AntimicrobialResistanceDetectioninIlluminaandNanopore SequencingSTEPHANIEHAO, B . OPENE, J . GURTOWSKI , M. SCHATZ, P. S IMNER, W. T IMP

JOHNSHOPKINSUNIVERSITY

AGBT2017

2

VancomycinResistanceEnterococci• IntheUnitedStates,VREiscommonlyacquiredinahealthcaresetting

• 20000rectalswabstestedforVREperyearatJHHS

• MajorOrganismsofInterest:• E.faecalis (vanB resistance)• E.faecium(vanA resistance)

O’Driscoll,Crank,2015.InfectionandDrugResistance

Species Europe US Canada Asia-Pacific Latin-America

E.faecium 8.8 79.4 22.4 14.1 48.1E.faecalis 1.0 8.5 0.1 0.01 3.1Allenterococci 4.0 35.5 6.0 11.9 12.9

%EnterococcithatareVancomycinResistant

TIMELINECOURTESYOFSIMNER,,VRECOSTSFROMHTTP://DX.DOI.ORG/10.1016/J.AJIC.2015.11.008 4

IdentificationofPathogenicOrganisms

LibraryPrepTime

Sequencing/GrowthTime

CostperSample

Culture N/A ~24-48hours $45*IlluminaMiseq 3-4hours ~14-16hours $400Oxford

Nanopore ~2 hours ~8hours $650

*CostisforChromAgar plate+PCRtestingforVRE

Procedure

OrganismDistribution

6

OrganismDistribution- VRE

7

VREdonotgenerallymakeupalargepartofthemicrobiome

OrganismDistribution- Human

8

Humancontaminationlevelsvary,butaretypicallylow.

OrganismDistribution– Other

9

Wecangetmoreinformationusingshotgunmetagenomicssequencingthanjustfromculturealone

10

Samples– Nanopore

11

KrakenwithcustomizedCARDdatabase

Klebsiella pneumoniaeCaseStudy

Hypervirulent (hypermucoviscous)K.pneumoniaeAnewvariantofKlebsiella pneumoniae

FirstdescribedintheAsianPacificRim1980s

Nowincreasinglyrecognizedinothercountries

• Definingclinicalfeatures:• Serious,life-threateningcommunity-

acquiredinfectioninyoungerhealthyhosts

• Liverabscess,pneumonia,meningitisandendophthalmitis

• Metastaticspread

Source:Shon,Rajinda,Russo2013CDChttp://www.cdc.gov/hai/organisms/cre/TrackingCRE.html#CREmapOXA

SIMNER,HAO,TIMP,INPREPARATION2017 14

SIMNER,HAO,TIMP,INPREPARATION2017 15

• Twostrains– HMVandXDR

• 9isolatessequencedonbothplatforms

16

Assemblywithcanu

17

Assemblywithsequencingfromv9.4flowcell

Nanopore Tree

18

19

GenesofInterest

Resistanceidentifiedwithin15minutes!

Sample OXA-181detection time

CTX-M-15detectiontime

4 4.8min 2.8min

8 10.3min 3.6min

9 10.73min 9.37min

10 0.99min 1.00min

12 13.07 min 6.04min

AntibioticResistanceDetection

ConclusionsSequencingcanprovidemoreinsightintoenvironmentalcontextoforganismsthanjustculturealoneNanopore sequencingcouldbeusefulasanaidinprovidingpropertreatmentforinfectiousdiseases◦ Getfullcoverageofpathogenicorganismswithoneflowcell◦ Rapidtimeofdetectionofgenesofinterest

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AcknowledgementsTimp Lab◦ Isac Lee◦ RachaelWorkman◦ Yunfan Fan◦WinstonTimp

JHMIInfectiousDiseases◦ PatriciaSimner◦ Belita Opene◦ AnnieAntar

SchatzLab◦MichaelSchatz◦ JamesGurtowski

Salzberg Lab◦ FlorianBretweiser