for studying dynamics of the bacterial community and metabolome in rumen

20 Pages·2017·4.76 MB·English

RESEARCHARTICLE
The application of rumen simulation
technique (RUSITEC) for studying dynamics of
the bacterial community and metabolome in
rumen fluid and the effects of a challenge
with Clostridium perfringens
StefanieU.Wetzels1☯,MelanieEger2☯,MarionBurmester2,LotharKreienbrock3,
AmirAbdulmawjood4,BeatePinior5,MartinWagner1,GerhardBreves2*,EvelyneMann1*
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a1111111111 1 InstituteforMilkHygiene,MilkTechnologyandFoodScience,DepartmentforFarmAnimalsandVeterinary
PublicHealth,UniversityofVeterinaryMedicineVienna,Vienna,Austria,2 InstituteforPhysiology,University
a1111111111
ofVeterinaryMedicineHannover,Hannover,Germany,3 InstituteforBiometry,Epidemiologyand
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InformationProcessing,UniversityofVeterinaryMedicineHannover,Hannover,Germany,4 Institutefor
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FoodQualityandFoodSafety,ResearchCenterforEmergingInfectionsandZoonoses(RIZ),Universityof
VeterinaryMedicineHannover,Hannover,Germany,5 InstituteforVeterinaryPublicHealth,Departmentfor
FarmAnimalsandVeterinaryPublicHealth,UniversityofVeterinaryMedicineVienna,Vienna,Austria
☯Theseauthorscontributedequallytothiswork.
*[email protected](GB);[email protected](EM)
OPENACCESS
Citation:WetzelsSU,EgerM,BurmesterM,
KreienbrockL,AbdulmawjoodA,PiniorB,etal. Abstract
(2018)Theapplicationofrumensimulation
technique(RUSITEC)forstudyingdynamicsofthe
Therumensimulationtechnique(RUSITEC)isawell-establishedsemicontinuousinvitro
bacterialcommunityandmetabolomeinrumen
fluidandtheeffectsofachallengewithClostridium modelforinvestigatingruminalfermentation;however,informationonthestabilityofthe
perfringens.PLoSONE13(2):e0192256.https:// ruminalbacterialmicrobiotaandmetabolomeintheRUSITECsystemisrarelyavailable.
doi.org/10.1371/journal.pone.0192256
Theavailabilityofhighresolutionmethods,suchashigh-throughputsequencingandmeta-
Editor:MichelR.Popoff,InstitutPasteur,FRANCE bolomicsimproveourknowledgeabouttherumenmicrobialecosystemanditsfermentation
Received:July18,2017 processes.Thus,weusedIlluminaMiSeq16SrRNAampliconsequencingandacombina-
tionofdirectinjectionmassspectrometrywithareverse-phaseLC-MS/MStoevaluatethe
Accepted:January18,2018
dynamicsofthebacterialcommunityandtheconcentrationofseveralmetabolitesina
Published:February7,2018
RUSITECexperimentasafunctionoftimeandinresponsetoachallengewithapathogenic
Copyright:©2018Wetzelsetal.Thisisanopen Clostridiumperfringens(C.perfringens)strain.Afterfourdaysofequilibration,samples
accessarticledistributedunderthetermsofthe
werecollectedondays5,6,7,10,12and15ofthesteady-stateandexperimentalperiod.
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and Fromatotalofsixfermenters,threenon-infectedfermenterswereusedforinvestigating
reproductioninanymedium,providedtheoriginal time-dependentalterations;threefermenterswereincubatedwithC.perfringensandcom-
authorandsourcearecredited.
paredwiththenon-infectedvesselsatdays10,12and15.Alongthetime-line,therewasno
DataAvailabilityStatement:IlluminaMiSeq statisticallysignificantchangeoftheoverallbacterialcommunity,however,somephylotypes
sequencingdataareavailableinBioProjectSRA
wereenrichedatcertaintimepoints.AdecreaseinFibrobacterandElusimicrobiaovertime
databaseundertheaccessionnumber
wasfollowedbyanincreaseinFirmicutesandActinobacteria.Incontrast,classicalfermen-
PRJEB15167.
tationmeasurementssuchaspH,redoxpotential,NH -N,shortchainfattyacidsandthe
3
Funding:Thispublicationwassupportedby
concentrationsofmetabolitesdeterminedbymetabolomics(biogenicamines,hexosesand
DeutscheForschungsgemeinschaftandUniversity
ofVeterinaryMedicineHannover,Foundation aminoacids)remainedstablethroughouttheexperiment.InresponsetoC.perfringens
withinthefundingprogrammeOpenAccess additiontheconcentrationsofseveralaminoacidsincreased.Althoughtheoverallbacterial
Publishing.Thefundershadnoroleinstudy
PLOSONE|https://doi.org/10.1371/journal.pone.0192256 February7,2018 1/20
Ruminalbacterialcommunitydynamics,metabolomics,andClostridiumperfringenschallengeinaRUSITECsystem
design,datacollectionandanalysis,decisionto communitywasnotalteredhereeither,someminorchangessuchasanenrichmentof
publish,orpreparationofthemanuscript. SynergistetesandBacteroidetesweredetectableovertime.Inconclusion,both,thebacte-
Competinginterests:Theauthorshavedeclared rialcommunitycompositionandthemetabolomeintheRUSITECsystemwererelatively
thatnocompetinginterestsexist. stableduringtheexperiment.
Introduction
Detailedknowledgeoftheruminaldynamic,anaerobicecosystemandrumenfermentation
processesaretheprerequisitetounderstandbasicrumenphysiologyandnutritionaswellas
gastrointestinaldiseasessuchasrumenacidosis.Studyingfactors,whichcontributetoamodu-
lationinvivounderlietimely,environmentalfluctuationsandhost-dependentphysiology[1].
Therumensimulationtechnique(RUSITEC)isawell-establishedinvitromethodtosimulate
andtoinvestigaterumenmicrobialprocesses,avoidinganimal’svariabilityinastandardized
environment[2].Thismethodiswidelyusedforstudyingeffectsofdifferentdietsorfeedaddi-
tivesonmicrobialfermentationpatterns,proteinsynthesisandmicrobialgrowth[3,4].
Althoughbeingahighlystandardizedmethod(e.g.intemperature,pHandbufferflow)the
systemisknowntodifferfrominvivoconditionsregardingabsorptiveprocesses,differences
intheratiobetweenliquidandsolidmaterials,lowershortchainfattyacid(SCFA)concentra-
tionsandprotozoalshiftscomparedtothedonoranimal[5–7].Itwasproposedthatthebacte-
rialdiversitydecreasesintheRUSITECandthatthedisappearanceofciliatescanbetraced
backtoalossofbalanceinthebacterialpopulations[7].Usingreal-timePCR,Lengowskiand
colleagues[8]demonstratedthatmostchangesduringtheadaptationoftheruminalmicrobial
communitytotheRUSITECsystemoccurwithininthefirst48hafterinoculation,however,
maycontinueforsomespecies.Theavailabilityofhigh-throughputsequencingmethodsoffers
theopportunitytoinvestigatealterationsinthemicrobialcommunityandmicrobialbiochem-
icalprocessesindetail.Belancheandcolleagueswerethefirst,whousednextgeneration
sequencingmethodstoevaluatetheimpactofdietarysupplementationintheRUSITECsys-
tem[9,10].Recently,Duarteandcolleagues[11]reportedaneffectofthesamplingdayonthe
liquid-associatedmicrobiotaintheRUSITECusingIlluminasequencing.
Therumenmicrobiotastronglyinfluencestheruminant’smetabolismbythepatternof
SCFAandproteinformation.WhileSCFAandNH -Nareassessedroutinelyinmoststudies
3
[3,12],metabolomictechniquesforthecomprehensiveanalysisoffurthermetabolites,e.g.
aminoacidsandbiogenicamines,haveonlybeenestablishedafewyearsagoforrumenfluid
[13].Subsequentstudiesindicatethatthelevelsofthesemetabolitesarealsolinkedtothefeed-
ingregimenandmightberelatedtopathogenicconditionsintherumen[14,15].Toour
knowledgethesetechniqueshavenotbeenusedintheRUSITECuntilnow.
Besideofautochthonousbacterialcommunitymembers,allochthonousmicrobescon-
stantlypasstherumenderivingfromfood,waterortheenvironment,ofwhichsomecanbe
pathogenicspecies.Suchpathogenicbacteriacancausealterationsinthegutmicrobiome[16,
17].Clostridiumperfringens(C.perfringens)causeshemorrhagicenteritisinneonatalrumi-
nants,enterotoxemia,jejunalhemorrhagesyndrome,abomasalulcersandtympanyorgas
gangrene[18,19].Inadultcattle,C.perfringensisfrequentlypresentinrumensamples[20].In
broilerchicken,C.perfringenshasbeenreportedtoaltertheintestinalmicrobiota[21],indicat-
ingthatitmightalsobeacandidateformodulatingtherumenmicrobiome.
Theaimofthestudyistwofold:First,toprovideadetailedinvestigationonthedynamicsin
thebacterialmicrobiotaandfermentationproductsduringa15-dayRUSITECexperiment
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Ruminalbacterialcommunitydynamics,metabolomics,andClostridiumperfringenschallengeinaRUSITECsystem
(fourdaysequilibrium,threedayssteady-stateandeightdaysexperimentalperiod)bycom-
bininghigh-throughputsequencingwithatargetedquantitativemetabolomicsapproach.
Second,toexaminewhetherthebacterialcommunityandthemetabolicprofileintheRUSI-
TECfermentersremainstableduringachallengewithC.perfringensasamodelpathogen.
Materialsandmethods
Ethicsstatement
AllproceduresinvolvinganimalswerecarriedoutinaccordancewiththeGermanlegislation
onanimalwelfare.ThefistulationofdonorcowswasapprovedbytheLowerSaxonyState
OfficeforConsumerProtectionandFoodSafety(approvalno.33.42502-05-07A480).
RUSITECexperiment
TheRUSITECexperimentwascarriedoutusingsixfermenters.Thetriplicate‘A-Cfermenter’
(samplesS1A-S6C)wasanalyzedinrespecttodeterminetime-dependentchangesofthe
microbiotaandthemetabolome,thetriplicate‘D-Ffermenter’(samplesS1D-S6F)wasused
forthechallengewithC.perfringensasamodelpathogen.Forallfermenters,rumencontent
wascollectedfromtwonon-lactatingruminalfistulatedGermanHolsteincattle(5yearsold,
bodyweightapprox.850kg)ownedbytheInstituteforPhysiology.Thedonoranimalswere
housedonstrawbeddingandfedhay(7.5kg/d),acommercialconcentrate(500g/d,Deuka
Schaffutter,DeutscheTiernahrungCremer,Du¨sseldorf)andamineralsupplement(75g/d,
VitaMiralTrockensteher,VitaVisGmbH,Mu¨nster,Germany).Rumencontentswerecol-
lected3haftermorningfeedingandseparatedintoliquidandsolidcontentbygauzefiltration.
Atthestartoftheexperimentallfermenterswereinoculatedwithtwonylonbags(poresize:
150μm,Gesellschaftfu¨rAnalysetechnikHLS,Salzwedel,Germany)intheinnervessel,one
containing70gofsolidrumencontentmixedfrombothanimals,onecontainingtheexperi-
mentalsubstrateof5ghay(2cmlength)and5gconcentrate,andapproximately750mlof
mixedrumenfluid.After24htherumencontentbagwasreplacedbyasubstratebag.Subse-
quently,bagswerechangedalternatelyasdescribedpreviously[2].Thefermenterswerekept
inawaterbathat39˚Candtheinnervesselwasslowlymovedupanddownbyanelectric
motor(sixtimesperminute).Buffersolution(S1Table)wasinfusedcontinuouslytoachievea
liquidturnoverofonceperdayandeffluentswerecollectedinconicalglassflaskskeptonice.
Dailyeffluentvolumeswererecorded(datanotshown).Fermentationgaswascollectedingas
bags(Plastigas,LindeAG,MunichGermany)toachieveanair-tightsystemandafterdaily
changeofthesubstratebagsandsamplecollectionglassflaskswereflushedwithnitrogento
maintainanaerobicconditions.
ExperimentaltimescheduleandRUSITECeffluentsampling
Theexperimentconsistedofanequilibrationperiodoffourdays,athreedayssteady-state
periodandanexperimentalperiodofeightdays(Fig1A).Throughoutthewholeexperiment
pHandredoxpotentialwereevaluateddaily(digitalpH-meter646,Knick,Berlin,Germany;
electrodes:InLabRoutineandInLabRedoxProORP,MettlerToledo,Gießen,Germany)to
ensureadequateenvironmentalconditionsformicrobialsurvival.Duringtheexperimental
period,10mlC.perfringensinoculumwereaddedtovesselsD-F1hafterfeedingatexperi-
mentaldays8,9,11,13and14ataconcentrationof108CFU/10ml(Fig1B).Inoculumwas
derivedfromfreshculturecontainingvegetativecells.Differentapplicationintervalswere
appliedtotestwhetherC.perfringensisabletocolonizethefermentersandwhetherrepeated
applicationresultsinanaccumulationofC.perfringensinthefermenters.Effluentsamples(60
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A
Equilibration Steady-state Experimental period
Days 1 to 4 Days 5 to 7 Days 8 to 15
B
Non-infected
C. perfringens
fermenters fermenters
Day A B C D E F
5 S S S S S S
1A 1B 1C 1D 1E 1F
6 S S S S S S
2A 2B 2C 2D 2E 2F
7 S S S S S S
3A 3B 3C 3D 3E 3F
8 x x x
9 x x x
10 S S S S S S
4A 4B 4C 4D 4E 4F
11 x x x
12 S S S S S S
5A 5B 5C 5D 5E 5F
13 x x x
14 x x x
15 S S S S S S
6A 6B 6C 6D 6E 6F
Fig1.Experimentalsetup.(A)4daysequilibrationperiod(yellow),3dayssteady-stateperiod(orange)and8days
experimentalperiod(red).(B)Threenon-infectedfermentersfromthesteady-state-andexperimentalperiodaredepicted
inA-CandthreeC.perfringensinfectedfermentersinD-F.S=RUSITECeffluentsampling,yellow“x”=C.perfringens
inoculation.TheredboxesindicateC.perfringens-spikedfermenters.
https://doi.org/10.1371/journal.pone.0192256.g001
ml)werecollectedduringthesteady-stateperiod(days5,6,7)andintheexperimentalperiod
atdays10,12and15fortheassessmentofSCFAproductionandammoniaconcentrationsas
wellasforbacterialcommunityandmetabolomeanalysis.Effluentsampleswerecollected
beforeintroducinganewfeedbag.Sampleswerefrozenimmediatelyat−20˚Ctominimize
metabolitedegradation.
C.perfringensisolate
C.perfringenstypestrainCCUG1795T(NCTC8237/ATCC13124)wasusedasinoculumfor
theRUSITECchallengeexperimentasamodelpathogen.Thestrainwasisolatedfrombovine
originandispositiveforthecpatoxingene.
Thebacterialsuspensionwaspreparedfrompurecultureofthetypestraininthioglycolate
broth.Thecolonyformingunits(CFU)wereestimatedusingserialdilutionoftheC.perfrin-
genscultureinsterileNaClsolution(0.85%).Thecolonynumberwasestimatedusingplate-
castingmethod.1mlofthedilutedculturewasmixedwith10–15mlmeltedSulfit-Cycloserin
(SC)Agar,(ThermoScientific,Wesel,Germany)at43–47˚C.ThenathinlayerofSCAgarwas
applied.Theplateswereincubatedanaerobicallyfor20h±2hat37˚C.Thespikingsuspen-
sion(10ml)wascomposedof107CFUC.perfringensperml.
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DNAextraction,Illuminaampliconsequencingandreadprocessing
TheRUSITECeffluentsamples(36samples)andnativerumenfluidsamplesofthedonorani-
mals(oneofeachofthetwocowsanda50:50%mixofboth)werethawedonice,vortexedand
genomicDNAwasextractedfrom0.25mlRUSITECeffluentinduplicateaftershakingthe
tuberigorously.ThePowerSoilDNAIsolationKit(MOBIOLaboratories,Inc.,California,
USA)wasusedaccordingtothemanufacturer’sprotocol(http://www.mobio.com)withfol-
lowingmodifications:Mechanicallysiswasdonefor15minonaMOBIOVortexAdapter
after10minincubationat70˚C.Afterextractiontheduplicatesampleswerepooled.DNA
concentrationwasdeterminedusingtheQubit2.0Fluorimeter(QubitdsDNABRAssayKit,
ThermoFisherScientific,Vienna,Austria).AnegativeextractioncontroloftheDNAextrac-
tionkitwasisolatedandprocessedlikeRUSITECsamples.Forsequencingof40samples(36
RUSITECsamples,threenativerumenfluidsamplesandonenegativeextractioncontrol),the
V3/4/5hypervariableregionofbacterial16SrRNAgeneswastargetedusingtheprimerset
341F(5’-CCTACGGGRSGCAGCAG-30)[22]and909R(50-TTTCAGYCTTGCGRCCGTA
C-30)[23].Librarypreparation,Nexteratwo-stepPCRamplification,equimolarpoolingof
samplesandsequencingwitha300bppaired-endreadsprotocolusinganIlluminaMiSeq
sequencingplatformwereperformedbyMicrosynth(Balgach,Switzerland).Sequencedata
wereanalyzedwiththesoftwarepackageQIIMEv1.9.1[24].Atotalof1,367,579demultiplexed
readswasproducedandqualitycontrolled.ChimericsequenceswerecheckedwithUSEARCH
6.1[25],bycomparingsequencesagainstthereferenceGolddatabase(http://drive5.com/
uchime/gold.fa).MicrobialtaxonomywasassignedbyclusteringreadswithUCLUST
(V.1.2.22q)usingGreengenes(V.13.8)asreferencedatabase(basedona97%similaritytres-
hold).OTUswith<10sequenceswereremoved,resultinginadefinitereadcountof997,650
foralldownstreamanalyses.Forevensamplinginbetadiversitycalculations,thedepthofcov-
eragewassettothelowestnumberofreadsobservedinaRUSITECsample(14,464reads).
Directflowinjection,LC-MS/MScompoundidentificationand
quantification,short-chainfattyacids(SCFA)andammoniaconcentration
measurements
Concentrationsofaminoacids,sugars,acylcarnitines,sphingolipids,glycerophospholipids
andbiogenicaminesweredeterminedusingatargetedquantitativemetabolomicsapproach.
Forthisacombinationofdirectinjectionmassspectrometrywithareverse-phaseLC-MS/MS
wasappliedusingtheAbsoluteIDQp150KitfromBIOCRATESLifeSciencesAG(Austria)as
describedbySaleemandcolleagues[26].OnemilliliterofallRUSITECeffluentsampleswas
senttoBIOCRATESLifeSciencesAG(Austria)formetabolomicsanalysis.Dailyproduction
ofSCFAwasassessedbymultiplyingtheeffluentvolumewithSCFAconcentrationsdeter-
minedbygaschromatographyasdescribedpreviously[27].Ammoniaconcentrationswere
measuredphotometricallyasdescribedbyRiedeandcolleagues[28].
qPCRofallbacterialcountsandartificiallyaddedmodelpathogenC.
perfringens
ThestandardforqPCRswaspreparedwithpooledDNAfrom12DNAsamples(twosamples
pertimepoint)withserialdilutionsofthepurifiedqPCRproductsaspreviouslydescribedby
Liandcolleagues[29].Standardcurves(range1e+1–1e+6genecopynumbers)wereincluded
ineachqPCRassay.Allamplificationreactionsandnegativecontrolswerepipettedindupli-
cates.qPCRreactionscontained10μl2×BrilliantIIIUltra-FastSYBRGreenqPCRMaster
Mix(Agilent,Vienna,Austria),2μlofeachprimer(2.5μMinitialconcentration),5μlof
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nuclease-freewaterand1μlDNAtemplate.Afterthereaction,ameltingcurvewitharangeof
70to95˚Cwithfluorescencemeasurementsat1˚Cintervalswasdone.Totalgenecopynum-
bersof16SrRNAgenesweredeterminedusingtheforwardprimer5’-CCTACGGGAGGC
AGCAG-3’(341F)andthereverseprimer5’-ATTACCGCGGCTGCTGG-3’(518R)[30]
(Microsynth,Balgach,Switzerland)withafinalconcentrationof0.25μMand1μlDNA.qPCR
includedaninitialdenaturationstepat95˚Cfor3min,followedby40cyclesof95˚Cfor5s
and61˚Cfor20swithafluorescencemeasurementatthelaststepofeachcycle.C.perfringens
wasquantifiedwithaplc-specificqPCRassay[31]usinganannealingtemperatureof60˚Cand
qPCRconditionsasdescribedinNagpal,etal.[31].AllqPCRswereperformedwithaStrata-
geneMx3000Preal-timePCRsystem(AgilentTechnologies,SantaClara,USA)andresults
wereanalyzedusingtheassociatedsoftware(StratageneMxPro,QPCRSoftware,version
2.00).InformationregardingqPCRasrecommendedbytheMIQEguidelines[32]areshown
inS2Table.
Statisticalanalysis
Todescribedifferencesregardingtosamplingtimepointsamongdiversityindices(Shannon
index,Chao1estimator)andqPCRresults,alinearregressionmodelwasappliedbyusingthe
openaccesssoftwareR[33].First,thesixsamplingtimepoints(days5,6,7,10,12,and15,fer-
mentersA-C)werecomparedamongeachother.Inasecondstep,fermenterswithartificially
addedC.perfringens(days10,12,and15,fermentersD-F)weretestedagainstnon-infected
fermenters(days10,12,and15,fermentersA-C)toverifywhetherC.perfringensinfluences
anyoftheseresiduals.Datawereassessedvisuallywithregardstonormaldistributionofresid-
uals(histogramsandquantileplots).Acontrastcoefficientamongthesixdifferenttimepoints
(fermentersA-C)andbetweentheC.perfringensandnon-infectedgroup(days10,12,and15)
werecalculatedforeachdiversityindexandqPCRresultsbyusingamultiplecomparisonof
means(Tukeycontrasts).ThecontrastcalculationwasimplementedinRusingthepackage
multcompwithasignificancelevelattainedatp(cid:20)0.05(https://cran.r-project.org/web/
packages/multcomp/multcomp.pdf).
Totestifthebacterialcommunitiesdifferbetweenthesixsamplingtimepointsandbetween
theC.perfringensandnon-infectedgroups,weightedandunweightedUniFracdistanceswere
calculatedandanalyzedwiththecompare_categories.pyscriptinQIIMEusingANOSIM.
Todefinebacterialphylotypesandmetabolitesthatweremostlikelytoexplaindifferences
betweentimepointsandbetweentheC.perfringensandnon-infectedgroup,themetagenomic
biomarkerdiscoverytoolLEfSewasapplied[34]usingtheKruskal-Wallissum-ranktestto
describedifferentabundancesbetweengroups[34].LEfSeanalysiswasperformedwitha
thresholdof2.0onthelogarithmicLDAscorefordiscriminativefeatureswithanalpha
value<0.05forthefactorialKruskal-Wallistestamongclassesandanall-against-allmulti-
classanalysisstrategy.
Accessionnumber
IlluminaMiSeqsequencingdataareavailableinBioProjectSRAdatabaseundertheaccession
numberPRJEB15167.
Results
OverallbacterialcommunitycompositionintheRUSITEC
Atotalof1,367,579demultiplexedreadswasproducedwiththe300bppaired-endreadspro-
tocolusingIlluminaMiSeqsequencing.Afterastringentqualitycontrol,adefinitereadcount
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of997,650(73%)remainedforalldownstreamanalysis.Consideringall36RUSITECsamples,
17bacterialphylawereidentifiedwithBacteroidetesandFirmicutesbeingmostabundant
(91.9%ofallreads).Intotal,4,071OTUswerebuiltbasedona0.03distancelevelandusedfor
allfurtherdownstreamanalyses.The50mostabundantOTUswereclassifiedagainsttype
strainsoftheGreengenesdatabaseandwerelistedinTable1.Thesetypestrainswereprevi-
ouslydescribedtobelongtothecommensalrumenmicrobiota,plants,orenvironmentalsam-
ples.ThetwomostabundantOTUs(7.5%and5.9%relativeabundance)wereclassifiedas
PrevotellabryantiiandPrevotellaruminicolawith100%sequencesimilaritycomparedtothe
bestGreengenestypestrainhit.
Additionally,wesequencedthreenativerumenfluidsamples.FirmicutesandBacteroidetes
werethemostabundantphyla(56.4%and38.5%,respectively).Intotal,91%oftheOTUs
detectedintheRUSITECsampleswerealsodetectedinthenativerumenfluidsamplesinclud-
ingallOTUswitharelativeabundanceof>0.8%.Atotalof70.5%oftheOTUswereoverlap-
pingbetweentheRUSITECsamplesandthenativerumenfluidsamples.Overallbacterial
communitycompositionofthenativerumenfluidsamplesdifferedstatisticallysignificantly
fromtheRUSITECsamples(ANOSIM;p=0.001).
Bacterialcommunitycompositionatdifferenttimepointsdidnotchangesignificantly
intheRUSITEC. Thebacterialcommunitieswithinthetriplicates(fermentersA-Corfer-
mentersD-F,testedforeachtimepoint)didnotdifferamongeachother(ANOSIM;p=
0.518).ThenumberofOTUsdecreasedslightlyattheendoftheexperiment(day5compared
withday12andday15,p=0.044andp=0.027,respectively),however,therewasnostatisti-
callysignificantdifferenceifcomparedalongthetimeline.Furthermore,theShannonindex
didnotdifferamongtimepoints.Therewasastatisticallysignificantincreaseofestimatedspe-
ciesrichness(Chao1estimator)fromday5today6(p=0.033)andastatisticallysignificant
decreasefromday7today10(p=0.048)(Table2).Bacterialcommunitycompositionsatdif-
ferenttimepointsdidnotchangeaccordingtotheweightedUniFracdistances(ANOSIMfor
day5comparedtoday6,day6comparedtoday7,day7comparedtoday10,day10compared
today12,andday12comparedtoday15,p=0.125,p=0.105,p=0.115,p=0.094,andp=
0.104,respectively,Fig2A).However,acontinuouscommunityshiftwasobservedinthe
unweightedUniFracanalysisatsamplingdays7to15(Fig2B),indicatingastablebacterial
communitycompositionfromdays5to7(steady-stateperiod)andashiftbetweenthelatter
threedayscomparedtodays10,12and15aswellaswithindays10,12and15.Thenative
rumenfluidsamplesclusteredseparatelyfromtheRUSITECsamples.
Toinvestigateshiftsalongthetimelineonlythenon-infectedfermentersA-Cwereused.In
total,54phylotypeswerefoundtobestatisticallysignificantlyenrichedalongthetimelineata
certaintimepoint(Fig3).Atday5,e.g.thephylumFibrobacteres,theclassesofAlphaproteo-
bacteriaandElusimicrobiaandthefamilyPrevotellaceaewereenrichedcomparedtoother
samplingdays.Atday6,theclassofBacilliandtheorderofPseudomonadaleswereincreased
andatday7,thefamilyParaprevotellaceae wasenrichedcomparedtoothersamplingdays.
Atday12,e.g.Lachnospiraceae wasincreased.Atday15,thehighestnumberofshiftswas
observed:ThephylaFirmicutesandActinobacteria,aswellasFlavobacterialesandSpirochae-
taleswereenrichedcomparedtoothersamplingdays.
Themetabolicprofile,pHandredoxpotentialweresummedupinTable3.From14amino
acidstested,11passedthedetectionlimitandwereanalyzable.Allofthemremainedstable
alongthesamplingperiod.From6biogenicamineswhichweretested,onlyputrescine
decreasedstatisticallysignificantlyatday15(p=0.048).SCFA,ammonialevels,redoxpoten-
tialandpHalsoremainedstableoverthetrial.
Totalgenecopynumbers,determinedbyqPCR,differedstatisticallysignificantlyamong
samplingtimepoints(Table4).Theincreaseofgenecopynumberswasstatisticallysignificant
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Table1. The50mostabundantOTUsareshownwiththerespectiverelativeabundanceintheRUSITECsamples,sequencesimilaritytoGreengeneshits,match
lengthandaccessionnumber.
OTU Rel.ab.1(%) Sim.2(%) Matchlength(bp) AccessionNo. Greengenesbesthit
2456812 7.50 100 543 NR_028866.1 Prevotellabryantiistr.B14;DSM113713
768947 5.92 100 543 AY699286.1 PrevotellaruminicolaL164
266210 2.19 100 552 EU728750.1 Megasphaeraelsdeniistr.DJF_RP06
312373 2.02 97.61 543 AB501154.1 Prevotellaruminicolastr.BP1-413
578085 1.77 97.24 543 AB501162.1 Prevotellaruminicolastr.BP1-803
811229 1.76 98.53 543 AB501173.1 Prevotellaruminicolastr.AC5-133
84193 1.67 82.14 543 AB255367.1 Galbibactermesophilusstr.Mok-17
579278 1.39 91.87 529 NZ_AAXG02000037.1 Bacteroidescapillosusstr.ATCC29799
576416 1.31 83.06 543 AY643076.1 Capnocytophagacynodegmistr.CIP103937
4358602 1.17 100 552 AB186315.1 Lactobacillusmucosaestr.DLS1003
209705 1.09 82.5 543 AB541983.1 Prolixibacterbellariivoransstr.JCM13498
813220 1.09 95.11 552 X81137.1 Succiniclasticumruminisstr.SE10
254969 1.04 83.79 543 AB554231.1 Alistipesonderdonkiistr.JCM16771
266445 1.00 100 552 EF120372.1 Lactobacillusamylovorusstr.LAB16
582088 0.86 97.42 543 AB501163.1 Prevotellaruminicolastr.BP1-903
581091 0.80 99.82 551 Y09434.1 Schwartziasuccinivoransstr.DSM10502T3
137580 0.78 98.36 550 EF120372.1 Lactobacillusamylovorusstr.LAB16
538223 0.77 99.82 552 EF120372.1 Lactobacillusamylovorusstr.LAB16
4438135 0.72 82.14 543 AY918928.1 Prolixibacterbellariavoransstr.F2
343181 0.66 82.99 541 AB078046.1 Flexibactercanadensisstr.IFO15130
New.Ref_OTU144 0.65 98.01 552 AB186315.1 Lactobacillusmucosaestr.DLS1003
559781 0.63 83.61 543 X97245.1 Capnocytophagacynodegmistr.ATCC49044
848968 0.59 92.08 543 AB501157.1 Prevotellaruminicolastr.BP1-603
213576 0.59 96.13 543 AB501162.1 Prevotellaruminicolastr.BP1-803
831922 0.58 86.2 529 U88891.1 Desulfotomaculumhalophilumstr.SEBR3139
324495 0.58 88.56 542 AB331896.1 Paraprevotellaclarastr.YIT11840
New.Ref.OTU379 0.58 98.19 552 EU163503.1 Streptococcuslutetiensisstr.907
New.Ref.OTU373 0.57 98.73 552 AB186315.1 Lactobacillusmucosaestr.DLS1003
1082539 0.57 100 552 EU163503.1 Streptococcuslutetiensisstr.907
738975 0.56 87.07 549 DQ833400.1 Sphaerochaetasp.str.TQ1
272580 0.55 93.74 543 AB501163.1 Prevotellaruminicolastr.BP1-903
592852 0.55 96.32 543 AB501173.1 Prevotellaruminicolastr.AC5-133
637375 0.54 89.25 549 DQ833400.1 Sphaerochaetasp.str.TQ1
635310 0.50 83.61 543 NR_025910.1 Rikenellamicrofususstr.Q-1;ATCC29728
New.Ref.OTU86 0.49 82.32 543 AF118419.1 Coenoniaanatinastr.726–82
New.Ref.OTU266 0.48 98.19 552 AB186315.1 Lactobacillusmucosaestr.DLS1003
New.Ref.OTU56 0.47 82.66 548 EU281854.1 Eubacteriumsp.str.F13
628517 0.45 86.09 532 DQ903989.1 Cytophagasp.PRPR22
330492 0.43 91.08 527 X76161.1 Clostridiumaminobutyricumstr.DSM2634
338757 0.42 98.91 552 AB186315.1 Lactobacillusmucosaestr.DLS1003
589852 0.42 100 526 NR_026315.1 Pseudobutyrivibrioruminisstr.DSM97873
546360 0.40 95.76 543 AB501173.1 Prevotellaruminicolastr.AC5-133
237728 0.40 82.5 543 GU470889.1 Capnocytophagasp.oraltaxon329str.F0087
144161 0.39 94.84 543 AF218619.1 Prevotellaruminicolastr.TC2-283
348764 0.39 86.74 528 AB186360.1 Clostridiumsp.str.EBR-02E-0046
New.Ref.OTU303 0.39 84.12 548 AF183405.1 Prevotellatanneraestr.89-9-1
(Continued)
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Ruminalbacterialcommunitydynamics,metabolomics,andClostridiumperfringenschallengeinaRUSITECsystem
Table1. (Continued)
OTU Rel.ab.1(%) Sim.2(%) Matchlength(bp) AccessionNo. Greengenesbesthit
New.Ref.OTU159 0.39 85.45 543 AB510702.1 Bacteroideshelcogenesstr.JCM6297
732053 0.38 84.67 548 EU281854.1 Eubacteriumsp.str.F13
548324 0.37 84.31 548 EU281854.1 Eubacteriumsp.str.F13
262849 0.37 96.01 552 NR_026450.1 Ruminobacteramylophilusstr.H18;DSM1361
353910 0.36 86.53 527 AJ229251.1 Clostridiumsp.str.FCB90-3
New.Ref.OTU97 0.36 85.5 545 AB547676.1 Prevotellabuccalisstr.JCM12246
99436 0.35 99.64 552 AB198428.1 Selenomonasruminantiumstr.S83
New.Ref.OTU512 0.35 98.55 552 EU163503.1 Streptococcuslutetiensisstr.907
1Rel.ab.=Relativeabundance
2Sim.=SequencesimilaritytobestGreengeneshit
3BestGreengeneshitisolatedfromtherumen.
4BestGreengeneshitisolatedfromthegastro-intestinaltractofruminants.
https://doi.org/10.1371/journal.pone.0192256.t001
atdays12and15comparedwithdays5,6,7and10(day12comparedtodays5,6,7and6:
p=0.007,p=0.019,p=0.004,p=0.001,andday11comparedtodays1,2,3and6:p=0.024,
p=0.038,p=0.019,p=0.022,respectively).
C.perfringensimpactscertainphylotypesbutnottheoverallbacterial
communitycompositionandmetabolome
TheC.perfringensstrain,whichwasaddedtofermentersD-Fduringtheexperimentalperiod,
wasdetectedtobethe113mostabundantOTUwitharelativeabundanceof0.80%,0.36%and
0.56%atdays10,12and15inthesequencingapproach.TheOTUwasnotdetectedinanyun-
spikedfermenter.
Bacterialcommunitycompositionsdidnotdifferbetweennon-infectedandC.perfringens
fermenters(S4A-S4CversusS4D-S4F,S5A-S5CversusS5D-S5F,S6A-S6CversusS6D-S6F;
ANOSIM;p=1.000,p=0.589,andp=0.101,respectively).Moreover,C.perfringensinfected
samplesdidnotclusterseparatelyintheweightedandunweightedUniFracanalyses(S1Fig)
andtheshiftsovertime,depictedinFig2Cand2D,weresimilartonon-infectedfermenters.
OTUrichnessanddiversitydidnotdifferbetweennon-infectedandinfectedfermentersat
anysamplingtimepoint(S3Table).Intotal,merely13,16and20phylotypeswerefoundtobe
differentiallyenrichedbetweenthenon-infectedandtheC.perfringensfermentersatdays10,
12and15,respectively(Fig4).Enrichmentsvariedbetweensamplingdays,exceptofLenti-
sphaeraewhichincreasedinC.perfringensinfectedsamplesatdays12and15andCoprococcus
Table2. StatisticalanalysisofOTUrichnessanddiversityofnon-infectedfermentersA-Cwithregardtosamplingday.
Samplingday
Steady-stateperiod Experimentalperiod
5 6 7 10 12 15 p-value
observedOTUs 1673±102ac 1690±44a 1704±116ac 1523±75bc 1486±46b 1390±103b <0.001(cid:3)
Chao1 2652±61bc 2807±58a 2735±43ab 2543±110ce 2362±77de 2241±99d <0.001(cid:3)
Shannon 8.01±0.27 8.07±0.09 8.08±0.24 8.03±0.29 8.17±0.14 7.77±0.39 NS
(cid:3)Timecontrastsweretestedbetweenallgroupsandnon-significantdifferencesweremarkedwithsameletters.
NS=notstatisticallysignificant.
https://doi.org/10.1371/journal.pone.0192256.t002
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Ruminalbacterialcommunitydynamics,metabolomics,andClostridiumperfringenschallengeinaRUSITECsystem
A B
Day 5
PC 2 (24.9 %) PC 2 (5.2 %)
Day 6
Day 7
Day 10
Day 12
Day 15
PC 1 (16.9 %)
PC 1 (34.6 %) PC 3 (4.5 %)
PC 3 (11.7 %)
C D
PC 2 (24.9 %) PC 2 (5.2 %)
PC 1 (16.9 %)
PC 1 (34.6 %) PC 3 (4.5 %)
PC 3 (11.7 %)
Fig2.UniFracdistancesofampliconsequencingdatadepictedasPCoAPlotwithsamplingdaysshownindifferentcolors.Thepercent
variationexplainedbyeachprincipalcoordinateisindicatedbytheaxes.A:weightedandB:unweightedUniFracwithonlynon-infected
fermenters(Fig1B,fermentersA-C).C:weightedandD:unweightedUniFracwithfermentersusedfortheC.perfringensspikingexperiment
(Fig1B,fermentersD-F).Onlyfermentersatdays10,12,and15arespikedwithC.perfringens.Nativerumenfluidsamplesofdonoranimals
areincludedineachpanel(Nativerumenfluid).
https://doi.org/10.1371/journal.pone.0192256.g002
whichwasenrichedinnon-infectedsamplesatdays12and15.OnphylumlevelSynergistetes
andBacteroideteswereenrichedatday15ininfectedfermenters,whileFirmicutesweremore
abundantinnon-infectedfermenters.
Differencesofsomemetabolitesbetweenthefermentersbeforespikingoffermenters(non-
infectedversusun-spikedinfectedfermenters:S1A-S1CversusS1D-S1F,S2A-S2Cversus
S2D-S2ForS3A-S3CversusS3D-S3F)werealreadystatisticallysignificant:acetate,propio-
nate,butyrate,isobutyrate,valerate,ammonia,methioninesulfoxide,phenylethylamine,
putrescine,aspartate,glutamate,spermidine,isoleucine,citrulline,lysine,proline,alanine,and
hexose.Forthestatisticalcomparisonofmetabolitesinnon-infectedversusinfectedfermen-
ters,thesemetaboliteswereexcludedfromtheanalyses.
PLOSONE|https://doi.org/10.1371/journal.pone.0192256 February7,2018 10/20