Propionibacterium acnes biofilm is present in intervertebral discs of patients undergoing

17 Pages·2017·2.06 MB·English

RESEARCHARTICLE
Propionibacterium acnes biofilm is present in
intervertebral discs of patients undergoing
microdiscectomy
ManuN.Capoor1,2*,FilipRuzicka3,JonathanE.Schmitz4,GarthA.James5,
TanaMachackova2,RadimJancalek6,MartinSmrcka7,RadimLipina8,FahadS.Ahmed2,
ToddF.Alamin9,NeelAnand10,JohnC.Baird2,NitinBhatia11,SibelDemir-Deviren12,
RobertK.Eastlack13,SteveFisher5,StevenR.Garfin14,JaspaulS.Gogia15,Ziya
L.Gokaslan16,CalvinC.Kuo17,Yu-PoLee11,KonstantinosMavrommatis18,ElleniMichu2,
HanaNoskova2,AssafRaz1,JiriSana2,A.NickShamie19,PhilipS.Stewart5,Jerry
a1111111111 L.Stonemetz20,JeffreyC.Wang21,TimothyF.Witham22,MichaelF.Coscia23,
a1111111111 ChristofBirkenmaier24,VincentA.Fischetti1,OndrejSlaby2*
a1111111111
1 LaboratoryofBacterialPathogenesisandImmunology,RockefellerUniversity,NewYork,NewYork,
a1111111111
UnitedStatesofAmerica,2 DepartmentofMolecularOncology,CentralEuropeanInstituteofTechnology
a1111111111
(CEITEC),MasarykUniversity,Brno,CzechRepublic,3 DepartmentofMicrobiology,FacultyofMedicine,
Masarykuniversity,St.Anne’sFacultyHospital,Brno,CzechRepublic,4 DepartmentofPathology,
MicrobiologyandImmunology,VanderbiltUniversitySchoolofMedicine,Nashville,Tennessee,UnitedStates
ofAmerica,5 CenterforBiofilmEngineering,MontanaStateUniversity,Bozeman,Montana,UnitedStatesof
America,6 DepartmentofNeurosurgery,St.Anne’sUniversityHospital,MasarykUniversity,Brno,Czech
Republic,7 DepartmentofNeurosurgery,UniversityHospitalBrno,MasarykUniversity,Brno,Czech
OPENACCESS
Republic,8 DepartmentofNeurosurgery,UniversityHospitalOstrava,OstravaUniversity,Ostrava,Czech
Citation:CapoorMN,RuzickaF,SchmitzJE, Republic,9 DepartmentofOrthopedicSurgery,StanfordUniversityMedicalCenter,StanfordUniversity,
JamesGA,MachackovaT,JancalekR,etal.(2017) Stanford,California,UnitedStatesofAmerica,10 Cedars-SinaiInstituteforSpinalDisorders,LosAngeles,
California,UnitedStatesofAmerica,11 DepartmentofOrthopaedicSurgery,UniversityofCaliforniaIrvine,
Propionibacteriumacnesbiofilmispresentin
SchoolofMedicine,Irvine,California,UnitedStatesofAmerica,12 SpineCenter,UCSFMedicalCenter,San
intervertebraldiscsofpatientsundergoing
Francisco,California,UnitedStatesofAmerica,13 ScrippsClinicDivisionofOrthopedicSurgery,SanDiego,
microdiscectomy.PLoSONE12(4):e0174518.
California,UnitedStatesofAmerica,14 DepartmentofOrthopaedicSurgery,UniversityofCaliforniaSan
https://doi.org/10.1371/journal.pone.0174518
Diego,SanDiego,California,UnitedStatesofAmerica,15 DepartmentofOrthopedicSurgery,Kaiser
Permanente—SanJoseMedicalCenter,SanJose,California,UnitedStatesofAmerica,16 Departmentof
Editor:HolgerBru¨ggemann,AarhusUniversitet,
Neurosurgery,TheWarrenAlpertMedicalSchoolofBrownUniversity,RhodeIslandHospital,Providence,
DENMARK
RhodeIsland,UnitedStatesofAmerica,17 DepartmentofOrthopedicSurgery,KaiserPermanente–Oakland
Received:January17,2017 MedicalCenter,Oakland,California,UnitedStatesofAmerica,18 CelgeneCorporation,Information
KnowledgeandUtilization,SanFrancisco,California,UnitedStatesofAmerica,19 Departmentof
Accepted:March10,2017 OrthopaedicSurgery,DavidGeffenSchoolofMedicine,UniversityofCalifornia,LosAngeles,California,
UnitedStatesofAmerica,20 DepartmentofAnesthesia,TheJohnsHopkinsHospital,Baltimore,Maryland,
Published:April3,2017
UnitedStatesofAmerica,21 DepartmentofOrthopedicSurgery,UniversitySouthernCalifornia,Los
Copyright:©2017Capooretal.Thisisanopen Angeles,California,UnitedStatesofAmerica,22 DepartmentofNeurosurgery,TheJohnsHopkinsHospital,
Baltimore,Maryland,UnitedStatesofAmerica,23 DepartmentofOrthopedicSurgery,OrthoIndyHospital,
accessarticledistributedunderthetermsofthe
Indianapolis,Indiana,UnitedStatesofAmerica,24 DepartmentofOrthopedics,PhysicalMedicine&
CreativeCommonsAttributionLicense,which
Rehabilitation,UniversityofMunich(LMU),Munich,Germany
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
*[email protected](MNC);[email protected](OS)
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthemanuscript. Abstract
Funding:DiscitisDx,Inc.providedsupportinthe
formofaresearchgrantwhichwasusedtopayfor
Background
culturing,microscopy,andmaterials.Neither
DiscitisDX,Inc.noranyothercommercial Inpreviousstudies,Propionibacteriumacneswasculturedfromintervertebraldisctissueof
organizationprovidedanysupportintheformof
~25%ofpatientsundergoingmicrodiscectomy,suggestingapossiblelinkbetweenchronic
salariesforauthorsanddidnothaveanyroleinthe
bacterialinfectionanddiscdegeneration.However,giventheprominenceofP.acnesasa
studydesign,datacollectionandanalysis,decision
topublish,orpreparationofthemanuscript.KM’s skincommensal,suchanalysesoftenstruggledtoexcludethealternatepossibilitythat
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Propionibacteriumacnesbiofilminintervertebraldiscsofpatientsundergoingmicrodiscectomy
commercialaffiliationwithCelgenedidnotplaya theseorganismsrepresentperioperativemicrobiologiccontamination.Thisinvestigation
roleinthestudy. seekstovalidateP.acnesprevalenceinresecteddisccultures,whileprovidingmicroscopic
Competinginterests:MNC,FSA,JCB,JLS,OS, evidenceofP.acnesbiofilmintheintervertebraldiscs.
JES,KM,andVAFhavestockownershiporoptions
inDiscitisDX,Inc.KMisanemployeeofCelgene
Methods
Corporation.Therearenootherfinancialornon-
financialcompetinginterests.Theseaffiliationsdo Specimensfrom368patientsundergoingmicrodiscectomyfordischerniationweredivided
notaltertheauthors’adherencetoPLOSpolicies
intoseveralfragments,onebeinghomogenized,subjectedtoquantitativeanaerobicculture,
onsharingdataandmaterials.
andassessedforbacterialgrowth,andasecondfragmentfrozenforadditionalanalyses.
ColonieswereidentifiedbyMALDI-TOFmassspectrometryandP.acnesphylotypingwas
conductedbymultiplexPCR.Forasub-setofspecimens,bacterialocalizationwithinthe
discwasassessedbymicroscopyusingconfocallaserscanningandFISH.
Results
Bacteriawereculturedfrom162discs(44%),including119cases(32.3%)withP.acnes.In
89cases,P.acneswasculturedexclusively;in30cases,itwasisolatedincombinationwith
otherbacteria(primarilycoagulase-negativeStaphylococcusspp.)Amongpositivespeci-
mens,themedianP.acnesbacterialburdenwas350CFU/g(12-~20,000CFU/g).Thirty-
eightP.acnesisolatesweresubjectedtomolecularsub-typing,identifying4of6defined
phylogroups:IA ,IB,IC,andII.Eightculture-positivespecimenswereevaluatedbyfluores-
1
cencemicroscopyandrevealedP.acnesinsitu.Notably,thesebacteriademonstrateda
biofilmdistributionwithinthediscmatrix.P.acnesbacteriaweremoreprevalentinmales
thanfemales(39%vs.23%,p=0.0013).
Conclusions
ThisstudyconfirmsthatP.acnesisprevalentinherniateddisctissue.Moreover,itprovides
thefirstvisualevidenceofP.acnesbiofilmswithinsuchspecimens,consistentwithinfection
ratherthanmicrobiologiccontamination.
Introduction
Althoughfirstmorethan15yearsago,therelationshipbetweenintervertebraldiscdegenera-
tionandchronic,primaryinfectionbyProprionibacteriumacnesremainscontroversial.In11
independentstudiespublishedbetween2001and2016,involving1188combinedpatients
undergoingdiscectomyormicrodiscectomy,thisGram-positivebacterialspecieswasisolated
from~25%ofresecteddiscspecimens[1–11].However,thesestudieshadsignificantmethodi-
caldifferencesandthemajoritywasstatisticallyunderpowered[1–9]Moreover,theuseof
qualitativemicrobiologictechniques—i.e.deemingaspecimenpositiveornegativeforbacterial
growthwithoutregardtocolonycounts—couldhaveledtoculturesthatwerefalselypositive
forP.acnesorotherorganisms.Thisisduetotheconcernforlow-levelbacterialcontamina-
tionfromthesurgicalenvironment,theclinicallaboratory,orthepatients’skin,asP.acnesisa
nearlyubiquitoushumancommensalthatcancolonizethefollicleandsebaceousunitathigh
levels[6,12].
Ontheotherhand,severalofthepreviousstudiesreportedalowprevalenceofculture-
positivityinresectedlumbardiscs[5,6,10].Inthislight,evenifP.acnesweretrulypresent
withinadisc,theorganism’smodeofgrowthandtheculturetechniquesemployedbythe
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Propionibacteriumacnesbiofilminintervertebraldiscsofpatientsundergoingmicrodiscectomy
laboratorymightunderestimatebacterialburden.P.acnescanbeslowtogrowandoften
proliferatesinvivoasaggregatedbiofilms,whichcanbechallengingtocultivateinvitroand
requirephysicalbiofilmdisassembly(e.g.throughsonicationorhomogenization)priorto
microbiologicplating[11–13].Asaresult,standardspecimenpreparationandculturetech-
niquescouldleadtofalsenegativeresults.Basedonthesemethodologicalissues,itisnot
surprisingthattheprevalencerateofculturedP.acnesinpreviouspublicationsrangesfrom
0to44%[11].
Inarecentstudyfromthecurrentauthors[11],P.acneswasidentifiedin40%of290
patientsundergoingmicrodiscectomyfordischerniation.Thatstudyutilizedquantitativecul-
turetechniquesanditrepresentedthelargestseriesofpatientsinvestigatedtodate.Neverthe-
less,severalmethodicalweaknessesremainedtobeaddressed,andwehadnotyetprovided
directevidenceofP.acnesbiofilmswithinthediscs.
Accordingly,inthecurrentstudy,wesoughttovalidateandextendourpreviousworkwith
asubsequent(andlarger)independentseriesofpatientsundergoingmicrodiscectomyforlum-
barherniation.ThisstudycharacterizestheprevalenceofP.acnesinresecteddisctissueviaan
improvedprotocolforquantitativebacterialculture,alongwithmolecularphylotypingofP.
acnesisolates.Moreover,weconductinsitufluorescenceimagingofasetofspecimenstodem-
onstratethedistributionofthebacteriaasabiofilmwithinthedisctissue.
Methods
Studydesignandpatientcharacteristics
AdultsscheduledformicrodiscectomyforsymptomaticlumbardischerniationbetweenJanu-
ary2016andNovember2016atSt.Anne’sUniversityHospital(Brno,CzechRepublic)and
UniversityHospitalBrno(Brno,CzechRepublic)wereprospectivelyscreenedimmediately
priortosurgeryandconsentedforpotentialparticipationinthisstudy.Tosummarize,inclu-
sioncriteriacoveredalladultsundergoingmicrodiscectomy.Exclusioncriteriawere:immuno-
logicalcompromise;traumaticherniations;thepresenceofanunknownradiographicmass;and
inflammatoryorrheumatologicdisease.Theinclusionandexclusioncriteriaaredescribedin
detailinCapooret.al.2016[11].Outof386patientsapproached,8declinedtoparticipateinthe
studyand5patientswerenotincludedduetorheumatoidarthritisorantibiotictreatmentfor
recurrenturineinfectionintheonemonthpriortosurgery.Sincewewerenotfocusedonthe
follow-upofpatients,theonlyreasonfordrop-outfromourstudywaszeroorinsufficient
amountofthedisctissueavailableformicrobiologyculture.Thissituationoccurredin5cases.
Theepidemiological/clinicaldatacollectionincluded:sex,age,intervertebralsegmentinvolved,
typeofherniation,historyofpreviousspinalsurgeries,priorepiduralsteroidinjections,pre-
operativepainscores(legpain[months],backpain[months],NRS(Numericalratingscalefor
pain)back,NRSleg,OswestryDisabilityIndex),anddevelopmentofpost-operativediscitis.Eth-
icsCommitteeapprovalswereobtainedforourstudyfromthefollowinghospitals:St.Anne’s
UniversityHospital(Brno,CzechRepublic)whereHospitalEthicsCommitteeapprovalwas
granted(Reference33V/2015)onJune10,2015;and,UniversityHospitalBrno(Brno,Czech
Republic)whereHospitalEthicsCommitteeapprovalwasgrantedonMay13,2015.Thisstudy
wasapprovedbytheInstitutionalReviewBoardsofthetwoparticipatinghospitalsandwritten
informedconsentwasobtainedfromeachpatient.
Surgicalspecimencollection
Alldiscsampleswereobtainedusingstandardoperatingpractices.Specimenswereasepti-
callyplacedintoaclosedsterilesamplecuptominimizethechancesofpost-resectioncon-
tamination[11].ThesecupswerelabeledandimmediatelytransportedtotheDepartment
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Propionibacteriumacnesbiofilminintervertebraldiscsofpatientsundergoingmicrodiscectomy
ofMicrobiologyatSt.Anne’sUniversityHospital(Brno,CzechRepublic).Sampleswere
sterilelydividedintotwofragments(processedwithin2–4hourspost-surgery):thefirstwas
utilizedimmediatelyforquantitativeanaerobicculture,whilethesecondwasfrozenfor
futuremicroscopyandotheranalyses.Thesizeoftheresecteddiscspecimensrangedfrom
3x3x5mmto10x5x5mm.
Microbiologicculture
Thediscfragmentforculturewasweighed,placedintoaMicroBag(Seward)containing4
mlofViande-Levuremedium,andhomogenizedwithaStomacher80(Seward)underasep-
ticconditions.100μloftheresultanthomogenatewasinoculatedontoWilkinsChalgren
AnaerobicAgarwith7%sheep’sbloodandvitaminK(HiMediaLaboratories).AnAnaero-
bicWorkStationConcept400(RuskinnTechnology)wasutilizedforculture;inoculated
plateswereincubatedfor14daysat37˚Cwithanatmosphereof80%N ,10%CO ,and
2 2
10%H .ThesameamountofthehomogenatewasalsoculturedaerobicallyonColumbia
2
BloodAgar(Oxoid)for7daysat37˚Cinordertodetectaerobicbacteria.Followingincuba-
tion,thebacterialcolonieswerecountedandthequantityofeachcolonialmorphotypewas
expressedascolonyformingunits(CFU)pergramoftissueusingtheMilesandMisra
method[14].
MALDI-TOFmassspectrometry
Taxonomicidentificationoftheabovecolonieswasconductedbymatrixassistedlaser
desorption/ionizationtime-of-flightmassspectrometry(MALDI-TOFMS).Thisanalysis
wasperformedonacommondiagnosticplatform,theMALDIBiotyperwithFlexControl
3.4software(BrukerDaltonik),accordingtomanufacturer’sinstructions.Tosummarize,
singlecolonieswereappliedasathinfilmontoasectionofaMALDI96-targetplate,over-
laidwith1μlof70%formicacid,anddriedatroomtemperature.Driedbacteriawereover-
laidwith1μlofmatrixsolution,saturatedα-cyano-4-hydroxycinnamicacidsolutionin
acetonitrile–water–trifluoroaceticacid(50:47.5:2.5,v/v),andallowedtodrybeforespectral
acquisition.MassspectrawereprocessedusingtheBioTyper3.1software,andmanufac-
turer-recommendedcut-offscoreswereemployedforidentification((cid:21)2indicatingspecies-
levelidentification;1.7–1.999indicatinggenus-levelidentification,and<1.7indicatingno
identification).Colonieswithisolatesaninitialscore<1.7wereretested,andthehighest
scorewasusedforidentification.
ConfocalscanninglasermicroscopyandSYTO9staining
Tenfrozendiscspecimenswereselectedprimarilybyasamplesize>0.5g.Theseincluded8
P.acnes-onlyspecimens(4frommenand4fromwomen)withabacterialload>103CFU/g
(outof38total),aswellas2negativecontrolswithnoculturegrowth.Frozenfragmentswere
transportedondryicetotheCenterforBiofilmEngineeringatMontanaStateUniversity
(CDCPHSPermit2016-05-119)forconfocallaserscanningmicroscopy(CSLM).Disctissue
wasdefrostedandfixedusing4%paraformaldehyde(ElectronMicroscopyScience);cryoem-
beddedinOptimalCuttingTemperaturemedium(SaukuraFinetek);cryosectionedat-20˚C
into7–10μmsections;andplacedontoSuperfrostPlusglassslides.Fornucleicacidlabeling,
slideswerestainedwiththefluorescentDNAmakerSYTO9(ThermoFisherScientific)and
imagedonaLeicaSP5confocalscanninglasermicroscope,with3-DreconstructionbyImaris
software.
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Propionibacteriumacnesbiofilminintervertebraldiscsofpatientsundergoingmicrodiscectomy
Fluorescentin-situhybridization
Twofluorescentin-situhybridization(FISH)probeswereemployedinthisstudy:thegeneral
eubacterial16SrRNAprobeEUB338(GCTGCCTCCCGTAGGAGT)andtheP.acnes-specific16S
rRNAprobePAC16s598(GCCCCAAGATTACACTTCCG).Theseoligonucleotides(Sigma-Aldrich)
werelabeledatboththe5’and3’endswitheitherCY5(forEUB338)orCY3(forPAC16s598).
ThetaxonomicspecificityoftheseprobeswasfirstvalidatedthroughexperimentswithculturedP.
acnes,Staphylococcusaureus,S.epidermidis,andPseudomonasaeruginosa.ForFISHanalysis,the
resecteddiscsweresectionedontoglassslides(asdescribedabove)andre-fixedwith4%parafor-
maldehyde(20minutesatroomtemperature),followedbyadeionizedwater(DIW)rinse.Slides
werenexttreatedwith1mg/mLlysozyme(AcrosOrganics)and30units/mLachromopeptidase
(Wako)for20minutesat37˚Cinahumidifiedchamber.AfteranotherDIWwash,thesections
weredehydratedinagradedethanolseries(50%,80%,100%;3minuteseach)anddriedwithfil-
teredcompressedair.
FISHprobes(2ng/μLeach,finalconcentration)wereaddedtoahybridizationbuffer
(900μLfilter-sterilizedH O,360μL5MNaCl,40μL1MTris/HCl,700μLformamide,and2μL
2
10%sodiumdodecylsulfate).Preparedslideswerestainedwiththeprobesovernightat46˚C,
followedby20minutesat48˚Cinwashbuffer(700μL5MNaCl,1mL1MTris/HCl,50μLof
10%SDS,madeupto50mLwithfilter-sterilizedH O).Slideswerethenrinsedwithice-cold
2
wateranddriedwithcompressedair.AdropofProlongGoldanti-fadesolution(LifeTechnol-
ogies)wasaddedtothesection,whichwascoveredwithacoverslipandallowedtodryover-
nightatroomtemperature.SectionswerevisualizedwithaNikonEclipseE800microscope
equippedwithaCY3andCY5filtercube.
P.acnesphylotypeanalysis
GenomicDNAfromP.acnesisolateswaspurifiedusingtheQIAampDNAminikit(Qiagen).
PhylotypingwasconductedaccordingtotheMLST8schemeanddeterminedbyarecently
describedmultiplexPCRassay[15].Refertothecitedpublicationforprimerinformationand
amplificationconditions.AC1000TouchTMThermalCycler(Bio-Rad)wasemployedfor
PCR.
Statisticalanalysis
Culturepositivity,bacterialidentity,andquantitativeorganismburden(CFU/gram)werecor-
relatedtothepatients’documentedclinicalparameterswiththeuseofthePrism5software
package(GraphPadSoftware,Inc.).Analysesofcategoricalvariableswereperformedwitha
Fisher’stwo-sidedexacttest,andanalysisofpatientagewasperformedwithanon-parametric
Mann-WhitneyUtest(significancelevelsetatp=0.05).
Results
Bacterialcultureanalysis
Resecteddiscspecimensfrom368patientswithlumbarherniationweresubjectedtoextended
anaerobicculture.BacterialgrowthwasobservedandidentifiedbyMALDI-TOFfor162of
368specimens(44%),withnocoloniesobservedfortheother206.Amongpositivespecimens,
P.acneswaspresentin119cases(32.3%);and,non-P.acnesbacteriawaspresentin42cases
(11.4%).S.saccharolyticuscomprised11(3%),S.epidermiduscomprised10(2.7%),andS.hae-
molyticus3(0.8%)ofthese42cases;and,alltheremainingspeciesrepresentedeitheroneor
twocases.ForspecimenswithgrowthofP.acnes,amajority(89/119,75%)yieldedonlythis
organism,withoutanyadditionalbacterialspeciespresent.Conversely,30specimens(34%)
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Propionibacteriumacnesbiofilminintervertebraldiscsofpatientsundergoingmicrodiscectomy
demonstratedmixedgrowthof2ormorebacterialspecies.OftheseP.acneswithS.epidermi-
dus,P.acneswithS.haemolyticus,P.acneswithS.hominis,andP.acneswithS.warnerieach
accountedfor4cases(1.1%).38P.acnescases(10.3%)werefoundtobe(cid:21)1000CFU/g:31
wherethespecieswasexclusive,4incombinationwithStaphylococcus(i.e.,2withS.warneri,1
withS.haemolyticus,and1withS.pasteuri),and1withP.granulosum.Also,13casesofother
bacteria(3.5%)werefound,inaggregate,tobe(cid:21)1000CFU/g,5ofwhichwereincombination
withP.acnes.Oftheremaining8cases(2.2%),4caseswereStaphylococcusonly(i.e.,2were
S.epidermidus,1ofS.epidermiduswithS.hominis,and1wasS.warneri),2wereprimarily
Streptococcus(i.e.,1ofS.mitisand1ofS.mitiswithS.parasanguinis.andA.graevenitz),1was
C.tuberculostearicum,and1wasEnterococcusgallinarum(refertoTable1foradetailedsum-
maryofthisinformation).Aerobiccultivationdidnotleadtoanyadditionalsignificantfind-
ings.Cultivationneitherincreasesthenumberofpositivesamplesnorextendsthespectrumof
detectedmicrobes.
QuantitativeburdenofP.acnesindiscspecimens
Theobservedorganismburdenfromthe119P.acnes-positivespecimensvariedsignificantly
fromcasetocase.QuantitativeP.acnescountsrangedfrom12to20952CFU/g,withamedian
of350CFU/gram.Wenoted38cases(32%)withP.acnescolonycounts(cid:21)103CFU/gram;46
cases(39%)with102–103CFU/gram;andtheremaining35cases(29%)with101–102(Fig1).
InsitudeterminationofbacteriabyCSLM
GiventhehighburdenofculturedP.acnesinmanyspecimens,wenextsoughttoinvestigate
theinsiturelationshipoftheorganismstothedisctissue.Asetof8specimenswith>1000
CFU/g(alongwith2culture-negativecontrols)wereselectedforvisualization,firstviaconfo-
calscanninglasermicroscopy(CSLM)andtheSYTO9DNAdye.Bacterialbiofilmswere
detectedin7outof8culture-positivespecimenstested(Table2).Athree-dimensionalrecon-
structionofatypicalbiofilmisprovidedinFig2A(fromSample4).Thebacteriaarepresent
withinthebodyoftheherniatedintervertebraldisc,notonthesurface(aswouldbeexpected
foraperioperativecontaminantthatbecameassociatedwiththetissueduringresection).No
bacteriawerevisualizedintheculture-negativecontrolsamples.
InsituidentificationofP.acnesbyFISH
TocomplementtheSYTO9images,furtherfluorescencemicroscopywasconductedwith16S
rRNAFISHprobestargetingeitheraubiquitouseubacterialsequence(EUB338)orP.acnes
specifically(PAC16s598).Theseprobeswereappliedtoall8ofthediscspecimensdescribed
above,forwhich>1000CFU/gofP.acneswereculturedandbiofilmswerevisualizedby
SYTO9stainingandCSLMwiththeexceptionofSample9.Thepresenceofbacterialbiofilms
wasconfirmedin6ofthe8specimenswiththeEUB338probe,whichalsostainedpositively
withPAC16s598.Twoexamplesofimages(withbothprobes)aredepictedinFig2andFig3.
Overall,thesedatafurtherdemonstratethepresenceofP.acnesbiofilm-aggregatesinresected
discspecimenswithcorrespondingculturepositivity.
PhylotypingofP.acnesisolatesfromintervertebraldiscs
Wephylotyped38isolatesofP.acnesfromdiscswith>1000CFU/gtogaugewhetherany
clade-specificassociationsexist.TypeIA P.acneswasidentifiedin15(39%)cases,typeIBin9
1
(24%)cases,typeICin1(3%)case,andtypeIIin13(34%)cases.Nodominantphylotypewas
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Propionibacteriumacnesbiofilminintervertebraldiscsofpatientsundergoingmicrodiscectomy
Table1. Summaryofthemicroorganismsisolatedin162casesfrom368patientintervertebraldiscspecimensbyanaerobicculture.
(cid:21)1000CFU/g
Aggregateof
PositiveCases P.acnes OtherBacteria
Isolatedmicroorganism N % N % N %
Propionibacteriumacnescases 119 32.3% 38 10.3% 5 1.4%
P.acnesonly 89 24.2% 31 8.4%
P.acneswithStaphylococcusonly 22 6.0% 5 1.4% 4 1.1%
P.acnes,S.aureus 1 0.3%
P.acnes,S.capitis 2 0.5%
P.acnes,S.epidermidus 4 1.1% 1 0.3%
P.acnes,S.haemolyticus 4 1.1% 2 0.5% 1 0.3%
P.acnes,S.hominis 4 1.1%
P.acnes,S.pasteuri 1 0.3% 1 0.3% 1 0.3%
P.acnes,S.(unspecified) 1 0.3%
P.acnes,S.warneri 4 1.1% 1 0.3% 2 0.5%
P.acnes,S.intermedius 1 0.3%
P.acneswithother 8 2.2% 2 0.5% 1 0.3%
P.acnes,P.granulosum 1 0.3% 1 0.3% 1 0.3%
P.acnes,P.granulosum,F.magna 1 0.3% 1 0.3%
P.acnes,P.acidifaciens,S.hominis 1 0.3%
P.acnes,S.epidermidus,S.viridans 1 0.3%
P.acnes,S.haemolyticus,S.oralis 1 0.3%
P.acnes,S.hominis,Paenibacillus 1 0.3%
P.acnes,S.mitis 1 0.3%
P.acnes,S.oralis,Veilonelladispar 1 0.3%
Non-P.acnesbacteriacases 43 11.7% 8 2.2%
Staphylococcusonly 32 8.7% 4 1.1%
S.capitis 1 0.3%
S.epidermidus. 7 1.9% 2 0.5%
S.epidermidus.S.haemolyticus 1 0.3%
S.epidermidus.S.hominis 1 0.3% 1 0.3%
S.haemolyticus 3 0.8%
S.hominis 2 0.5%
S.lugdunensis 1 0.3%
S.pasteuri 2 0.5%
S.saccharolyticus 11 3.0%
S.(unspecified) 2 0.5%
S.warneri 1 0.3% 1 0.3%
Staphylococcuswithother 3 0.8%
S.epidermidus,A.odontolyticus 1 0.3%
S.haemolyticus,Arthrobacter(unspecified) 1 0.3%
S.haemolyticus,Rothiaamarae 1 0.3%
Streptococcus 3 0.8% 2 0.5%
S.mitis 1 0.3% 1 0.3%
S.viridans 1 0.3%
S.mitis,S.Parasanguinis.,A.graevenitz 1 0.3% 1 0.3%
Corynebacterium 2 0.5% 1 0.3%
C.minutissimum 1 0.3%
(Continued)
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Propionibacteriumacnesbiofilminintervertebraldiscsofpatientsundergoingmicrodiscectomy
Table1. (Continued)
(cid:21)1000CFU/g
Aggregateof
PositiveCases P.acnes OtherBacteria
Isolatedmicroorganism N % N % N %
C.tuberculostearicum 1 0.3% 1 0.3%
Other 3 0.8% 1 0.3%
Agromyces(unspecified) 1 0.3%
Enterococcusgallinarum 1 0.3% 1 0.3%
Rothiadentocariosa 1 0.3%
Total 162 44.0% 38 10.3% 13 3.5%
Notes:forP.acnes(cid:21)1000CFU/gappliestothatspecies;forallotherbacterialisted,anaggregateofthespeciesfoundisused.“(unspecified)”referstoa
speciesnotidentifiedwithintheprecedinggenus.
https://doi.org/10.1371/journal.pone.0174518.t001
identifiedineitherthetotalgroupof38strainsorthesubsetwithvisualizedbiofilmsinre-
secteddiscs.
PatientcharacteristicsandPropionibacteriumacnespositivity
Atotalof368patientswereenrolled(222malesand146females)inthestudy,atanaverage
ageof49.3±13.6yearsandanagerangeextendingfrom20to83.Betweenthetwosurgical
Fig1.DistributionofP.acnescolonycountsinculture-positivedisctissuespecimens.
https://doi.org/10.1371/journal.pone.0174518.g001
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Propionibacteriumacnesbiofilminintervertebraldiscsofpatientsundergoingmicrodiscectomy
Table2. Characteristicsofthediscsamplesevaluatedbymicroscopicmethods.
Previous Sample SYTO-9 FISH
Sample CFU/ Sample Spine Weight Biofilm Biofilm
Number gram Description Surgery mg observed observed
1 0 Control No 950 No No
2 0 Control No 1070 No No
3 1097 P.acnesonly No 2480 Yes No
4 9016 P.acnesonly No 1930 Yes Yes
5 3977 P.acnesonly No 1770 Yes Yes
6 1378 P.acnesonly No 900 Yes Yes
7 2069 P.acnesonly No 580 Yes Yes
8 1193 P.acnesonly No 570 Yes Yes
9 3482 P.acnesonly No 1700 No* Yes
10 4073 P.acnesonly No 550 Yes No
*Individualbacteriawereobserved.
https://doi.org/10.1371/journal.pone.0174518.t002
centers,245patientswereenrolledatUniversityHospitalBrnoand123atSt.Anne’sUniver-
sityHospital;therewerenosignificantdifferencesbetweenthetwocenterswithregards
patientage,sex,ordisease-specificclinicaldata.Amedicalhistoryofpreviousspinalsurgery
wasnotedin58(16%)ofcases,thoughnoneofthepatientsdevelopedclinicallyevidentpost-
operativediscitisafterthesesurgeries.
Fig2.VisualizationofbacterialbiofilminthedisctissuebyCSLMandconfirmationofP.acnesby
FISH.A.ThreedimensionalreconstructedCSLMimageofbiofilmbacteriastainedwithaDNAstain(SYTO9,
green)inadisctissuesample(#4,Table2).B-C.ThepresenceofP.acnesbiofilmsinthissampleverified
usingFISH.EpifluorescencemicrographsofabiofilmclustershowingredfluorescencefromtheCY5-labeled
EUB338generaleubacterialprobe(B)andgreenfluorescencefromtheCY3-labledP.acnes-specificprobe
(C).Co-localizationoftheredandgreenfluorescenceindicatesthatallofthebacteriainthisbiofilmwereP.
acnes.
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Propionibacteriumacnesbiofilminintervertebraldiscsofpatientsundergoingmicrodiscectomy
Fig3.VisualizationofP.acnesbiofilminthedisctissuebyuseofFISH.A.Thiscolor-combinedimage
showsthe“pocket”ofgreenfluorescentP.acnescells(biofilm)nearthecenterrightoftheimage(disctissue
sample#8,Table2).ThepresenceofP.acnesbiofilmsinthissamplewasverifiedusingFISH.B-C.Red
fluorescenceisthegeneraleubacterialprobe(B)andgreenistheP.acnesprobe(C).TheB/Cimageisa
zoomofAshowingfluorescencefromtheredandgreenchannelsseparately.AlmostallofthecellsinAare
emittingbothredandgreenfluorescenceindicatingthattheyareP.acnes.
https://doi.org/10.1371/journal.pone.0174518.g003
Toprovideclinicalcontexttothemicrobiologiccultureresults,wesoughttocorrelateP.
acnespositivitytothepatients’clinicalparameters.P.acnespositivepatientsweresignificantly
youngerthanP.acnesnegativepatients(46.7versus50.5years;p=0.0380).Roughly39%of
222maleswereP.acnespositivecomparedto23%of146females(p=0.0013).Therewereno
significantdifferencesbetweenP.acnespositiveandnegativepatientsinprevalenceofprevious
spinalsurgery,distributionofherniationtype,affectedintervertebrallevel,orpre-surgical
painscores.ResultsaresummarizedinTable3.
Discussion
Overthelast15years,ithasbeenhypothesizedthatintervertebraldiscdegenerationandthe
accompanyingchroniclowerbackpainmaybeassociated—atleastinsomecases—with
chronicsubclinicalbacterialinfectionbyP.acnes[16].Arecentlydescribedanimalmodel
demonstratestheabilityofP.acnestoinducediscdegeneration[17,18].However,inmicrobi-
ologicculturedata,contaminationisdifficulttoexcludeentirely,asP.acnesisacommonskin
commensal.Thecurrentstudyseekstoresolvethisdilemmathroughanimprovedprotocol
forquantitativebacterialculture,insituimagingofresecteddisctissue,andmolecularphylo-
typingofP.acnesisolates.
Inourpreviousstudy[6],wewerefirsttoutilizequantitativebacterialculturefordetection
ofP.acnesindiscsamples.Hereweaddressedseveralweaknesses:(i)weighingthedisctissue
priortohomogenizationtoallowforapreciseCFU/gramdetermination,ratherthanCFU/ml
homogenate;(ii)utilizingaStomacher80fordisctissuehomogenization,ratherthanamanual
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