University of Groningen
Application of next generation sequencing in clinical microbiology and infection prevention
Deurenberg, Ruud H.; Bathoorn, Erik; Chlebowicz, Monika A.; Couto, Natacha; Ferdous,
Mithila; Garcia-Cobos, Silvia; Kooistra-Smid, Anna M. D.; Raangs, Erwin C.; Rosema, Sigrid;
Veloo, Alida C. M.
Published in:
Journal of Biotechnology
DOI:
10.1016/j.jbiotec.2016.12.022
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Deurenberg, R. H., Bathoorn, E., Chlebowicz, M. A., Couto, N., Ferdous, M., Garcia-Cobos, S.,
Kooistra-Smid, A. M. D., Raangs, E. C., Rosema, S., Veloo, A. C. M., Zhou, K., Friedrich, A. W., & Rossen, J. W. A.
(2017). Application of next generation sequencing in clinical microbiology and infection prevention. Journal
of Biotechnology, 243, 16-24. https://doi.org/10.1016/j.jbiotec.2016.12.022
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JournalofBiotechnology243(2017)16–24
ContentslistsavailableatScienceDirect
Journal
of
Biotechnology
jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / j b i o t e c
Application
of
next
generation
sequencing
in
clinical
microbiology
and
infection
prevention
Ruud
H.
Deurenberg
a,
Erik
Bathoorn
a,1,
Monika
A.
Chlebowicz
a,1,
Natacha
Couto
a,1,
Mithila
Ferdous
a,1,
Silvia
García-Cobos
a,1,
Anna
M.D.
Kooistra-Smid
a,b,1,
Erwin
C.
Raangs
a,1,
Sigrid
Rosema
a,1,
Alida
C.M.
Veloo
a,1,
Kai
Zhou
c,1,
Alexander
W.
Friedrich
a,
John
W.A.
Rossen
a,∗aDepartmentofMedicalMicrobiology,UniversityofGroningen,UniversityMedicalCenterGroningen,TheNetherlands
bCerte,DepartmentofMedicalMicrobiology,Groningen,TheNetherlands
cStateKeyLaboratoryforDiagnosisandTreatmentofInfectiousDiseases,CollaborativeInnovationCenterforDiagnosisandTreatmentofInfectious
Diseases,TheFirstAffiliatedHospitalofMedicineSchool,ZhejiangUniversity,Hangzhou,China
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received31October2016
Receivedinrevisedform
27December2016
Accepted28December2016
Availableonline29December2016
Keywords:
Infectionprevention
IonPGMTM
Clinicalmicrobiology
MiSeq
Nextgenerationsequencing
Wholegenomesequencing
a
b
s
t
r
a
c
t
Currentmoleculardiagnosticsofhumanpathogensprovidelimitedinformationthatisoftennot suffi-cientforoutbreakandtransmissioninvestigation.Nextgenerationsequencing(NGS)determinestheDNA sequenceofacompletebacterialgenomeinasinglesequencerun,andfromthesedata,informationon resistanceandvirulence,aswellasinformationfortypingisobtained,usefulforoutbreakinvestigation. Theobtainedgenomedatacanbefurtherusedforthedevelopmentofanoutbreak-specificscreening test.Inthisreview,ageneralintroductiontoNGSispresented,includingthelibrarypreparationandthe majorcharacteristicsofthemostcommonNGSplatforms,suchastheMiSeq(Illumina)andtheIonPGMTM (ThermoFisher).AnoverviewofthesoftwareusedforNGSdataanalysesusedatthemedicalmicrobiology diagnosticlaboratoryintheUniversityMedicalCenterGroningeninTheNetherlandsisgiven. Further-more,applicationsofNGSintheclinicalsettingaredescribed,suchasoutbreakmanagement,molecular casefinding,characterizationandsurveillanceofpathogens,rapididentificationofbacteriausingthe 16S-23SrRNAregion,taxonomy,metagenomicsapproachesonclinicalsamples,andthedetermination ofthetransmissionofzoonoticmicro-organismsfromanimalstohumans.Finally,weshareourvision ontheuseofNGSinpersonalisedmicrobiologyinthenearfuture,pointingoutspecificrequirements.
©2016TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Identification and characterization of micro-organisms that causeinfectionsarecrucialforsuccessfultreatment,recoveryand safetyofpatients.However,noteverybacterialspeciescanbe suc-cessfullyculturedinthediagnosticlaboratory, andtheavailable moleculartestsareunabletodetectemerginggeneticfeaturesin successfullyevolvingpathogens thatspreadinhumans,animals andtheenvironment. Unrecognizedpathogens caneasilycause hospitaloutbreaks,puttingpatientsatriskduringtheirhospital admissions.
During the last two decades, molecular diagnostic methods haveexperiencedarapiddevelopmentandplayedanincreasingly
∗ Correspondingauthor.
E-mailaddress:j.w.a.rossen@rug.nl(J.W.A.Rossen).
1 Theseauthorshavecontributedequallytothiswork.
importantroleinmedicalmicrobiologylaboratories(Buchanand Ledeboer,2014).Thesemethodshavereducedtheturnaroundtime fromreceivingthesampletothefinalresult,andmadeitpossible todetectnon-cultivablepathogens.However,molecularmethods needaprioriknowledgeofthelikelypathogenicspeciesthatcould bepresent in thesample. One of the molecularmethods used inmedicalmicrobiologylaboratoriesisthesequenceanalysesof genesorthewholegenomeofpathogens.
Sequenceanalysescanbeusedtoanswerdifferentdiagnostic questions,suchas thegeneticrelationshipofeither bacteriaor viruses,thedetectionofmutationsinviralorbacterialgenomes leadingtoresistanceagainstantiviralsorantibiotics,identification offungithroughsequenceanalysesofthe18Sribosomal deoxyri-bonucleicacid(rDNA)oftheinternaltranscribedspacer(ITS)region and identificationof bacteriathrough sequence analysesofthe 16SrDNA (Bush,2013; Deurenbergand Stobberingh,2008; Liu etal.,2012;Reissetal.,2000).In general,Sangersequencingis
http://dx.doi.org/10.1016/j.jbiotec.2016.12.022
Fig.1. AschematicoverviewofthegeneralworkflowofdiagnosticproceduresincludingNGSinourlaboratory.
usedforthis,precededbyamplificationofeachgeneorgenomic regionusingspecificprimers.Thesame methodcan beapplied fortheidentificationofpathogensinclinicalmaterial.However, thisapproachbecomesproblematicwhenclinicalmaterialismore complexandcontainsmultiplespecies,suchasfaecalsamples.In suchcases,resultsobtainedbySangersequencingarenotreliable andmakeithardorevenimpossibletoidentifyspecificpathogens. Furthermore,thecostofSangersequencingforthesetasksishigh, andtheturnaroundtimeislong.
TheUniversityMedicalCenterGroningen(UMCG)isoneofthe largestuniversityhospitalsinTheNetherlandswith1339bedsand morethan12,000employees.Theclinicalmicrobiology diagnos-ticlaboratoryattheUMCGreceivesaround5750samplesperyear fordetailedmolecularanalysis,ofwhichapproximately1500are subjectedtonextgenerationsequencing(NGS)usingtwoIllumina MiSeqandoneLifeTechnologiesIonPGMTMsequencers.NGSwas
introducedfor routinediagnostics in 2014,andthemajority of indicationsare outbreakinvestigationand genotypingofhighly resistant micro-organisms.NGS is requested by clinical micro-biologistsor infectious disease specialistsin collaboration with molecularmicrobiologistsandinfectioncontrolpractioners.
2. Nextgenerationsequencing
NGS allows sequencing of the whole genome of numerous pathogens in one sequence run, either from bacterial isolates of (different) patients, or from multiple species present in patient materialfrom one individual(metagenomics). Boththe investment-andtherunningcostsofNGShavedecreased dramat-icallyduringthelastdecade(Dark,2013;Sboneretal.,2011).A greatadvantageofNGSisthat,incontrasttoSangersequencing,a singleprotocolcanbeusedforallpathogensforbothidentification andtypingapplications.Therefore,thistechnologyhasbeenproven tobeusefulinmedicalmicrobiologylaboratoriesandforinfection preventionmeasures(Zhouetal.,2016).Aschematicoverviewof
thegeneralworkflowusedforNGSanalysesattheUMCGisshown inFig.1.
ForNGS,thereisnoneedfortargetspecificprimers,whichare neededforSangersequencing.Inasinglerun,thewholegenomeof apathogenissequencedatrandom.Beforesequencing, fragmen-tationofthegenomeisperformed,sincethemaximumlengtha benchtopsequencer cansequencevariesbetween100and1000 bases and thus the genome cannot be sequenced in one part (Junemannetal.,2013;Lomanetal.,2012).Anexceptiontothis arethethirdgenerationofsequencers,suchastheMinION(Oxford Nanopore)andtheSequel(PacificBiosciences),whichcangenerate largerfragments(morethan200kb).However,thesesequencers arenotyetusedintheclinicalmicrobiologylaboratory,duetotheir lackofaffordability, thelowerqualityofthesequences,andthe lowthroughput.Therefore,NGSstillrequiresthepreparationof libraries,inwhichfragmentsofDNAorRNAarefusedtoadapters andbarcodestodistinguishtheDNAofthesequencedisolatesafter sequencing,followedbyaclonalamplification,normalizationand sequencing.Forthis,arobustpreparationofthelibraries,which containsarepresentativesourceoftheDNAorRNAofthegenome underinvestigation,isneeded(Headetal.,2014).
Fragmentation can be performed in several ways, either mechanical,using,e.g.,theAdaptiveFocusedAcoustics(AFA) tech-nologyfromCovaris,followedbyadaptorligation,orenzymatic, suchaswithtransposonsasusedintheNexteraXTLibrary Prepa-ration kit from Illumina. This method has the advantage that fragmentationandfusionoftheadaptorstotheDNAorRNA frag-mentsareperformedinonestep,whichmakesiteasiertoautomate it.Besidesthat,lessinputDNAisneeded.Mechanicalfragmentation hastheadvantagethatthegenerationoftheappropriatefragment lengthislessinfluencedbyfactorspresentinthesamplethatinhibit theenzymesusedduringthelibrarypreparation,andistherefore verysuitableforlibrarypreparationsofdirectsamplematerial,such asbiopsiesandfaecessamples(Headetal.,2014).
NGSlibrariesmaycontainerrorsthatdecreasethedataquality, andthuscandisruptthedatainterpretation.Detailedknowledgeof
18 R.H.Deurenbergetal./JournalofBiotechnology243(2017)16–24
Table1
PropertiesofcurrentNGSplatforms.
Company Equipment Output/run(Gb) Maximumreadlength(bp) Reads(x106) Runningtime
Illumina MiniSeq 0.6–7.5 2×150 25 4–24h
Illumina Miseq 0.3–15 2×300 25 5–55h
Illumina NextSeq 20–120 2×150 130/400 12–30h
Illumina HiSeq3000 125–700 2×150 2500 <1–3.5days
ThermoFisher IonPGMTM 0.03–2 200–400 0.4–5.5 2–7h
ThermoFisher Ion5STM 0.6–15 200–400 3–80 2.5–4h
ThermoFisher Ion5STMXL 0.6–15 200–400 3–80 <24h
OxfordNanopore MinION 21–42 230,000–300,000 2.2–4.4 1min–48h
PacificBiosciencesa Sequel 0.75–1.25 >20,000 370,000 30min–6h
PacificBiosciencesa RSII 0.5–1 >20,000 55,000 30min–4h
aThePacificBiosciencesdataarepersmartcell;boththeSequelandtheRSIIcanrun1–16smartcellsinonerun.
thekindoferrorsisimportanttofindwaystoavoidthe introduc-tionofsucherrorsandforthecorrectinterpretationoftheNGSdata. Almostallseparatestepsinthesequenceprocedurecanintroduce errors.ThisisespeciallytruewithRNAsequencingthatis techni-callymorechallengingcomparedtoDNAsequencing(Junemann etal.,2013;Lomanetal.,2012).
Atthemoment,anumberofNGSplatformsareavailable.The mostimportantpropertiesofseveralNGSplatforms,suchas out-putand fragmentlength,arepresentedin Table1(Bertelli and Greub,2013;Dark,2013;Junemannetal.,2013;Lomanetal.,2012). ThedifferentNGSplatformsusedifferentsequencingtechnologies. Illuminasequencersusesequencingbysynthesis offluorescent, reversibleterminators,and ThermoFisher sequencersuse semi-conductorsequencing that measurea change inpH duringthe incorporationof nucleotides. PacificBiosciencesusefluorescent nucleotidesintheirsinglemoleculereal-time(SMRT)technology, andOxfordNanoporeplatformsuseioniccurrentsensing,inwhich DNAisguidedthroughnano-pores,therebychangingthecurrentin awaythatisspecificforthetypeofnucleotide.Extensive informa-tiononthedifferentNGSplatformsandtheirmethodofsequencing areavailableonthecompanies’websites.Duetothetechnological developments,thecostofNGSdecreasedbetween2001and2015, whilethespeedofsequencinghasincreased(Dark,2013;Sboner etal.,2011).
3. Softwarefordataanalyses
ThebiggestchallengeconcerningtheintroductionofNGS in theclinicalmicrobiologylaboratoryisthedataanalyses. Nonethe-less,even withlittleknowledgeof bioinformatics,it ispossible toperformNGSdataanalysesfordiagnosticpurposes,usingthe numerous user-friendly software packages available (Edwards andHolt,2013).However,formore in-depthanalysis,scientific knowledgeisrequiredonthegenomicfeaturesandthebiological backgroundofthemicro-organismunderinvestigation.
Aftersequencing,thesequencedfragments(reads)canbede novoassembled(genomeassembly).Hereby,thereadsarealigned againsteachotherwithouttheuseofareferenceorganism.In gen-eral,thelargerthefragments,theeasierand moreaccuratethe genomeassembly willbe.Softwarepackages(Table2), suchas CLCGenomicWorkbench(Qiagen),SPAdesandVelvet,areusedin ourlaboratorytoassemblethegenomes.Thegeneticrelationship betweenisolatescanbeinvestigatedbyusingagene-by-gene com-parisonusingamulti-locussequencingtyping(MLST)approach, eitherbystudyingtheconserved coregenome(cgMLST),orthe wholegenome(wgMLST),whichincludesasetofvariable acces-sorygenes.Severalsoftwarepackages,suchasSeqSphere(Ridom) and BioNumerics (Applied Maths, Biomérieux), or online tools, suchasEnteroBaseandBIGSdb(BacterialIsolateGenomeSequence Database)(JolleyandMaiden,2010),canbeusedforthisapproach.
Furthermore,theuseofanestablishedcgMLSTschemeallowsthe introductionof a commonnomenclaturefor genetically related strains(deBeenetal.,2015; Kohletal.,2014;Ruppitsch etal., 2015).Atthemoment,itisnotclearhowmanyallelestwogenomes maydiffertocallthem(closetobeing)identical.Thesame prob-lemapplieswhencomparingtwogenomesusingsingle-nucleotide polymorphisms(SNP)typing(Maidenetal.,2013).However,the termgeneticdistance(theproportionofdifferentalleles,calculated bydividingthenumberofalleledifferencesbythetotalnumber ofgenessharedbytwosequences)hasbeenrecentlyintroduced, andenablesunbiasedcomparisonsfordifferentcgMLSTorwgMLST schemesaswellasthedefinitionofthresholdsbystudying col-lectionsof epidemiologically and non-epidemiologically related strains(Kluytmans-vandenBerghetal.,2016b).Anadvantageof cgMLSTandwgMLSTisthatthereiscompatibilitybetweencgMLST, wgMLSTandoldertypingmethods,sincebothBioNumericsand SeqSpheregivethesequencetype(ST)asdeterminedby conven-tionalMLSTandthespatype(incaseofStaphylococcusaureus).
SeveralpossibilitiestoanalyseNGSdatacanbefoundonthe websiteoftheCenterforGenomicEpidemiology.Forthedetection ofvirulence-andresistancegenes,VirulenceFinderandResFinder, canbeused. Alternatively,theComprehensive Antibiotic Resis-tanceDatabase(CARD)andtheVirulenceFactorDatabase(VFDB) canbeusedtoobtaindataonresistanceandvirulencegenes.With theseonlinetools,boththenon-assembledsequencedataandthe assembledgenomecanbeuploaded.However,theresultsobtained throughthesewebsitesneedsconfirmationusingothermethods. IncaseofS.aureus,ithasbeenreportedthatthereisagood corre-lationbetweenthepresenceofresistancegenesanditsphenotypic resistancepattern(Aanensenetal.,2016).
FurthercomparativegenomestudiesarepossibleusingArtemis ComparisonTool(ACT)(Carveretal.,2005),Artemis(Carveretal., 2012),andDNAplotter(Carveretal.,2009)fromtheSanger Insti-tute.
SpecificresearchquestionsrequireknowledgeofUnix-systems andtohandlethelargediversityofbioinformaticssoftware pack-ages available for it. Furthermore, software for the analysesof metagenomicsdataisavailable,suchastheMEGANAlignmentTool (Husonetal.,2007).Incontrasttothedecreasedsequencingcosts, thecostsfordatastorageanddataanalyseshaveincreasedduethe generationoflargeamountsofdata,andthecomplexityofit.
4. NGSinclinicalmicrobiology
NGSisalreadyappliedinseveralmedicalmicrobiology labo-ratories,includingourlaboratoryattheUMCG,whereitisused foroutbreakmanagement,molecularcasefinding,characterization andsurveillanceofpathogens,rapididentificationofbacteriausing the16S-23SrRNA region,taxonomy, metagenomicsapproaches
Table2
SoftwarepackagesfrequentlyusedforNGSdataanalysesinourlaboratory.
Application Software Link Note
Annotation Prokka www.vicbioinformatics.com
RAST http://rast.nmpdr.org
Assembly BioNumerics www.applied-maths.com Commercialsoftware
CLCGenomicWorkbench www.clcbio.com Commercialsoftware
SeqSphere www.ridom.de Commercialsoftware
SPAdes http://bioinf.spbau.ru/spades Unix-based
Velvet www.ebi.ac.uk/∼zerbino/velvet Unix-based
Dataqualitycheck BaseSpace https://basespace.illumina.com Commercialsoftware
BioNumerics www.applied-maths.com Commercialsoftware
CLCGenomicWorkbench www.clcbio.com Commercialsoftware
FastQC www.bioinformatics.babraham.ac.uk
Identification K-merFinder www.genomicepidemiology.org
NCBIBLAST www.ncbi.nlm.nih.gov/blast
Metagenomics MEGAN http://ab.inf.uni-tuebingen.de/software/malt
Phylogeny FastTree www.microbesonline.org/fasttree
RAxML http://sco.h-its.org/exelixis/software.html
SeqSphere www.ridom.de Commercialsoftware
SNPTree www.genomicepidemiology.org
Resistance ARDB https://ardb.cbcb.umd.edu
CARD https://card.mcmaster.ca
ResFinder www.genomicepidemiology.org
SNPcalling BioNumerics www.applied-maths.com Commercialsoftware
CLCGenomicWorkbench www.clcbio.com Commercialsoftware
Samtools www.htslib.org
SeqSphere www.ridom.de Commercialsoftware
Typing(wgMLST) BIGSdb http://bigsdb.readthedocs.io
BioNumerics www.applied-maths.com Commercialsoftware
CLCGenomicWorkbench www.clcbio.com Commercialsoftware
EnteroBase https://enterobase.warwick.ac.uk
SeqSpere www.ridom.de Commercialsoftware
Virulence VFDB www.mgc.ac.cn/VFs
VirulenceFinder www.genomicepidemiology.org
Visualisation& ACT www.sanger.ac.uk/science/tools
comparativestudy Artemis www.sanger.ac.uk/science/tools
BRIG https://sourceforge.net/projects/brig/
ClustalW www.genome.jp/tools/clustalw
DNAplotter www.sanger.ac.uk/science/tools
WebACT www.webact.org
onclinicalsamples,andthedeterminationofthetransmissionof zoonoticmicro-organismsfromanimalstohumans.
4.1. Outbreakmanagement
The advantages of using whole genome sequencing (WGS)-based typing is promoting the implementation of NGS for epidemiologicalstudiesandpublichealthinvestigations.Itis espe-ciallyimportantandhelpfulinoutbreakdetectionandmonitoring the evolution and dynamics of multi-drug resistant pathogens (ECDC,2016).Severalstudiesillustratedtheusefulnessof WGS-based typing for disclosing and tracing the dissemination of emergingpathogens.Indeed,itwasusedinourhospitalto charac-terizeanewlyemergingCTX-M-15producingK.pneumoniaeclone withsequencetype(ST)1427(Zhouetal.,2015b).Inaddition,the transmissionofaCTX-M-15-producingST15Klebsiellapneumoniae betweenpatientstreated ina singlecentreand thesubsequent inter-institutionalspread bypatientreferralhasbeentracedby genomicphylogeneticanalysis(Fig.2).Theinvestigationallowed theearlydetectionofaK.pneumoniaehigh-risk-clone(HiRiC)with prolongedcirculationintheregionalpatientpopulation(Zhouetal., 2016).Furthermore,thisstudyshowedtheusefulnessofaunique markerapproach, inwhich a clone-specificPCR wasdeveloped toinvestigatethedisseminationoftheHiRiCbetweenhealthcare centres.
In additiontooutbreaktracingand characterization,theuse of WGSalsoallows theimplementationofcontrol measuresto avoid thespread ofresistant bacterialclones.An outbreakof a colistin-resistantcarbapenemase-producingK.pneumoniae(KPC) withinter-institutionalspreadinTheNetherlands,wascontrolled bytransferringallpositive residentstoaseparatelocation out-sidetheinstitution,whereadedicatedteamcaredforthepatients (Weteringsetal.,2015).
Apartfrommultidrug-resistantbacteria,WGSisalsousefuland applicabletocharacterizehighly-virulentbacteria,suchasshiga toxin-producingEscherichiacoli(STEC)O104:H4.Thisbacterium hasemergedasanimportantpathogenandhasbeenresponsiblefor largeoutbreaks.However,therehasbeenlittleinformationabout theevolutionaryhistoryandgenomicdiversityofit.Phylogenetic analysisofoutbreakandnon-outbreakrelatedisolatesusingWGS provided an evolutionarycontext and revealed lineage-specific markers, indicative for selective pressure and niche adaptation (Zhouetal.,2015a).Inaddition,core-genomephylogenetic analy-sisofshigatoxin-producingEnteroaggregativeE.coli(EAECStx2a+) O104:H4showeddifferentclusteringanddifferentresistanceand virulence patternsdepending onthetime ofisolation (Ferdous etal.,2015).ThesestudiesreflecttheimportanceofNGSasahigh discriminatory powertool todifferentiate betweenclones with specificpropertiesandtousetheobtainedknowledgeforpatient management,infectionpreventionandevolutionarystudies.
20 R.H.Deurenbergetal./JournalofBiotechnology243(2017)16–24
Fig.2.Thetransmissionroutewasreconstructedbyepidemiologicalandgenomicdata.Eachnoderepresentsapatient,andanarrowindicatesapossibletransmission
eventfromonepatienttoanother.Thebluearrowwithsolidlinerepresentsadirecttransmissioneventsupportedbybothepidemiologicaldataandgeneticdata,theblue
arrowwithdashlinerepresentsanindirecttransmission(e.g.viaenvironment)supportedbyepidemiologicaldata,andtheredarrowindicatestheequallyparsimonious
transmissionlinkwhichcannotberesolvedbyneitherepidemiologicaldatanorgeneticdata.Theinter-institutionaltransferofthepatientisshownbydashlines,onwhich
thedistancebetweeninstitutionsisindicated.Theredstarrepresentsanoutbreakatasecondaryhospital,buttheisolateswereunavailableforfurtherresearch(Zhouetal.,
2016).(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
4.2. Molecularcasefinding
Molecularcasedefinitionsofoutbreakisolatesarecommonly usedinoutbreakinvestigations.NGSdatabasescanretrospectively besearchedforcasesincomplexandcomprehensiveoutbreaks. Thismayresultindetectionofcasesthatwouldnothavebeenfound bytraditionalepidemiologicalinvestigation.Inastudy,aNewDelhi Metallo-ß-lactamase-5 (NDM-5)-producing K. pneumoniae ST16 strainwasisolated froma Dutchpatientfroma long-termcare facilitywithoutarecenthistoryoftravelabroad.Molecularcase findingshowedthattheDutchstrainwasclonallyrelatedtostrains isolatedfromfourpatientsinDenmarkin2014(Bathoornetal., 2015),buttherewerenoobviousepidemiologicallinksbetween thecasesintheDanishand Dutchhospitals.Europeannational surveillancecentreswerecontactedformolecularcasefindingin theirNGSdatabases,butnoadditionalcasesweredetected.
Otherexamplesof molecular case findingin NGS databases havebeenreportedafterthediscoveryoftheplasmidresistance genemcr-1, responsiblefor colistinresistance, in life-stock and hospitalisedpatients.ItsintroductioninEuropeancountrieswas investigatedusingaretrospectivelysearchforthemcr-1genein NGSdatabases.ThisresultedinthedetectionofcasesinDenmark, Germany,andTheNetherlands(Falgenhaueretal.,2016;Hasman et al., 2015; Kluytmans-van den Bergh et al., 2016a). In The Netherlands, more than 2000Dutch Enterobacteriaceae isolates werescreenedwithinafewhourstorevealthepresenceofthe mcr-1gene(Kluytmans-vandenBerghetal.,2016a).So,NGSdata already presentcan beusedtoscreen for thepresence ofnew (antibioticresistance)genes(insilicoscreening).
4.3. Characterizationandsurveillanceofpathogens
Thecurrentroutineprocedureforpathogencharacterizationis basedonalargevarietyofbacteriological,biochemicaland molec-ularmethods,makingthisprocedurelaborious,time-consuming, andexpensive.NGSmayserveasaperfectone-steptooltostudy abroadrangeofpathogencharacteristicsandisapplicableona widerangeofpathogens (Aanensenetal.,2016;Fournieretal.,
2014;Hasmanetal.,2014).Knowledgeofthevirulenceprofileofa pathogeniscrucialtopredictthediseaseseverity,outcomeofthe infectionandtoallowriskassessmentduringtheearlyonsetofthe disease.WGShasthepotentialtomakeasubstantialcontribution todeterminethepresenceofvirulencefactorsusingseveralonline tools,sinceitisnotrestrictedtoaspecificgene(Franzetal.,2014; Laabeietal.,2014).
Inalargecohortstudy,WGSwasusedformolecular charac-terizationofSTEC, resultingin aclearunderstandingaboutthe populationstructureandgenomicplasticityofSTECintheregions aroundthecities Groningenand Rotterdamin TheNetherlands (Ferdousetal.,2016).Allrelevantinformationcouldbeextractedin silicofromthesequencedata,includinggenotype,serotype,MLST profile,virulenceandantibioticresistanceprofiles,andthe phylo-geneticbackgroundtoobtaintheoverallmolecularfeatureswith ahighdiscriminationamongcloselyrelatedstrains.NGSallowed tocharacterizeandcomparemanystrainsindetailwithina rela-tivelyshorttimespan.Thus,forarapidandimprovedmolecular epidemiologicalsurveillanceofpathogensatregionalandnational scale,theroleofWGSisundeniable.
NGS isalsohelpfulindetection ofnovelresistancegenesin bacteria,bothincurrentaswellasinhistoricalstraincollections. Novelvariantsofantibioticresistancegenes(ARG)canbeidentified usingNGS,andfurtherexperimentscanbeperformedtodetermine ifthesegenesareindeedresponsiblefortheobservedantibiotic resistancepattern(Nijhuisetal.,2015).
4.4. TargetedNGSofthe16S-23SrRNAclusterregionforrapid bacterialidentificationinclinicalspecimen
NGSallowsculture-freedetectionofatheoreticallyunlimited numberofpathogensandthusprovidesinsightinthefull micro-biome.Metagenomicswillbetheultimateapproachindetectingall micro-organisms(e.g.bacteria,viruses,fungi)inaclinicalsample (Hasmanetal.,2014).However,analysisoflargedatasetsrequires acombinationofbioinformaticsskillsandcomputationalresources thatisnowadaysmostlyabsentindiagnosticmedical microbiolog-icallaboratories.Furthermore,metagenomicsapproachesaretime
consumingastheturnaroundtimeisapproximatelyfourtofive days.
Tofillthegapbetweentheconventionalmethods(cultureand PCR)andmetagenomics,aculture-free approachusingtargeted NGSappearstobeanexcellentapproachtodetectand identify bacterialspecies.Comparedtometagenomics,itisfaster,less com-plicatedandcheaper,andthereforemorelikelytogetimplemented indiagnosticlaboratorieswithinashorttimeframe.The16SrRNA genesequencehasbeenproventobeareliablegeneticmarkeras itispresentinallbacteria,andthefunctionhasnotchangedover time(Patel,2001).Itcanbeapplieddirectlyonclinicalmaterials andhasproventobeavaluablesupplementarytestindailyclinical practice(Schuurmanetal.,2004;Srinivasanetal.,2012).However, highsequencesimilaritiesinthisgenebetweencertainbacterial speciesdonotalwaysleadtoanunequivocalidentification(Kalia etal.,2016).
Recently, we developed an innovative culture-free 16S-23S rRNANGSapproachforthedetectionandidentificationof bacte-rialspeciesinclinicalsamples.Themethodprovedtobesuperior toothercommonlyusedidentificationmethodsandcorrectly iden-tifiedpathogensinurinesamplesthatwerealsoidentifiedasthe causeforurinarytractinfectionswithconventionalculture(Sabat etal.,2016).Furthermore,themethodallowssimultaneous iden-tificationofseveralpathogensinclinicalmaterialsthatpreviously wouldhaveremainedunculturedandPCRnegative.Clearly,this willhaveanenormousclinicalimpactandwillhaveconsequences for patient treatment, including improvedantibiotic treatment. Finally,thismethodwillallowclinicalmicrobiologylaboratoriesto implementNGSintheirroutinediagnosticlaboratoryandtokeep upwithtechnologicalandbioinformaticsdevelopmentsrequired tobeabletoimplementmetagenomicsindiagnosticsinthefuture. 4.5. WGSandtaxonomy
Intheeighteenthcentury,Linnaeus(Linnaeus,1735)provided guidelinesforclassificationoflivingcreaturesbasedontheir phe-notypicfeatures.Acenturylater,Darwinaddedthephylogenetic component to thetaxonomy (Darwin, 1859).The taxonomy of bacterialspecieschangeddramaticallybytheintroductionof16S rRNAgenesequencing.Nowadays,WGSisalsousedtoidentifyand describenewspecies.Bycomparingthewholegenomesequences ofdifferentspecieswitheachother,thelimitedtaxonomic reso-lutionofonlythe16SrRNAgenecanbeovercome(Tindalletal., 2010).AnotherchangeintaxonomymaybeexpectedwhenWGS isusedforrevealingthetaxonomyofbacteria.
Indeed,usingWGSfor taxonomypurposesallowstoinclude more genes to delineate between species than the classical DNA–DNAhybridizationor16SrRNAsequencingmethodsthereby improvingtheresolution. Furthermore,as WGScan beusedto calculatetaxonomictreesbasedonthewholegenome-sequence alignment ofallthegenes presentin thecoregenome,a more robusttreewillbeobtained(Daubinetal.,2001).Ithasalready beenproposedthatdescriptionsofnewtaxashouldalsoincludea draftgenomesequence,withatleast20timescoverage(Thompson etal.,2013).
4.6. Metagenomicsinclinicalmicrobiology
Asalreadymentioned,NGScanbeapplieddirectlytoclinical specimens.NotonlybyusingatargetedNGSapproach,butalso bysequencingtheDNAorRNAfrompatientsamplesbyshotgun metagenomicssequencing(Fig.3).Usingthismethod,itis possi-bletoinvestigatethepresenceofpathogensandthepresenceof virulenceand/orresistancegenesinonesequencerun.
A recentstudy compared thedetection ofviruses inknown respiratory virus-positive samples and not previously analysed
nasopharyngealswabsbyanRNAsequencing-basedmetagenomics approachwithamoreconventionalmolecularmethod.The data-analyseswasperformedusingTaxonomer,arapidandinteractive, web-basedmetagenomicsdata-analysestool(Flygareetal.,2016). Overall,themetagenomicsapproachhadahighagreementwith themolecularmethod,detectedvirusesnottargetedbythe molec-ularmethod,andyieldedepidemiologicallyandclinicallyrelevant sequenceinformation(Grafetal.,2016).
Apartfromidentifyingpathogens,a metagenomicsapproach canalsobeusedtostudytheresistome.Thegutisaknownreservoir forantibioticresistancegenes(ARG),andtreatmentwith antibi-oticshasanimpactontheintestinalresistome,whichcanleadto horizontalgenetransferandtheselectionofresistantbacteria.A studyattheUniversityofTübingeninvestigatedthepresenceof ARGsinthegutoverasix-dayperiodofciprofloxacintreatmentin twoindividualsusingmetagenomics.Furthermore,thisstudy pre-sentedanovelmethodforanalysingthedeterminationofantibiotic selectionpressure,whichcanbeusedinhospitalstocompare ther-apeuticregimensandtheireffectontheintestinalresistome.This informationisimportantforclinicianstochooseantibiotictherapy withalowselectiveantibioticpressureonthepatient’sbacteriain thegut,possiblyresultinginadecreaseddisseminationofantibiotic resistantbacteria(Willmannetal.,2015).
4.7. Determiningthetransmissionofzoonoticmicro-organisms fromanimalstohumans
NGSwillalsorevealmoreknowledgeonzoonotictransmission ofmicro-organisms.Thefirststudiesonthistopicwerebasedon low discriminatorymethods,suchasserotyping(Tenoveretal., 1997).Morerecently, studiesusinghigher discriminatory tech-niques, such as pulsed-field gel electrophoresis or multi-locus variablenumbertandemrepeatanalysis,wereusedtodetect spe-cificbacterialclonesinanimalsandhumans(Sabatetal.,2013). However, much remains to be understood, especially when it comes to the frequency of transmission (e.g. single contact or repeatedcontact withanimalsor animalproducts), risk factors associatedwiththeacquisitionofazoonoticmicroorganism(e.g. riskconducts,suchasanimalkissingincompanionanimals,orstool handlinginfarmanimals)andhowtheuseofantibioticsinanimals affectsthetransferofpathogenicbacteriatohumans.
NGSbringsanewperspectivetothesetopics.Ahigher discrimi-natorypowerwillrevealdifferencesinpreviouslyindistinguishable animalandhumanbacterialstrains.Thistogetherwith epidemi-ological information allowssourcetracing ofpotentialzoonotic infections(Harrisonetal.,2013).Inaddition,NGSallowsa com-prehensive analysis of how antibioticuse manipulates specific microbiotaandtheconsequencesforinterspeciestransmissionand willincreasetheknowledgeonmicrobial evolutionthroughthe analysisofbacterialgenomes,namelythevariableregions,which usuallydeterminehost-adaptationandthepotentialofspreadto differenthosts(Harrisonetal.,2014;Priceetal.,2012).
Asthepatients’safetyisdependingonitsenvironment, includ-ing their contact with food and animals, research projects are currently performedin the UMCGto understandthedynamics of transmission of bacteria between humans, animals and the environment.Thesestudiesareperformedincollaborationwith veterinaryresearchgroupsandfocusonanti-microbialresistant bacteria.Inonesuchstudy,themcr-1genewasdetectedbyWGS tobepresentinthreeE.colistrainsisolatedfromretailchicken meat.Althoughnoneofthehumanstrainscarriedthisgene,two ofthethreestrainsbelongedtoST117,acommoncloneinboth poultryandhumans,representingapotentialpublichealthconcern (Kluytmans-vandenBerghetal.,2016a).
22 R.H.Deurenbergetal./JournalofBiotechnology243(2017)16–24
Fig.3. Anexampleofanoutputofametagenomicsapproachofafaecalsample.Thedifferentcoloursrepresentdifferentbacterialfamilies.
5. Conclusionandoutlook
ForgeneratingNGSdatafromsamplesoriginatingfromhumans, animals,foodandtheenvironment, thesamelaboratory proto-colforlibrarypreparationcanbeused,and,afterdataanalyses, informationonthepresenceofspecificantibioticresistanceand virulencegenesisobtained.Furthermore,NGSmakesitpossible tostandardisetypingmethodsforpathogens(“onetestfitsall”). TheroleofNGSinmedicalmicrobiologylaboratorieswillincrease duringthenextyears,not onlyfor research,butalso,andmore importantly,formoleculardiagnostics,infectionprevention,the investigationofoutbreaksbytheuseofauniqueoutbreakmarker approach,thecharacterizationandsurveillanceofpathogens,the
detectionofnovelresistancegenesand fortheapplicationof a metagenomicsapproachonclinicalsamples.
However,furtherstudiesarerequired toimprovethe work-flowfor NGS,in particularshortentheturnaroundtimeforthe librarypreparation and therunsonthe NGSplatforms, and, at thesametime,furtherreducingcosts.Next,automaticpipelines fordata-analysesandeasy-to-usesoftwareformetagenomicshave tobedeveloped. Additionally,moreestablishedtypingschemes forpathogens andcut-offvalues forthesetypingschemeshave to be established, leading to reference databases with genetic andmetadata,and(inter)regionalandinternationalcollaborations. Importantly,externalqualitycontrolsforproficiencytestinghave tobedeveloped.Onlythenwillpatientguidanceandinfection con-trolmanagementatlocal,(inter)regionalandinternationallevel,
aswellastargetedantibiotictherapy usingNGSdatabecomea possibility,leadingtopersonalisedmicrobiology.
Conflictofinterest
Theauthorsdeclarethattheyhavenoconflictofinterest.
Fundingsources
Forthewritingofthis reviewnospecificgrantfromfunding agenciesinthepublic,commercial,ornot-for-profitsectorswas received.
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