Prevalence of Propionibacterium acnes in Intervertebral Discs of Patients Undergoing Lumbar

12 Pages·2016·0.41 MB·English

RESEARCHARTICLE
Propionibacterium acnes
Prevalence of in
Intervertebral Discs of Patients Undergoing
Lumbar Microdiscectomy: A Prospective
Cross-Sectional Study
ManuN.Capoor1,3*,FilipRuzicka2,TanaMachackova3,RadimJancalek4,MartinSmrcka5,
JonathanE.Schmitz6,MarketaHermanova7,JiriSana3,ElleniMichu3,JohnC.Baird3,
FahadS.Ahmed3,KarelMaca5,RadimLipina8,ToddF.Alamin9,MichaelF.Coscia10,Jerry
a11111 L.Stonemetz11,TimothyWitham12,GarthD.Ehrlich13,14,15,ZiyaL.Gokaslan16,
KonstantinosMavrommatis17,ChristofBirkenmaier18,VincentA.Fischetti1,
OndrejSlaby3*
1 LaboratoryofBacterialPathogenesisandImmunology,RockefellerUniversity,NewYork,NY,United
StatesofAmerica,2 DepartmentofMicrobiology,FacultyofMedicine,Masarykuniversity,St.Anne’sFaculty
Hospital,Brno,CzechRepublic,3 DepartmentofMolecularOncology,CentralEuropeanInstituteof
Technology(CEITEC),MasarykUniversity,Brno,CzechRepublic,4 DepartmentofNeurosurgery,
OPENACCESS St.Anne’sUniversityHospital,MasarykUniversity,Brno,CzechRepublic,5 DepartmentofNeurosurgery,
UniversityHospitalBrno,MasarykUniversity,Brno,CzechRepublic,6 DepartmentofPathology,
Citation:CapoorMN,RuzickaF,MachackovaT, MicrobiologyandImmunology,VanderbiltUniversitySchoolofMedicine,Nashville,TN,UnitedStatesof
JancalekR,SmrckaM,SchmitzJE,etal.(2016) America,7 1stDepartmentofPathologicalAnatomy,St.Anne’sUniversityHospital,MasarykUniversity,
PrevalenceofPropionibacteriumacnesin Brno,CzechRepublic,8 DepartmentofNeurosurgery,UniversityHospitalOstrava,OstravaUniversity,
IntervertebralDiscsofPatientsUndergoingLumbar Ostrava,CzechRepublic,9 DepartmentofOrthopedicSurgery,StanfordUniversityMedicalCenter,
StanfordUniversity,Stanford,CA,UnitedStatesofAmerica,10 DepartmentofOrthopedicSurgery,
Microdiscectomy:AProspectiveCross-Sectional
OrthoIndyHospital,Indianapolis,IN,UnitedStatesofAmerica,11 DepartmentofAnesthesia,TheJohns
Study.PLoSONE11(8):e0161676.doi:10.1371/
HopkinsHospital,Baltimore,MD,UnitedStatesofAmerica,12 DepartmentofNeurosurgery,TheJohns
journal.pone.0161676
HopkinsHospital,Baltimore,MD,UnitedStatesofAmerica,13 CenterforGenomicSciences,Drexel
Editor:HolgerBrüggemann,AarhusUniversitet, UniversityCollegeofMedicine,Philadelphia,Pennsylvania,UnitedStatesofAmerica,14 Departmentof
MicrobiologyandImmunology,DrexelUniversityCollegeofMedicine,Philadelphia,Pennsylvania,United
DENMARK
StatesofAmerica,15 DepartmentofOtolaryngologyHeadandNeckSurgery,DrexelUniversityCollegeof
Received:July18,2016 Medicine,Philadelphia,Pennsylvania,UnitedStatesofAmerica,andInstituteofMolecularMedicineand
InfectiousDisease,DrexelUniversityCollegeofMedicine,Philadelphia,PA,UnitedStatesofAmerica,
Accepted:August9,2016 16 DepartmentofNeurosurgery,TheWarrenAlpertMedicalSchoolofBrownUniversity,RhodeIsland
Hospital,Providence,RI,UnitedStatesofAmerica,17 CelgeneCorporation,InformationKnowledgeand
Published:August18,2016
Utilization,SanFrancisco,CA,UnitedStatesofAmerica,18 DepartmentofOrthopedics,PhysicalMedicine
Copyright:©2016Capooretal.Thisisanopen &Rehabilitation,UniversityofMunich(LMU),Munich,Germany
accessarticledistributedunderthetermsofthe
*[email protected](OS);[email protected](MNC)
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
credited. Abstract
DataAvailabilityStatement:Allrelevantdataare
withinthemanuscript.
Funding:DiscitisDxprovidedsupportintheformofa Background
researchgrantwhichwasusedtopayforculturing
TherelationshipbetweenintervertebraldiscdegenerationandchronicinfectionbyPropio-
andPCRreagentsandmaterials.NeitherDiscitiDX,
Inc.noranyothercommercialorganizationprovided nibacteriumacnesiscontroversialwithcontradictoryevidenceavailableintheliterature.
anysupportintheformofsalariesforauthorsanddid Previousstudiesinvestigatingtheserelationshipswereunder-poweredandfraughtwith
nothaveanyroleinthestudydesign,datacollection methodicaldifferences;moreover,theyhavenottakenintoconsiderationP.acnes’abilityto
andanalysis,decisiontopublish,orpreparationof
formbiofilmsorattemptedtoquantitatethebioburdenwithregardtodeterminingbacterial
themanuscript.Thespecificrolesoftheseauthors
arearticulatedinthe‘authorcontributions’section. counts/genomeequivalentsascriteriatodifferentiatetrueinfectionfromcontamination.The
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P.acnes'PrevalenceinHerniatedDiscs
CompetingInterests:Theauthorshavereadthe aimofthisprospectivecross-sectionalstudywastodeterminetheprevalenceofP.acnesin
journal'spolicyandtheauthorsofthismanuscript patientsundergoinglumbardiscmicrodiscectomy.
havethefollowingcompetinginterests:MNC,FSA,
JLS,CB,OS,JES,K.Mavrommatis,andVAFhave
stockownershiporoptionsinDiscitisDX,Inc.There MethodsandFindings
arenootherfinancialornon-financialcompeting
Thesampleconsistedof290adultpatientsundergoinglumbarmicrodiscectomyforsymp-
interests.Thisdoesnotaltertheauthors'adherence
toPLOSpoliciesonsharingdataandmaterials. tomaticlumbardischerniation.Anintraoperativebiopsyandpre-operativeclinicaldata
weretakeninallcases.Onebiopsyfragmentwashomogenizedandusedforquantitative
anaerobiccultureandasecondwasfrozenandusedforreal-timePCR-basedquantifica-
tionofP.acnesgenomes.P.acneswasidentifiedin115cases(40%),coagulase-negative
staphylococciin31cases(11%)andalpha-hemolyticstreptococciin8cases(3%).P.
acnescountsrangedfrom100to9000CFU/mlwithamedianof400CFU/ml.Thepreva-
lenceofintervertebraldiscswithabundantP.acnes((cid:1)1x103CFU/ml)was11%(39cases).
Therewassignificantcorrelationbetweenthebacterialcountsobtainedbycultureandthe
numberofP.acnesgenomesdetectedbyreal-timePCR(r=0.4363,p<0.0001).
Conclusions
Inalargeseriesofpatients,theprevalenceofdiscswithabundantP.acneswas11%.We
believe,disctissuehomogenizationreleasesP.acnesfromthebiofilmsothattheycanthen
potentiallybecultured,reducingtherateoffalse-negativecultures.Further,quantification
studyrevealingsignificantbioburdenbasedonbothcultureandreal-timePCRminimizethe
likelihoodthatobservedfindingsareduetocontaminationandsupportsthehypothesisP.
acnesactsasapathogeninthesecasesofdegenerativediscdisease.
Introduction
Propionibacteriumacnes(P.acnes)isafacultativeanaerobic,Gram-positive,slow-growing,fas-
tidiousbacteria[1,2]prevalentonhumanskinandsebaceousductsaspartofthenormalflora
[3,4,5]andisimplicatedinthedevelopmentofacnevulgaris[6]aswellaspostoperativeand
device-relatedinfections[7].P.acneshavealsobeenidentifiedintheoralcavity,gastrointesti-
naltract,genitourinarytract,eye(conjunctiva),andexternalearcanal[1].P.acnesmayplaya
roleinotherconditions,includingprostatitis[8],SAPHO[9],sarcoidosis[10],anddegenera-
tivediscdisease[11].
Thepioneeringstudypublishedin2001byStirlingetal.inLancet,reportedthat53%ofthe
patientswithseveresciaticahadGram-positiveanaerobicmicroorganismsintheirdisctissue
ofwhich84%wereP.acnes[12].ConsideringthepotentialclinicalconsequencesofaP.acnes
infectionasanetiologicalfactorindegenerativediscdisease,thesefindingsareindicativeof
potentiallysignificantsimilaritiestothediscoveryofHelicobacterpyloriinvolvementinpatho-
genesisofpepticulcersandtheshiftitledtointhewaypepticulcersaretreated[13].Subse-
quently,severalindependentstudieshavedemonstratedthepresenceofinfectedextrudeddisc
tissuefromfirst-timedischerniations,withP.acnesbeingthemostcommonlyinvolvedorgan-
ism[14–18].Accordingtoameta-analysisofthesestudiesfrom2015,P.acneswasthemost
prevalentbacteriaindisctissue,withthepooledestimateoftheproportionwithpositivesam-
plesbeing34%[19].Morerecently,thefirstexperimentalevidencewasprovidedshowingthat
P.acneshastheabilitytoinducediscdegenerationinarabbitmodel[20].Otherstudies,
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P.acnes'PrevalenceinHerniatedDiscs
however,reportedveryloworzeroprevalenceofbacterialinfectioninsamplesfrompatients
withlumbardischerniation,quotingcontaminationasthepredominantsource[21–23].
Contributingtothislackofconsensus,significantmethodicaldifferencesexistamongstud-
ies.Themostnotableofwhichisthelackoflaboratoryproceduresthattakeintoaccountapos-
siblebiofilmmodeofgrowthassociatedwithP.acnesinfection.Thistranslatesinto
inconsistenciesinregardtotissuehomogenizationprocedurestobreakupbiofilmaggregates,
leadingtonegativecultureresults.Asaconsequence,P.acnespositivitywaslikelyunderesti-
matedinsomeofthepreviousstudies[21,23].Noneofthestudiesimplementedquantitative
cultureorreal-timePCR-basedquantificationofP.acnesgenomes.Webelievethatquantita-
tionofthebacterialbioburdenisanimportantcriterionthatcancontributetodiscrimination
betweentrulyinfectedandskin-contaminatedcases.Thus,alackofquantitativemethodsto
identifyP.acnescouldhaveledtooverestimatingthepositivityratesinpreviousstudies.These
methodicalweaknessestogetherwiththesmallsamplesizes(onlyonestudyenrolledmore
than100patientsforbacterialculture[23])haveleftthesestudiesunderpowered,withlimited
applicabilityoftheirresultstowardsclinicaldecision-makingoranydefinitiveconclusions.
Inthisprospectivecross-sectionalstudyonthecurrentlylargestseriesofpatientsundergo-
ingmicrodiscectomyforlumbardischerniation,weimplementeddisctissuehomogenization,
quantitativemicrobiologicculture,real-timePCR-baseddetectionofP.acnesgenomes,and
proceduralDNAcontaminationcontrolstodetermineP.acnescountsindisctissuesamples
andevaluatedtheprevalenceofintervertebraldiscswithanabundanceofP.acnes.
Methods
Studydesignandpatients
BetweenMay2015andDecember2015,adultsscheduledformicrodiscectomyforsymptom-
aticlumbardischerniationatUniversityHospitalBrno(Brno,CzechRepublic)andSt.Anne’s
UniversityHospital(Brno,CzechRepublic)wereprospectivelyscreenedforpotentialpartici-
pationinthiscross-sectionalstudy.Inclusioncriteriaincluded:lumbarorlumbosacralradicu-
lopathywithorwithoutsensorydeficitsbutwitheitheramatching,clinicallyrelevantmotor
deficitincorrelatinglumbarorsacralnerverootdistributions(seeimagingcriteria)orwith
radicularpain(sciaticaorfemoralgia)thatwasintractablebyconservativemeans;matching
physicalexaminationfindingsincludingpositivestraightlegraisetest,dermatomalsensory
deficits,myotomalmotordeficitsand/oradiminisheddeeptendonreflexes;currentmagnetic
resonanceimagingorcomputedtomographyimagingofthelumbosacralspineshowingafree
nucleuspulposussequestrationoradischerniation/protrusioninadistributioncorrelating
withtheclinicallyaffectednerverootsandwiththephysicalexamination.Exclusioncriteria
included:coexistentinfectionorimmunologicallycompromisedconditions;corticosteroidor
antibioticsuseinthemonthbeforesurgery;trauma;unknownradiographicmass;diagnosisof
inflammatoryarthritisorotherrheumatologicdiseases.Thefollowingepidemiologicaland
clinicaldatawerecollected:Gender,age,intervertebralsegmentinvolved,typeofherniation,
prevalenceofpreviousspinalsurgeries,priorepiduralsteroidinjections,anddevelopmentof
post-operativediscitis.Awritteninformedconsentwasobtainedfromeachpatient.Thestudy
wasapprovedbytheInstitutionalReviewBoardsofUniversityHospitalBrnoandSt.Anne’s
UniversityHospitalBrno.
Collectionofintraoperativesamples
Thesurgicalsitewasscrubbedwithtriplepreparationofpovidoneiodineanddrapedusing
standardsteriletechnique.Standardperioperativeantibioticsweregivenbeforetheskininci-
sioninallcases.Cefazolinwasthestandardantibioticgiveninmostcases.Inpenicillin-allergic
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P.acnes'PrevalenceinHerniatedDiscs
patients,eithervancomycinorclindamycinwasadministered.Thepreciselocationfortheskin
incisionwasguidedbyintraoperativefluoroscopyandaposteriormidlineapproachusing
sharpdissectionandelectrocauterywasperformed.Afterplacementofaself-retainingretrac-
tor(Caspartype)andunderanoperatingmicroscope,ligamentumflavumwasresectedas
requiredbymeansofPenfielddissectorsandKerrisonrongeurs.Thedischerniationwas
exposedbygentleretractionofthetraversingnerverootandthenremovedtogetherwiththe
remnantloosefragmentsofthenucleuspulposusinthediscspaceneartheannulardefect.All
tissuesampleswerehandledinsuchawayastominimizetheircontamination,retainedina
closedsterilesamplecup,andthenpassedofftothefieldforlabelingandtransporttothe
DepartmentofMicrobiologyatSt.Anne’sUniversityhospital(Brno,CzechRepublic),where
thedisctissuessamplesfrombothcenterswerefurtheranalyzed.Thesamplesizeswere
approximately3x3x5-10x5x5mmandnotmeasuredaccuratelyasweattemptedtoobtainas
muchmaterialasthepossiblefromthesurgicalspecimen.Sampleswerenotfrozenpriorto
processingandculturewasestablishedwithin2to4hourspost-surgery.
Microbiologicalculture
Freshdisctissuesampleswerecutintosmallerfragmentsusingasterile,individuallypackaged,
gamma-irradiatedscalpel;and,asterile,gamma-irradiatedpetridish.Oneofthesefragments
wasplacedintoa2mLmicrocentrifugeDNA-freetubeandstoredat-80°Cuntilprocessedfor
P.acnesDNAanalysis.Tissueprocessingandhomogenizationwascarriedoutinasterilepestle
andmortarwithsterilequartzsand(sizeparticle0.1–0.5mm;Penta,CzechRepublic)and
salinesolutioninasepticconditions,inaclass2biologicalsafetycabinet.Thehomogenizedtis-
suesampleswereinoculatedontoWilkinsChalgrenAnaerobicAgar(HiMediaLaboratories,
India)with7%ofsheep’sbloodandvitaminK.Inoculatedplateswereincubatedanaerobically
(80%nitrogen,10%CO and10%H )inanAnaerobicWorkStation(RuskinnTechnology,
2 2
UK)at37°Cfor14daysandassessedforbacterialgrowth.Thequantityofbacteriainthesam-
plewasexpressedascolonyformingunits(CFU)in1mlofthehomogenate.Identificationof
bacteriawascarriedoutbiochemicallyusingtheRapidANAIISystem(Remel,USA).
Quantitativereal-timePCR
Frozentissuesampleswerethawed(medianwetweightwas130mg,range20–180mg),cut
intosmallfragmentsandtransferredtoasterile2mLmicrocentrifugetubebyuseofnewly
openedsterilesetsofneedle,scalpelandtweezers.Smallfragmentsofthetissuesampleswere
furthersuspendedin500μlofATLbuffer(Qiagen,Germany)with50μlofproteinaseK(20
mg/ml)(Qiagen)anddigestedat56°Cand650rpminathermomixerovernight.Toeachsetof
thesamplesthatwereprocessedinparallel,weusedatubewithsterilewaterasalaboratory
contaminationcontroltofollowtheentirelaboratoryprocessfromdigestionofthetissueand
DNAisolationtoreal-timePCRanalysis.DNAwasextractedbyuseoftheQIAampUCPPath-
ogenMiniKit(Qiagen)asdescribedinthemanufacturer’sinstructions.Concentrationof
DNAweremeasuredspectrophotometricallyusingaNanodrop2000(Thermofischer,USA);
orwithfluorescentdyeandaQubit3.0fluorometer(LifeTechnologies,USA)forsampleswith
DNAconcentrationslessthan5ng/μl.Apreviouslydescribedreal-timePCRassaywasper-
formedusingprimerstoamplifya131-bpregionofthe16SrRNAgeneofP.acnes:forward
primer5’-GCGTGAGTGACGGTAATGGGTA-3’,reverseprimer5’-TTCCGACGCGAT
CAACCA-3’andTaqManprobe5’-AGCGTTGTCCGGATTTATTGGGCG-3’[24].The15-μl
PCRreactionmixturecontained6,75μlofDNAsample,5pmolofeachprimerand2pmolof
TaqManprobe,and1XTaqManGeneExpressionMasterMix(LifeTechnologies,USA).The
QuantStudio12KFlexsystem(LifeTechnologies,USA)wasusedwiththethermalcycling
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P.acnes'PrevalenceinHerniatedDiscs
profileof50°Cfor2min,95°Cfor10minand50cyclesof95°Cfor15sand60°Cfor1min.
TheP.acnesgenomeequivalentsinsampleswereestimatedwithaninternalstandardcurve
preparedwithfivereplicatesofsixconcentrations(10–106copies)ofsynthesizedP.acnes
amplicon(131bp)(IntegratedDNATechnologies,USA).Laboratorycontaminationcontrols
describedaboveandPCRnegativecontrolswereincludedineveryPCRreaction.Assayswere
doneinduplicateforeachsample,andthemeannumberofthe16SrRNAgenecopieswascal-
culated.Toeliminatelaboratorycontamination,16SrRNAcountsdetectedinlaboratorycon-
taminationcontrolweresubtractedfromthecopiesnumberinthetissuesamples.Thenumber
ofbacterialgenomesineachsamplewasfinallycalculatedusingtheknownnumberofcopies
ofthe16SrRNAoperon(3copies/cell)inP.acnes[4]andrepresentedasthenumberofbacte-
rialgenomesin500ngoftotalDNAextractedfromthedisctissuesample.Humanß-globin
genewasincludedasaninternalcontroltoallowassessmentofthespecimenqualityandthe
nucleicacidextractionaswellastheinhibitionamplificationprocessasdescribedpreviously
[24].
Dataanalysis
Adescriptionofidentifiedbacterialfrequencieswasperformedandtherelationshipbetween
presence/countsofP.acnesandclinicalparameterswasevaluated.Categoricalvariableswere
analyzedusingatwo-sidedFisher’sexacttest,continuousvariables(genomecounts,age)with
thenon-parametricMann-WhitneyUtest.Spearman’scorrelationwasusedtotesttheassocia-
tionbetweenbacterialcountsreachedinthesamesamplesbydifferentmethods.Thesignifi-
cancelevelwassetat0.05.ThestatisticalsoftwareusedwasPrism5(GraphPadSoftwareInc.,
USA).
Results
Patientcharacteristics
Weprospectivelyenrolled290patients(171malesand119females)withanaverageageof
47±13years.Onehundredninety-twocaseswereenrolledatUniversityHospitalBrno,ninety
eightcasesatSt.Anne’sUniversityHospital.Thedegenerativeintervertebraldiscsegments
wereL2/L3in8(3%),L3/L4in21(7%),L4/L5in137(47%)andL5/S1in124(43%)cases.For
8patientswhounderwentadouble-leveldisksurgery,onlythecaudaldiskwasselectedto
avoidastatisticalbias.Discherniationtypesincludeddiscprotrusionin23(8%),discextrusion
in126(44%),anddiscsequestrationin141(48%)patients.Medicalhistoryofpreviousspinal
surgerywasnotedin51(17%)cases,priorepiduralsteroidinjectionwasreceivedby14(5%)
patients.Noneofthepatientsdevelopedclinicallyevidentpost-operativediscitis.Therewere
nosignificantdifferencesinpatientcharacteristicsbetweenthetworecruitmentcentersregard-
ingage,genderanddisease-specificclinicaldatawhichwerecollectedandusedinourstudy.
Resultsofbacterialculture
Bacteriawereidentifiedin130of290disctissuessamples,theother160biopsiesresultedinno
bacterialcolonies.Thefollowingbacteriawereisolated:P.acnesin115cases(40%),coagulase-
negativestaphylococci(CoNS)in31cases(11%)andalpha-hemolyticstreptococci(AHS)in8
cases(3%)(Fig1A).Twenty-fourpatients(8%)hadtwomicroorganismspresentinthedisc.
Nopatientshadmorethantwotypesofbacteriaisolated.Weobservedsignificantvariabilityin
P.acnescountsamongpositivediscsamplesrangingfrom100to9000CFU/ml(median400
CFU/ml).
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P.acnes'PrevalenceinHerniatedDiscs
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P.acnes'PrevalenceinHerniatedDiscs
Fig1.ResultsofanaerobiccultureanddetectionofP.acnesgenomecountsindisctissuesamples.Prevalence
rateofthebacteriaisolatedfromdisctissuesamples(A),stratificationofthesamplesaccordinglytobacterialcounts
reachedbyculture(B)anddifferencesinthebacteriagenomecountsdetectedbyreal-timePCRingroupofpatientswith
abundantP.acnesbyculture,non-abundantP.acnesandculture-negativecases(C).
doi:10.1371/journal.pone.0161676.g001
Athresholdwasdefinedas(cid:1)1x103CFU/ml(75thpercentile)forthediscswithabundantP.
acnes.PrevalenceofthediscsampleswithabundantP.acneswas11%(39cases);intheremain-
ing29%(76cases)ofpositivediscsamplesP.acneswasnotabundant(<1x103CFU/ml).
Propionibacteriumacnesgenomecountsindisctissuesamples
DNAwassuccessfullyextractedfromallsamplesincludedinthestudywithconcentration
rangingfrom2to45ng/μl(median13ng/μl).ThemeanCt(thresholdcycle)valueforthe
internalcontrol,humanß-globingene,was24.03±1.81.Eighteencasesofthe290caseswere
excludedfromstatisticalevaluationduetonegativeofß-globingene(Ct>35).ThemeanP.
acnesgenomescountinthelaboratorycontaminationcontrolsampleswas4±4genomes.P.
acnesgenomeswereundetectableinonly13(4%)cases.ThenumberofP.acnesgenomesin
the259P.acnes-positivedisctissuesrangedfrom2to5831withamedianof260genomesper
500ngoftotalDNA.ThereweresignificantdifferencesinthenumberofP.acnesgenomes
detectedinthesampleswithabundantP.acnesbyculture(1039±244genomes),whencom-
paredtosampleswithnon-abundantP.acnes(448±98genomes)andsamplesnegativebycul-
ture(415±57genomes)(forbothP<0.0001,Fig1C).WealsofoundsignificantPearson
correlationbetweenCFU/mlandP.acnesgenomecountsdetectedbyreal-timePCRinpatients
positivebyculture(r=0.4363,p<0.0001).TherewasnodifferenceinthenumberofP.acnes
genomesbetweendisctissuesampleswithnon-abundantP.acnesandsamplesnegativebycul-
ture(p=0.6973).
RelationshipsofPropionibacteriumacnesandpatientcharacteristics
TheaverageageofpatientshavingdiscswithabundantP.acnesbyculturewassignificantly
lower(42versus48years;p=0.0086).Therewerenosignificantdifferenceswhencomparing
thegroupsofpatients(i.e.,abundantP.acnes,non-abundantP.acnes,ornegativecultures)in
gender,prevalenceofpreviousspinalsurgery,prevalenceofpriorepiduralsteroidinjection,
distributionofherniationtype,oraffectedintervertebrallevel(summarizedinTable1).With
theexceptionoftheage,therewerenosignificantassociationsfoundbetweenP.acnespreva-
lenceandclinicalcharacteristicsincludingthesituationinwhichallpatientswithapositive
culture(abundant+non-abundant)wereconsideredpositiveforstatisticalevaluationortheP.
acnesgenomecountswerecomparedbetweengroups(allp>0.05).
Discussion
Traditionally,mechanical,degenerative(age-related),traumaticandgenetics-basedtheories
havebeenproposedaspossibleetiologiesofdegenerativediscdisease[25],butevidencehas
emergedoverthelastdecadeshowingthatlow-gradeinfections,mostlybyP.acnes,playinga
potentialcausativeroleindiscdegenerationandresultantdisc-herniationandradicularpain
[11,19].However,thepotentialrelationshipbetweenintervertebraldiscdegenerationand
chronicinfectionbylow-virulenceP.acnesremainscontroversial,withcontradictoryevidence
intheliterature.WesuggestthatforaccuratedetectionofP.acnes,itiscriticaltoanticipateits
existenceinitsbiofilmform[5,26,27]andtousequantitativemethodstodeterminebacteria
counts.Unfortunately,thisapproachhasnotbeenutilizedinanyofthepublishedstudiesin
thisfield.Inthecurrentstudy,wehomogenizeddiscsamplestodisruptbiofilmaggregates
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P.acnes'PrevalenceinHerniatedDiscs
Table1. RelationshipsbetweenstrongpositivityofP.acnesinthediscandepidemiological/clinicalcharacteristicsofthepatients.
Parameters AbundantP.acnesbycultureN(%) Non-abundantP.acnesornegativecultureN(%) P-value
Gender
Male 28(16%) 144(84%) 0.1144
Female 11(9%) 108(91%)
Age
Mean±SD 42±12y 48±13y 0.0086
Previousspinalsurgery
Yes 8(16%) 43(84%) 0.5019
No 30(14%) 209(86%)
Priorepiduralinjection
Yes 3(21%) 11(79%) 0.4128
No 111(40%) 165(60%)
Typeofherniation
Protrusion 3(13%) 20(87%) 0.6445
Extrusion 14(11%) 112(89%)
Sequestration 21(15%) 119(85%)
Intervertebrallevel
L2/L3 1(13%) 7(87%) 0.6313
L3/L4 1(5%) 20(95%)
L4/L5 15(16%) 81(84%)
L5/S1 23(14%) 142(86%)
CoNS,coagulase-negativestaphylococci.
doi:10.1371/journal.pone.0161676.t001
(whichifundisruptedoftenleadtonegativecultureresults[27])toeliminatefalsenegativity,
andusedquantitativeanaerobiccultureandreal-timePCRtodeterminebacterialcounts/
genomesasoneofthecriteriatoeliminatepotentialcontaminationandfalsepositivity.
Weprospectivelyenrolled290patientsundergoingmicrodiscectomyforlumbardischerni-
ation,whichisthelargestseriesofpatientstodate,andexaminedtheirintraoperativedisc
biopsiesbyquantitativecultureandreal-timePCR.Weisolatedthreebacterialspeciesbycul-
ture:P.acnes,CoNSandAHSin40%,11%and3%ofcases,respectively.Ourdataareinagree-
mentwithamajorityofthepreviousreportsdescribingP.acnesandCoNSasthemost
frequentlyisolatedbyanaerobicculture[12,14–17].OveralltheP.acnespositivityrate
observedinourstudyissimilartotheresultsofStirlingetal.[12],Arndtetal.[16]andAlbert
etal.[17],whoobserved44%,35%and40%positivityrate,respectively.Analogicallytoour
results,5%,14%and31%prevalenceofCoNSwasdescribedinthreeothersstudies[12,14,16].
Weusedstandardsurgicalcareprotocols,includingtheuseofprophylacticperioperative
antibiotics(cefazolin),thusensuringpatientsafetywhileconductingourresearch.Werea-
sonedthatcephalosporinprophylacticantibioticswouldnotaffecttheresearchresultsbecause
theseantibioticsdonotpenetrateintotheintervertebraldiscsandbecauseP.acnesbiofilm
withinthediscisantibioticresistantatthetypicallyuseddoses[28].Intwoprotocols,prophy-
lacticantibioticswereadministeredaftercollectionofthebiopsiestoavoidinhibitionofany
bacteriainthesample[17],orwerenotadministeredatall[22].Inthefirstcase,thepositivity
rateofP.acneswasidenticalwithourstudy(40%inboth),inthesecondstudyP.acnespositiv-
itywasonly4%,indicatingthatprophylacticcefazolinedoesnothaveanyinhibitoryeffectson
theP.acneswithinthedisc.
NoneofthepreviousstudiesdeterminedP.acnescountsinthediscsanddistributionofthe
bacteriacountswithintheircaseseries.WedescribedtherangeofP.acnescountsobtainedby
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P.acnes'PrevalenceinHerniatedDiscs
cultureandobservedan11%prevalenceofintervertebraldiscswithabundantP.acnes
((cid:1)1x103CFU/ml).Wesuggest,thatthisquantity-adjustedprevalenceislessbiasedbycon-
taminationthanoverallpositivity.SimilarprevalenceoftheP.acnespositivediscsampleswere
observedbyAgarwaletal.[15],Cosciaetal.[14]andZhouetal.[18]describing13%,19%and
24%positivityrate,respectively.Asquantitativedatafromotherstudiesarenotavailable,and
differencesinthepositivityratesdiffersignificantly(from4to44%),itislikelythatsome
authorsevaluatedweakpositivityascontaminationandothersaspositivity.
However,therearealsostudiesdescribingloworzeroP.acnespositivityofdiscsintheir
caseseries[21–23].Forinstance,P.acneswasnotisolatedfromanyof30discsamplesinthe
studybyBen-Galimetal.[21],whoimplementedinoculationofculturesdirectlyintheoperat-
ingtheatretominimizetheriskofcontamination.Authorsofthisstudysuggestthatthehigh
prevalenceofP.acnesobservedinotherstudiesmainlyreflectscontamination.Wespeculate
thatthisunusualapproachdidnotprovideforsufficienthomogenizationofthetissueanddis-
ruptionofP.acnesbiofilms,whichcombinedaccountsfortheirnegativecultureresults.Simi-
larly,inthelarge-scalestudyonpatientswithchroniclowerbackpain,discfragmentswere
cultureddirectlywithouttissueprocessingorhomogenization,andP.acneswereisolatedin
only2of313cases[23,29].
Weimplementedareal-timePCRassayandlaboratorycontaminationcontrolstoaccu-
ratelyquantifyP.acnesgenomesdirectlyinthedisctissuesamples.ThenumbersofP.acnes
genomesinthediscs,whichwereP.acnesabundantbyculture,weresignificantlyhigherwhen
comparedtonon-abundantorP.acnesnegativediscs.Therewasnodifferenceinthenumbers
ofP.acnesgenomesbetweenthediscswithnon-abundantP.acnesandnegativesamplesby
culture,supportingapotentialcontaminationoriginofthediscsampleswithnon-abundantP.
acnesandaccuracyoftheempiricalthreshold((cid:1)1x103CFU/ml)forthesampleswithabun-
dantP.acnes.Onlyin13(4%)cases,werenoP.acnesgenomesdetected.Thisisinagreement
withoutputsoflarge-scalestudiesbasedonnext-generationsequencingofP.acnesDNAina
varietyofclinicalspecimensconcludingthatwhenhighlysensitivemolecularmethodsare
employed,P.acnesDNAcouldbedetectedinpracticallyallsampletypes[30].Moreover,it
wasshownthatP.acnesDNAisafrequentcontaminantofcommercialTaqpolymerasesand
PCRsolutions[31],highlightingthenecessityofquantitativeinformationtodistinguish
betweencontaminationandpositivity[32].Untilnow,onlyoneteamfocusedondirectdetec-
tionofP.acnesDNAindisctissuebyqualitativeend-pointPCR[17].OthersusedPCRonly
forverificationofP.acnesDNAinpurifiedcolonies[18].Albertetal.observedconsistent
resultsofcultureandPCRmethods;and,surprisingly,thecasesnegativebyculture(60%)were
negativebyPCR[17],whichcouldbeexplainedbythedecreasednumberofcycles(only35)in
theirassay,probablytoartificiallydecreasethesensitivityoftheirmethod.
WedidnotobserveanysignificantassociationbetweenP.acnespositivityandprevalenceof
previousspinalsurgeryandpriorepiduralsteroidinjectionindicatingendogenousoriginofP.
acnesandtheirroleindegenerativediscdiseasemorethantheiriatrogenicoriginthroughdisc
contaminationinthepreviousspinalsurgeries.P.acnespositivityrateswereindependentof
gender,typeofherniation,andaffectedspinalsegment.Theaverageageofpatientshaving
discswithabundantP.acneswerelower(42versus48years).Wehypothesize,thatP.acnes
infectioncouldaccelerateregularmechanical,age-related,degenerativeprocessinaffected
individualsandresultinthenecessityofearliersurgicalintervention.
Alsoofnoteisthetwomostcommoncommensals,P.acnesandcoagulase-negativestaphy-
lococci,werealsothetwomostcommonbacteriafoundinthedisctissuesamples,which
reflectsthecompellingneedtogainanabilitytodiscriminatebetweenacontaminationandan
infection.
PLOSONE|DOI:10.1371/journal.pone.0161676 August18,2016 9/12
P.acnes'PrevalenceinHerniatedDiscs
Thisstudyhasseverallimitations.Aerobiccultureswerenotperformedgivendisctissue
originatesinanotablyanaerobicenvironment.Asweconsidertissuehomogenizationacritical
stepoftheexperimentalprotocol,morestandardizedhomogenizationmethodsthanmortar
andpestleshouldbeimplemented.WeassumethatthebiofilmformofP.acnesistypically
presentwithinthedisc,butdonotprovidedirectevidenceofP.acnesbiofilmsfromthedisctis-
suebymicroscopicmethods.However,inthefieldoforthopedicinfections,thereisstrongevi-
denceofP.acnesformingabiofilm[26,27].Asdisctissuefromage-matchedhealthy
individualsisnotavailable,theidentificationofP.acnesinbiopsiesfromcomparablehealthy
individualscannotberuledout.Further,thelackofcontrolculturescannotexcludethepossi-
bilityofcontaminationfromthelaboratory,operatingroom,surgicalfield,orskin.With
exceptionofone[22],mostofthepreviousstudiesthatincludednegativecontrolsprovedthat
theyarelargelysterile[12,14,18].Sincepositiveendogenouscontrolsdonotnecessarilycon-
notecontaminationofthediscsample,wedonotbelievethatendogenouscontrolsaretheopti-
malsolutiontoidentifycontaminateddiscsamples.Inourstudy,P.acnescountsandthe
numberofP.acnesgenomeswereimplementedasoneofthecriteriatoeliminatepotential
contamination.ThefinalanswerwithregardtodistinguishingendogenousP.acnesinfection
ofthediscfromskincontaminationwillprobablyutilizecomparativegenomicstodifferentiate
P.acnesgenomesfromtheskinanddisctissuefromagivenindividual[33,34].
Inconclusion,toourknowledgethisisthefirststudythatprovidesquantitativeinformation
aboutP.acnesindisctissuesamplesinthelargestseriesofpatientswithlumbardischernia-
tion–wedemonstratedan11%prevalenceofthedischerniationsampleswithanabundanceof
P.acnes.Because“findingabundantmicroorganisminorganismssufferingfromthedisease”is
oneofthemostimportantofKoch’spostulates,injudgingtherelationshipbetweenaspecific
microbeandaspecificdisease,webelievethatourfindingssupportthetheorythatP.acnesis
involvedinthepathogenesisofatleastasubsetofpatientswithdegenerativediscdisease.
FuturestudiesareneededtoevaluaterelationshipbetweenP.acnesinfectionandclinicalout-
comesofthesepatients.
AuthorContributions
Conceptualization:MNCFRRJJESMSK.MavrommatisFSAJLSCBVAFOS.
Datacuration:MNCFSAOSTMJSCB.
Formalanalysis:MNCOS.
Investigation:FRFSATMEMJSMH.
Methodology:MNCFRRJJESMSK.MavrommatisFSAJLSCBVAFOS.
Projectadministration:MNCOSFRCBRJ.
Resources:FROSRJRLMSK.Maca.
Supervision:VAF.
Visualization:MNCOSJCBFSA.
Writing-originaldraft:MNCFSAOSJCB.
Writing-review&editing:MNCFRTMRJMSJESMHJSEMJCBFSAK.MacaRLTAMC
JLSTWGDEZLGK.MavrommatisVAFCBOS.
PLOSONE|DOI:10.1371/journal.pone.0161676 August18,2016 10/12