The Human Microbiome

1. The Human Microbiome John Lunz, PhD Assistant Professor Department of Pathology

2. Microbiome • Generalized term describing the microbial populations that inhabit various anatomical niches. • The anatomic location is the primary determinant of the microbial variation. • Tissues/organs exposed to the environment are host sites for microbes. • The population of microbes out numbers our own cells 10 to 1.

3. Not Just Bacteria • Microbiome can include: • Bacteria • Viruses • Fungi • Others • Bacteriophages

4. Microbiome Cho and Blaser 2012, Nat Rev Genetics

5. The Many Influences of the Microbiome • Recognized that microbes have profound effects at every anatomical location. • Local • Direct contact of the microbiome with tissues • Helicobacter pylori stomach infection associated with gastric ulcer • Gut flora changes linked to inflammatory bowel disease • Global • Changes or perturbations in the microbiome effecting other systems • Germ-free mice – altered hematopoietic composition • Altered gut flora associated with global immunological changes • Gut bacterial products impacting the liver

6. Autoimmune diseases associated with microbial disruption. Vrieze et al (2013) Best Practice and Research Clinical Gastroenterology

7. Identifying the Constituents of the Microbiome • Microbiome can be classified according to: • Phylogeny • Using classical taxonomy • Function • Defining the metagenetics of the microbiome

8. Bacterial diversity in twins segregated by phylum Cho and Blaser 2012, Nat Rev Genetics

9. Cluster of Orthologous Group pathway analysis of the microbiome metagenetics Cho and Blaser 2012, Nat Rev Genetics

10. EnterotypesCombining phylogeny and genetics • Arumugam et al (Nature 2011) Phylogenetic profiles combined with metagenetic analysis of 33 gut microbial samples. • Sequencing results were used for principal component analysis and multi-dimensional cluster analysis. • 33 samples were from a number of ethnicities. • The goal was to see if gut flora across populations and individuals could be clustered, like blood groups.

11. Phylogenetic diversity in the gut microbiome Arumugam et al (Nature 2011)

12. Metagenomic distribution within the gut microbiome Arumugam et al (Nature 2011)

13. 3 enterotypes Analysis were able to divide samples into 3 enterotypes based on phylogeny and function. Unrelated to nationality or host characteristics (body mass, age, gender). Arumugam et al (Nature 2011)

14. Constant Competition in the Microbiome • In any anatomical niche, the microbial population is a diverse and complex ecosystem. • Maintenance of this ecosystem is dependent on interactions with the host and with other members of the population. • One microbe population might actively suppress others, and vice versa. Disruption of the ecosystem can lead to problems.

15. Studying the microbiome • Almost as complex as the microbiome itself! • Multi-disciplinary approach • Microbiology • Ecology/Evolutionary • Physiological • Immunological • Bioinformatics • Pathology • Organ/disease specialties

16. Human Microbiome Projecthttp://commonfund.nih.gov/hmp • NIH sponsored research consortium to investigate everything microbiome related.

17. Methods to identify microbiome components • Culture • Genetic

18. Methods to identify microbiome components • Culture • Classical microbiology approach to microbe identification • Isolate microbes from a niche and grow it in vitro • Used as the primary method of identification until the 1990’s • Limited approach – if it doesn’t grow, it doesn’t exist • <30% of the gut microbiome has been cultured • Permissible culture conditions may not be known for most gut bacteria populations • Some gut bacteria can only grow within a symbiotic community. These cannot be grown in isolation.

19. Methods to identify microbiome components • Genetic Approaches • Most of the diversity of the microbiome has been identified through genetic techniques. • Majority examine differences among microbes in the 16S rRNA gene.

20. Bacterial rRNA • rRNA is highly conserved among bacterial species. • But it does have variable regions that are useful in phylogenetic identification. • 16s rRNA has the best combination of conserved sites for universal primers interlaced with variable regions providing diversity. Fraher et al, 2012 Nat. Rev. Gastro

21. Utilizing 16S rRNA for bacterial identification • Many methods have probed the 16S rRNA to quantify global bacteria in a niche. • PCR • Mainly used in combination with other post-PCR techniques • Quantitative PCR (qPCR) • Quantitative property is ideal for monitoring changes in bacteria • Both of these can use “universal” primers or primers specific for a more defined phyla or even species. • Gene Sequencing

22. Utilizing 16S rRNA for bacterial identification Fraher et al, 2012 Nat. Rev. Gastro

23. DNA Fingerprinting • Methods that use denaturing gel electrophoresis or restriction enzyme digestion to establish the amount of diversity in a population. • Typically uses 16S rRNA amplification products to determine the breadth of bacteria within a sample. • Qualitative, but limited quantification. http://www.knowledgescotland.org/briefings.php?id=157

24. Gene/Genome Sequencing • Most common technique currently used to examine microbiome diversity. • Majority of techniques are based on 16S rRNA sequencing. • Sanger sequencing • Cloning bacterial 16S rRNA • Sequencing each clone • Next generation sequencing • becoming the choice method for identifying microbiome constituents • High-throughput • more quantitative

25. Pyrosequencing the Microbiome Sequences large number of DNA bases in a single run with >99% accuracy. Allows phylogenic identification and semi-quantization of bacteria in a large, mixed microbiome population. Generates a very large amount of data that necessitates a bioinformatics approach to define sequences. Fraher et al, 2012 Nat. Rev. Gastro

26. Fraher et al, 2012 Nat. Rev. Gastro

27. How to study the microbiome • Identify microbes • Association of microbe populations with disease states • Rodent models • Antibiotic treatment • Deplete microbes • Typically used in gut flora • Gnotobiotic animals • “germ-free” or selective colonization • Expensive, difficult to maintain germ-free status • But very useful • Gene knockout mice • Knockout mice can be used to study the effect of host genetics on microbiome diversity and dysbiosis.

28. Dysbiosis • Alterations of the “normal” microbiome • Dysbiotic state can be disease-causing. • Multiple ways this can occur: • Antibiotic use • Interpersonal transfer • Lifestyle change (diet) • Genetics

29. Cho and Blaser 2012, Nat Rev Genetics

30. Influence of microbiome on inflammatory processes Greer and O’Keefe 2011 Frontiers in Physiology

31. Gut microbiome • Approximately 1014 bacterial cells • 10 times more than our own cells! • Comprised of 400-500 individual species • Might be an underestimation • Initially was only thought to contribute to intestinal diseases, but now recognized as a microbial population that can effect multiple organs throughout the body.

32. Development of the gut flora Development starts at birth. Initial maternal exposure is important and is mainly lactobacilli. Post-natal development depends on multiple factors: Environment Exposure Food Antibiotic use Fully developed gut flora is evident by about 3 years of age. Development of gut flora is hypothesized to contribute to multiple aspects of development.

33. Gut Microbiome Stomach/Small Intestine Bacteria load Large Intestine/Rectum Modified from Cho and Blaser 2012, Nat Rev Genetics

34. Holmes et al 2012, Science Translational Medicine

35. Importance of the gut microbiome • Extraction of nutrients from food • Assist in digestion • Normal gut flora can be protective • Antibiotic use associated with hospital-borne infections, C. difficile

36. Transition in the gut microbiome Manichanh et al, Nature Reviews Gastroenterology and Hepatology 2012

37. Microbiome and Cancer • Some tumors arise from an “inflammatory” environment. • Potential for the microbiome to contribute to tumor development. • Predominantly studied in the gut.

38. Colonization of germ-free IL-10-/- mice with E. faecalis or E. coli

39. Some E. coli stains harbor a polyketide synthase pathogenic island that can produce a genotoxin, Colibactin. • Investigated this as a cause of tumorigenesis.

40. This study concluded/proposed a 2-hit model whereby inflammation creates an environment supportive of carcinogenesis in the host and the microbiota. • Inflammation supports growth of bacteria that may have genotoxic potential and can lead to tumor formation.

41. Interaction between host and microbiome • Illustrated the interaction between host and microbiome. • Demonstrated transferability of disease phenotype by gut bacteria.

42. TLR5 KO Mice • TLR5 senses bacterial flagellin • TLR5 KO Mice spontaneously develop “metabolic syndrome” • overweight, high blood pressure, high fasting glucose, high cholesterol, etc… • TLR5 KO mice have altered gut bacteria

43. Transferring gut bacteria from TLR5 KO to Germ Free mice transferred the disease

44. Altered gut flora Transfer to WT GF mouse Confer “disease state” by gut bacteria transfer

45. Response to injury • To determine if the changes in the cellular composition lead to altered responses to liver injury, we examined the effect of gut bacteria depletion on cold ischemia and NK T cell activation (alpha galactosylceramide).

46. 24 hr Alpha Gal treatment in mice with and without oral antibiotics Antibiotic Rx Control Drinking water contained: 1g/L neomycin, metronidazole, ampicillin 0.5g/L vancomycin

47. 40x 40x 2x 2x Control Antibiotic-treated

48. Syngeneic mouse liver transplantImpact of the gut flora on liver cold ischemia Drinking water contained: 1g/L neomycin, metronidazole, ampicillin 0.5g/L vancomycin Corbitt et al, Am. J. Pathology, In press

49. Conclusions • The microbiome is a critical part of multiple sites in and on the human body. • Microbiome contributes vastly to many physiological and pathological functions. • Studies of the microbiome are just beginning, and so far show a complex interaction. • Therapeutic modification of the microbiome is currently being considered as a strategy for treating disease resulting from dysbiosis.