest to the DoE due to its ability to convert CO2 and hydrogen into methane (7). This was not simply a prelude to the start of the Human Genome Project (8), reported as largely complete in 2001, but a sign of bigger things. Some thought that the subsequent Venter group boat trips beginning in the Sargasso Sea, down the Atlantic seaboard, through Caribbean, the Panama Canal and across the Pacific to French Polynesia via the Galapagos Islands were an excuse for an extended sailing sabbatical. Actually, the Sorcerer II Global Ocean Sampling Expedition harvested, characterized and sequenced 7.7 million Global Ocean Sampling (GOS) sequences” of oceanic bacteria and through assembly predicted 6.12 million proteins (9), (10). New techniques and accuracy surrounding next-generation sequencing were introduced, and it was now possible to be able to simultaneously obtain the full genome of most of the individual members of a mix containing hundreds of bacteria per sample. A total of 3,995 medium and large-sized clusters consisting of only GOS sequences were identified, out of which 1,700 have no detectable homology to known families and were presumably from previously unidentified bacterial strains. Further, several archae and microalgae strains of interest to renewable energy were discovered, which might provide a better understanding of, for example, bacteriorhodopsin vs. chlorophyll based photosynthesis.

Others had already begun using these tools such as deep 16S rRNA sequencing to investigate microbiome populations and the stage was now set to map out the details of the human microbiome via the HMP (1), (11).

The details revealed by the HMP are staggering (11). Each individual, who has ~ 10^12 human cells, lives in commensal synergy with more bacterial cells than they have human cells (currently thought to be about 1-3 times, although estimates vary, previously it was often overstated with the historically inaccurate number of 10 to 100 times). Around 1000 different kinds of bacteria can be identified and the bulk of them reside in the GI tract. Semi quantitation of the mix of bacteria reveals that, for example, within the GI tract of an individual, 100 to 200 classes of bacteria predominate, and within a healthy population, there can be multiple combinations and iterations of these which are doing a similar job. In other words, my healthy assembly of 200 bacteria can be completely different from your healthy 200. Important connections to different disease states are being discovered. The obese microbiome and the Type II diabetic microbiome are different from the “healthy” microbiome. Other microbiomes have been associated with other health problems. The obvious ones are diseases of the GI tract, such as IBD, IBS and Crohn’s disease (12); less obvious are Rheumatoid arthritis, atherosclerosis and other cardiovascular diseases, and cancer. Still more of a stretch but apparently real are CNS problems including autism and depression. Important observations linking C-section births to issues in later life have been attributed to a lack of exposure of the newborn to the vaginal microbiome. The interplay between food intake, prebiotics and probiotics has also begun to be analyzed as well as the molecules that bacteria use to signal each other, some from one strain to another, some within the same strain. Quorum sensing (13), an important process whereby harmless bacterial populations transition in numbers and become harmful, can now be better understood. The role toll-like receptors and other PAMPS (Pathogen-Associated Molecular Patterns) as bacterial sensors linked to the innate immune system can also be better understood (14). Other receptors which line the GI tract, including all of the taste receptors and the short, medium and long chain fatty acid receptors, can be allocated to a role not just as a food sensor but also as a regulator of bacterial populations (15). One can think about whether we are predominantly feeding ourselves with our microbiome coming along for the ride versus if we are predominantly feeding our microbiome with ourselves just coming along for the ride. The truth may be in the middle, but it is worth mentioning that the prokaryotes predated eukaryotes on the evolutionary timeline. It should also be mentioned that the current promise of microbiome interventions is graphically illustrated by the clinically effective FMT (fecal matter transplant) procedure authorized for hospital use to treat C. difficile infections including antibiotic resistant C. difficile … success rates are in the high 90 percents which is way better than the current best practice antibiotic treatments of vancomycin plus metronidazole (16).

So, where are we today ? (17) We are at the tip of the iceberg. First of all, we understand little of the details underlying the various observations which connect the microbiome to health and disease, and we hope that the food and dietary supplement people, who have habitually made exaggerated claims regarding product benefits based on little to zero data, don’t cloud the process of further knowledge acquisition that needs to occur. The opportunities for new pathways and interventions to treat a variety of challenging diseases are boundless. The pathways themselves are also boundless and range from traditional pharmaceutical industry intervention involving molecule/receptor or molecule/enzyme interactions, through to prebiotics which favor a particular bacterial mix all the way to potential probiotic treatments where extraneous bacteria are added back into the mix, through to development of modified bacterial strains to address a particular set of problems, through to the “transplantation” of entire microbial communities to correct an imbalance within an individual’s microbiome. However, life scientists will need to adjust their thinking. The one molecule, one target paradigm which has dominated pharmaceutical research thinking over the past 50 years has actually rarely held 100% true when molecules got into the clinic, once ADME/Tox, off target effects and tissue distribution came into the picture. Attrition is high enough with singular molecular entities, so the bar is high when considering mixtures of microbiome to show reproducible benefits, but as the FMT process shows, it is entirely possible. It looks like a number of scenarios for microbiome intervention can involve multiple targets and combinatorial mixtures of molecules. This also opens up potentially exciting opportunities for combination therapies between small molecules, biologicals, and probiotics. Just like personalized medicine, the possibilities must be balanced with the cost and challenge of creating double blind, placebo controlled, statistically significant clinical trials. There is also the virome (the virology equivalent of the microbiome) (18) and the mycobiome (the fungal equivalent of the microbiome) (19) to consider, where work is only just beginning to understand the complexity of that aspect of what we live with. What better way to track all of that diversity than software provided by CDD!!

References

1) en.wikipedia.org/wiki/Human_Microbiome_Project
www.ted.com/talks/rob_knight_how_our_microbes_make_us_who_we_are
2) Nair P. (2012) “Woese and Fox: Life, rearranged”. Proc Natl Acad Sci U S A. 109 (4):1019-1021
Woese CR, Fox GE. (1977). “Phylogenetic structure of the prokaryotic domain: The primary kingdoms“. Proc Natl Acad Sci U S A. 74 (11): 5088–5090
Fox GE, Magrum LJ, Balch WE, Wolfe RS, Woese CR. (1977) “Classification of methanogenic bacteria by 16S ribosomal RNA characterization”. Proc Natl Acad Sci U S A. 74(10): 4537-4541
3) en.wikipedia.org/wiki/Koch’s_postulates
4) Lessa FC, Gould CV, McDonald LC (2012). “Current status of Clostridium difficile infection epidemiology“. Clinical Infectious Diseases. 55 Suppl 2: S65
5) en.wikipedia.org/wiki/Gnotobiosis
Reyniers JA (1959). “Germfree Vertebrates: Present Status“. Annals of the New York Academy of Sciences. 78 (1): 3
6) Ley RE, Bäckhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI. (2005) “Obesity alters gut microbial ecology”. Proc Natl Acad Sci U S A. 102 (31): 11070-11075
Bäckhed F, Manchester JK, Semenkovich CF, Gordon JI. (2007) “Mechanisms underlying the resistance to diet-induced obesity in germ-free mice”. Proc Natl Acad Sci U S A. 104 (3):979-984
7) en.wikipedia.org/wiki/Methanocaldococcus_jannaschii
J. Craig Venter et al (1996). “Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii“. Science. 273 (5278): 1058–1073.
8) www.genome.gov/10001772/all-about-the–human-genome-project-hgp
9) Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, Yooseph S, Wu D, Eisen JA, Hoffman JM, Remington K, Beeson K, Tran B, Smith H, Baden-Tillson H, Stewart C, Thorpe J, Freeman J, Andrews-Pfannkoch C, Venter JE, Li K, Kravitz S, Heidelberg JF, Utterback T, Rogers YH, Falcón LI, Souza V, Bonilla-Rosso G, Eguiarte LE, Karl DM, Sathyendranath S, Platt T, Bermingham E, Gallardo V, Tamayo-Castillo G, Ferrari MR, Strausberg RL, Nealson K, Friedman R, Frazier M, Venter JC. (2007) “The Sorcerer II Global Ocean Sampling expedition: northwest Atlantic through eastern tropical Pacific”. PLoS Biol. 5(3):e77
10) Yooseph S, Sutton G, Rusch DB, Halpern AL, Williamson SJ, Remington K, Eisen JA, Heidelberg KB, Manning G, Li W, Jaroszewski L, Cieplak P, Miller CS, Li H, Mashiyama ST, Joachimiak MP, van Belle C, Chandonia JM, Soergel DA, Zhai Y, Natarajan K, Lee S, Raphael BJ, Bafna V, Friedman R, Brenner SE, Godzik A, Eisenberg D, Dixon JE, Taylor SS, Strausberg RL, Frazier M, Venter JC. (2007) “The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families”. PLoS Biol 5(3):e16
11) hmpdacc.org
12) en.wikipedia.org/wiki/Crohn’s_disease
13) en.wikipedia.org/wiki/Quorum_sensing
www.ted.com/talks/bonnie_bassler_on_how_bacteria_communicate
14) en.wikipedia.org/wiki/Toll-like_receptor
15) Covington DK, et al. (2006) “The G-protein-coupled receptor 40 family (GPR40-GPR43) and its role in nutrient sensing”. Biochem Soc Trans. 34:770-773
16) en.wikipedia.org/wiki/Fecal_microbiota_transplant
www.youtube.com/watch?v=C9bYKd_Ffgc
17) Waldor MK, Tyson G, Borenstein E, Ochman H, Moeller A, Finlay BB, Kong HH, Gordon JI, Nelson KE, Dabbagh K, Smith H. (2015) “Where next for microbiome research ?” PLoS Biol. 13 (1):e1002050
18) en.wikipedia.org/wiki/Human_virome
19) en.wikipedia.org/wiki/Mycobiome


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