bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
GastrointestinalcarriageisamajorreservoirofK.pneumoniaeinfectionin
intensivecarepatients
ClaireLGorrie1,2,3,MirjanaMirceta4,RyanRWick1,2,DavidJEdwards1,2,RichardA
Strugnell3,NigelPratt5,JillGarlick5,KerrieWatson5,DavidPilcher6,7,Steven
McGloughlin6,7,DenisWSpelman8,AdamWJJenney8,KathrynEHolt1,2
1.CentreforSystemsGenomics,TheUniversityofMelbourne,Victoria,Australia
2.DepartmentofBiochemistryandMolecularBiology,Bio21MolecularScienceand
BiotechnologyInstitute,TheUniversityofMelbourne,Melbourne,Victoria,Australia
3.DepartmentofMicrobiologyandImmunologyatthePeterDohertyInstitutefor
InfectionandImmunity,TheUniversityofMelbourne,Melbourne,Victoria,Australia
4.MicrobiologyUnit,AlfredHealth,Melbourne,Victoria,Australia
5.InfectiousDiseasesClinicalResearchUnit,TheAlfredHospital,Melbourne,
Victoria,Australia
6.IntensiveCareUnit,TheAlfredHospital,Melbourne,Victoria,Australia
7.AustralianandNewZealandIntensiveCare–ResearchCentre,SchoolofPublic
HealthandPreventiveMedicine,MonashUniversity,Victoria,Australia
8.MicrobiologyUnit&DepartmentofInfectiousDiseases,TheAlfredHospital,
Melbourne,Victoria,Australia
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
Abstract
Background
Klebsiellapneumoniaeisanopportunisticpathogenandaleadingcauseofhospitalassociated(HA)infections.Patientsinintensivecareunits(ICUs)areparticularlyat
risk,andoutbreaksarefrequentlyreportedinICUs.K.pneumoniaeisalsopartofthe
healthyhumanmicrobiome,providingapotentialreservoirforHAinfection.
However,thefrequencyofK.pneumoniaegutcolonizationanditscontributionto
HAinfectionsarenotwellcharacterized.
Methods
Weconductedone-yearprospectivecohortstudyofICUpatients.Participants
(n=498)werescreenedforrectalandthroatcarriageofK.pneumoniaeshortlyafter
admission,andclinicalinformationwasextractedfromhospitalrecords.
K.pneumoniaeisolatedfromscreeningswabsandclinicaldiagnosticsampleswere
characterizedusingwholegenomesequencing.Genomicandepidemiologicaldata
werecombinedtoidentifylikelytransmissionevents.
ResultsandConclusions
K.pneumoniaecarriagefrequencieswereestimatedat6%(95%CI,3%-8%)amongst
ICUpatientsadmitteddirectfromthecommunity,and19%(95%CI,14%-51%)
amongstthosewhohadrecentcontactwithhealthcare.Gutcolonisationon
admissionwassignificantlyassociatedwithsubsequentK.pneumoniaeinfection
(infectionrisk16%vs3%,OR=6.9,p<0.001),andgenomedataindicatedamatch
betweencarriageandinfectionisolatesinmostpatients.Fivelikelytransmission
chainswereidentified,resultinginsixinfections(12%ofK.pneumoniaeinfectionsin
ICU).Incontrast,49%ofK.pneumoniaeinfectionswerecausedbyastrainthatwas
uniquetothepatient,and48%ofpatientswithK.pneumoniaeinfectionswho
participatedinscreeningwerepositiveforpriorcolonisation.Thesedataconfirm
K.pneumoniaecolonisationisasignificantriskfactorforsubsequentinfectioninICU,
andindicatethathalfofallK.pneumoniaeinfectionsresultfrompatients’own
microbiota.Screeningforcolonisationonadmissioncouldlimitriskofinfectionin
thecolonisedpatientandothers.
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
Introduction
Klebsiellapneumoniaeisasignificantcauseofmorbidityandmortalityworldwide
andcausesarangeofopportunisticinfectionsinsusceptiblehostsincludingthe
young,elderly,immunocompromised,hospitalizedandparticularlythoseinintensive
careunits(ICUs)1-3.InfectionswithK.pneumoniaeareincreasinglyrefractoryto
antibiotictreatmentduetorisingratesofantibioticresistance4-6,andK.pneumoniae
isamongsttheESKAPEpathogensresponsibleforthemajorityofhospital-associated
(HA)infections7.TheUSCentreforDiseaseControlandPrevention(CDC)have
identifiedcarbapenemase-producing(CP)Enterobacteriaceaeandextended
spectrumbeta-lactamase(ESBL)producingEnterobacteriaceaeassignificantthreats
toglobalhealth,andfoundKlebsiellaspeciescomprised11%and23%ofhealthcareassociatedCPandESBLinfectionsintheUS,respectively4.Theemergenceof
multidrugresistant(MDR)K.pneumoniaehasresultedinadramaticincreasein
researchintoreservoirsandriskfactorsforHAK.pneumoniaeinfections,largely
focusedonESBLorCPbacteriaisolatedfrominfectionsandintra-hospitaloutbreaks.
ThesestudieshavedemonstratedtransmissionofESBLorCPK.pneumoniae
betweenpatients,andshowthatgastrointestinal(GI)tractcolonisationwithESBLor
CPK.pneumoniaecanbeariskfactorforinfection6,8.YetwhilstthemajorityofK.
pneumoniaeHAinfectionsarenotESBLorCP3,9,thereislittledataonthefrequency
andclinicalrelevanceofcolonisationwithK.pneumoniaemoregenerally.
K.pneumoniaeisknowntoasymptomaticallycolonisetheskin,mouth,respiratory
andGItractsofhumans.Analysisof16SrRNAgenedatafromhealthyvolunteersin
thehumanmicrobiomeprojectdetectedK.pneumoniaeinapproximately10%of
samplesfromthemouth,naresandskin,and3.8%ofstoolsamples10.Tworecent
studiesinvestigatedcarriageofK.pneumoniaeintherespiratorytractsofhealthy
individualsincommunitiesinAsia,usingbacteriologicalculture.A2010studyin
Indonesiadetectednasopharyngealcarriagein15%ofadultsand7%ofchildren11,
whilea2014studyinVietnamdetectednasopharyngealcarriagein2.7%ofadults
andthroatcarriagein14%12.Inaddition,ithasrecentlybeenshownthatpatients
sufferingfromCPK.pneumoniaeinfections(typicallyclonalgroup258),orfrom
pyogenicliverabscesscausedbyhypervirulentK.pneumoniae(typicallyclonalgroup
23),frequentlycarrytheirinfectingstrainintheirGItractforbetween30days
(≤74%)andsixmonths(<30%)followingdischargefromhospital13.
IfthegutmicrobiomeisacommonreservoirforHAK.pneumoniaeinfection,then
theriskofsuchinfectionscouldpotentiallybemitigatedthroughscreeningandGI
decontamination14,asisoftendoneforvancomycinresistantEnterococcus(VRE).
ThequestionofwhetherK.pneumoniaecolonisationposesariskforsubsequent
infectionhasbeeninvestigatedinjusttwostudiestodate.A1971studyfound18.5%
ofpatientsadmittedtovariouswardsintheDenverVeteransAdministration
HospitalwerepositivebycultureforrectalcarriageofK.pneumoniae,andthat
carriagewassignificantlyassociatedwithriskofsubsequentHAinfection(45%vs
11%)15.A2016studyattheUniversityofMichiganHealthSystemtertiarycare
hospitalreportedsimilarcolonisationrates(23%)andincreasedriskofinfection
followingcolonisation(5.2%incolonisedvs1.3%innon-colonised)16.
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
Inthepresentstudy,weassessedtheprevalenceofK.pneumoniaecolonisationin
anat-riskcohortinanICUwithinamodern,well-equipped,andwell-managed
tertiaryteachinghospitalinAustralia.Additionally,weinvestigatedwhether
colonisationonadmissionenhancesriskofsubsequentK.pneumoniaeinfection
amongICUpatientsandtherelativecontributionofpatients’owngutmicrobiota
andintra-hospitaltransmissiontotheburdenofK.pneumoniaecarriageand
infectionintheICU.
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
Methods
Ethics
EthicalapprovalsforthesestudiesweregrantedbytheAlfredHospitalEthics
Committee(Projectnumbers#550/12and#526/13).
Settingandrecruitment
TheKlebsiellaAcquisitionSurveillanceProjectatAlfredHealth(KASPAH)was
conductedfromApril1,2013toMarch31,2014.Thestudyincludedmonitoringfor
allclinicalisolatesidentifiedasK.pneumoniaeinfectionsbythehospitaldiagnostic
laboratory(detailsinSupplementaryMethods)andrecruitmentofadultpatients
(≥18years)intheAlfredHospitalICUforK.pneumoniaecarriagescreeningviarectal
andthroatswabs.Verbalconsentwasneededfrompatients(oranadultresponsible
forthepatient)forthefirstninemonths(consent-basedcollection).Forthelast
threemonths(universalcollection)aMDRsurveillancestudy(#526/13)was
concurrentlyconductedintheICUbyourgroupanddidnotrequireverbalconsent.
AdditionaldetailsareprovidedinSupplementaryMethods.Asnodifferenceswere
observedinthedistributionsofage,sexorK.pneumoniaecarriagebetweenthe
patientsenrolledduringconsent-basedanduniversalcollectionprotocols
(SupplementaryFigure1),resultsfromthesetwogroupswerecombinedforall
analyses.
Sampleanddatacollection
Baselinescreeningswabsandaccompanyingpatientdatawascollectedforallstudy
participantsassoonaspossibleafterrecruitment.Follow-upswabsanddata
collectionwererepeatedeach5-7daysafterbaselineforthedurationofICUstay
andupto4daysfollowingtransfertoanotherward.Detailsofswabbingprocedures,
bacterialcultureandantimicrobialsusceptibilitytestingaregiveninSupplementary
Methods.Briefly,samplesweregrownonselectivemediaandanywiththe
appearanceofKlebsiellaspecieswereinvestigatedusingmatrix-assistedlaser
desorptionionization-timeofflight(MALDI-TOF)andsubjectedtoantimicrobial
profilingusingVitek2.Informationonage,gender,datesofhospitalandICU
admission/s,surgeryinthelast30days,andantibiotictreatmentinthelast7days
wereextractedfromhospitalrecords.Datesofdischargeand/ordeathwere
extractedfromhospitalrecordsattheconclusionofthestudy.Allclinicalisolates
recoveredfromICUpatientsandidentifiedasK.pneumoniaeinfectionsbythe
hospitaldiagnosticlaboratoryaspartofroutinecarewereincludedinthestudy.
Communityassociatedvshealthcareassociatedcarriage
Individualsinthecommunityassociated(CA)screeninggroupincludepatientswho
wereboth(i)admittedtotheAlfredHospitalICUeitherdirectly(day0)orvia
anotherwardonday0,1or2oftheoriginalHospitaladmission;and(ii)first
swabbedonday0,1or2ofthatadmission.Patientsfirstswabbedonday3orlater
oftheirHospitaladmissionareincludedintheHA/Day3+screeninggroup.
IndividualsreferredtotheAlfredHospitalICUbythetraumawardofanother
hospitalwereassumedtobeemergencyadmissionsfromthecommunity,andwere
assignedtotheCA/Day0-2orHA/Day3+screeninggroupsaccordingtothedayof
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
firstswabrelativetotheirAlfredHospitaladmission.Allotherpatientstransferred
fromanotherhospitalwereincludedintheHA/D3+screeninggroup.Assuch,the
CA/Day0-2screeninggroupsrepresentindividualsadmitteddirectlytothehospital
fromthecommunity,whereastheHA/D3+groupincludesindividualswithrecent
hospitalexposure.
DNAextractionandsequencing
DNAwasextractedfromovernightculturesusingaphenol:chloroformprotocoland
phaselockgeltubesandsequencedviaIlluminaHiSeqtogenerate125bppairedendreads(seeSupplementaryMethods).Followingqualitycontrolchecksofthe
sequencedata,148isolatesfrom106patientsweresubjectedtocomparative
genomicanalysis.Dataandaccessionnumbersonallisolatesincludedareavailable
inSupplementaryTable1.Amaximumlikelihoodphylogenetictreewasinferred
fromanalignmentofallSNPsidentifiedwithincoreK.pneumoniaegenesusing
FastTreev2.1.817,18.LineagesweredefinedbasedonthistreeusingRAMI19and
multi-locussequencetypeswereassignedusingSRST220.Isolatesfallingwithinthe
samelineagewerefurtherinvestigatedtoidentifypairwiseSNPsviaassemblyand
readmapping.FulldetailsofgenomicanalysesaregiveninSupplementary
Methods.
Statisticalanalysis
AllstatisticalanalyseswereconductedusingR(v3.3.1),includingFisher’sexacttest
forassociationandleastsquareslinearregressionformodellingtherelationship
betweenpairwisegeneticdistanceandtimebetweenisolation(detailsin
SupplementaryMethods).
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
Results
K.pneumoniaecarriage
Atotalof498ICUpatientswererecruitedandscreenedforK.pneumoniaecarriage.
ThisrepresentsonefifthofallICUadmissionsinthestudyperiod,and64%ofadults
spendingmorethan72hoursinICU.Fifty-fourpatients(10.8%)testedpositiveat
baselinescreening(50GIcarriageonly,2throatcarriageonly,2both).Carriagewas
detectedatthesamefrequencyinmalesandfemales(11.0%vs10.4%;p=0.9)and
therewasatrendtowardshighercarriageratesamongolderindividuals
(SupplementaryTable2).
WeestimatedtherateofCAK.pneumoniaeGIcarriage,amongpatientsrecruited
andswabbedintheICUwithintwodaysoftheirfirstrecordedadmissiontothe
Hospital(CA/D0-2group,seeMethods),tobe5.9%(95%confidenceinterval(CI)[3%
–8%],Table1,SupplementaryTable3).TheHAGIcarriagerate,amongstpatients
whowerefirstswabbedintheICUonorafterthethirddayofadmissiontothe
Alfredhospitalorfollowingreferralfromanotherhospital(HA/D3+group,see
Methods),wassignificantlyhigherat19%(95%CI[13.6%–25.7%],OR=3.75,
p=0.00001usingFisher’sexacttest,Table1).
OnethirdoftheparticipantsintheICUscreeningstudy(n=170)contributedoneor
morefollow-upscreeningswabs(Table2).TheoverallGIcarriagerateatfollow-up
was15.3%(n=26/170),similartotheHAGIcarriagerateof19%(95%CI[0.71–
2.38],OR=1.3,p=0.39).Participantstestingpositiveonfollow-uprectalswabs
included19whotestednegativeforK.pneumoniaeontheirbaselinerectalswab,
yieldingaconversionrateof12%.
Noneofthe19CAbaselinecarriageisolateswereMDR(Table1).GIcarriageofMDR
strainswasdetectedatsimilarratesamongHAbaselineisolates(18%,including4
ESBLand2CPisolates,allinpatientswhohadreceivedantibioticsinthelast7days)
andfollow-upscreeningisolates(16%ofpatients,including4withESBLand1with
CPisolates).Atotalofsevenpatientscontributedbothbaselineandfollow-upGI
carriageisolates(Table2).For4/7patientstheresistanceprofilesforthefollow-up
isolateremainedthesameasthebaselineisolate,for2/7patientsthefollow-up
isolatewasmoreresistant(onehadincreasedresistancetoamoxicillin/clavulanic
acid,ticarcillin/clavulanicacid,piperacillin/tazobactam,ceftazidineanddecreased
resistancetonitrofurantoin;theotherhadincreasedresistancetocefoxitinand
nitrofurantoin),andfor1/7thefollow-upisolatewaslessresistant(decreased
resistancetonitrofurantoin).
KlebsiellaGIcolonisationisasourceofinfectioninICU
Atotalof49patients(1.8%ofalladultICUadmissions)whospenttimeintheICU
duringtheirhospitalstay,wereidentifiedashavingK.pneumoniaeinfections(11
ESBL,ofwhich3werealsoCP).Mostofthesepatients(n=39)wereintheICUwhen
theK.pneumoniae-positivediagnosticspecimenwastaken,andtenwereinanother
wardshortlyaftertransferfromtheICU.Pneumoniawasthemostfrequentformof
K.pneumoniaeinfectioninICUpatients(60%),followedbywoundinfections(15%),
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
non-disseminatedUTI(10%)andbacteraemiawithsepsis(8%).MDRwasmost
commonamongwoundandbloodisolates(>50%)(Table3).
InordertoassesswhetherK.pneumoniaeGIcarriageonadmissiontoICUwasarisk
factorforsubsequentK.pneumoniaeinfectionduringhospitalstay,weexamined
thesubsetof491individualswhosebaselinescreeningswabwasobtainedatleast
twodayspriortocollectionofanyclinicalspecimenfromwhichK.pneumoniaewas
isolated(Figure1).TherateofK.pneumoniaeinfectionwassignificantlyhigher
amongstpatientswhowereculture-positiveforGIcarriageatbaselinecomparedto
thosewhowereculture-negative(16%vs3%;OR=6.9,95%CI[2.3–19.7],p<0.001,
seeFigure1).OfallICUpatientswhodevelopedK.pneumoniaeinfectionsand
contributedbaselinescreeningswabs,half(n=13/27,48%)testedpositivefor
K.pneumoniaeGIcarriageatbaseline(includingeightwhowerescreened>2days
priortodevelopingtheinfection).
TheseresultsshowK.pneumoniaeGIcolonisationwassignificantlyassociatedwith
K.pneumoniaeinfectioninICUpatients.Todeterminewhetherinfectionswere
causedbypatients’owncolonisingbacteriaandtoidentifytransmissionbetween
ICUpatients,wesequencedthegenomesofallK.pneumoniaeisolatedfrompatients
whohadspentanytimeintheICUduringtheirhospitalstay(s).Atotalof143high
qualitywholegenomesequenceswereobtainedfrom106patients,including56
clinical,80GIcarriageand7throatcarriageisolates.Coregenomephylogenetic
analysis(Figure2)revealedthepresenceof61lineagesofK.pneumoniaesensu
stricto(n=111isolates)and24lineagesoftwocloselyrelatedspecies:20K.variicola
lineages(n=28isolates)andfourK.quasipneumoniaelineages(n=4isolates).The
latterarerecently-describedspeciesthatarecloselyrelatedtoK.pneumoniaeand
aretypicallyidentifiedasK.pneumoniaeusingbiochemicalorproteomics-based
methods21.TheaveragedistancebetweenK.pneumoniaelineageswas0.5%
nucleotidedivergence,representingthousandsofyearsofevolutionaryseparation.
MostKlebsiellalineages(n=69/85,81%)wereidentifiedinjustonepatient,and60%
ofpatients(n=64)hadtheirownuniquelineagenotobservedinanyotherpatients
(Figure2).Halfofinfections(24/49patients,49%)werecausedbyalineageunique
tothatpatient.FifteenpatientshadbothGIcarriageandinfectionisolatesavailable
forgenomecomparison,and12ofthesepairsmatchedatthelineagelevel
(includingsixpatientswhosecarriageisolatewascollected>2dayspriortothe
infection).Notably,eachofthesepatientscarriedtheirownuniqueK.pneumoniae
lineage,exceptforAH0214andAH0255whosharedlineageST323carriageand
infectionisolates.
KlebsiellatransmissionintheICU
SixteenKlebsiellalineagesweredetectedinmorethanonepatient(darkshading,
Figure2).Lineagesharingbetweenpatientscouldresultfromrecenttransmissionof
bacteriawithinthehospital(strainsharing),orbyindependentacquisitionofa
lineagethathasbeencirculatinginthecommunity(lineagesharing).Todistinguish
thesepossibilitieswecomparedpairwiseSNPdistancesbetweenisolatesfromthe
samepatientwiththosefromdifferentpatients(Figure3A).Intra-patientgenetic
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
distanceswerenearlyall(97%)lessthan25SNPsper5Mbpandmost(82%)were
lessthan10SNPs,whilebetween-patientgeneticdistancesrangedfrom0to>5,000
SNPs(Figure3A).Using25and10SNPsper5Mbpascut-offstoindicatelikelyand
verylikelystrainsharingbetweenpatients,weidentifiedfivegroupsofICUpatients
thatlikelysharedKlebsiellastrains.Strikingly,eachofthesegroupscomprised
patientswithoverlappingadmissions,makingthemepidemiologicallyplausibleintrahospitaltransmissionchains(Figure4).Weidentifiedsixpatientswithinfectionsthat
wereattributabletotheseintra-hospitaltransmissionchains(n=4ST681(K.
variicola),n=1ST323,n=1ST231;Figure4),representing12%(n=6/49)ofallICU
patientswithinfections.Theseinfectionsincludedtwoepisodesofpneumonia,two
woundinfectionsandtwoUTIs,oneofwhichdisseminatedtocausebacteraemia
withsepsis.Ofthefourdonorsinthetransmissionchains,threehadpneumoniaand
onehadawoundinfection.Mostoftheinfectionsassociatedwithtransmissionwere
MDR(3/4donorsand3/6recipients),indicatingastrongassociationbetweenMDR
infectionsandtransmissionintheICU(OR13.6,p=0.002).Inaddition,weobserved
fourpatientswhosecarriageofK.pneumoniaewasattributabletointra-hospital
transmissionchainsbutdidnotresultinanyrecordedK.pneumoniaeinfection
duringhospitalstay(n=1ST323(MDR),n=2ST215(non-MDR),n=1ST15(MDR)),
representing5%ofallpatientsinwhomcarriagewasdetected.
Afurther11lineagesweredetectedinmorethanoneICUpatienteach,butwere
consideredunlikelytorepresentstrainsharingduetoexcessivepairwiseSNP
distances.ThepatientscarryingtheselineagesalsolackedoverlappingICU
admissions(SupplementaryFigure2).Suchlineagesharingcouldresultfrom
independentacquisitioninthecommunitypriortohospitaladmission,butcouldalso
potentiallybeexplainedbycrypticlonger-termtransmissionchainsinvolving
bacteriathatpersistwithinthehospitalbetweenadmissions(forexample,in
reservoirssuchassinksordrains,orcolonisationofhealthcareworkers22-24).Inthe
lattercase,wewouldexpectthegeneticdistancesbetweenisolatesintheseclusters
tobeconsistentwiththeaccumulationofgeneticvariationovertimeinanevolving
reservoirortransmissionchain,whichcouldexceedthe25-SNPcut-offifsufficient
timehaspassedbetweenisolations.Toassessthis,wecomparedgeneticdistances
withtime-between-isolationforpairsofisolatesfromthesamepatientsandfrom
withinthefiveepidemiologicallysupportedintra-hospitaltransmissionchains
(Figure3B),whichshowedasignificantlog-linearrelationship(R2=0.25,p=4x10-8)
betweengeneticdistance(SNPaccumulation)andtime.Observedgeneticdistances
forisolatepairsfromthe11multi-patientlineageswithnon-overlappingstays
exceededthe99%predictionintervalofthelinearregressionmodelwiththe
exceptionoftwocasesthatcouldpotentiallyreflectplausiblecryptictransmission
chains(SupplementaryFigure2):(i)patientAH0352’sfourthscreeningswabwas
positiveforST641tenmonthsaftertheisolationofaST641isolatefrompatient
KC0038atageneticdistanceof125SNPsper5Mbp(valuewithinthe95%
predictioninterval),and(ii)patientAH0620’sclinicalwoundisolatewas83SNPsper
5MbpdistantfromthatisolatedfrompatientAH0416onemonthearlier(value
exceedsthe95%predictioninterval,butfallswithinthe99%predictioninterval).
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
DISCUSSION
Weestimateda5.9%CArateforculture-positiveGIcarriageofK.pneumoniae,
similartothe3.9%estimatedamongsthealthyindividualsinthehumanmicrobiome
projectbasedon16SrRNAampliconsequencingofstoolsamples10.HAcarriage
amongstICUpatientswasestimatedtobemuchhigherat19%,with12%ofpatients
convertingfromculture-negativeatbaselinetoculture-positiveonfollow-up.This
likelyreflectsacquisitionofbacteriainthehospital(transmissionofstrainswas
directlyobservedin5%ofcases)and/orselectionforgrowthofpre-existing
K.pneumoniaeintheGImicrobiomeduringhospitalisation(MDRratewas18%inHA
baselinecarriageand15%atfollow-up,butMDRwasnotdetectedinCAbaseline
carriage).TheHArateestimatedhereissimilartotheculture-positiveGIcarriage
ratesestimatedinotherhospitalstudies15,16.Throatcarriagewasnegligibleinour
ICUpopulation,muchlowerthantheratesofCAnaso/oropharyngealcarriage
estimatedinbothculture-basedandculture-freestudies10,12.Thisispossiblydueto
highratesofintubationinICUpatients,whichmaysubstantiallyalterthelocal
microbialcommunities.
OurdatademonstratethatK.pneumoniaeisacommoncomponentofthehumanGI
microbiomeandofclinicalsignificanceintheICUsetting,as:(i)K.pneumoniae
carriageonadmissiontoICUwassignificantlyassociatedwithsubsequent
K.pneumoniaeinfection(OR6.9,p=0.0003),consistentwiththeresultsreported
fromthe1970sDenverstudy(OR4.0,p=0.000915)andthe2016Michiganstudy(OR
4.1,p=0.0000216);and(ii)theWGSdataconfirmedadirectlinkbetweencolonising
andinfectingstrainsin13patients(80%ofthosewithpairedisolatesavailablefor
testing),alsoconsistentwiththeMichiganstudy16.Importantly,wefoundstrong
agreementbetweentwomethodsforestimatingtheproportionofICU
K.pneumoniaeinfectionsthatareattributabletoinfectionwithpatients’ownGI
microbiota:(i)ofall49K.pneumoniaeinfectionsdiagnosedinICUpatientsduring
thestudyperiod,49%wereassociatedwithK.pneumoniaelineagesuniquetothe
patient;and(ii)ofthe27K.pneumoniaeinfectionsdiagnosedinICUpatientsfrom
whomscreeningswabswereobtained,48%occurredinpatientswhotestedpositive
forpriorGIcolonisationwithK.pneumoniae.Incontrast,only12%ofinfections
showedevidenceofresultingfromintra-hospitaltransmissioninthissetting.This
suggeststhatwhilemeasurestoreducecross-contaminationbetweenpatientsare
necessary,theyarenotsufficienttoeliminateK.pneumoniaeinfectionsin
hospitalisedpatients,andmeasurestominimisetheriskofinfectionwiththe
patients’ownmicrobiomedeservessignificantattention25-27.
KeystrengthsofthisstudyaretheprospectivecohortdesignandtheuseofWGSto
confirmspeciesidentificationandstrainrelatednessforallK.pneumoniaeisolated
fromICUpatients,regardlessofantimicrobialsusceptibility.Mostpreviousstudiesof
K.pneumoniaecolonisationinhospitalshavefocusedonESBLand/orCPisolates
only,andwhileinfectionsassociatedwithsuchstrainsaremostcomplicatedtotreat,
theydonotrepresentthemajorburdenofK.pneumoniaeinfections.WGS
demonstratedthatsomeisolatesidentifiedasK.pneumoniaebyMALDI-TOFactually
belongedtocloselyrelatedgroupsthathaverecentlybeendescribedasseparate
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
species21,K.quasipneumoniaeandK.variicola(Figure2).Sincethesespeciesare
verycloselyrelatedtoK.pneumoniaesensustricto(~4%nucleotidedivergence),are
clinicallyindistinguishable,andaretypicallyidentifiedasK.pneumoniaeindiagnostic
laboratories,comparablestudiesreportingK.pneumoniaecarriageencompassthe
entireK.pneumoniaecomplex.Henceweincludedallthreespeciesinour
summariesofresults.WGSalsodemonstratedthatasmallnumberofisolates
identifiedasK.pneumoniaewereeithernotKlebsiella(n=3),werecontaminated
withalargeproportionofDNAfrommultipleKlebsiella(n=2)ornon-Klebsiella(n=3),
orhadverylowKlebsiellamixture(indicatedinFigure2).Thesemayreflectcoinfectionfollowedbyselectionfordifferentsubpopulationsinthelaboratoryused
forthedifferenttests,ormisidentificationbyMALDI-TOF.Thesesampleswere
thereforeexcludedfromhigh-resolutiongenomicanalysisforattributionpurposes
(dataFigure1,SupplementaryTable4)butwereincludedincalculationofoverall
ratesofinfectionandcarriage,whichthereforereflectsolelytheroutinelaboratory
identificationandaredirectlycomparablewithotherreportedresults.
Themainlimitationsofthestudyarisefromtheswabbingproceduretoidentify
K.pneumoniaecolonisationonadmission.Effortsweremadetocollectswabsas
soonaspossiblefollowingadmission,howevertheneedtoobtaininformedconsent
fromtheparticipantorapersonresponsibleandnotdisruptpatientcaremeantthat
itwasoftennotpossibletoobtainscreeningswabsonthedayofadmission.
Notably,similarculture-positiverateswereobservedforswabscollectedonday0,1
or2ofadmission(SupplementaryTable3),soitisunlikelythatthishadasignificant
impactonresults.WeusedrectalswabculturetodetermineK.pneumoniaeGI
colonisationstatus,howeverwhilethisapproachisstandardforpathogencarriage
screening28-30,itssensitivitytodetectK.pneumoniaeisnotwellcharacterisedand
likelydependsontheK.pneumoniaestrain,GImicrobiomecompositionandrecent
antimicrobialexposures.Thereislikelyasignificantfalsenegativerate,particularly
forthedetectionofCAcarriageofsusceptibleorganismsintheabsenceofrecent
antibiotictreatment.Henceourstudyprobablyunderestimatestherateof
colonisationinthecommunityandthecontributionofcolonisationtosubsequent
infection.
OurconclusionthattheGImicrobiomeisasourceofK.pneumoniaeinfectionsinICU
patientsechoessimilarfindingsthatcolonisingstrainsofS.aureus,A.baumannii,
Enterococcus,andEnterobacteriaceaeareacommonsourceofHAinfections16,22-24.
RoutinescreeningfornasalcarriageofmethicillinresistantS.aureus(MRSA)orgut
carriageofvancomycinresistantEnterococcus(VRE)orESBL/CPEnterobacteriaceae
hasbeenintroducedinvarioushospitalsettings28-31.ArecentstudyofCP
K.pneumoniaeinIsraelsuggestedscreeningandisolationofcarrierscouldhelpend
currentoutbreaksandpreventfutureones6.Asimilarstudyintroducedscreeningfor
EBSLK.pneumoniaeinordertolimitandpreventcurrentandfutureoutbreaks32.
WhilstthosestudiesfocusonscreeningforCPorESBLK.pneumoniae,ourresults
indicatethatroutinescreeningforgeneralK.pneumoniaecarriageintheICUcould
alsobeavaluabletool.Inparticular,foreknowledgeoftheantimicrobial
susceptibilityprofilesofK.pneumoniae,aswellasotheropportunisticpathogens
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
residentinthemicrobiome,couldguidethechoiceofprophylacticandtherapeutic
antimicrobialtreatment25,26,28.
Acknowledgements
ThisworkwasfundedbytheNHMRCofAustralia(project#1043822andFellowship
#1061409toK.E.H)andC.L.G.wassupportedbyanAustralianGovernment
ResearchTrainingProgramScholarship.Theauthorsgratefullyacknowledgethe
contributionandsupportofJanineRoney,MellissaBryant, JenniferWilliams,Iain
AbbottandNoeleneBrowneattheAlfredHospital,andthesequencingteamatthe
WellcomeTrustSangerInstitute.
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Table1.K.pneumoniaeGIcarriagedetectedatbaselinescreeningofAlfred
Hospitalpatients.Patientgroups:ICUCA/Day0-2,rectalscreeningswabobtained
onday0,1or2ofadmissiontoAlfredHospitalandnotreferredfromanother
hospital(exceptfromtraumaunit);ICUHA/Day3+,rectalscreeningswabobtained
onday3orlaterofadmissiontoAlfredHospitalorreferredfromanotherhospital.
K.pneumoniaescreeningswabresult
Patientgroup
Totalscreened
Culturepositive(%)
MDR(%)
ICU
CA/Day0-2
324
19(5.9%)
0(0%)
HA/Day3+
174
33(19.0%)
6(17.6%)
Total
498
52(10.4%)
6(11.3%)
Table2.K.pneumoniaeGIcarriagedetectedinfollow-upscreeningofICUpatients.
K.pneumoniaedetectiononfollow-up
K.pneumoniaeculture
Total
resultatbaseline
screened Culturepositive(%)
MDR(%)
Positive
16
7(37%)
2(29%)
Negative
154*
19(12%)
2(11%)
Total
170
26(15%)
4(15%)
*2patientshereweretypedasK.pneumoniaefollow-upswab-positivebutWGS
latershowedtheseisolatesasE.coli,andarenotcountedinthe19follow-up
positive.
Table3.Patientswithinfection(s)andtimeintheICU.Typeofinfectionandsource
ofinfectionoutlined(position/presenceintransmissionchain,priorcolonisation,
unknown).Note3patientshadUTIandbacteraemiawithsepsis;theyare
representedhereinthebacteraemiawithsepsiscolumn.
Pneumonia
Wound
Other
2
UTI
(non-invasive)
1
Total
0
Bacteraemia
withsepsis
1
Recipientin
transmission
chain
Donorin
transmission
chain
PriorGI
colonisation
Priorthroat
colonisation
Unknown
source
(unique
lineage)
Total
2
3
0
1
0
0
4
6
1
1
0
0
8
1
0
0
0
0
1
17(12)
3(3)
3(1)
3(2)
3(1)
29
29(5MDR)
5
7(4MDR)
3
4(2MDR)
48
6
OneICUpatienthadaninfectiondiagnosedasK.pneumoniaebutgenomesequencingofthe
isolateidentifieditasA.baumannii;thispatientincludedintotalreportedinfectionsinthe
textbutisnotrepresentedinthistable.‘Unknownsource’includesthoseinfectionsfor
whichthereisnogeneticorepidemiologicalevidencetoindicatewhethertheinfectionhas
arisenfromapatient’sowncarriagestrainsorthroughtransmissionfromanothersource;
numbersinbracketsindicatethenumberofsuchinfectionsassociatedwithalineagethat
wasuniquetothatpatient.
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
Screenedforbaseline
carriage
n=498
Excludedasbaselinescreening
occurred≤2dayspriorto
infec1on
n=7
Screenedforbaseline
carriagebeforeany
infec1on
n=491
Baselinecarriage
n=49
Infec1on
Nobaselinecarriage
n=442
Infec1on
n=8(16%)
n=12(3%)
MDRinfec1on
MDRinfec1on
n=5
n=3
Figure1.Flowchartoutliningnumberofpatientsincludedineachpartofrates
analyses.
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
Figure2.GenomediversityofisolatesfromICUpatientsidentifiedasKlebsiella
pneumoniae.
AlltreesaremaximumlikelihoodtreesinferredfromcoregenomeSNPalignments.
Scalebarsindicateaveragenumberofsubstitutionspersiteacrossthegenome.Tip
coloursindicateisolatesourceasperinsetlegend.*Mixedisolate,sequencereads
includesubstantialnon-Klebsiellasequences;positionintreeshownisbasedon
KlebsiellaSNPs.PhylogeneticlineagestowhichmorethanoneICUisolatebelongs
arehighlightedandlabelledwiththeircorrespondingmulti-locussequencetype(ST)
andthetotalnumberofSNPsidentifiedbetweenisolatesinthelineage;darker
shadingindicatesmultiplepatientscontributedisolatesinthatcluster,asperinset
legend.(A)Unrootedtreeofallisolates,revealingthreedistinctspeciesthatare
typicallyidentifiedasK.pneumoniaeindiagnosticlaboratories.(B)Midpointrooted
speciestreeforK.variicolaisolates.(C)Midpointrootedspeciestreefor
K.pneumoniaesensustrictoisolates.
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
Figure3.Pairwisegeneticdistancesbetweenisolatesbelongingtothesamelineage,
expressedasSNPsper5Mbpofgenomeinordertonormalisefordifferencesin
sharedgenecontentbetweenstrainpairs.(A)Violinplotsshowingdistributionof
pairwisegeneticdistanceswithinandbetweenpatients;blackbarsindicatethe
mediandistribution;notelog10scale.(B)Scatterplotofpairwisegeneticdistances(yaxis)ontimebetweenisolation(x-axis),colouredaccordingtoinsetlegend;bothon
log10scale.Solidline,least-squareslinearregressionlinefittoallisolatepairs
involvedinplausibletransmissionchains(intra-patientpairs,purplepoints;and
inter-patientpairsassociatedwithoverlappingadmissions,filledblackpoints);
dashedlinesindicate99%predictionintervalforthelinearmodel.
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
Figure4.Timelinesforalllineagesdetectedinmultiplepatientsthatshowany
inter-patientpairwisegeneticdistancebetweenisolatesof≤25SNPsper5Mbp.
Lineagesareboxedandlabelledwiththeirmulti-locussequencetype(ST).Each
horizontaldashedlineindicatesthetimelineforapatient,labelledtotheleft
(crossesindicatedateofdeathwhereapplicable).PeriodsofAlfredHospital
admissionareindicatedaswhiteboxes,periodsinICUaspinkshading.Circles
indicateK.pneumoniaeinfectionisolates(red,belongingtothelineage;black,other
lineage);trianglesindicaterectalscreeningswabs(red,K.pneumoniaebelongingto
thelineage;black,K.pneumoniaeofanotherlineage;unfilled,negativeforK.
pneumoniae).Orangeboxesindicategroupsofisolatesforwhichallpatientshaveat
leastonepairwisegeneticdistanceof≤10SNPsper5Mbpwithanotherinthe
group;similarlyforyellow(≤25SNPs)andblue(≤100SNPs)boxes.
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
SupplementaryTable1.Carriageandinfectionsampledataandaccession
numbers.
[seesupplementarydocumentcalled
SupplementaryTable1_SampleDataAndAccessionNumbers.csv]
SupplementaryTable2.K.pneumoniaecarriageratesdetectedfrombaseline
swabsofICUpatients.Lasttwocolumnsshowcomparisonofratesinfemalesvs
males:OR,oddsratio;P,p-value.
Associationwithfemalesex
Female
Male
(Fisher’sexacttest)
Carriage carriers carriers
Age Total
N(%)
N(%)
N(%)
OR(95%CI)
p-value
≤25
28
3(11%)
1(14%) 2(10%)
1.6(0.0,35.2)
1
26-35
44
5(11%)
1(6%)
4(15%)
0.3(0.0,3.8)
0.6
36-45
47
4(9%)
1(5%)
3(8%)
0.4(0.0,4.8)
0.6
46-55
85
5(6%)
1(4%)
4(7%)
0.6(0.0,6.7)
1
56-65 107
9(8%)
3(10%)
6(8%)
1.4(0.2,7.0)
0.7
66-75
89
11(12%) 3(10%) 8(14%)
0.7(0.1,3.3)
0.7
76-85
77
15(19%) 7(25%) 8(14%)
1.7(0.5,6.2)
0.4
>85
21
2(10%)
0(0%)
2(13%)
0(0.0,13.8)
1
All
498 54(10.8%) 17(10%) 37(11%)
0.9(0.5,1.8)
0.9
SupplementaryTable3.BaselineGIcarriagerates,withCAcohortbrokendown
intoindividualdays.
Totalswabbed(n) Kppositive(%)
Kpnegative(%)
CA/Day0-2
324
19(5.9%)
305(94.1%)
-Day0
24
1(4%)
23(96%)
-Day1
206
15(7%)
191(93%)
-Day2
94
3(3%)
91(97%)
HA/Day3+
174
33(19.0%)
141(81.0%)
Total
498
52(10.4%)
446(89.6%)
SupplementaryTable4.PatientswithtimeintheICU,withcarriageisolates,
infectionisolates,orbothcarriageandinfectionisolates.
[seesupplementarydocumentcalledSupplementaryTable4_ICUpatientData.csv]
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
SupplementaryFigure1.DistributionofageandK.pneumoniaeculturepositive
rates,bygender,amongindividualsscreenedforbaselinecarriageofK.pneumoniae
overthefirstninemonths(panelsAandB)andthefinalthreemonths(panelsCand
D)ofthestudyperiod.Lightercolours,culturenegativeindividuals;darkercolours,
culturepositive.
bioRxiv preprint first posted online Dec. 23, 2016; doi: http://dx.doi.org/10.1101/096446. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
SupplementaryFigure2.Timelinesforalllineagesdetectedinmultiplepatients
thatdidnotshowanyinter-patientpairwisegeneticdistancebetweenisolatesof
≤25SNPsper5Mbp.Lineagesareboxedandlabelledwiththeirmulti-locus
sequencetype(ST).Eachhorizontaldashedlineindicatesthetimelineforapatient,
labelledtotheleft(crossesindicatedateofdeathwhereapplicable).Periodsof
AlfredHospitaladmissionareindicatedaswhiteboxes,periodsinICUaspink
shading.CirclesindicateK.pneumoniaeinfectionisolates(red,belongingtothe
lineage;black,otherlineage);trianglesindicaterectalscreeningswabs(red,
K.pneumoniaebelongingtothelineage;black,K.pneumoniaeofanotherlineage;
unfilled,negativeforK.pneumoniae).Blueboxesindicategroupsofisolatesfor
whichallpatientshaveatleastonepairwisegeneticdistancefromanotherinthe
groupthatfallsbelowthecut-offindicatedintheinsetlegend.