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Children’s Hospital Pediatric Grand Rounds
May 16, 2014
“The Role of the Microbiome: Current
Concepts“
Robert J. Genco, DDS, PhD
SUNY Distinguished Professor of
Oral Biology, Microbiology and Immunology
[email protected]
“The Human Microbiome”
I.
Composition
II. Factors Affecting Composition
III. The Microbiome in Health and
Disease
IV. Treatment and Manipulation of
the Microbiome
In 2000, Nobel Laureate: Joshua Lederberg
called for an end to the “We
good, they
evil” thinking “we should think of
each host and its parasites as a
superorganism with the
respective genomes yoked into
a chimera of sorts”.
References:
Johnson C and Versalovic J. The Human
Microbiome and Its Potential Importance to
Pediatrics. Pediatrics 129(5):950-960, 2012.
The Human Microbiome Project Consortium.
Structures, Function, and Diversity of the Healthy
Human Microbiome. Nature 486:207-214, 2012.
Petrof EO, et al., Microbial Ecosystems
Therapeutics: A New Paradigm in Medicine?
Beneficial Microbes 4(1):53-65, 2013.
Urbaniak C, et al., Microbiota of Human Breast
Tissue. Appl. Environ. Microbiol. 80(10):3007, 2014.
The Human Microbiome Project
• NIH project, launched in 2007
• Goals – identify and characterize
microorganisms associated with healthy and
diseased humans
• Methods – culture-independent, sequencing
of bacterial 16S rRNA genes
• Whole genome sequencing of entire microbial
community (microbiome)
• Informatics for data analysis
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Taxonomy Annotation
16S rRNA Gene
Annotated database is a collection of sequences
whose taxonomy assignments are already known.
Bacterial Clusters
Findings
• Enrolled 300 healthy humans
• Specimens collected from
oral cavity (6 sites)
nares
skin
GI tract
vagina
• 16SrRNA sequenced-describes diversity of
organisms 52 phyla discovered mostly uncultured
• Oral flora 1000 bacteria species found
• 100 trillion microbiological cells They out number
us 10:1
• 10 trillion mammalian cells
(Koren et al., PLOS Computational Biology 9:1, 2013)
Predominant Bacterial Phyla in the Human Body
Phylum
Characteristics
Examples
Firmicutes
Gram-positive; diverse
morphology, mainly
beneficial and
commensal
Lactobacillus,
Ruminococcus,
Clostridium,
Staphylococcus,
Enterococcus
Bacteroidetes
Gram-negative; in soil,
seawater, animal
intestines
Bacteroides,
Prevotella
Proteobacteria
Gram-negative; variety of
pathogens
Escherichia,
Pseudomonas
Actinobacteria
Gram-positive; major
antibiotic producers
Bifidobacterium,
Streptomyces,
Nocardia
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Women’s Health Initiative-Subgingival Microbiome
Healthy
Periodontal Disease
(Caporaso et al., Genome Biology 12:R50, 2011)
Predominant Organisms in Periodontitis – Previously Undetected
Species Highlighted
Bacteroides oral taxon 274, 271
Camplyobacter species
Capnocytophaga species
Cantonella morbi
Chlorotrexi
Clostridiales
Comamonadacea
Desulfovibrio SRI
Dialister species
Eubacterium species
Filifactor alocis
Fusobacterium species
Gemella morbillorum
Johnsonella species
Lachnospiraceae species
Leptotrichia species
Mogibacterium timidum
Neisseria species
Peptococcus
Peptoniphilus
Porphyromonas species
Prevotella species
Selenomonas species
Streptococcus species
Synergistes species
TM7 species
Tannerella species
Treponema species
Veillonella species
Study of the oral microbiome from the Mesolithic
period (10,000-8,000 b.c.; before farming) to the
medieval period.
(Adler CJ, et al., Nature Genetics 45:450-455, 2013)
• Calculus from 34 prehistoric skulls was
harvested.
• Sequencing of extracted bacterial DNA.
• Controlled for environmental, contaminating
DNA.
Prehistoric Plaque and the Gentrification of Europe’s Mouth
Results
• Oral microbiota shift with introduction of
farming in Neolithic period (4500-2000 BC).
• Early hunter-gatherer had fewer caries and
periodontal disease associated bacteria.
• Agricultural period led to introduction of
cariogenic and periodontal bacteria.
• Phylum-level classification of microbiome
carried out.
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Hunter-gather (Mesolithic)
Modern Samples (500-1300 AD)
• Clostridiales
• Ruminococcaceae
Farming-Population (Neolithic)
Veillonellacea (decay-cavity)
Porphyromonas gingivalis,
Tannerella and
Treponena genera
Higher level of Streptococcus mutans-decay
causing (Industrial Revolution, associated
with refined sugar from beets and sugar
cane).
Periodontal
Disease
Conclusion
Major changes in carbohydrate intake
in human history – appears to have
affected the oral microbiome leading
to shift to cariogenic and
periodontopathic flora.
Pathophysiologic Significance of the Human
Microbiome
•
•
•
•
•
•
•
•
•
•
Keep pathogens in check
Regulates immune system
Digest food
Synthesize vitamins
Linked to mood and behavior
Associated with gut disorders
Eczema
Chronic sinusitis
Dental caries, and periodontal disease
In dysbiotic state may harbor one or more of 1200
pathogens
“The Human Microbiome”
Microbiome Acquisition and Composition
I.
Composition
II. Factors Affecting Composition
III. The Microbiome in Health and
Disease
IV. Treatment and Manipulation of
the Microbiome
Mode of Birth Delivery
Nature of Feeding
Hospitalization and Gestational Age
Effects of Antibiotics
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Mode of Birth Delivery
Vaginally delivered – newborn
acquires maternal vaginal and
intestinal microbiota.
Cesarean delivered – newborn
acquires maternal skin microbiota
eg., Staphylococcus species.
Nature of Feeding
Breast Milk
Human milk oligosaccharides (HMO)
stimulate growth of Bifidobacterium
sp.
Also HMOs act as decoys → inhibit
pathogen binding to epithelial cells.
Breast-fed infants
Higher levels of Bifidobacterium and
Lactobacillus
Formula-fed infants
Higher levels of C. difficile
- Source of gut microbiome
Breast feeding may provide protection
from
Breast-fed, vaginally delivered term
infants
Reduced C. difficile and E. coli, and
enhanced colonization by beneficial
eg. Bifidobacterium spp.
allergies,
diarrhea,
necrotizing entercolitis,
obesity,
and T2 Diabetes
What is the role of beneficial microbes in
this protection?
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Effects of Diet of Microbiome
Possible Significance?
Major shifts in taxa observed in weaning to solid
foods (Koenig JE, et al., PNAS 108(S1):4578-4585,
2011).
Bacteroides – produce beneficial
molecules like polysaccharide A and
short-chain FA
Alterations in fiber and fat content of diet of
children and adults → changes in microbiome in
24 hrs (Wu GD, et al., Science 334:105, 2011).
Bacteroides – associated with animal protein and fat
Prevotella – associated with carbohydrates.
Polysaccharite A – protective in mouse
colitis
Short-Chain Fatty Acids – maintenance
of colonic epithelium, energy, reg.
immunity
Developing Infant Gut Microbiome
- Full-term, vaginally delivered healthy male.
- Monitored gut microbial composition of one
infant over 2.5 years.
↓
↓
Results
- Majority of sequences bacterial; fungi, and
viruses at low levels.
- Diversity increased over time.
- At 2.5 yr. old, microbiome resembles the
adult.
Koenig JE, et al., PNAS 108:1107-1109, 2011
Hospitalization and Gestational Age
Intestinal microbiota of preterm
infants – reduced bacterial diversity
(Rougie et al., Anaerobe, 2010),
Jacquiot et al., J. Pediatric, 2011).
Antibiotics can lead to reduction in
microbial diversity in days, and with
some antibiotic recovery is rarely
achieved!
eg. fluoroquinolone ciprofloxacin
Impact is significant in infants
Additional risks
- selection of antibiotic resistant strains
eg. C. difficile diarrhea/colitis
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“The Human Microbiome”
I.
Composition
II. Factors Affecting Composition
III. The Microbiome in Health and
Disease
IV. Treatment and Manipulation of
the Microbiome
Putative differences in the microbiome of seroconverted
versus high-risk non-diabetic persons
Role in Type I Diabetes
- Alterations in intestinal
microbiome preceed Type I
Diabetes
Property
Seroconverted
subjects
High-risk control
subjects
Dominant phylum
Bacteroidetes
Firmicutes
SCFA producers
Succinate, acetate
Butyrate
Bacterial diversity
Low
High
Functional diversity
Low
High
Genus differences
Bacteroides
Bifidobacterium
Clostridium
Faecalibacterium
Community stability
Veillonella
Lactobacillus
Low
High
(Dunne J et al., 10.1111/cei.12321 in press, 2014)
Intestinal Environment and Type I Diabetes
Gut Microbiota and Metabolic Disorders
Human Studies
Obese – more Firmicutes (Lactobacillus)
lower Bacteroidetes than lean
Firmicutes → convert indigestible
carbohydrates to short chain
fatty acids → obesity?
Vaarala O, et al., Diabetes 57:2555-2562, 2008
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Weight loss in Adolescents
Weight loss associated with increase in
High fat diet leads to inflammation and metabolic
disorders.
Bacteroides (Prevotella)
propionate
decreased lipids
lean phenotype
(Nadal et al., Int. J. Obesity, 2009)
“The Human Microbiome”
I.
Composition
II. Factors Affecting Composition
III. The Microbiome in Health and
Disease
IV. Treatment and Manipulation of
the Microbiome
Prebiotics – oligosaccharides stimulate
one or multiple beneficial gut
microbes
Probiotics – food supplements that
contain living bacteria eg.
Bifidobacteria, Lactobacilli,
Streptococci, non-pathogen
E. coli
Mostly weak and transient effects.
Cani and Delzenne. Current Pharmaceutical Design 15:1546-1558, 2009.
Can we significantly change
the flora?
Prebiotics
Probiotics
Microbial Transplantation
Microbial Transplantation
Fecal transplants – in treating C. difficile
pseudomembranous colitis.
(Eiseman et al., Surgery 1958; Borody and Khoruts,
Nat Rev Gastroenterol Hepatol, 2011)
31 studies, 376 patients
90% (65-100%) cure rate
Major Challenges
- Donor selection
- Long-term safety
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Making it better
Synthetic therapeutic probiotic mixture
MET – Microbial Ecosystem Therapeutics
- 33 cultured species from healthy donor
- favorable antibiotic resistance
- reliable in continuous culture
- administer via colonoscopy
- 2 cases of CDI “cured”
(Petrof et al., Beneficial Microbes, 2013)
Other diseases where MET may be useful
• Ulcerative colitis in children and
adolescents (Qin et al., 2010, Borody and
Campbell, 2012).
• Obesity (Greenbaum et al., 2012, Vrieze et
al., 2012) lean to obese fecal transfer
resulted in insulin sensitivity.
• Necrotizing enterocolitis in infants,
mixtures of probiotics show protection
(Hoyus 1999; Lin et al., 2005, 2008; BinNun et al., 2005).
Microbial ecosystems play a crucial role in
human health.
What do we need to know before we can
manipulate the flora to design healthy
microbiomes?
Healthy
What is healthy microbiota? How does it
vary over time?
How does ecosystem replacement occur?
What are the long-term effects?
Alcoholic
Network-based modeling to identify a core
healthy microbiome.
Breaking News!
Microbiota of Human Breast Tissue
Urbaniak C, et al., Applied and Environmental
Microbiology 80:3007, March 7, 2014.
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Summary
Breast tissue not sterile; diverse
microbiome
Proteobacteria most prominent in
Canadian and Irish samples.
Also principal phylum in human milk.
Organisms cultured from breast tissue.
Likely DNA is from viable bacteria.
Collaborators
Further study needed to determine:
a) How breast microbiome is established.
Jean Wactawski-Wende - Women’s Health
Jo Freudenheim – Breast Cancer
Mike LaMonte – Metabolic Syndrome
b) Why infections do not accompany breast
colonization.
Amy Millen – Diet
c) The extent to which the breast microbiome
contributes to the breast-fed infant flora.
Michael Buck – Molecular Genetics
d) The role of the endogenous breast
microbiome.
Karen Falkner – Clinical Trials
Yijun Sun – Bioinformatics
Ashu Sharma – Molecular Genetics
Jeff Lackner – Irritable Bowel Syndrome
Tim Murphy – Respiratory Infection
Frank Scannapieco – Respiratory Infection
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Who are we?
…. a chimera of sorts
Thank you!
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