Objectives The Human Microbiome and Infectious Disease Understand - PDF document

2/3/2017 Objectives The Human Microbiome and Infectious Disease • Understand the advances in technology that allow culture ‐ independent study of the microbiome • Understand limitations of microbiome studies Microbiomology 101 • Describe what we have learned, esp as it relates to ID Joanne Engel, M.D., Ph.D. Chief, Division of Infectious Disease Director, Microbial Pathogenesis and Host Defense Program UCSF Thanks to Drs. Susan Lynch and Michael Fischbach for some of the slides Technical advance: PCR ‐ based 16S The human microbiome rRNA gene sequencing • Most of the human microbiota are not culturable • 16S rRNA most highly conserved bacterial gene • New technologies have allowed us to quantify & – But conserved and variable regions within gene classify microbiota Universal primers – Sequence highly conserved gene (16s rRNA) with amplification C V – “Deep” sequencing directly of patient samples C – Bioinformatics • Your microbiome is your friend – Gut microbiota necessary for gut development, metabolism, Data nutrient acquisition, immune system development and function analysis • Changes in gut microbiota associated with many diseases – Obesity, IBD, AAD, C. diff, malnutrition, cancer, neurologic disease Bioinformatics: Extract DNA Amplify 16S Sequence rRNA community profile rRNA genes amplicon • Abx (temporarily) disrupt your microbiota! 1

2/3/2017 Technical advance: Inexpensive gigh ‐ Metagenomic sequencing throughput shot ‐ gun sequencing Deep sequencing/next generation sequencing • Cost has decr dramatically Log scale! • Number and length of reads improved • Sequence communities or single cell • Bioinformatics – Massive and cumulative data basis allow analysis and cataloguing of sequences Metagenomics has improved how we Metagenomics classify microorganisms • Before metagenomics – I can’t describe what I can’t culture • After metagenomics – I can sequence everything including the kitchen sink 2

2/3/2017 What sequencing can tell us New terminology • Qualitative versus quantitative changes • How many different things (taxa, lineages, OTUs • Phylotype: Environmental DNA sequence within one sample) and which ones are shared or group of sequences sharing more than between samples an arbitrarily chosen level of similarity • How many of each per sample based on a specific marker – Richness – number of observed OTU’s in a sample – Most commonly based on rRNA gene – Evenness – distribution of OTUs within a sample • Operational taxonomic unit (OTU) – Cluster of microorganisms grouped by >97% DNA similarity (rRNA gene) – OTU= ≠ species More “omics” Gnotobiotic (germ ‐ free) mice: Animal model to study microbiome • Controlled reconstitution of gut microbiome • Instill microbiota from different groups or pure cultures, feed controlled diet 3

2/3/2017 NIH Human Microbiome Project Fun things we have learned • NIH Human Microbiome Project • How many microbes on our body, • Gut microbiome in health and disease spatio ‐ temporal issues • Factors influencing gut microbiome including • How do they differ between site and/or antibiotics between individuals • Fecal transplants from the microbiome • How do they change over time or in viewpoint response to environmental changes • Antibiotic “resistome” • Is there a conserved “core” microbiome How many and what kind of bacteria: Methods The human super organism “We the People” or “we the people and microbes” • Bacteria predominate (euks 0.5%, archaea 0.8%, viruses <5.8%) • How many? – 10 bacteria for every human cell? 1:1? • What kinds – >10,000 microbial species occupy human ecosystem – Each human harbors ~ 1000 OTUs • How many different genes • 300 healthy subjects – Humans: 20,000 genes • 15 or 18 body sites – Microbiome: >100,000 genes • >11,000 primary specimens – Metabolic functions often contributed by • 1,900 reference strains Proctor, Cell Host Microbe (2011) 10 , 287 rarer phyla Proctor, Cell Host Microbe (2011) 10 , 287 4

2/3/2017 Variation within skin sites Variation between sites Grice et al, Science (2009) 324 , 1190 Grice & Segre, Nat Rev Microbiol (2011) 9 , 244 Class and order Proctor, Cell Host Microbe (2011) 10 , 287 Intrapersonal variation > Gut microbiome interpersonal variation • GI tract houses several trillion microbial cells • 9.9 million microbial genes • > 1 billion years of mammalian ‐ microbial evolution has led to interdependency • 4 main phyla: Firmicutes, Bacteroidetes >> Proteobacteria, Actinobacteria Class N Engl J Med 2016; 375: 2369 5

2/3/2017 Physiologic functions of the gut Gut microbiome is essential for microbiome health • Provide source of energy biogenesis • Maturation and continued education of the host immune response • Biosynthesize vitamins, neurotransmitters, others • Protection against pathogen overgrowth (barrier function) • Metabolize bile salts • Influence host ‐ cell proliferation and • Affect drug metabolism vascularization • Eliminate exogenous toxins • Regulate intestinal endocrine functions, neurologic signaling, bone density N Engl J Med 2016; 375: 2369 N Engl J Med 2016; 375: 2369 Microbiota: Largest endocrine organ? Microbiome as host defense McKenney and Pamer, 2015 Donia & Fischbach, Science (2015) 349 , 395 6

2/3/2017 Factors Influencing Gut Microbiome Factors Influencing Gut Microbiome Composition Composition Lifestyle/Diet Lifestyle/Diet Host genotype Environment/ Host genotype Environment/ Geography Geography Antimicrobials Antimicrobials Age Age Microbiome Microbiome Host immunity Host immunity Ecological/anatomi Ecological/anatomi al niche al niche Gut microbiome changes with age Functional capacity is “conserved” Stable in healthy adults (phylum level) Phylum Function • Metabolism • Fermentation Oral • Methanogenesis • Oxidative phosphorylation • Lipopolysaccharide Fecal biosynthesis Human Microbiome Project, Nature, 2012 N Engl J Med 2016; 375: 2369 7

2/3/2017 Gut microbiomes: 3 “enterotypes” Environment > genetics Animal fat/protein Carbohydrate ? Arumugam et al, Nature (2011) 473, 174 • Each enterotype dominated by different species and is associated with diet • Not nation ‐ or continent ‐ dependent • Suggests several preferred, balanced, and stable communities Monozygotic = dizygotic or “equilibrium states” Turnbaugh et al, Nature (2009) 457, 480 Microbiome is altered in lean vs obese Gut microbiome and obesity mice and humans Body fat accumulation Humans on diets Mice Lean humans: diverse Conventionally Conventionalized Germ-free microbiome, altered gene raised representation Germ-free mice eat more but weigh less. Ley et al. Nature. 2006 Ley et al PNAS 2005 Turnbaugh et al, Nature 2009 Backhed et al. PNAS. 2004. 8

2/3/2017 Microbiome can modulate obesity Role of microbiome in starvation • Healthy gut microbiome is required for healthy postnatal development • Renutrition may Altered Energy not be sufficient Metabolism • Reconstitute nl microbiome? – probiotics Short term changes in diet affect Pathogenesis of IBD gut microbiota • Rapid, reproducible changes as assessed by short term intervention studies with dietary restrictions – Meat consumption: incr bile ‐ metabolizing bacteria – Vegetable consumption: plant polysaccharide ‐ fermenting bacteria David et al, Nature 2014 9

2/3/2017 Gut microbiota is disrupted in IBD Short ‐ term abx in healthy pts • Sequenced rRNA from stool samples of 3 healthy pts over 10 • Dysbiosis: increase in mos period who received 2 x 5 D pathogenic bacteria Cipro Rx 6 mos apart w/concomitant decrease in • Cipro affected richness (# of beneficial bacteria different OTUs), diversity, • Decrease in diversity & evenness (# of each OTU) of stability community – Decr in anti ‐ inflammatory • Returned to baseline < 4 wks firmicutes and increase in pro ‐ after Abx stopped, but some taxa inflammatory species ( R. gnavus ) still missing > 6 mos • Dysregulated GI immune • Gut microbiome is mostly response towards microbiota “resilient” – Genetic component: defect in • But, repeat course resulted in innate immunity Dethlefsen et al, 2008, 2011 Ahmed et al, 2016 stable distinct state Fecal Microbiota Transplantation Short ‐ term Abx in hospitalized pts Garbage in =? Garbage out • Stool collected from 21 pts admitted to hospital for non ‐ digestive diseases pre and post 7 D course Abx (b ‐ lactams, FQ) • 16S rRNA sequencing quantitative and qualitative • Lots of variability in pt population and none were healthy • Did not control for previous abx exposure • Global change in community structure: abundance and composition altered – Abx increased bacterial load! – Decr in taxa complexity, Incr in Bacteroidetes Pancha et al, 2014 10