Download The Human Microbiome and Infectious Disease Objectives The

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
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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