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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MAB - Between Patient SNPs

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M. abscessus Genetic Diversity

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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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