Sputum Bacterial and Fungal Dynamics during Exacerbations of Severe COPD

13 Pages·2015·3.64 MB·English

RESEARCHARTICLE
Sputum Bacterial and Fungal Dynamics
during Exacerbations of Severe COPD
JinSu1,2☯,Hai-yueLiu1☯,Xi-lanTan1,3,YongJi1,Yun-xiaJiang1,MPrabhakar1,Zu-
huaRong1,Hong-weiZhou1,Guo-xiaZhang1*
1 StateKeyLaboratoryofOrganFailureResearch,DepartmentofEnvironmentalHealth,Guangdong
ProvincialKeyLaboratoryofTropicalDiseaseResearch,SchoolofPublicHealthandTropicalMedicine,
SouthernMedicalUniversity,Guangzhou,China,2 DepartmentofRespiratoryPhysicians,Nanfang
Hospital,SouthernMedicalUniversity,Guangzhou,Guangdong,China,3 DepartmentofHospitalInfection
Management,ZhujiangHospital,SouthernMedicalUniversity,Guangzhou,Guangdong,China
☯Theseauthorscontributedequallytothiswork.
* [email protected]
Abstract
Thechangesinthemicrobialcommunitystructureduringacuteexacerbationsofsevere
OPENACCESS chronicobstructivepulmonarydisease(COPD)inhospitalizedpatientsremainlargely
uncharacterized.Therefore,furtherstudiesfocusedonthetemporaldynamicsandstructure
Citation:SuJ,LiuH-y,TanX-l,JiY,JiangY-x,
PrabhakarM,etal.(2015)SputumBacterialand ofsputummicrobialcommunitiesduringacuteexacerbationofCOPD(AECOPD)wouldstill
FungalDynamicsduringExacerbationsofSevere benecessary.Inourstudy,theuseofmolecularmicrobiologicaltechniquesprovidedinsight
COPD.PLoSONE10(7):e0130736.doi:10.1371/
intobothfungalandbacterialdiversitiesinAECOPDpatientsduringhospitalization.Inpar-
journal.pone.0130736
ticular,weexaminedthestructureandvarietiesoflungmicrobialcommunityin6patients
Editor:YiguoHong,CAS,CHINA
withsevereAECOPDbyamplifying16SrRNAV4hyper-variableandinternaltranscribed
Received:March11,2015 spacer(ITS)DNAregionsusingbarcodedprimersandtheIlluminasequencingplatform.
Accepted:May24,2015 Sequenceanalysisshowed261bacterialgenerarepresenting20distinctphyla,withan
averagenumberofgeneraperpatientof>157,indicatinghighdiversity.Acinetobacter,Pre-
Published:July6,2015
votella,Neisseria,Rothia,Lactobacillus,Leptotrichia,Streptococcus,Veillonella,andActi-
Copyright:©2015Suetal.Thisisanopenaccess
nomyceswerethemostcommonlyidentifiedgenera,andtheaveragetotalsequencing
articledistributedunderthetermsoftheCreative
CommonsAttributionLicense,whichpermits numberpersputumsamplewas>1000018SITSsequences.Thefungalpopulationwas
unrestricteduse,distribution,andreproductioninany typicallydominatedbyCandia,Phialosimplex,Aspergillus,Penicillium,Cladosporiumand
medium,providedtheoriginalauthorandsourceare
Eutypella.OurfindingshighlightthatCOPDpatientshavepersonalizedstructuresandvari-
credited.
etiesinsputummicrobialcommunityduringhospitalizationperiods.
DataAvailabilityStatement:Allrelevantdataare
availablefromtheEuropeanNucleotideArchive
(accessionnumbersERS687383toERS687493).
Funding:ThisworkwassupportedbytheNational
ScienceFoundationofChina(NSFC31270152, Introduction
31322003)(http://www.nsfc.gov.cn/),whoprovided
supportforthelabmaterials;theEducational Chronicobstructivepulmonarydisease(COPD)ischaracterizedbypersistentlimitedairflow
CommissionofGuangdongProvinceofChina thatisusuallyprogressiveandnotfullyreversible[1].COPDisbecomingaleadingcauseof
(2012KJCX0031),(http://www.gdhed.edu.cn/),who
mortalityinChinaandimposesaheavyeconomicburden,withthetotalexpenditureper
providedsupportfortheIlluminasequencing;anda
patientamountingto40%and~33.3%oftheaverageannualfamilyincomeinurbanandrural
grantfromSchoolofPublicHealthandTropical
areasofChina,respectively[2].AcuteexacerbationofCOPD(AECOPD)[3]oftendevelops
MedicineofSouthernMedicalUniversity,China
(grantno.GW201435,GW201431)(http://portal.smu. intoemergencysituationsthatareassociatedwithhighmorbidityandmortality,andregular
PLOSONE|DOI:10.1371/journal.pone.0130736 July6,2015 1/13
SputumBacterialandFungalDynamics
edu.cn/gwxy/),whoprovidedsupportfortheAJE treatmenthasnoprovenvalueduringtheseemergencies[4].Arecentreviewnotedthatnearly
costs. halfofAECOPDcasesareduetobacterialinfections[5].AECOPDresultsinworseningof
CompetingInterests:Theauthorshavedeclared healthstatus;limitationofactivities;andanincreaseincomorbidities,includingcardiovascular
thatnocompetinginterestsexist. disease,osteoporosisandneuropsychiatriccomplications.Exacerbationsalsoleadtoapoorer
prognosisbyincreasingtherateofdiseaseprogressionandtheannualdeclineinlungfunction
[6].
Previousstudieshavedemonstratedthatinbothhealthyanddiseasedstates,therespiratory
tractiscolonizedbydiversecommunitiesofbacteria[7,8].Duetomountingapplicationsof
high-throughputsequencingtechniques,thenumberofstudiesonthemicrobialcommunityof
COPDpatientsisgrowingdramatically,andthesestudieshaveyieldedexcitingobservations
regardingtherelationshipbetweenthelungmicrobiotaandrespiratorydisease[1,7].Many
investigationshavereporteddifferentstructuresofmicrobiotadiversitiesbetweenorgansand
betweenCOPDpatients[9,10].Therelationshipsbetweeninfectionbyspecificmicrobialspe-
ciesandAECOPDaswellastherelationshipsbetweenCOPD-associatedpathogensandother
microbeshaveinformedourunderstandingofairwaymicrobiology[11].Manypreviousstud-
ieshavefocusedonbacterialdiversity,andveryfewhavereportedonfungaldiversity[12,13].
Furthermore,ithasbeenfoundthattreatmentwithantibioticsaloneprimarilydecreasesthe
abundanceofProteobacteriaandcanresultinprolongedsuppressionofcertainmicrobial
communitymembersinpatientswithAECOPD[11].Whereastheroleofspecificbacterial
speciesinCOPDhasbeenextensivelystudied,othermicrobialspeciesthatmaycolonizethe
airwaysinCOPDpatientshavenotbeenanalyzed.Therefore,thetemporaldynamicsofthese
communitiesandtheirresponsestoperturbationsarenotwellunderstood.Untilnow,we
couldnotclearlydeterminethetreatmentofAECOPDaccordingtothelungmicrobiotacom-
position.Thisissuedeservesintensestudytocharacterizethedynamicsofthelungmicrobiota
duringthehospitalizationofAECOPDpatients.
Asshowninacohortstudyperformedin2014,thefrequencyoffungalinfectionsin
AECOPDpatientspeaksat4.4%[14].Inparticular,associationsbetweenAspergilluscoloniza-
tionandCOPDhavebeennoted[15],andsensitizationtoAspergillusfumigatushasbeenasso-
ciatedwithreducedlungfunctioninCOPDpatients.However,whetherfungalcolonization
canserveasamarkerofmoreseverelungdiseaseandwhetheraggressivetherapyisneeded
remainunknown[16].Previousstudieshaveshownthatfungalinfectionisanimportant
aspectofAECOPDthatisworthyofintenseresearch.Furthermore,colonizationbyviruses,
ChlamydophilaandfungihasbecomeincreasinglyprevalentinAECOPDpatients[17,18].
Nevertheless,fewreportsonthepotentialroleoffungiinhospitalizedAECOPDpatientsas
wellasonthespecialmicrobialcommunitythatthesefungiareapartofhavebeenpublished.
Whetheralterationinthefungalcommunitywilldisturbtheentiremicrobialstructureand
howitwillfurtheraffectAECOPDthusremaintobestudied.
Weemployedhigh-throughputsequencingofbacterial16SrRNAV4hyper-variableand
fungalinternaltranscribedspacer(ITS)DNAregionstoevaluatehowthesputummicrobiota
ofCOPDpatientschangeseachdayduringhospitalization.Ourprimaryobjectiveinthepres-
entstudywastodeterminewhetherand,ifso,howtherapyinfluencesthestructureandcom-
positionofthebacterialandfungalcommunitiesinthesputumofCOPDpatientsduring
hospitalization.Additionally,wesoughttoexaminehowthediversityofthemicrobialfloraof
sputumischangedbythepresenceoffungi.
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SputumBacterialandFungalDynamics
MaterialsandMethods
EthicalStatement
ThisstudywasapprovedbytheEthicalCommitteeofSouthernMedicalUniversity.Allpartici-
pantsprovidedwritteninformedconsent.
Subjects
PatientsexperiencinganacuteexacerbationofsevereCOPDandreceivingmedicaltreatment
atNanfangHospital,SouthernMedicalUniversity,wererecruitedforthestudy.Allsixsubjects
exhibitedacutesymptoms,suchascough,dyspnea,fatigue,andsputumproduction.
SampleCollection
Sputumsampleswereobtainedfromsixmaleindividualsranginginagefrom73to83years
old,withanaverageageof77years.Foraperiodof7to16days,eachpatientprovidedself-col-
lectedsputumsamplesuntildischargefromthehospital;thefirst3samples,whichwerecol-
lectedonthefirstday,werecollectedbeforeantibioticconsumption.Allsampleswereobtained
fromJune2012toSeptember2012andwerestoredat-80°CuntilDNAextraction.
DNAExtraction
DNAwasextractedfromthesputumofthepatientsassoonasthepatientswerediagnosed
withsevereCOPD.Afterstorageat-80°C,thesputumsampleswerethawedunderventilation
for20min,and2μLfromtheinsideportionofeachsputumsamplewasplacedina2-mL
Eppendorftube.GenomicDNAwasextractedfromeachsputumsampleusingtheForensic
SampleNucleicAcidExtractionKit(BioeasyTechnology,Inc.,China)accordingtothemanu-
facturer’sinstructions.
Bacterial16SrRNAandFungalITSGeneAmplification
The16SrRNAgeneswereamplifiedusingbarcodedV4primersandwerethenpurifiedand
pooledasdescribedbyHeetal.[19].TheITSgeneswereamplifiedusingbarcodedITSF:5´
CTTGGTCATTTAGAGGAAGTAA3´andITSR:5´GCTGCGTTCTTCATCGATGC3´
primers.Each20-μLreactionconsistedof10μLofMaximaHotStartPCRMasterMix
(Thermo,USA),2μLoftemplateDNA(approximately100ng),0.5μLofthebarcodedITSF
primer(10μM),0.5μLoftheITSRprimer(10μM),and7μLofnuclease-freewater.Thecon-
ditionsforPCRincludedaninitialhot-startincubation(15minat94°C),followedby5cycles
ofdenaturationat95°Cfor30s,annealingat50°Cfor30sandextensionat72°Cfor1min;
35cyclesofdenaturationat95°Cfor30s,annealingat65°Cfor30sandextensionat72°Cfor
1min;andthenafinalextensionat72°Cfor15min.The16SrRNAandITSPCRproducts
weresequencedattheBeijingGenomicsInstitutionusingpaired-endsequencingwithan
IlluminaHiSeq2000platform;154bpofthe16SrRNAand70bpoftheITSsequencewere
specificallysequencedfromeachend,withmismatchessetatlessthan10bp,andusedforlater
analysis.ThesequencesweredepositedintheEuropeanNucleotideArchive(ENA),andthe
accessionnumbersfromERS687383toERS687493.
SequenceProcessingandAnalysis
TheV4hyper-variableregionwassequencedbyoverlappingusingthePE250sequencingstrat-
egy.Forquality-controlpurposes,weremovedallofthesequencesthatcontainedambiguous
reads(N)andanysequencesthatcontainedmismatchesintheforwardorreverseprimers.
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SputumBacterialandFungalDynamics
Theseclean,non-continuoussequenceswerethenscreenedforchimerasusingUCHIME[20].
Atotalof3034133high-qualitysequencingreadsofthe16SrRNAgeneweregeneratedafter
UCHIMEuse,517850ofwhichbelongedtofungi.Thesubsequentanalyseswereimplemented
usingQIIME(1.5.0).Each16SrRNAgenesamplewasnormalizedto22000sequences,andwe
usedthenon-normalizeddatatoperformlaterfungalanalyses.Beforeweanalyzedthefungal
data,wetransformedthedatausingtheconstituentratio.Thesequenceswerethenclustered
usingUCLUST,withathresholddistancesetto0.03,whichcorrespondedtothespecieslevel.
TheRibosomeDatabaseproject(RDP)algorithmwasappliedtoclassifytherepresentative
sequencesofeachoperationaltaxonomicunit(OTU)[21],theShannonandPD_whole_tree
indiceswereusedtoanalyzealphadiversity,andPCoAanalysisusingQIIMEbasedonthe
UniFracdistancewasimplemented[22].Thesequenceswereclassifiedatthegenusleveland
groupedintothenextlowesttaxonomiclevel.Thesequencesthatwereclearlyfungalwereused
inanadditionalBLAST[23]analysisusingtheNCBIdatabank.Beta-diversityanalyseswere
implementedbasedontheUniFracdistance,andallstatisticalanalyseswereperformedusing
SigmaPlot12.0.
Results
PatientInformation
Atotalof65sputumsampleswereobtainedfrom6subjectswithsevereCOPDwhohadexpe-
riencedoneacuteexacerbation.Theclinicaldataofthepatientswerecollectedfromreviewsof
eachpatient’shospitalmedicalrecords(S1Table.).C-reactiveprotein(CRP)levelsandthe
FEV1%weremeasuredforallpatientsasaroutinesurveillanceprocedureatNanfangHospital,
exceptforpatientN3,forwhomtheFEV1%couldnotbemeasuredbecausethepatientdidnot
havethepowertoreleasesufficientairformeasurement.
MicrobialDiversityintheSputum
Thesputumbacterialcommunitystructurevariedamongthedifferentparticipants;Fig1
showsthealpha-andbeta-diversityresults.WeusedthePD_whole_treeandShannonindices
tocomparethealphadiversitiesofthedifferentsamples.Therewasconsiderablevariabilityin
thealphadiversityofeachsubject;however,noconsistentpatternswereobserved.Insubjects
N1,N5,andN12,thePD_whole_treeandShannonindicesinitiallydeclinedandthengradu-
allyincreasedovertime.InsubjectsN3andN10,therewasanincreaseintheShannonindex
throughouttheobservationperiod,whereasthePD_whole_treeindexshowedtheopposite
trend.ForsubjectN7,thePD_whole_treeindexshowedadescendingtrend,withamild
increaseovertime,whereastheShannonindexshowedaconsistentlydescendingtrend.We
additionallystudiedthePCoAoftheUniFracdistancesamongthedifferentsubjects.The
PCoAanalysisofthesampledistributionbasedontheunweightedUniFracdistanceofeach
patientshowedthatthesampleswereseparated,revealingthatpatientswithsevereCOPD
haveauniquemicrobialcommunitystructure.Thesesamplescouldbedividedintotwosepa-
rategroupsbasedontheCRPvalue.
Thefungalalpha-diversityanalysisshowednoconsistentpatterns(Fig2),similartothebac-
terialalpha-diversityresults.TheShannonindicesofpatientsN3andN12werehigheronthe
firstday,followedbyaquickdecreaseandthenamoderateincreaseattheendoftheobserva-
tionperiod.TheoppositetrendwasobservedfortheN1andN5samples.Similartrendswere
alsoobservedforthePD_whole_treeindices.
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BacterialCommunities
Phylum-levelcompositionandtemporaldynamics. Twentyphylaweredetectedinthe
sputumsamples.Firmicutes(37.57%),Proteobacteria(29.12%),Bacteroidetes(17.91%),Acti-
nobacteria(11.31%),Fusobacteria(2.93%)andCyanobacteria(1.02%)werefoundathighper-
centages.Incontrast,Spirochaetes,Euryarchaeota,Planctomycetes,Deinococcus-Thermus,
Acidobacteria,Aquificae,Chlamydiae,Nitrospira,Chloroflexi,Crenarchaeota,Euryarchaeota,
Synergistetes,SR1,TenericutesandTM7appearedatlowpercentages.FirmicutesandProteo-
bacteriaweredistributedwidelyandabundantlyamongallsamples,andthepercentageofPro-
teobacteriaremainednearlystable,exceptinsamplesN10andN12.SubjectN10hadan
increasedpercentageofNeisseriaandadecreasedpercentageofMoraxella,whereassubject
N12haddecreasedpercentagesofNeisseriaandMoraxella.Firmicuteswerefoundatdecreased
percentagesinsubjectsN1,N3,andN7,whereassubjectsN5,N10,andN12showedincreased
percentages;theseresultswereattributedtochangesintheabundanceofthegenusStreptococ-
cus.TheamountofFusobacteriaalsochangedsignificantlyovertime,andthisresultwas
Fig1.Distributionsoftheindices.(A)PD_whole_treeindex,i.e.,evennessindex.(B)Shannonindex,i.e.,sampledistributionindex.(C,D)PCoAof
unweightedUniFracdistance.(C)Communitycomparisonofthe6differentsubjects.(D)TwodifferentgroupsbasedontheCRPvalue.
doi:10.1371/journal.pone.0130736.g001
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Fig2.Distributionsoftheindices.(A)PD_whole_treeindex,i.e.,evennessindex.(B)Shannonindexoffungaldiversityinthesputumsamplesofthe6
patients.
doi:10.1371/journal.pone.0130736.g002
reflectedatalltaxonomiclevels,downtothegeneraLeptotrichiaandFusobacterium.Strepto-
phytawasfoundtobethemostabundantamongmembersofthephylumCyanobacteria,and
RothiaandActinomyceswerethemostunstablegeneraamongmembersofthephylumActino-
bacteria(Fig3).
HighlyabundantOTUsandtheirtemporaldynamicsineachpatient. Subjectswith
severeCOPDshoweduniquemicrobialdiversityatthegenuslevel.Streptococcus,Acinetobac-
ter,Prevotella,Rothia,Veillonella,Pediococcus,Neisseria,Leptotrichia,Capnocytophaga,
Porphyromonas,andActinomyceswereregularlydetectedinallofthesubjects(Table1).Addi-
tionally,StenotrophomonaswasregularlydetectedinallsubjectsotherthanN12.Certaingen-
erawerehighlyabundant(>1%oftheirsequences)inseveralpatientsbutdidnotregularly
appearinotherpatients.Forexample,LeptotrichiaregularlyappearedonlyinsubjectsN5,N7,
andN10;AcinetobacterwasonlyhighlyabundantinsubjectsN1,N3andN10;andActinomy-
ceswashighlyabundantinsubjectsN3andN10.
InsubjectN1,thepercentageofAcinetobacterincreasedthroughouttheobservationperiod,
thepercentagesofPasteurellaceaeandPrevotellaquicklydecreasedovertime,andthepercent-
ageofStreptococcusincreasedonthe2ndand3rddaysoftheobservationperiodandthen
decreased.ThepercentageofEnterobacterremainedstableuntilday9,afterwhichitincreased
untiltheendoftheobservationperiod.RothiaandNeisseriawerestablethroughouttheobser-
vationperiod.InsubjectN3,thepercentageofRothiaincreasedquicklyonthe2ndday,whereas
thepercentageofStreptococcusdecreased,andthepercentagesofActinomycesandAcinetobac-
terincreaseduntilthe6thdayandthendecreased.InsubjectN5,thepercentageofPrevotella
decreaseduntilthe9thdayandthenquicklyincreased,andthoseofStreptococcusandRothia
increaseduntiltheendoftheobservationperiod.InsubjectN7,thepercentagesof
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Fig3.ComparisonofthebacterialcommunitiesatthephylumlevelandofrepresentativegenerawithhighlyabundantOTUs.(A)Taxonomic
identificationatthephylumlevelshowingdifferentphylathatvariedwiththeperiodofhospitalization,the“other”representedforthephylathatdidnotbeing
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displayeddirectly.PatientswithseveralsignificantchangesintheOTUproportionovertime:(B1)subjectN1,(B2)subjectN3,(B3)subjectN5,(B4)subject
N7,(B5)subjectN10,and(B6)subjectN12.Thestandardizationofsequencingdepthforeachsamplewas22000.N,casenumber;D,admissiondate.
doi:10.1371/journal.pone.0130736.g003
StreptococcusandLeptotrichiaincreasedbeforethe6thdayandthenslightlydecreased,theper-
centageofVeillonellashowedthecompletelyoppositetrend,thepercentagesofPseudomonas
andPrevotellaquicklydecreasedstartingonday1,andthepercentageofRothiadecreased
slightlyduringthefirsttwodaysandthenincreased.InsubjectN10,thepercentagesofPas-
teurellaceaeandMoraxelladecreasedbeginningonday1,thepercentagesofStreptococcusand
Prevotellaincreased,andthepercentageofNeisseriaincreasedslightlystartingonthe2ndday.
InsubjectN12,Neisseria,LeptotrichiaandPasteurellaceaenearlydisappearedstartingonthe
firstday,whereasthepercentagesofPrevotella,StreptococcusandVeillonellarosesteadily,
exceptonthe4thand5thdays,whentheydecreasedslightly.
Temporaldynamicsoffungalcommunities. Thefungalcommunityinthelungmicro-
biomeofeachpatientwasanalyzedbyITSgenesequencing,andsystemclusteringwasper-
formedtoobtainavisualrepresentationoftheoverallsimilarityamongthesputumsamples.
Fig4showstherelativeabundanceandsystemclusteringtreesofthesamplesbasedontheper-
centageoffungaloperationalunitsineachcommunity.Eachpatientexhibitedanunstable
microbialcommunityduringtheobservationperiod.ThelungmicrobiomesofsubjectsN5,
N7,andN12weredominatedbyAspergillus,whereastheothertwosubjectshadintermediate
orlowlevelsofAspergillus.ThemicrobiomeofsubjectN1wasdominatedbyPhialosimplex
andCandida,whereasthemicrobiomeofsubjectN3wasdominatedbyAspergillusandPhialo-
simplex.ThemicrobiomeofsubjectN10wasdominatedbyTeratosphaeriaandSterigmato-
mycesafterthepatiententeredthehospitalandthenbyAureobasidiumafterward.
Discussion
Inthisstudy,weanalyzedvariationsonthebacteriaandfungiinthesputumofpatientshospi-
talizedwithsevereCOPD.Incontrasttomostpreviousstudies,whichdescribedparticular
communitycompositionsduringexacerbation[10,24],theverticalsamplingemployedinour
experimentaldesignallowedustomorecloselyfollowtheday-to-daychangesinsputum
microbiotaduringhospitalstays.
Severalchangeswerecommon,i.e.,theamountsofFirmicutes,ProteobacteriaandFusobac-
teriachangedsignificantlyovertime,andthesechangeswerenotedatalltaxonomiclevels,
Table1. Generaaccountingformorethan1%ofthesequencesforeachsubject.
N1 N3 N5 N7 N10 N12
Acinetobacter Acinetobacter Campylobacter Capnocytophaga Acinetobacter Capnocytophaga
Brevibacillus Actinomyces Capnocytophaga Leptotrichia Actinomyces Corynebacterium
Enterobacter Enterococcus Lactobacillus Neisseria Capnocytophaga Leptotrichia
Enterococcus Lactobacillus Leptotrichia Porphyromonas Leptotrichia Neisseria
Prevotella Pediococcus Neisseria Prevotella Neisseria Porphyromonas
Rothia Rothia Prevotella Pseudomonas Pediococcus Prevotella
Stenotrophomonas Rothia Rothia Porphyromonas Rothia
Streptococcus Streptococcus Streptococcus Prevotella Streptococcus
Veillonella Rothia Veillonella
Stenotrophomonas
Streptococcus
Veillonella
doi:10.1371/journal.pone.0130736.t001
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Fig4.Systemclusteringoffungalcompositionatthegenuslevel.Thenamesofseveralofthemostabundantgenerathatincludedtheterminaltaxa
shownintheheatmaparelistedontherightofthefigure.Thesamplenamesarelistedatthetopoftheheatmap.“N”representssamplesfromdifferent
subjects,andthecolorbaratthetopoftheheatmapshowsthepercentagesof<5%ofthesequencesineachsputumsample.
doi:10.1371/journal.pone.0130736.g004
downtothegeneraStreptococcus,Lactobacillus,Veillonella,Moraxella,Neisseria,Leptotrichia
andFusobacterium.ChangesinthephylaActinobacteriaandBacteroideteswerealsoreflected
bychangesinthegeneraRothia,Corynebacterium,Actinomyces,Capnocytophagaand
Prevotella.
About50%ofCOPDexacerbationsinvolvebacteria,withthemostfrequentlyisolatedspe-
ciesbeingHaemophilusinfluenzae,Pseudomonasaeruginosa,Moraxellacatarrhalis,Streptococ-
cuspneumoniae,Staphylococcusaureus,Haemophilushaemolyticus,andHaemophilus
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parainfluenzae[25].Ourresultsshowedthatthedynamicpatternsofchangeinsputummicro-
bialcommunitiesshowedsignificantvariationamongpatients,andthesepatternswerealmost
alwayspersonalizedtoeachpatient.Mostofthepatientsexperiencedsomechangesincommu-
nitycompositionduringtheirhospitalstay;however,nogeneraltrendswereapparent.
Inourresearch,ashiftintherelativeabundanceofafewpopulationswasnoted;however,
nosinglepopulationbecamedominant.Althoughthesamplinglocationsweredifferent,the
sameresultswerefoundinastudybySzeetal.[10].Theresultsofourbacterialcommunity
compositionanalysis(Fig2)alsoagreedwithfindingsreportedbyHuang,whostatedthatindi-
vidualswithgreaterbacterialcommunitydiversityalsosupportedalargenumberofFirmicutes
andthatthosewithlessbacterialdiversityhadagreaternumberofProteobacteria[26].Our
resultsshowedthatLeptotrichiaandStreptococcusarethemaingeneraassociatedwithFuso-
bacteriaandFirmicutes,respectively,insevereCOPDpatients.Thesefindingshavegotsome
differenceswiththepreviousresearchbySze,whoseresearchindicatingthat7in8samples,
LactobacillusisthemaingenusassociatedwithanincreasedamountofFirmicutes,inonesam-
ple,StreptococcusbeingthemaingenusassociatedwithanincreasedamountofFirmicutes
[10].TheAspergillusgenuswasfoundinthesputumsamplesofallsixpatients.Inaprevious
study,apositiveAspergillusculturewasreportedtoaffectgrade3or4COPD[27].
OurstudyfocusedonsevereCOPDandrevealedthatthemostcommonbacterialgenera
foundinCOPDpatientswereAcinetobacter,Prevotella,Neisseria,Rothia,Lactobacillus,Lepto-
trichia,Streptococcus,Veillonella,Pasteurella,KlebsiellaandActinomyces.Amongthese,Strep-
tococcus,PrevotellaandNeisseriahavealsobeenfoundinhealthysubjects[28].Ourdata
clearlydisagreewiththehypothesisthatthebronchialmicrobiomesofCOPDpatientscontain
anincreasedpresenceofHaemophilus,MoraxellaandPseudomonasspp.[29–31].Sethietal.
foundthatthepresenceofH.influenzaandM.catarrhaliswasassociatedwithincreased
inflammatorymarkersinthesputum,butthisfindingwasnotsupportedbythedatainthe
presentstudybecausesubjectN1,whohadthehighestCRPlevel,hadnegligibleabundancesof
H.influenzaandM.catarrhalis[32].Themicrobiomesclusteredintotwodifferentregions
basedonthelevelofCRP,asclearlydemonstratedbyunweightedUniFracanalysis.Similar
researchwasperformedbyClarketal.,whofoundthatCRPlevelsarecloselyassociatedwith
thevirusandmixedvirus/bacteriadetectionratesinCOPDpatients[33].Additionally,CRP
hasbeenreportedtosteadilyincreasetheeffectsofantibiotictreatments[34].
Toourknowledge,wearethefirsttocombinebacterial16SrRNAandfungalITSsequence
analysestocharacterizesputummicrobiotainsevereCOPDpatientsandtoanalyzesamples
fromeachdayofhospitalizationduringCOPDexacerbation.Theimportanceofdailydynam-
icsresearchintohowbacterialandfungalcommunitiesvaryamongAECOPDpatientswas
unknownuntilnow.SeveralimportantstudiespreviouslycollectedsamplesfromsevereCOPD
patients,buttheywerelimitedintheirabilitytocharacterizethebacteriathatwerepresentin
thesesamples.Nonetheless,therearecertainlimitationstoourstudythatimpedeusfrom
drawingbroadconclusions.Thenumberofindividualsenrolledinthispreliminarystudywas
small,whichlimitedourabilitytoconductrigorousstatisticalanalyses.Additionally,theculti-
vation-independentmethodsemployedherecouldnotproduceestimatesofthequantitative
changesobservedinpreviousstudies.Furthermore,wesuspectthatmanyfactorsmayinflu-
encethedynamicsofsputumcommunitiesduringexacerbations,suchasthetypesofantibiot-
icsused,complications,andelementsofthehostimmunesystem,thelevelofCRP,the
mucosalenvironment,androutinemonitoringindexes,amongothers.Toassesstheimpor-
tanceofthesevariablesinsputumcommunitydynamics,largerstudiesthatreplicatetreat-
mentsoverconsecutiveexacerbationperiodswillbenecessary.Additionally,whetherthe
specificcompositionalorpotentialbiomarkergenerathatweredetectedintheairwaymicro-
biomecouldbeusefulindicatorsofexacerbationremainstobedeterminedinfurtherstudies.
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