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2/3/2017 The Human Microbiome and Infectious Disease Microbiomology 101 Objectives • Understand the advances in technology that allow culture‐independent study of the microbiome • Understand limitations of microbiome studies • 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 The human microbiome • Most of the human microbiota are not culturable • New technologies have allowed us to quantify & classify microbiota Technical advance: PCR‐based 16S rRNA gene sequencing • 16S rRNA most highly conserved bacterial gene – But conserved and variable regions within gene Universal primers – Sequence highly conserved gene (16s rRNA) with amplification – “Deep” sequencing directly of patient samples – Bioinformatics C V C • Your microbiome is your friend – Gut microbiota necessary for gut development, metabolism, nutrient acquisition, immune system development and function Data analysis • Changes in gut microbiota associated with many diseases – Obesity, IBD, AAD, C. diff, malnutrition, cancer, neurologic disease • Abx (temporarily) disrupt your microbiota! Extract DNA Amplify 16S rRNA genes Sequence rRNA amplicon Bioinformatics: community profile 1 2/3/2017 Technical advance: Inexpensive gigh‐ throughput shot‐gun sequencing Metagenomic sequencing • Cost has decr dramatically • Number and length of reads improved • Sequence communities or single cell • Bioinformatics Log scale! Deep sequencing/next generation sequencing – Massive and cumulative data basis allow analysis and cataloguing of sequences Metagenomics Metagenomics has improved how we 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 New terminology • Phylotype: Environmental DNA sequence or group of sequences sharing more than an arbitrarily chosen level of similarity based on a specific marker – Most commonly based on rRNA gene What sequencing can tell us • Qualitative versus quantitative changes • How many different things (taxa, lineages, OTUs within one sample) and which ones are shared between samples • How many of each per sample – Richness – number of observed OTU’s in a sample – 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 Fun things we have learned • NIH Human Microbiome Project • Gut microbiome in health and disease • Factors influencing gut microbiome including antibiotics • Fecal transplants from the microbiome viewpoint • Antibiotic “resistome” Methods NIH Human Microbiome Project • How many microbes on our body, spatio‐temporal issues • How do they differ between site and/or between individuals • How do they change over time or in response to environmental changes • Is there a conserved “core” microbiome How many and what kind of bacteria: 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 • • • • 300 healthy subjects 15 or 18 body sites >11,000 primary specimens 1,900 reference strains – Each human harbors ~ 1000 OTUs • How many different genes Proctor, Cell Host Microbe (2011) 10, 287 – Humans: 20,000 genes – Microbiome: >100,000 genes – Metabolic functions often contributed by Proctor, Cell Host Microbe (2011) 10, 287 rarer phyla 4 2/3/2017 Variation between sites Variation within skin 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 > interpersonal variation Class Gut microbiome • 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 N Engl J Med 2016; 375: 2369 5 2/3/2017 Gut microbiome is essential for health • Maturation and continued education of the host immune response • Protection against pathogen overgrowth (barrier function) • Influence host‐cell proliferation and vascularization • Regulate intestinal endocrine functions, neurologic signaling, bone density N Engl J Med 2016; 375: 2369 Microbiota: Largest endocrine organ? Donia & Fischbach, Science (2015) 349, 395 Physiologic functions of the gut microbiome • Provide source of energy biogenesis • Biosynthesize vitamins, neurotransmitters, others • Metabolize bile salts • Affect drug metabolism • Eliminate exogenous toxins N Engl J Med 2016; 375: 2369 Microbiome as host defense McKenney and Pamer, 2015 6 2/3/2017 Factors Influencing Gut Microbiome Composition Factors Influencing Gut Microbiome Composition Lifestyle/Diet Lifestyle/Diet Environment/ Geography Host genotype Antimicrobials Host immunity Microbiome Age Ecological/anatomi al niche Environment/ Geography Host genotype Antimicrobials Host immunity Gut microbiome changes with age Ecological/anatomi al niche Functional capacity is “conserved” Stable in healthy adults (phylum level) Phylum Metabolism Fermentation Methanogenesis Oxidative phosphorylation • Lipopolysaccharide biosynthesis • • • • N Engl J Med 2016; 375: 2369 Age Microbiome Function Oral Fecal Human Microbiome Project, Nature, 2012 7 2/3/2017 Gut microbiomes: 3 “enterotypes” Animal fat/protein Carbohydrate Environment > genetics ? 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 or “equilibrium states” Monozygotic = dizygotic Turnbaugh et al, Nature (2009) 457, 480 Microbiome is altered in lean vs obese mice and humans Humans on diets Gut microbiome and obesity Body fat accumulation Mice Lean humans: diverse microbiome, altered gene representation Conventionally raised Conventionalized Germ-free 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 not be sufficient • Reconstitute nl microbiome? Altered Energy Metabolism – probiotics Short term changes in diet affect gut microbiota Pathogenesis of IBD • 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 • Dysbiosis: increase in pathogenic bacteria w/concomitant decrease in beneficial bacteria • Decrease in diversity & stability – Decr in anti‐inflammatory firmicutes and increase in pro‐ inflammatory species (R. gnavus) • Dysregulated GI immune response towards microbiota – Genetic component: defect in innate immunity Ahmed et al, 2016 Short‐term abx in healthy pts • Sequenced rRNA from stool samples of 3 healthy pts over 10 mos period who received 2 x 5 D Cipro Rx 6 mos apart • Cipro affected richness (# of different OTUs), diversity, evenness (# of each OTU) of community • Returned to baseline < 4 wks after Abx stopped, but some taxa still missing > 6 mos • Gut microbiome is mostly “resilient” • But, repeat course resulted in stable distinct state Dethlefsen et al, 2008, 2011 Fecal Microbiota Transplantation Garbage in =? Garbage out Short‐term Abx in hospitalized pts • 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 2/3/2017 Not exactly new… • B. Eiseman. “Fecal enema as an adjunct in the treatment of pseudomembranous enterocolitis”, Surgery 1958:44:854 to • “re‐establish the balance of nature”…”3/4 pts had immediate and dramatic responses”…”this simple yet rational therapeutic method should be given more extensive clinical evaluation” FMT restores diversity • Phylum analysis of 3 controls, 4 pts CDI, 3 pts recurrent CDI • Stool flora largely intact during initial CDI infxn • Pts w/recurrent CDI lost diversity, esp bacteroides phylum Less diversity = recurrent CDI Gut “resistome” Diversity • Total number of antibiotic resistance (ABR) genes harbored by gut microbiome • Reservoir for spread of ABR by horizontal gene transmission (transformation, transduction, conjugation with your neighbors) • Hypothesis: pts with recurrent CDI would have increased ABR genes which will be reduced after repopulating colon microbiome by FMT van Nood E et al. N Engl J Med 2013;368:407-415 Millan B et al. Clin Infect Dis 2016;62:1479-1486 11 2/3/2017 Methods • Stool samples collected from donors and from pts pre and post FMT • DNA extracted, shot‐gun sequencing, sequences aligned against bacterial taxonomy dataase and comprehensive ABR data base • >17 million reads • Presence of ABR genes confirmed by custom microarray Millan B et al. Clin Infect Dis 2016;62:1479-1486 Microbiomes Results • Pre‐FMT RCDI pts: – Proteobacteria (E. coli, Klebsiella) – number and diversity of ABR genes compared to donors and healthy controls • B‐lactamases, efflux pumps, fluoroquinolone resistance • Post‐FMT RCDI pts: – change in phylum: bacteroidetes and Firmicutes, ↓ proteobacteria – ↓number and diversity of ABR genes • Improved ABR profiles maintained for > 1 yr Millan B et al. Clin Infect Dis 2016;62:1479-1486 Pre‐FMT ABR profiles Phylum Genus Millan B et al. Clin Infect Dis 2016;62:1479-1486 Millan B et al. Clin Infect Dis 2016;62:1479-1486 12 2/3/2017 Pre and Post‐FMT ABR profiles Millan B et al. Clin Infect Dis 2016;62:1479-1486 New therapies from the human microbiota Sonnenburg & Fischbach, Sci Transl Med, 2011 Summary • Metagenomics etc allow study of unculturable human microbiome • Your microbiome is your friend – Gut microbiota necessary for gut development, metabolism, nutrient acquisition, immune system development and function • Changes in gut microbiota associated with many diseases – Obesity, IBD, AAD, C. diff, malnutrition, cancer, neurologic disease • Abx (temporarily) disrupt your microbiota! • New avenues for therapeutics 13