Next Generation Phylogenetics Phylogenetics of Pathogenic ...

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Translational Genomics Research Institute | www.tgennorth.org

David M. Engelthaler, PhD

Associate Professor

Director, TGen North

Translational Genomics Research Institute

Phylogenetics of

Pathogenic Environmental Fungi


Next GenerationPhylogenetics

• Advances in sequencing technology allow for:

– Complete survey of genome phylogenetic markers

• SNPs, indels, genes, CNVs, LGT, etc

– Rapid and relatively inexpensive WGS allows for dozens to hundreds of genomes

– Use of multiple tools for phylogenetic understanding

• Pop structure, hybridization/introgression, recombination, incomplete lineage sorting, etc


From Genotyping to Phylotyping

• Genotyping: Linkage of isolates based on genetic similarity

– MicroSats, MLST, AFLP, RFLP, etc.

• Phylotyping: Linkage of isolates based on evolutionary relationships

– Whole Genome SNP Typing, wgMLST, cgMLST

– needs context: whole genomes and entire populations


Phylogenetics Applied

• Ancient Dispersal and Emerging Fungi

• Previously unseen and unexplored environmental fungi

• Transplant Outbreaks

• Healthcare product contamination

Population GenomicsGenomic Epidemiology

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Coccidioides

Cryptococcus

ApophysomycesExserohilum

FungalPlayers

Sarocladium


Coccidioides posadasii and C. immitis


Global DistributionCoccidioides sp. grows in the soil in arid, hot areas


Coccidioides phylogeny from microsatellite analysis

Fisher et al, PNAS 2002 8

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Translational Genomics Research Institute | www.tgennorth.orgposterior

1

0.25

0.01.02.03.04.05.06.0

SJV_6

730332_Guatemala

Tucson_17Tucson_22

SJV_8

730334_Guatemala

Nuevo_Leon_2

Phoenix_2

B0858_Guatemala

Tucson_18

Tucson_8

Tucson_15

Michoacan_1

730333_Guatemala

Washington_1

San_Antonio_1

SJV_9

Coahuila_2

GT017_ParaguayB10813_Texas

SJV_7

Tucson_11

SJV_4

Sonora_2

Phoenix_4

Phoenix_5

Tucson_6

Tucson_7

Michoacan_2

Tucson_2

Tucson_16

Tucson_10

Tucson_19

Nuevo_Leon_1

Tucson_1

Tucson_24

SJV_11

GT002_Texas

WA221_Washington_soil

Tucson_9

SJV_10

SJV_1

B0727_Argentina

B5773_Brazil

Colorado_Springs_1

San_Diego_1

Tucson_3

Phoenix_3

SJV_2

SJV_3

Tucson_13

B10757_NevadaTucson_14

Tucson_20

Phoenix_6

Tucson_23

Tucson_12

Tucson_21

B1249_Guatemala

Phoenix_7

Guerrero_1

Tucson_4

Phoenix_1

Phoenix_8

Tucson_5

Sonora_1

Phoenix_9

Coahuila_1

SJV_5

MYA

C. immitis

C. posadasii

BEAST: GTR + GStrict Constant

405,244 total SNPs

Whole Genome SNP Phylogeny

(n=70)

Engelthaler et al mBio 2016


C. posadasiiWhole GenomeSNP PhylogenyBEAST



Fixed K=3

C. posadasiiAdmixture results of BAPS population structure analysis:

Best population structure:K=3

Admixture

Arizona

Guatemala

Tex-Mex-SA



FinestructureC.p. and C.i.


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800 kya 600 400 200 Present

GABI 3 GABI 4

Drain of Cen CA Basin

Central Am.South Am.Tex-MexCentral AZSouth AZ-Sonora

Central CA ValleySouth CA-Baja MxSE Washington

Geo-spatial PhylogeneticsThe Dispersal of Coccidioides



The Washington Cocci Cases

A new endemic zone?


Field Investigation:Coccidioides identified in soil

2010: soil was collected from two locations in SE WA where patients reported potential exposures

No reliable test for identifying Coccidioides in soil existed at the time

▪ 2013: a novel, real-time PCR assay develop

▪ Detected DNA in 6/22 soil samples

• CDC improved ability to isolate from soil

• C. immitis isolated from 4/6 positive samples

Marsden-Haug et al, MMWR 2014Litvintseva, et al CID 2015

Coccidioides identified WA Patients, 2010


Whole Genome SNP Typing of C. immitis isolates

Clinical isolate was genetically indistinguishable from environmental isolates

Litvintseva et al. CID 2015

Case

Soil

Soil

Soil

Soil

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CDC202_soil

Washington_1


CDC212_soil

CDC211_soil

CDC205_soil

3000048581_CDC

3000050036_WA

San_Joaquin_Valley_2





San_Diego_1_ref

3000373807_WA

6194

209

6255

115

9464

7037

6513

7374

6543

1

0

2

2

0

3

1524

2001

2595

1946

2061

795

6652

39

159

2000

The Washington Clone

Patient

Patient

Patient


CDC202_soil

Washington_1


CDC212_soil

CDC211_soil

CDC205_soil

3000048581_CDC

3000050036_WA

1

3

0

2

2

0

115

209

159

39

5 0

The Washington “Clone”

Patient

Patient

Patient


Cryptococcus gattiiin Pacific Northwest


Cryptococcus gattiiin Pacific Northwest

• Emerged on Vancouver Island in 1999 (VGIIa)

• 2nd outbreak US: human cases in Oregon and Washington (VGIIc)

• Peripatric Black Swan Events

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Molecular divergence between the major haploid molecular types of the Cryptococcus species complex

18.5

37

9.5

C. neoformans var. grubii

C. n. var. neoformans

C. gattii

24.5

49

12.5

4.5

8.5

11.7

VNI

VNII

VNIV

VGI

VGIII

VGIV

VGII

A

D

B

C ACT1, IDE, PLB1, URA5mtRNA, ITS1/2, OMP, DOX

Molecular Ecology 2000, Vol 9(10) 1471-1481

Multiple gene genealogies reveal recent dispersion and hybridization in the human pathogenic fungus Cryptococcus neoformans

Jianping Xu1,2,*, Rytas Vilgalys2, Thomas G. Mitchell1


80000.0

WM_3032_Australia_Clinical_VGII

WM

_08_309_Australia_V

eterinary_VG

II

CD

C_

32

43

WM

_1

1_

13

9_

VG

III

B2_

Bra

zil_

Anim

al_V

GII

WM_05_530_Brazil_Clinical_VGII

WM

_05_525_B

razil_

Clin

ical_

VG

II

B9764_WA_cat_VGII

WM

_0

5_

52

7_

Bra

zil_

Clinic

al_

VG

II

B8

825

_O

R_

hum

an_

VG

IIc

A5_Brazil_Environmental_VGII

WM

_11_941_V

GIII

WM

_05_547_Brazil_C

linical_VG

II

WM

_0

5_

46

2_

Bra

zil_

Clin

ica

l_V

GII

WM_179_VGI

WM

_05_456_B

razil_

Enviro

nm

enta

l_V

GII

WM

_1846_VGIII

WM_04_78_Colombia_Clinical_VGII

WM

_182

6_V

GIII

WM_11_32_VGIII

R265

WM_779_VGIV

B9563_WA_porpoise_VGIIb

WM

_11_

950_V

GIII

WM

_0

5_

45

2_

Bra

zil_

Clin

ica

l_V

GII

WM_05_528_Brazil_Clinical_VGII

CDC_3225

WM_1802_VGIII

B11_Greece_Human_VGII

WM

_1

85

1_

Ve

ne

zu

ela

_C

lin

ica

l_V

GII

WM

_05_533_B

razil_

Clin

ical_

VG

II

WM

_05_529_B

razil_

Clin

ical_

VG

II

WM_05_274_Colombia_Clinical_VGII

WM

_05_

275_C

olombia

_Clinica

l_VG

II

WM

_04_

84_B

razi

l_C

linic

al_

VG

II

WM_09_94_Australia_Veterinary_VGII

WM

_166

7_VG

III

CD

C_

32

34

A4_Uruguay_Environmental_VGII

B8973_H

I_hum

an_V

GIIW

M_08_311_Austra

lia_Vete

rinary

_VGII

WM

_05_419_Brazil_Clinical_VGII

VGII$

VGIII$

VGIV$

VGI$

C. Gattii major molecular types VGI-VGIVWhole Genome SNP Phylogeny

1,282,876 total SNPs 544,881 p.i.CI of 0.729


PNW C. gattii – MLST vs WGST

VGIIa

VGIIb

VGIIc

Gillece et al PLoS One 2011


Cryptococcus gattii in PNW

Gillece et al PLoS One 2011

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0.01

WM

05.462 BR ST15

WM 05.461 BR ST14

IAL-3234 BR ST137

WM

05.525 BR ST41

IAL-3225 BR ST264

HL_B

11 GR

ST35

HL_

A5

BR

ST12

4W

M 0

5.45

6 B

R S

T17

WM

05.

452

BR

ST

16

WM

05.

457

BR S

T19

HL_

B12

GR

ST1

8

B89

73 H

I ST5

0

WM 08.309 AU ST48

WM 09.83 AU ST48

WM

09.152 AU ST48

B9764 WA ST21

WM

178 AU ST21

HL_A8 BR

ST185

WM

05.533 BR ST11

IAL-3243 B

R S

T283

WM

05.536 BR ST9

WM

1851 VE S

T45

HL_A

2 BR

ST28

WM

05.527 BR

ST

28

WM

04.84 BR

ST

34H

L_B2 B

R S

T248

WM

08.311 AU

ST5

WM

3032 AU

ST

33

WM

11.65 AU

ST

33

WM

05.419 BR

ST

39

WM

05.

547

BR

ST

22

WM

05.

546

BR

ST

23

WM

05.

545

BR

ST

24

HL_

A4

UY

ST

3

WM

05.

274

CO

ST

29

WM

05.

528

BR

ST

40

WM

05.

530

BR

ST

13

WM

09.

94 A

U S

T38

WM

04.

78 C

O S

T31

WM

05.

529

BR

ST2

7

WM 04.78 CO ST31

WM 05.339 CO ST43

WM 06.12 VE ST37

WM 1850 VE ST25

WM 05.342 CO ST25

WM 05.275 CO ST25

HL_A6 AW ST25

ICB-107 BR ST252

B7390 ID ST49

WM 06.10 AR ST20

B8577 BC ST20

B9757 BC ST20

B9759 BC ST20

HL_A1 BC ST20

HL_A11 BC ST20

HL_A3 BC ST20

HL_B5 BC ST20

R265 BC ST20

WM 03.697 BC ST20

WM 05.554 BR ST20

B7436 CA ST20

CA-1014 CA ST20

CBS-7750 CA ST20

HL_B6 DK (BC Travel) ST20

WM 05.432 JP (BR Travel) ST20

B6864 OR ST20

B7422 OR ST20

B7467 OR ST20

B8793 OR ST20

B8849 OR ST20

B9457 OR ST20

B9458 OR ST20

B7395 WA ST20

B8555 WA ST20

NIH-444 WA ST20

WM 03.27 AU ST7

WM 04.71 AU ST7

B9758 BC ST7

R272 BC ST7

WM 05.465 BR ST7

B9588 FL ST7

WM 2552 MY ST7

B7735 OR ST7

B8554 OR ST7

WM 06.634 TH ST7

WM 06.636 TH ST7

B7394 WA ST7

B8828 WA ST7

B9157 WA ST7

B9552 WA ST7

B9563 WA ST7

WM 04.75 TH ST30

B6863 OR ST6

B7432 OR ST6

B7434 OR ST6

B7466 OR ST6

B7491 OR ST6

B7493 OR ST6

B7641 OR ST6

B7737 OR ST6

B7765 OR ST6

B8210 OR ST6

B8214 OR ST6

B8510 OR ST6

B8788 OR ST6

B8798 OR ST6

B8825 OR ST6

B8833 OR ST6

B8843 OR ST6

B9816 OR ST6

B9933 OR ST6

GT-11-7650 OR ST6

HL_B3 OR ST6

HL_B4 OR ST6

B8838 WA ST6

B8571 WA ST6

BA

C

= South America= North America

= Australia= Europe= Asia/Oceana

VGIIa

VGIIc

Phylogenetic Network of C. gattii VGII


VGIIb


Hagen et al Plos One 2013

Souto et al. Plos NTD 2016

Billmyre et al mBio 2014


7.0

Sample_HL_B5

Cg-B7436

Cg-B8793

Cg-B7422

Cg-B6864

R265_velvet_new

Sample_HL_A11

Cg-NIH-444

Cg-B8577

Cg-B8555

Sample_HL_A3

Sample_HL_A1

Cg-B8849

Cg-CBS-7750

Sample_HL_B6

Cg-B7467

Cg-B7395

21.1008

25.1273

23.388

67.62

18.5283

22.8537

27.0509

23.1263

22.6873

22.3022

18.7634

17.5867

23.9504

9.1512

20.1359

39.0664

1971 (1967-1972)

1983(1978-1988)

Time to Most Recent Common Ancestor

VGIIa BEAST Strict Clock Analysis


Root to Tip Distance Method

y = 0.7532x - 12.593R² = 0.5599

0

5

10

15

20

25

30

35

40

45

50

0 10 20 30 40 50 60 70

Ro

ot

to T

ip D

ista

nce

fro

m N

IH44

4 n

od

e

Time after 1950

tMRCA for PNW IIa

Series1

Linear (Series1)

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Modern IIa Samples Only

y = 0.6274x - 1242R² = 0.2626

0

5

10

15

20

25

30

1960 1970 1980 1990 2000 2010 2020

Dis

tan

ce f

rom

Ou

tbre

ak N

od

e

Year

SNPs

Linear (SNPs)

The modern outbreak node is estimated at 1980 (x intercept).


9000.0

B6_Denmark_BC_visit_human_VGIIa_2007

B8838_WA_human_VGIIc_2010

B9552_WA_porpoise_VGIIb_2011

WM_09_144_Denmark_BC_visit_human_VGIIa_2006

B7735_OR_Clin_VGIIb_2009

WM_06_12_Ven_Clin_1996

WM_05_456_Brazil_Env_1994

A11_BC_human_VGIIa_2001

WM_05_525_Brazil_Clin_1997

B8554_OR_dog_VGIIb_2008

B7422_OR_cat_VGIIa_2009

B9457_OR_human_VGIIa_2011


B12_Greece_Clin_1998

B9757_BC_environment_VGIIa_2002

IAL3243_Brazil_Clin_2000

B8214_OR_human_VGIIc_2009


WM_04_75_Thailand_human_VGIIb_1993


WM_09_83_Aus_Clin_1985

B9816_OR_cat_2012

WM_06_13_USA_environment_VGIIa_1990


WM_05_554_Brazil_human_VGIIa_2002

B11766

B8833_OR_cat_VGIIc_2010

A1_BC_veterinary_VGIIa_2002



B11442


B11566B11569

B9759_BC_cat_VGIIa_2003

B11460


WM_05_77_Greece_Clin_1998


B7395_WA_dog_VGIIa_2008

B9758_BC_environment_VGIIb_2002


B11352


B8849_OR_environment_VGIIa_2010

NIH_444_WA_human_VGIIa_1972


B11468

B8555_WA_human_VGIIa_2006


B2_Brazil_Vet_2000

B11562

WM_178_Aus_Clin_1991






WM_11_65_Aus_2011

WM_05_465_Brazil_human_VGIIb_1997


B5_BC_human_VGIIa_2002



B7467_OR_porpoise_VGIIa_2009


B11_Greece_Clin_1996


WM_05_275_Colombia_Clin_2001

R265_BC_human_VGIIa_2001

B11591

B7735_OR_human_VGIIb_2009


WM_09_152_Aus_Env_2009

IAL3234_Brazil_Clin_1998



WM_09_94_Aus_2001


A4_Uruguay_Env_1989


WM_06_10_Argentina_human_VGIIa_2000

B8577_BC_environment_VGIIa_2009


WM_03_27_Australia_Env_VGIIb_1992


B8_BC_human_VGIIa_2002

B8973_HI_Clin_2010

B7394_WA_cat_VGIIb_2008

B11428

WM_04_84_Brazil_Clin_VGII_1986

B7436_CA_alpaca_VGIIa_2009




B11567

B11560

B7390_ID_human_VGIIc_2008

B8825_1_OR_human_VGIIc_2010


WM_04_78_Colombia_Clin_VGII_1998

B9588_FL_human_VGIIb_2012

B7493_OR_sheep_VGIIc_2009

WM_2552_Malaysia_human_VGIIb_1997

IAL3225_Brazil_Clin_1994WM_05_529_Brazil_Clin_1997

WM_05_274_Colombia_Clin_2002

B11441

B11473

B9764_WA_cat_2012

B9157_WA_horse_VGIIb_2011

B7765_OR_dog_VGIIc_2009

WM_06_33_Aruba_1953

WM_05_76_Greece_Clin_1996

B8793_OR_dog_VGIIa_2010

B11592

A3_BC_environment_VGIIa_2002

WM_05_432_Japan_Brazil_human_VGIIa_2000

CBS_7750_CA_environment_VGIIa_1990


WM_06_8_Uru_Env_1996


Crypto gattii VGII. R265 reference.Rooted with WM11.6586.04% quality breadth, Reference is 17,531,765 bases

VGIIc

VGIIb

VGIIa

2016 PNW Isolates


3.0

051 01 52 02 53 0

B11473


B11468









B7390_ID_human_VGIIc_2008

B11442

B9816_OR_cat_2012



B7493_OR_sheep_VGIIc_2009


B11592





B7765_OR_dog_VGIIc_2009


B11352




VGIIc BEAST AnalysisBased on 10,000 trees,1,000 were burn-inMCC tree =Maximum clade credibilitymid-point rootedTMRCA: 28.7285 years ago95% HPD Interval:[18.7836, 40.9205]


Phylogenetics of a Novel Fungus

Apophysomyces

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Apophysomyces

Joplin, MissouriMay, 2011


Killer Tornado - Joplin, MO May 22, 2011

• EF5 Tornado

• 156 Killed

• 1,100 Hospitalized

• 30-50% of Joplin destroyed

Translational Genomics Research Institute | www.tgennorth.org Translational Genomics Research Institute | www.tgennorth.org

Neblett Fanfair et al. 2012 NEJM

Day 10

PhotosCourtesyGinaWeddle,RN,MSN,CPNP-AC,TheChildren’sMercyHospital

Day 24 Day 102

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Apophysomyces spp.

28k SNPs

CI = 0.62

Joplin

Outbreak

Etienne, et al. 2012 PLoS One


Genetic vs Geographic Distance

Etienne, et al. 2012 PLoS One


Phylogenetics of a Transplant and Medical Contamination OutbreaksCoccidioides, Exserohilum and Sarocladium


Valley Fever Transplant Outbreak

• Cocci is opportunistic in organ recipients• In 2010, three solid organ recipients from a

common donor in LA all developed severe Valley Fever – two died

• No samples from donor existed for testing• Needed empirical evidence to determine if all

three were donor-derived or naturally infected

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Engelthaler et al 2011 EID

Transplant Outbreak with Coccidioides immitis


Fungal Meningitis Outbreakvia Contaminated Injectables


Fungal Meningitis - Sept 2012Exserohilum rostratum

• 13,000 people were exposed to three lots of methylprednisolone acetate (MPA)

• >750 cases of fungal meningitis or localized epidural, paraspinal, and peripheral joint infections

• 64 deaths

• Exserohilum found in patients and vials of MPA


Whole Genome SNP Typing(WGST)

• 22 E. rostratum isolates from 19 case patients

• 3 isolates from each of two contaminated lots of MPA

• 7 unrelated control E. rostratum isolates

WGST done in parallel at CDC and TGen on different platforms (GAIIx, MiSeq, HiSeq, PacBio) and with

bioinformatic tools (NASP, kSNP)

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Exserohilum meningitis from injectable steroid

Litvintseva, et al 2014. JCM

• No more than 2 SNPs between any two outbreak isolates

• ~136K SNPs to the nearest control strain


Sarocladium kilienseBlood Stream Infections


Sarocladium BSI Outbreak• In January 2014, the Chilean Ministry of Health

reported a cluster of 64 (56 peds) cases of Sarocladium kiliense bloodstream infections

– 8 different hospitals in Santiago, Chile

• All patients had received post-chemotherapy anti-nausea drug Ondansetron

• Subsequently S. killiense was cultured from vials of Ondansetron

• Concurrently, the Columbian Instituto Nacional de Salud identified 16 cases of S. kiliense, originally thought to be Fusarium


Sarocladium kiliense

3000.0

B10652, Clinical, Chile

B10971, Clinical, Colombia


B10661, Vial, Chile





B10979, Vial, Colombia

B10766, Clinical, USA







CBS155.61, B######, Soil, India







B10660, Vial, Chile




B10762, Clinical, Germany





ATCC64672, B#####, Dog, Costa Rica

CBS157.61, B#####, Soil, India


Reference

0

0

1

1693

1

0

0

0

0

2

398

0

17066

3

0

0

0

0

0

11996

13372

0

3461

13273

7171

0

0

3490

4928

1

15195

7550

0

0

01

0

0

2

1

2

4

0

0

199

3

0

2543

0

0

200

1

0

0

2

2

0

12942

0

0

581

1

0

1

440

1

12503

7838

0

0

Etienne, et al. EID 2016

• No more than 4 SNPs between any two outbreak isolates (n=25)

• Except one control (800 SNPs away), ~13K SNPs to the nearest control strain

ATCC64672, Dog, Costa Rica

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Must Haves for This Type of Research

• Global Context for Local Analysis• Multiple Tools for Phylogenetic Understanding• Extensive Resource Access

– Global collaborators– Comprehensive sample repositories– Cutting edge genomic and bioinformatic tools– Highly experienced research team with genomic,

biological, epidemiological, ecological, clinical, and informatics expertise

– Excellent funding sources


Translational Genomics Research Inst, USA

Nathan D Hicks

Chandler C Roe

John D Gillece

James M Schupp

Elizabeth M Driebe

Bridget Barker

Marcus Teixeira

Northern Arizona University, USA

Paul S. Keim

Jason Sahl

Centers For Disease Control, USA

Shawn R. Lockhart

Anastasia Litvintseva

Tom Chiller

Julie Harris

Mary Brandt

University of California Davis, USA

George R Thompson III

Arizona Department of Health Services

Victor Waddell

Kenneth Komatsu

Washington State Health Dept

Nicola Marsden-Haug

Westmead Hospital, University of Sydney, AUS

Carolina Firacative

Felix Gilgado

Tania C Sorrell

Wieland Meyer

Australian National University, AUS

Åsa Pérez-Bercoff

Gavin Huttlley

Institut Agronomique néo-Calédonien, NC

Fabian Carriconde

FIOCRUZ, Brazil

Luciana Trilles

Marcia dos Santos Lazera

Instituto Adolfo Lutz, São Paulo-SP, Brazil

Marcia S.C. Melhem

Instituto Nacional de Salud, Bogotá, Colombia

Elizabeth Castañeda

Mahidol University, Bangkok, Thailand

Popchai Ngamskulrungroj

Imperial College, UK

Matthew C. Fisher

University of Birmingham, UK

Kerstin Voelz

Robin C. May

COLLABORATORS


Thank You!

“Valley Fever”R. Dillon, 2008

Next Generation Phylogenetics Phylogenetics of Pathogenic ...

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