Objectives The Human Microbiome and Infectious Disease Understand - - PDF document

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The Human Microbiome and Infectious Disease

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

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

The human microbiome

• Most of the human microbiota are not culturable
• New technologies have allowed us to quantify &

classify microbiota

– Sequence highly conserved gene (16s rRNA) with amplification – “Deep” sequencing directly of patient samples – Bioinformatics

• 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!

Technical advance: PCR‐based 16S rRNA gene sequencing

Extract DNA Amplify 16S rRNA genes Sequence rRNA amplicon Data analysis Bioinformatics: community profile

• 16S rRNA most highly conserved bacterial gene

– But conserved and variable regions within gene

V C C Universal primers

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Technical advance: Inexpensive gigh‐ throughput shot‐gun sequencing

Deep sequencing/next generation sequencing

• Cost has decr dramatically
• Number and length of

reads improved

• Sequence communities or

single cell

• Bioinformatics

– Massive and cumulative data basis allow analysis and cataloguing of sequences

Log scale!

Metagenomic sequencing Metagenomics

• Before metagenomics

– I can’t describe what I can’t culture

• After metagenomics

– I can sequence everything including the kitchen sink

Metagenomics has improved how we classify microorganisms

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New terminology

• Phylotype: Environmental DNA sequence
• r group of sequences sharing more than

an arbitrarily chosen level of similarity based on a specific marker

– Most commonly based on rRNA gene

• Operational taxonomic unit (OTU)

– Cluster of microorganisms grouped by >97% DNA similarity (rRNA gene) – OTU=≠species

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

More “omics”

• Controlled reconstitution of gut microbiome
• Instill microbiota from different groups or pure cultures, feed

controlled diet

Gnotobiotic (germ‐free) mice: Animal model to study microbiome

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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”

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

Methods

• 300 healthy subjects
• 15 or 18 body sites
• >11,000 primary specimens
• 1,900 reference strains

Proctor, Cell Host Microbe (2011) 10, 287

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

– Each human harbors ~ 1000 OTUs

• How many different genes

– Humans: 20,000 genes – Microbiome: >100,000 genes – Metabolic functions often contributed by rarer phyla

Proctor, Cell Host Microbe (2011) 10, 287

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Variation between sites

Proctor, Cell Host Microbe (2011) 10, 287

Variation within skin sites

Grice et al, Science (2009) 324, 1190 Grice & Segre, Nat Rev Microbiol (2011) 9, 244 Class and order

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

Physiologic functions of the gut microbiome

• Provide source of energy biogenesis
• Biosynthesize vitamins, neurotransmitters,
• thers
• Metabolize bile salts
• Affect drug metabolism
• Eliminate exogenous toxins

N Engl J Med 2016; 375: 2369

Microbiota: Largest endocrine organ?

Donia & Fischbach, Science (2015) 349, 395

Microbiome as host defense

McKenney and Pamer, 2015

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Microbiome

Host genotype Lifestyle/Diet Ecological/anatomi al niche Host immunity Environment/ Geography

Factors Influencing Gut Microbiome Composition

Age Antimicrobials

Microbiome

Host genotype Lifestyle/Diet Ecological/anatomi al niche Host immunity Environment/ Geography

Factors Influencing Gut Microbiome Composition

Age Antimicrobials

Gut microbiome changes with age

Stable in healthy adults (phylum level)

N Engl J Med 2016; 375: 2369

Functional capacity is “conserved”

• Metabolism
• Fermentation
• Methanogenesis
• Oxidative

phosphorylation

• Lipopolysaccharide

biosynthesis

Oral Fecal Human Microbiome Project, Nature, 2012 Phylum Function

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• Each enterotype dominated by different species and is

associated with diet

• Not nation‐ or continent‐dependent
• Suggests several preferred, balanced, and stable communities
• r “equilibrium states”

Gut microbiomes: 3 “enterotypes”

Animal fat/protein Carbohydrate ?

Arumugam et al, Nature (2011) 473, 174

Turnbaugh et al, Nature (2009) 457, 480

Environment > genetics

Monozygotic = dizygotic

Microbiome is altered in lean vs obese mice and humans

Ley et al. Nature. 2006 Ley et al PNAS 2005 Humans on diets Turnbaugh et al, Nature 2009 Mice

Lean humans: diverse microbiome, altered gene representation

Gut microbiome and obesity

Backhed et al. PNAS. 2004.

Body fat accumulation

Conventionally raised Conventionalized Germ-free

Germ-free mice eat more but weigh less.

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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?

– probiotics

Altered Energy Metabolism

Short term changes in diet affect 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

Pathogenesis of IBD

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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

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

Fecal Microbiota Transplantation Garbage in =? Garbage out

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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”

• 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

van Nood E et al. N Engl J Med 2013;368:407-415

FMT restores diversity

Diversity

Gut “resistome”

• 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

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

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

Microbiomes

Phylum Genus

Millan B et al. Clin Infect Dis 2016;62:1479-1486

Pre‐FMT ABR profiles

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