Genomic Approaches to Investigate Nontuberculous ......Suspected TB cases that are actually NTM...
Transcript of Genomic Approaches to Investigate Nontuberculous ......Suspected TB cases that are actually NTM...
Presented by:
Michael Strong, Ph.D.
Associate Professor
Center for Genes, Environment, and Health
National Jewish Health and University of Colorado, Denver
Genomic Approaches to Investigate
Nontuberculous mycobacteria
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Michael Strong, Ph.D. declares that he has no conflicts of interest.
Current Funding:
Cystic Fibrosis Foundation
National Science Foundation
State of Colorado, Bioscience Initiative
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Learning Objectives
To better understand how next generation sequencing can be
used to characterize the genomic diversity of clinically
important NTM species and subspecies.
To better understand how NTM genomics can facilitate:
Outbreak analysis,
Putative transmission analysis,
Infectious disease control, and
Genotype to phenotype inferences and investigationsProp
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Question: How many species of nontuberculous
mycobacteria (NTM) are named and taxonomically
recognized?
A. 1 to 10
B. 11 to 50
C. 51 to 100
D. 101 to 150
E. 150 to 200
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Question: How many species of nontuberculous
mycobacteria (NTM) are named and taxonomically
recognized
E. 150 to 200 !
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M.avium
M. chimaera
M.intracellulare
M. abscessus
Red – slowly growing mycobacteria
Blue – rapidly growing mycobacteria
Web-Accessible Database of hsp65 Sequences from Mycobacterium
Reference Strains. Jianli Dai, Yuansha Chen, and Michael Lauzardo.
J Clin Microbiol. 2011 Jun; 49(6): 2296–2303
This phylogeny based on
hsp65 gene sequence
M. tuberculosis
Phylogenetic Diversity of Nontuberculous mycobacteria
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3. ,
4. ,
5.
. . Am J Respir Crit Care Med ;:-
Adjemian J, Olivier KN, Seitz AE, Holland SM, Prevots DR.
Prevalence of nontuberculous mycobacterial lung disease
in U.S. Medicare beneficiaries. Am J Respir Crit Care Med
2012;185:881-6.
Increasing Incidence of Nontuberculous
Mycobacteria, Taiwan, 2000–2008 Chih-
Cheng Lai, Che-Kim Tan, Chien-Hong Chou,
Hsiao-Leng Hsu, Chun-Hsing Liao, Yu-Tsung
Huang, Chuan-Liang Kao, Kwen-Tay Luh, and
Po-Ren Hsueh. Emerging Infectious Diseases
NTM – Increasing Prevalence
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Suspected TB cases that are actually NTM
Leveraging Advances in Tuberculosis Diagnosis and Treatment to Address Nontuberculous Mycobacterial
Disease. Raju RM, Raju SM, Zhao Y, Rubin EJ. Emerg Infect Dis. 2016 Mar;22:365-9
“NTM-associated disease is much more common than previously thought: more
common than TB in the industrialized world and likely increasing in prevalence
globally” Emerg Infect Dis. 2016 Mar;22:365-9
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NTM TransmissionSuggestive genomic evidence of transmission of NTM impacting the Cystic Fibrosis population
Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium.
Bryant JM et al. Science. 2016 354:751-757.
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Genome Proteome Interactome
OMIC approaches to Mycobacteria
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Next Generation Sequencing Platforms
Next Generation sequencing enables the rapid and robust sequencing of whole bacterial genomes.
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We can take advantage of the decrease in next-
generation sequencing costs
Cost to Sequence Human Genome
http://www.genome.gov/sequencingcosts/
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As we approach affordable bacterial genome sequencing, the challenge
remains with streamlining high throughput workflows, analysis
strategies, data archiving, and genomic data sharing .
Data
Generation
Data
Analysis
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Bacterial Preparation
Genomic DNA isolation
Genomic Library Construction
Next Generation Sequencing and Genomic Analysis
Phylogenomic Comparisons
SNP and INDEL analysis
Plasmid Analysis
Phenotypic Implications
Sequence Analysis Pipeline
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How many base pairs (A , T, C, G ) does the M. abscessus subspecies
abscessus reference genome have?
A. 1 to 10,000
B. 10,001 to 100,000
C. 100,001 to 1,000,000
D. 1,000,001 to 5,000,000
E. Over 5,000,000
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How many base pairs (A , T, C, G ) does the M. abscessus subspecies
abscessus reference genome have?
E. Over 5,000,000 !
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Mapping Sequence Reads to a Reference Genome
Verification of deletion
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NTM Phylogenomics – based on whole genome sequence comparisons
Phylogenomics of Brazilian epidemic isolates of Mycobacterium abscessus subsp. bolletii reveals relationships of global outbreak strains.
Davidson RM, Hasan NA, de Moura VC, Duarte RS, Jackson M, Strong M.
Infection Genetics and Evolution Sep 18;20C:292-297. 2013
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Genome sequencing of Mycobacterium abscessus isolates from patients in the United States and comparisons to globally diverse
clinical strains.
Rebecca M. Davidson, Nabeeh A. Hasan, Paul R. Reynolds, Sarah Totten, Benjamin Garcia, Adrah Levin, Preveen Ramamoorthy,
Leonid Heifets, Charles L. Daley, and Michael Strong. Journal of Clinical Microbiology, 52:3573-3582 (2014).
Genomic Comparisons
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NTM Whole Genome Sequencing at NJHThe Colorado CF NTM Research and Development Program
https://www.nationaljewish.org/cocfrdp
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NTM Whole Genome Sequencing at NJHThe Colorado CF NTM Research and Development Program
https://www.nationaljewish.org/cocfrdp
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Phylogenomics of M. abscessus isolates from CF patients
Population Genomics of Nontuberculous Mycobacteria Recovered from United States Cystic Fibrosis Patients.
Nabeeh A. Hasan*, Rebecca M. Davidson*, L. Elaine Epperson, Sara M. Kammlade, Rachael R. Rodger, Adrah R. Levin, Alyssa
Sherwood, Scott D. Sagel, Stacey L. Martiniano. Charles L. Daley, Max Salfinger, Jerry A. Nick, Michael Strong. BioRxiv (2019)
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Phylogenomics of M. avium isolates
5 lineages
Population Genomics of Nontuberculous Mycobacteria Recovered from United States Cystic Fibrosis Patients.
Nabeeh A. Hasan*, Rebecca M. Davidson*, L. Elaine Epperson, Sara M. Kammlade, Rachael R. Rodger, Adrah R. Levin, Alyssa
Sherwood, Scott D. Sagel, Stacey L. Martiniano. Charles L. Daley, Max Salfinger, Jerry A. Nick, Michael Strong. BioRxiv (2019)
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NTM Genomic Surveillance
Defining thresholds of similarity
SNPs
Frequency
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NTM Genomic Surveillance
Identification of
Genetically Similar
Clusters in
M. abscessus
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Outbreak / Transmission
Investigations
Notes from the Field: Mycobacterium chimaera Contamination of Heater-Cooler Devices Used in Cardiac Surgery - United States.
Kiran M. Perkins, Adrian Lawsin, Nabeeh A. Hasan, Michael Strong, Alison L. Halpin, Rachael R. Rodger, Heather Moulton-Meissner, Matthew B. Crist, Suzanne Schwartz, Julia
Marders, Charles L Daley, Max Salfinger, Joseph F. Perz. Morbidity and Mortality Weekly Report (MMWR) , 65(40);1117-1118 (2016).
Genomic Analysis of Cardiac Surgery-Associated Mycobacterium chimaera Infections, United States.
Nabeeh Hasan, L. Elaine Epperson, Adrian Lawsin, Rachael Rodger, Kiran Perkins, Alison Laufer, K. Perry, Heather Moulton-Meissener, Daniel Diekema, Matthew Crist, Joseph Perz,
Max Salfinger, Charles Daley, Michael Strong. Emerging Infectious Diseases 25:559-563 (2019).
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A. B.
Contigs: 113
Connections: 86
Length: 5,403,311 bp
Contigs: 3
Connections: 2
Length:
5,463,379 bp
Toward Complete NTM genomeswith plasmid identification
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Genome sequencing of Mycobacterium abscessus isolates from patients in the United States and comparisons to globally diverse
clinical strains.
Rebecca M. Davidson, Nabeeh A. Hasan, Paul R. Reynolds, Sarah Totten, Benjamin Garcia, Adrah Levin, Preveen Ramamoorthy,
Leonid Heifets, Charles L. Daley, and Michael Strong. Journal of Clinical Microbiology, 52:3573-3582 (2014).
Phenotypic Characterization of NTM strains
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Rifampin
z
Rifampin Binding Region
RpoB homology model
RpoB homology model with rifampin bound
A B C
F439
D441H451
I497
S456
L436
L458
Genotype to Phenotype in TB
Tuberculosis Drug Resistance Mutation Database .
Andreas Sandgren, Michael Strong, Preetika Mutukrishnan,
Brian K. Weiner, George M. Church, Megan B. Murray.
PLoS Medicine 6:2 (2009).
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Genotype to Phenotype in TB
Tuberculosis Drug Resistance Mutation Database .
Andreas Sandgren, Michael Strong, Preetika Mutukrishnan,
Brian K. Weiner, George M. Church, Megan B. Murray.
PLoS Medicine 6:2 (2009).
A74A90
Fluoroquinolone
Structure of the GyrA Dimer with DNA and quinolone modeled
Quinolone
Resistance
Determining
Region (QRDR)
A B CT80
A74
A90G88
S91
D94
S95
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Genotype to Phenotype Characterization of NTM strains
RNA polymerase B
rpoB
Gyrase A
gyrA
Beta lactamase
blaC
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Systems Biology of NTM
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Systems Biology of NTM
M. abscessus M. avium
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Human Side of Mycobacterial Disease
Network Analysis of Human Genes Influencing Susceptibility to Mycobacterial Infections
Ettie Lipner, Benjamin J. Garcia, Michael Strong. PLOS ONE. 11: e0146585, (2016).
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We actively sequence mycobacterial genomes to better
understand the diversity of clinically important NTM species.
Even within the same species and subspecies of NTM, there is
often quite a bit of genetic diversity (SNPs, plasmids, gene
content)
Phylogenomic characterization facilitates outbreak analysis,
putative transmission analysis, and infectious disease control.
Genotype to phenotype structural analysis can help us
understand genetic features influencing phenotypes, including
acquired or innate drug resistance
Systems Biology provides the “blueprint” of how the genomic
pieces (genes and proteins) work together
Summary
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Thanks to:
Nabeeh Hasan PhD,
Elaine Epperson PhD,
Sean Beagle
Rebecca Davidson PhD,
Cody Glickman
Josephina Hendrix
Jenn Honda PhD,
Jerry Nick MD
Max Salfinger MD,
Chuck Daley MD
www.StrongLab.orgProp
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