Microbial hitchhikers

And the leaky gut

Suzanne Devkota Ph.D. Cedars-Sinai Medical Center

Gut Microbiome Conference September 25. 2016

Defining Microbiome Research

Evolutionary Biology

(The principle)

Microbiology (Microbe)

Physiology (Host)

Ecology

Mucosal immunology

Microbiome Research

Gastroenterology

Genetics

Infectious diseases

Global Prevalence of

Diabetes- 2014

52M

7.9%

22M

5.1%

138M

8.5%

39M

11.4%

75M

8.7%

25M

8.1%

Source data: IDF Atlas 6th Edition Update, 2014 and CDC.gov

2011

2005

2000

1995

1990

1985

8% 6% 4% 0% 2%

2.7%

2.7%

3.1%

4.4%

5.6%

6.9%

2.5% 1980

Diabetes incidence in the U.S.

1980-2011

0

200,000

400,000

600,000

800,000

Denmark Canada U.K. China United States

Crohn's Disease Ulcerative Colitis

Trends in Incidence Rate of CD and UC in the US 1970-2011

6

9

12

15

Rate

of

new

ly d

iagnosed

cases p

er1

00,0

00 p

eople

1970-1979 1980-1989 1990-1999 2000-2009

Crohn’s

Disease

Ulcerative

Colitis

Prevalence and

Incidence of IBD-

Crohn’s and

Ulcerative Colitis

Nations with highest prevalence of IBD

Tota

l dia

gnosed c

ases

Source data: CCFA.org

Highest rates are in industrialized nations

• A move away from agrarian diets

• Higher energy density of foods

• More food additives

• Hyper-cleanliness

• Overuse of antibiotics

• Lack of physical activity

A shifting microbial landscape

Dietary Inputs

Fatty acids, Fibers, Amino

acids, Sugars, Food

additives, Choline, Carnitine,

Gluten, Fasting, Starvation

Gut Microbial Reactions

B. wadsworthia, B. fragilis,

SFB, A. muciniphila,

M.smithii, Clostridium cluster

XIV, Desulfovibrio

(Fungi and Viruses too!)

Mucosal Immune Responses

T- cell differentiation,

Foxp3+Treg pool and IL10

production, antimicrobial

peptides, serum amyloid A

Disease State

Byproducts/Metabolites

H2S, methane, SCFAs,

indoles, biotin, deoxycholic

acid, polysaccharide A

The mucosal barrier: our only protection from the outside world

Food particles

Bacteria, viruses, fungi Toxins

Microbial byproducts

Amerindia

United States

Malawi

Yatsunenko et al, Nature (2012)

Amerindia

United States

Malawi

Malawian

Yatsunenko et al, Nature (2012)

U.S.

Malawian

U.S.

Amerindian

Amerindian

Malawian

Sonnenburg, et al. Nature, 2016

Characteristics of the American diet

(NHANES, 2011)

• 2,618 kcal/d

• 49% carbohydrate

• 15.5% protein

• 32.8% fat

• 10.9% saturated fat (of

total kcal)

Study Diets Low-fat

(AIN-93M)

Saturated-

Milk Fat

Saturated-

Lard Fat

PUFA

(Safflower oil)

Fat (%kcal)

-Saturated

(%total)

-PUFA

Protein

Carbohydrate

Micronutrients/

Fiber

5

0.6

3.9

19

66

*ND

38

65

3.5

16

47

*ND

38

39

11

16

47

*ND

38

9

78

16

47

*ND

Fatty acid composition of common dietary fats

Milkfat Lard Safflower

Oil

4:0 Butyric 3.8

6:0 Caproic 2.3

8:0 Caprylic 1.1

10:0 Capric 2

12:0 Lauric 3.1

14:0 Myristic 11.7 1.5

16:0 Palmitic 26.2 24.8 6.5

16:1 Palmitoleic 1.9 3.1

17:0 Margaric 0.7

18:0 Stearic 12.5 12.3 2.4

18:1 Oleic 28.2 45.1 13.1

18:2 Linoleic 2.9 10 78

20:0 Arachidic 0.2

20:4 Arachadonic 0.1 0.4

20:5 EPA

22:5 DPA

22:6 DHA

Devkota et al., Nature 2012

Sartor, Nat Rev Gastro Hep. 2012

High Fat Feeding

Change Gut Flora

Increased Permeability

Increased systemic LPS/Endotoxemia

Inflammation

Diet-induced gut permeability

Metabolic Disorders

Samples: Macaques, Mice, & Humans

Unpublished (Macaque: Lean vs. Obese)

Subcut aneous

fa t

Inguina l

LN

M esent eric

LNM esent eric

Fat

Liver

Serum

St ool

Int est ine

Om ent a l

Fat

Skin

Unpublished (Mouse: Normal chow vs. HFD)

Epididym al

fa t

Subcut aneous

fa t

M esent eric

Fat

St ool

Int est ine

Unpublished (Human: Healthy vs. T2DM)

Subcut aneous

fa t

Om ent a l

Fat

Adipose tissue bacterial culture

16 weeks

High Fat Diet

Animals: (n = 22)

Adipose depot 1,000 ul PBS

+

sterile beads

Homogenize

800 ul PBS

Plate

Aerobic Anaerobic

5 days

(37oC)

Readout 1:

- CFU

Readout 2:

- Pool colony to ID taxa via 16S sequencing

High Fat Diet

vs.

Normal Chow

Sample Collection

Gut contents:

- Stool

Adipose:

- Mesenteric

- Inguinal

- Subcutaneous

Live, culturable bacteria in adipose! Mesenteric > subcutaneous > inguinal

Marta Wlodarska (Broad/Novartis) & Koji Yasuda (Harvard School of Public Health)

Unpublished

108 – 1010 / gram of stool Gut

102 – 104 / gram of fat Adipose

MES SQ ING1

10

100

1000

10000

100000

CF

U/g

ram

fa

t

Aerobic

anaerobic

Could Bifidobacteria be conferring beneficial effects in adipose?

Greater enrichment of anaerobic species in HFD adipose

anaerobic aerobic

Bifidobacterium

Bacteroides

Parabacteroides

Bacteroidets

Staphylococcus

Enterococcus

Lactobacillus

Lactococcus

Streptococcae

Clostridiacae

Peptostreptococcae

Allobaculus

Sutterella

Enterobacteraceae

Pasturellaceae

Hemophilus

Adipose tissue 16s sequencing

16 weeks

High Fat Diet

Animals: (n = 16)

High Fat Diet

vs.

Normal Chow

Sample Collection

Gut contents:

- Stool

- Colon

- Cecum

- Ileum

Adipose:

- Mesenteric

- Inguinal

- Subcutaneous

Adipose depot

DNA extraction

PCR amplification

(V4 region of 16S)

Miseq sequencing

AGCTAGCT

…GTC

TTCGATTC…

AAA

TCGATCGG

…GCG

CCGATCGC

…

Figure S4, related to Experimental Procedures. Study design and survey of primate gut microbial biogeography and microbial functional

potentials with and without Helicobacter A) Paired intestinal mucosal and lumenal contents were collected from the ileum, ascending, transverse,

and descending colon of 15 clinically-healthy rhesus macaques, in addition to stool and a sample of jejunal mucosa. The microbiome of the samples

was profiled by sequencing the V4 region of the 16S rRNA gene. B) After sequencing, community structure, function, and covariation with

biogeography were characterized by ordination (Caporaso et al., 2010), univariate (Segata et al., 2011a) and multivariate (Morgan et al., 2012)

association testing, metagenomic inference (Langille et al., 2013), and logistic regression. C) Comparison of the gastrointestinal tracts of humans and

rhesus macaques. In contrast to macaques, humans lack a prominent cecum. The total length of the GI tract is 6-7 m for an adult human and 1.5-2m

for an adult rhesus macaque. Comparison of intestinal microbial mass (Solnick et al., 2006; Walter and Ley, 2011), pH (Mercier et al., 2007; Walter

and Ley, 2011) and transit time (Dubois et al., 1977; Mercier et al., 2007). Percent of intestinal length is normalized to an intestinal length of 6.5 m

for humans and 1.75 m for a macaque, for comparison purposes. D) PICRUSt (Langille et al., 2013) was used to infer community function, and

LEFSe (Segata et al., 2011b) was used to determine which functions were most differential between the mucosa and lumen and LI and SI. Due to the

high abundance of Helicobacter, this analysis was repeated with Helicobacter removed. The ten largest LDA effects are shown here. The top and

bottom two panels are derived from 16S data including Helicobacter, and excluding Helicobacter OTUs, respectively.!

Microbial communities are determined primarily by diet and location

HFD

Control Gut

Adipose

High-fat vs. Low-fat Gut vs. Adipose

Pronounced HFD-induced changes in genus across body sites R

ela

tive a

bundance (

%)

Ileum Cecum Colon Stool Mesenteric Fat Inguinal Fat SQ Fat

In humans, diabetic adipose microbiota appear indistinguishable from healthy

T2DM

Normal

Omental fat (PC1 vs. PC2)

Subcutaneous fat (PC1 vs. PC2)

T2DM

Normal

Frozen samples, no diet records, no gut comparison

Extr

acti

on

P

CR

P

osit

ive

No

rmal o

men

tal

T2D

M o

men

tal

No

rmal sq

T2D

M s

q Human fat

Pre

-t2D

M(5

), u

nko

wn

(3),

T1 (

1),

om

Pre

-t2D

M(5

), u

nko

wn

(3),

T1 (

1),

sq

Macaque fat Macaque gut

Liv

er

ILN

ML

N

Seru

m

Skin

Macaque Mouse fat gut

Genus level relative abundance without contaminant

without Ralstonia

24

Prevalence of oligotypes among different tissue types and

hosts

HUMAN: Bacteria_unclassified_phylum MACAQUE: Streptococcus

Bacterial taxa absent in controls but are unique to host species MACAQUE:

Christensenellaceae

25

Prevalence of oligotypes among different tissues and hosts

Turicibacter oligotype 1 Turicibacter oligotype 2

Two similar taxa behaving differently across sample types

Blood

Adipose and stool collection from gastric bypass patients

Study Workflow

Human Adipose Tissue Microbiome – 2016P- 000259 Color designation = study staff responsible: =AV =GC =AV+GC =KY =Misc.

Visit 1:

Pre-Op: Input Description: Output Follow-Up

Source document - questionnaire

2 copies, consent form Pre-op sample collection

kit: Saliva & Stool kit Instructions Biohazard bag Freezer packs

Blood work CBC, A1C, CMP, lipids

Identify potential patients Fill out questionnaire Consent patients Provide collection kit Order pre-op blood work

if not typical

Scan consent Scan questionnaire Input data into

REDCap

Visit 2:

Morning of:

Input Description Output Follow-Up

Verify pre-qualifying exclusionary criteria questions

Blood work CBC, A1C, CMP, lipids

Post-op sample collection kit: Saliva kit & Stool kit Instructions Biohazard bag Freezer packs

Order pre-op blood work if not typical

Collect saliva & stool samples from patient

Pre-op samples Saliva sample Stool sample Blood sample

Input sample collection data into REDCap

Joslin Diabetes Ctr: Whole blood + serum: -

80°C freezer

Harvard School of PH: RNA+DNA (bact)

Intra-Op:

Input Description Output Follow-Up

Intra-op sample collection Aerobic culture tube Anaerobic culture tube Tissue collection cup

PBS, RNA later, liquid nitrogen, cryotubes

Mesenteric adipose [1-3] Near and/or far

Omental adipose [1-3] Near and/or far

Subcutaneous adipose [1-3]

Adipose tissue samples (#3-12)

PBS, RNA later, Liquid Nitrogen, cryotubes

Input sample collection data into REDCap

BWH Microbiology: Culture

Joslin Diabetes Ctr: Tissue: -80°C freezer

Harvard School of PH:

RNA+DNA (bact)

Visit 3:

Post-Op:

Input Description Output Follow-Up

Blood work CBC, A1C, CMP, lipids

Order pre-op bloodwork if not typical

Collect saliva & stool samples from patient

Post-op samples Saliva sample Stool sample Blood sample

Input sample collection data into REDCap

Joslin Diabetes Ctr: Whole blood + serum: -

80°C freezer

Harvard School of PH:

RNA+DNA (bact)

Cedars cultures

Subject Metadata

Record ID

0259-001 AGES

0259-002 BOAT

0259-003 LACE

0259-004 TOWN

0259-005 MEMO

0259-006 OATS

Gender Diabetic HbA1c BMI Waist (") Surgery DOS Probiotics Date, probx EtOH

F No 5.2 54.24331361 Sleeve 5/9/2016 No No

F No 6.1 42.86369363 Sleeve 5/25/2016 No Yes

F No 5.4 53.99506836 35 RYGB 6/8/2016 No No

F No 5.2 58.36455042 48 Sleeve 5/16/2016 No No

M Yes 7.3 76.42325685 71 Sleeve 6/6/2016 Yes 5/17/2016 No

F No 5.1 42.92883379 49 Sleeve 6/13/2016 No Yes

1 M : 5 F

1 DM : 5

No

0 Lean : 6

Obese

[2 not

obtaine

d pre-

op]

1 RYGB :

5

sleeve

1 probiotics : 5

NONE

2

EtOH

: 4

No

Smoking Stop Date PPD Age, wt gain MAX wt Pre-op Body type PRE Saliva HH:MM elapsed PRE Stool HH:MM elapsed

No 8 340 Apple Yes 0:04 Yes 4:00

Yes Yes 2009 0.25 30 260 Apple Yes 0:05 Yes 14:00

No 7 315 Apple Yes 2:00 Yes 11:00

No 9 311 Pear No No

Yes Yes 2009 1.5 1 521 Apple Yes 0:01 No

No 12 274 Apple Yes 13:00 Yes 13:00

2 Past

smoker :

4 Never

1 No PRE :

5 Yes PRE

SALIVA All w/in 24 HRS

2 No PRE

: 4 Yes

PRE

STOOL All w/in 24 HRS

Amt/Freq

4/mo

1/mo

Record ID

0259-001 AGES

0259-002 BOAT

0259-003 LACE

0259-004 TOWN

0259-005 MEMO

0259-006 OATS

Growth so far…

DOS Subject Subject Acronym BWH core ID Location BMI Sx Type Diabetic GROWTH

5/9/2016 001 AGES 1 SQ 54.33 SLEEVE

5/9/2016 001 AGES 2 Gastro-colic 54.33 SLEEVE

5/16/2016 004 TOWN 3 SQ 58.34 SLEEVE

5/16/2016 004 TOWN 4 OM 58.34 SLEEVE

5/16/2016 004 TOWN 5 Gastro-colic 58.34 SLEEVE

5/25/2016 002 BOAT 6 SQ 42.95 SLEEVE

5/25/2016 002 BOAT 7 Gastro-colic 42.95 SLEEVE

5/25/2016 002 BOAT 8 OM 42.95 SLEEVE

6/6/2016 005 MEMO 9 SQ 76.44 SLEEVE YES

6/6/2016 005 MEMO 10 GC 76.44 SLEEVE YES YES

W 6/6/2016 007 ANTS 11 SQ 25.87 CHOLECYSTECOMY

W 6/6/2016 007 ANTS 12 GC 25.87 CHOLECYSTECOMY

6/8/2016 003 LACE 13 OM 55.74 R-YGB

6/8/2016 003 LACE 14 GC 55.74 R-YGB

6/13/2016 006 OATS 15 GC 43.00 SLEEVE

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

YES

Acknowledgements

Cedars-Sinai

Connie Ha

Anthony Martin

Joslin Diabetes Center

Allison Goldfine

Brigham and Women’s Hospital

Lynn Bry

Ashley Vernon

Gemma Carter

Harvard School of Public Health

Curtis Huttenhower

Koji Yasuda

Broad Institute/Novartis

Marta Wlodarska

Funding: