MONITORING AND MODIFYING THE MICROBIOME IN

THE IMMUNOCOMPROMISED HOST

Ami S. Bhatt, MD, PhD

Stanford | bhattlab.com

4th HIV Microbiome Workshop – Rockville, MD

October 16, 2018

Disclosures

• Scientific Advisory Board: ArcBio, January.ai

• Paid Consultant: Kaleido Biosciences, Janssen Human Microbiome Institute

• Honoraria: Illumina

• Research collaboration without funding support: 10x genomics, Illumina

• Research funding support: Agilent

• Nonprofit Boards: Global Oncology, Inc

• CLINICAL MICRO

• DEEP METAGENOMICS

• MICROBES ARE ALIVE & EVOLVING

• RNA WORLD

• A JOURNEY INTO THE UNKNOWN

CLINICAL MICROIN THE SEQUENCING ERA…

#1

STRAINS MATTER

WHO WE STUDY

• Patients with leukemia and lymphoma

• Being treated with cellular therapies– Hematopoietic Cell (Bone marrow) Transplant

– CAR-T cell therapies*

• Patients with neurodegenerative and cardiometabolic disorders

Mystery Case #1

39F with B-cell acute lymphoblastic leukemia underwent a “bone marrow” transplantation.

• 37days after transplantation, she developed fevers and chills.

• A blood culture grew Staphylococcus epidermidis.

Where did this infection come from?

nci.gov

Images from cdc.gov & healthcatalyst.com

CLABSI (Central line associate blood stream infection) COSTLY, PENALIZED

“Recovery of a pathogen from a blood culture in a patient who had central line at the time of infection or within 48 hours before development of infection…” (CDC)

Mystery Case #1, cont.

• At significant cost, the central venous catheter is removed and then replaced 2 days later.

• The patient’s hospital stay is extended by 2 days.

• She eventually clears her infection with IV antibiotics.

ARE WE SURE THE SKIN WAS THE SOURCE?

INFECTIOUS DISEASE SOURCE IDENTIFICATION

SKIN -or- GUT

?

StrainSifter

https://github.com/bhattlab/StrainSifter

• A straightforward bioinformatic tool

• Identifies highly concordant microbial strains between samples

• Compares metagenomes to bacterial isolate sequencing

S. epidermidis infection likely originated from the gut microbiome – not the skin

Tamburini, Andermann et al, Nature Medicine, in press

S. epidermidis infection likely originated from the gut microbiome – not the skin

Patient 3 stool day −32

Patient 3 stool day −33

Patient 3 stool day −27

Patient 3 stool day −41

Patient 23 BSI

Patient 12 stool day −21

Patient 12 stool day −14

Patient 14 stool day 19

Patient 25 stool day 3

Patient 25 BSI

0.1

Enterococcus faecium

Patient 3 stool day −27

Patient 3 stool day −33

Patient 3 stool day −32

Patient 3 BSI

Patient 8 stool day 0

Patient 11 BSI

Patient 9 BSI

Patient 18 stool day −16

Patient 2 stool day 0

Patient 19 stool day 0

Patient 14 stool day −9

Patient 12 stool day −42

Patient 12 stool day −21

Patient 12 stool day −14

Patient 7 stool day −51

Patient 7 BSI

0.1

Escherichia coli

Patient 3 stool day −27

Patient 19 stool day 0

Patient 13 stool day −1

Patient 4 stool day 4

Patient 4 stool day −24

Patient 24 stool day 31

Patient 14 stool day −9

Patient 14 BSI

Patient 2 stool day 0

Patient 2 BSI

0.1

Klebsiella pneumoniae

Patient 27 BSI

Patient 3 BSI

Patient 5 BSI

Patient 4 BSI

Patient 1 BSI

Patient 12 BSI

Patient 10 BSI

Patient 21 BSI

0.1

Staphylococcus aureus

Patient 15 BSI

Patient 18 BSI

Patient 13 stool day −1

Patient 13 BSI

Patient 3 stool day −33

Patient 7 stool day −9

Patient 7 stool day −16

0.1

Staphylococcus epidermidis

Patient 26 BSI

Patient 19 stool day −7

Patient 30 BSI

Patient 16 BSI

Patient 17 BSI

Patient 13 stool day −1

Patient 29 BSI

Patient 22 stool day −8

Patient 22 BSI

0.1

Streptococcus mitis

Tamburini, Andermann et al, Nature Medicine, in press

DEEP METAGENOMICS:ILLUMINATING THE DARK MATTER

#2

https://tinyurl.com/ycn4s9e5

https://tinyurl.com/y7qrrcsw

https://tinyurl.com/y9gfom9x

https://tinyurl.com/ycn4s9e5

https://tinyurl.com/y7qrrcsw

https://tinyurl.com/y9gfom9x

de novo ASSEMBLY: GENOMES from METAGENOMES

de novo ASSEMBLY: CONTIGS from METAGENOMES

• Commercially available technology• Requires only 1ng of HMW DNA• Short read accuracy• Long range information

READ CLOUDS:A PROMISING SOLUTION TO OUR GENE TRANSFER MEASUREMENT

PROBLEM

ATHENA – CUSTOMIZED SOFTWARE FOR METAGENOMIC

ASSEMBLY

HUMAN GUT MICROBIOMES: READ CLOUD SEQUENCING & ATHENA ASSEMBLY OUTPERFORMS

THE COMPETITION

Phascolarctobacterium sp.

Bishara, Moss et al, Nature Biotech, in pressIn collaboration with Serafim Batzoglou

GENOME PLASTICITY:MICROBES ARE ALIVE & EVOLVING

#3

Mystery Case #2

• 46 year-old man with myelodysplastic syndrome

• Underwent a hematopoetic cell transplantation

• Became oligodominated with blue bug

• Why???A B C D

HCT

Athena generates a complete B. caccae genome

The complete genome reveals STRUCTURAL VARIATION over time

GENE TRANSFER OF THE TRANSPOSON (IS) TURNS ON norM, AN ANTIBIOTIC RESISTANCE GENE

GENE TRANSFER OF THE TRANSPOSON (IS) TURNS ON norM, AN ANTIBIOTIC RESISTANCE GENE

RNASeq

TRANSPOSON MOVES TO TURN ON norM, AN ANTIBIOTIC RESISTANCE GENE – ENABLING BLUE BUG DOMINATION

Bishara, Moss et al,submitted

A B C D

HCT

RNA WORLD: THE TRANSLATOME & THE WILD WORLD OF STRUCTURED RNA

#4

RNA WORLDmetaRiboSeq

Fremin & Bhatt*Patent pending

RNA WORLDthe wild world of structure RNAs

Fremin & Bhatt*Patent pending

RNA WORLDthe wild world of structured RNAs

Fremin & Bhatt*Patent pending

RNA WORLDthe wild world of structured RNAs

Fremin & Bhatt*Patent pending

Fremin & Bhatt*Patent pending

RNA WORLDMassive CRISPR repeats (n=84)Ruminococcus sp. UNK.MGS-30

A JOURNEY INTO THE UNKNOWN: THE PHAGEOME

#5

Bacteriophages are tremendously abundant but frequently

overlooked in the gut microbiome

crAssphage is a highly abundant human gut phage

cross-Assembly phage discovered in silico

97 kilobase double-stranded DNA genome

Up to 22% of total community reads

Found in 73% of fecal metagenomes in MG-

RAST

Highly human gut-specific

22%

97kb

Dutilh et al, 2014

crAssphage is a highly abundant human gut phage

cross-Assembly phage discovered in silico

97 kilobase double-stranded DNA genome

Up to 22% of total community reads

Found in 73% of fecal metagenomes in MG-

RAST

Highly human gut-specific

22%

Dutilh et al, 2014

crAssphage is a highly abundant human gut phage

cross-Assembly phage discovered in silico

97 kilobase double-stranded DNA genome

Up to 22% of total community reads

Found in 73% of fecal metagenomes in MG-

RAST

Highly human gut-specific

22%

97kb

Dutilh et al, 2014

crAssphage is a highly abundant human gut phage

cross-Assembly phage discovered in silico

97 kilobase double-stranded DNA genome

Up to 22% of total community reads

Found in 73% of fecal metagenomes in MG-

RAST

Highly human gut-specific

22%

97kb

Dutilh et al, 2014

crAssphage is a highly abundant human gut phage

cross-Assembly phage discovered in silico

97 kilobase double-stranded DNA genome

Up to 22% of total community reads

Found in 73% of fecal metagenomes in MG-

RAST

Highly human gut-specific

22%

97kb

Dutilh et al, 2014

crAssphage is a highly abundant human gut phage

cross-Assembly phage discovered in silico

97 kilobase double-stranded DNA genome

Up to 22% of total community reads

Found in 73% of fecal metagenomes in MG-

RAST

Highly human gut-specific

22%

97kb

Dutilh et al, 2014

Why is crAssphage so successful and specific?

Open questions

Open questions

Where do you get

crAssphage from?

Open questions

What are the dynamics

of crAssphage

colonization?

Where do you get

crAssphage from?

Open questions

What are the dynamics

of crAssphage

colonization?

Where do you get

crAssphage from?

How does the genome

structure vary across

individuals and

populations?

crAssphage can be vertically transmitted

Cohort: Backhed et al., 2015

Stool microbiome samples from 100 mother and

infant pairs

Cohort: Backhed et al., 2015

Stool microbiome samples from 100 mother and

infant pairs

Mom and infant share phage

crAssphage can be vertically transmitted

crAssphage can be vertically transmitted

Cohort: Backhed et al., 2015

Stool microbiome samples from 100 mother and

infant pairs

Mom and infant share phage

Phage only in infant

crAssphage can be vertically transmitted

Cohort: Backhed et al., 2015

Stool microbiome samples from 100 mother and

infant pairs

Mom and infant share phage

Phage only in infant

Mom and infant phages differ

crAssphage can be vertically transmitted

Cohort: Backhed et al., 2015

Stool microbiome samples from 100 mother and

infant pairs

CrAssphage only present in infants at 4 month,

12 month timepoints

Mom and infant share phage

Phage only in infant

Mom and infant phages differ

crAssphage can be acquired via fecal microbiota transplantation

Gut microbiome data from FMT

donors and recipients

Cohort: Smillie et al., 2018

crAssphage can be acquired via fecal microbiota transplantation

Gut microbiome data from FMT

donors and recipients

Cohort: Smillie et al., 2018

crAssphage can be acquired via fecal microbiota transplantation

Gut microbiome data from FMT

donors and recipients

Donor crAssphage persists

through sampling time points

Cohort: Smillie et al., 2018

crAssphage strains are maintained over time

20 of 59 (34%) of bone marrow

transplant (BMT) patients have

crAssphage

Patients maintain the same

crassphage over time

Cohort: Bhatt, unpublished

crAssphage strains can be transmitted

Cohort: Bhatt, unpublished

Clade 1

Clade 2

crAssphage strains can be transmitted

Cohort: Bhatt, unpublished

Conserved crAssphage genome structure

Reference - Missouri

Boston 1

Boston 2

Stanford healthy

South Africa 1

South Africa 2

South Africa 3

South Africa 4

South Africa 5

Stanford BMT 1

Stanford BMT 2

Stanford BMT 3

Stanford BMT 4

Stanford BMT 5

Sam

ple

Global crAssphagedistribution

crAssphage phylogeny in six

geographically distributed

populations

Bhatt, unpublished; Smillie et al., 2018; Backhed et al., 2015; Li et al., 2016; Chu et al., 2017

Global crAssphagedistribution

crAssphage phylogeny in six

geographically distributed

populations

Bhatt, unpublished; Smillie et al., 2018; Backhed et al., 2015; Li et al., 2016; Chu et al., 2017

Global crAssphagedistribution

crAssphage phylogeny in six

geographically distributed

populations

Bhatt, unpublished; Smillie et al., 2018; Backhed et al., 2015; Li et al., 2016; Chu et al., 2017

crAssphage can be

acquired vertically and

through FMT

crAssphage strains are

stable over time & may be

transmitted in the hospital

Genome structure is

conserved across

populations

CLINICAL MICROIN THE SEQUENCING ERA…

#1

STRAINS MATTER

DEEP METAGENOMICS:ILLUMINATING THE DARK MATTER

#2

GENOME PLASTICITY:MICROBES ARE ALIVE & EVOLVING

#3

RNA WORLD: THE TRANSLATOME & THE WILD WORLD OF STRUCTURED RNA

#4

A JOURNEY INTO THE UNKNOWN: THE PHAGEOME

#5

Collaborators

Serafim Batzoglou

Andrew Rezvani

Sally Arai

Courtney Greene

Hanlee Ji

Stephen Montgomery

Mike Snyder

Elizabeth Hohmann

Anshul Kundaje

Rob Negrin

Federico Simonetta

David Miklos

Rebecca Culver

Harmony Folse

Edgar Asiimwe

Matt Buckley

Scott Hazelhurst

Venessa Sahibdeen

Ovokeraye Oduaran

Shane Norris

Steve Tollman

Michele Ramsay

The Lab

Ben Siranosian

Brayon Fremin

Chris Severyn

Eli Moss

Eunice Yang

Fiona Tamburini

Hila Sberro

Jessica Ribado

Karen Andrade

Matt Durrant

Michelle Li

Paulina Chamely

Ryan Brewster

Soumaya Zlitni

Summer Vance

Tessa Andermann

Recent “grads”Gavin Sherlock (visiting prof)Katia TkachenkoJoyce KangAlex Bishara

Our Patients

• CLINICAL MICRO – StrainSifer

• DEEP METAGENOMICS - Read Clouds + Athena

• MICROBES ARE ALIVE & EVOLVING - Transposons

• RNA WORLD – MetaRiboSeq, structured RNAs

• A JOURNEY INTO THE UNKNOWN - crAssphage