monitoring and modifying the microbiome in the immunocompromised...
TRANSCRIPT
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