DTIC ADA619246: Development of a Novel, Highly Quantitative In Vivo Model for the Study of Biofilm-Impaired Cutaneous Wound Healing

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WoundRepairandRegeneration
Development of a novel, highly quantitative in vivo model for
the study of biofilm-impaired cutaneous wound healing
AnandevN.Gurjala,MD,MS1;MatthewR.Geringer,BS1;AkhilK.Seth,MD1;SeokJ.Hong,PhD1;
MarkS.Smeltzer,PhD2;RobertD.Galiano,MD1;KaiP.Leung,PhD3;ThomasA.Mustoe,MD1
1.DivisionofPlasticSurgery,FeinbergSchoolofMedicine,NorthwesternUniversity,Chicago,Illinois,
2.DepartmentofMicrobiologyandImmunology,UniversityofArkansasMedicalSciences,LittleRock,Arkansas,and
3.MicrobiologyBranch,USArmyDentalandTraumaResearchDetachment,InstituteofSurgicalResearch,FortSamHouston,Texas
Reprintrequests: ABSTRACT
ThomasA.Mustoe,DivisionofPlastic
Surgery,FeinbergSchoolofMedicine, A growing body of evidence suggests that in addition to hypoxia, ischemia-
NorthwesternUniversity,675N.St.Clair, reperfusioninjury,andintrinsichostfactors,bacterialbiofilmsrepresentafourth
Suite19250,Chicago,IL60611. majorpillarinchronicwoundpathogenesis.Giventhatmoststudiestodaterely
Tel:[phone]; oninvitroorobservationalclinicaldata,ouraimwastodevelopanovel,quan-
Fax:[phone]; titativeanimalmodelenablingfurtherinvestigationofthebiofilmhypothesisin
Email:[email protected] vivo.DermalpunchwoundswerecreatedinNewZealandrabbitears,andused
as uninfected controls, or inoculated with green fluorescent protein-labeled
Manuscriptreceived:September24,2010 Staphylococcusaureustoformwoundswithbacteriapredominantlyintheplank-
Acceptedinfinalform:January25,2011 tonic or biofilm phase. Epifluorescence and scanning electron microscopy re-
vealedthatS.aureusrapidlyformsmaturebiofilminwoundswithin24hoursof
DOI:10.1111/j.1524475X.2011.00690.x inoculation, with persistence of biofilm viability over time seen through serial
bacterial count measurement and laser scanning confocal imaging at different
time points postwounding and inoculation. Inflammatory markers confirmed
thatthebiofilmphenotypecreatesacharacteristic,sustained,low-gradeinflam-
matory response, and that over time biofilm impairs epithelial migration and
granulation tissue in-growth, as shown histologically. We have established and
validated a highly quantitative, reproducible in vivo biofilm model, while pro-
viding evidence that the biofilm phenotype specifically contributes to profound
cutaneous wound healing impairment. Our model highlights the importance of
bacterialbiofilmsinchronicwoundpathogenesis,providinganinvivoplatform
forfurtherinquiryintothebasicbiologyofbacterialbiofilm–hostinteractionand
high-throughputtestingofantibiofilmtherapeutics.
Bacterialbiofilms,sessilecommunitiesofbacteriaencased woundhealingprocess.PhilStewartfromMontanaState
in a protective polysaccharide matrix, can find harbor in Universityinparticularhasputforthaconsiderableseries
manyparts of thebody, having beenreportedonbiolog- ofpapersarticulatingthishypothesis.Inaclinicalstudyof
icalsurfacesasdisparateasmyocardium,nasalepithelium, humanwounds,hisgroupconvincinglydemonstratedus-
anddentalenamel.1Oneachofthesesurfacestherelation- ing light and scanning electron microscopy (SEM) that
ship between bacteria and host may range from purely bacteriainbiofilm-phasearepresentinalltypesofchronic
commensal (as in the intestinal gut) to highly destructive wounds,establishingakeylinkbetweenbiofilmandnon-
when established on microbiologically naıve tissues (such healing.3Studiesbyothergroupsusingsophisticatedmo-
asinendocarditisorcysticfibrosis).Whetherondamaged lecular techniques for bacterial recovery have further
heartvalvesorpoorlyfunctioningrespiratoryepithelium, established that a diverse array of bacterial species exists
the pathophysiology of bacterial biofilms remains con- inhumanchronicwounds,andthatthesebacteriapersist
served:hostdefensesarerenderedunabletoclearbiofilm over time.4,5 Moreover, a variety of in vitro models have
duetoitsadaptivesurvivalmechanisms,andyetarecon- made essential contributions, including demonstration of
stantly stimulated by its physical presence, shedding of robustbiofilmresistancetoantimicrobials,6importantge-
planktonic bacteria, and elaboration of virulence factors. netic pathways for bacterial communication including
The result is a chronic inflammatory cycle causing injury quorum sensing,7 and even inhibitory effects of biofilms
tohosttissuesovertime.2 againstculturedhumankeratinocytes.8
Inrecentyearsthewoundbedofcompromisedskinhas While the aforementioned studies have been crucial to
been thought to offer yet another biological surface onto developinganunderstandingofbiofilminwounds,equiv-
which bacterial biofilms can take hold. The susceptibility alentstrideshavenotbeenmadeintheareaofinvivoex-
ofopenwoundstobacterialseedingandthemoist,nutri- perimentation,principallyduetothelackofanestablished
tionally supportive microenvironment of the wound ma- animalmodel.Giventhecomplexityofwoundhealing,ex-
trixcreateanidealsettingforbiofilmtointerferewiththe trapolation from in vitro biofilm studies to the clinic has
400 WoundRepReg(2011)19400 410 (cid:2)c 2011bytheWoundHealingSociety
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1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED
01 MAY 2011 N/A -
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Development of a novel, highly quantitative in vivo model for the study of
5b. GRANT NUMBER
biofilm-impaired cutaneous wound healing.
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
Gurjala A., Geringer M. R., Hong S.-J. Smeltzer M. S., Galiano R. D.,
5e. TASK NUMBER
Leung K. P., Mustoe T. A.,
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
United States Army Institute of Surgical Research, JBSA Fort Sam REPORT NUMBER
Houston, TX
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11. SPONSOR/MONITOR’S REPORT
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Approved for public release, distribution unlimited
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14. ABSTRACT
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Gurjalaetal. Animalmodelforbiofilmimpairedwoundhealing
been challenging, because only animal models can take plasmid, pCN57, was introduced to UAMS-1 by phage
intoaccountsuchcriticalinvivofactorsastheinterplayof transduction as described by Charpentier et al.11 with
the host immune response and wound bed components modifications.Briefly,strainRN9623(recentlydesignated
withbacteria.Secondly,humanstudieshavebeenonlyob- as NARSA strain ID# NRS623, which contains pCN57)
servational and correlative, lacking the experimental and wasculturedtoexponentialgrowthphaseinTSBcontain-
causative data necessary to fully establish the biofilm hy- ing 5mM CaCl (Sigma-Aldrich, St. Louis, MO) and
2
pothesis.Thereisthusawideconsensusamongthebiofilm 10mg/mL erythromycin (Sigma-Aldrich). To prepare
research community on a need for more in vivo biofilm phage lysates, strain RN9623 was infected with phage 11
models,andthattheywillbenecessarytoolsforfurthering at a multiplicity of infection (MOI) 0.5 and cultured at
ourunderstandingofbiofilm–hostbiology. 371Cuntilcelllysisoccurred.Lysateswerecentrifugedto
Inthepresentstudyweaimto buildonpreviouswork removecelldebrisandfilteredthrougha0.45mmfilter.For
by developing a novel, in vivo biofilm model in rabbit, transduction, phage lysates at a MOI 0.2 were incubated
capableofpreciselydefiningtheimpactofbiofilmsoncu- withUAMS-1thathasbeenresuspendedinTSBcontain-
taneouswoundhealing.Throughrigoroustesting,weval- ing5mMCaCl .After20minutesincubationat371C,the
2
idate that our model allows for biofilm formation within reactionmixturewaswashedwithice-cold20mMsodium
woundsandmaintenanceofitsstructureandviabilityover citrate and plated in TSA containing 10mg/mL erythro-
time.Althoughwoundinfectionsrepresentaspectrumof mycin.ThepresenceofpCN57inUAMS-1wasconfirmed
bacterialphenotypes,involvingbacteriainboththeplank- byplasmidisolation,restrictionanalysis,andfluorescence
tonicandbiofilmphases,weuseourmodeltostudydiffer- microscopy.
ences in planktonic- and biofilm-dominant infections,
which are classically associated with acute and chronic
Woundprotocolandbacteriabiofilmmodel
wound pathogenesis, respectively. In particular, we hy-
pothesize that both planktonic-dominant, or active, and Forcreationofthedermalwounds,therabbitswereanes-
biofilm-dominant wound infections will show severe thetized with an intramuscular injection of ketamine
wound healing deficits when compared with uninfected (22.5mg/kg)andxylazine(3.5mg/kg)mixturebeforesur-
wound beds. Furthermore, we propose that phenotypic gery.Earswereshavedandsterilizedwithabetadinescrub
differencesbetweenthesetwotypesofinfectionwillleadto and70%ethanol.Followingintradermalinjectionof1%
distinct host inflammatory responses even under circum- lidocaine with epinephrine local anesthetic, six 6-mm-
stances where both infections have similar bacterial bur- diameterfull-thicknessdermalwoundswerecreateddown
dens. Through these experiments, we hope to further totheperichondriumontheventralsurfaceoftheearand
definetheintricaciesofwoundbacterialbiofilms,whilein- dressed with semiocclusive transparent film (Tegaderms,
troducing and establishing an in vivo biofilm model that 3MHealthCare,St.Paul,MN).Forthecontrolarmofthe
willserveasafoundationforfurthermechanisticandclin- experiments, wounds were redressed with sterile Tega-
icallyrelevantinvestigation. derms on postoperative days (PODs) 3, 5, 6, 8, and 10.
For the infected wound groups, bacteria grown on agar
plates were harvested and proliferated in culturemedium
MATERIALSANDMETHODS broth, followed by suspension in phosphate buffer solu-
tion (PBS), from which a concentration was measured.
Animals Approximately106colony-formingunits(CFUs)ofplank-
tonicbacteriawereinoculatedfromsuspensionontoeach
Young, adult New Zealand white rabbits (3–6 months,
woundbedonPOD3andallowedtoproliferateunderthe
(cid:3)3kg) were acclimated to standard housing and fed ad Tegadermsdressing.Fortheactiveinfectionarm,plank-
libitum under a protocol approved by the Animal Care
tonic bacteria were allowed to continue active prolifera-
and Use Committee at Northwestern University. All ani- tion, with Tegaderms dressing changes on PODs 5, 6, 8
malswerehousedinindividualcagesunderconstanttem-
and10.Forthebiofilmarm,topicalmupirocin(2%)anti-
perature (221C) and humidity with a 12-hour light–dark
biotic ointment (Teva Pharmaceuticals, Sellersville, PA)
cycle.Atotalof82rabbitswereusedinthedevelopmentof
wasappliedonPOD4toeliminateplanktonic-phasebac-
this model after a series of pilot studies manipulating the teria and create a predominantly biofilm-phase infection.
followingvariablesindifferentpermutations:woundsize,
In order to prevent seroma formation and maintain
woundlocation,numberofwoundsperear,wounddepth
biofilm-phase infection by preventing regrowth of plank-
(partialvs.fullthickness),timingofinoculation,lengthof tonic bacteria, an antimicrobial absorbent dressing
antibiotictreatment,typeofabsorptivedressing,timingof containing polyhexamethylene biguanide (Telfas AMD,
dressingchanges,andlengthofwoundhealingperiodbe-
Tyco Healthcare Group, Mansfield, MA) was applied to
fore harvest. Following exhaustive characterization of
biofilmwoundsonPODs5,6,8,and10.Alldressingswere
thesepermutationsthefinalmodelprotocoldescribedbe- checkeddailythroughouttheentireprotocol.
lowwaschosenbasedonrecapitulationofessentialaspects
ofthehumanbiofilm-infectedchronicwound.
Harvestingofwounds
Aftereuthanizingtheanimalsbyanintracardiaceuthasol
Bacterialstrains,plasmid,andphagetransduction
injection,woundswereharvestedforvariousanalyses.For
Staphylococcus aureus strain UAMS-19,10 was grown on the biofilm time course experiment, wounds were har-
tryptic soy agar (TSA) and in tryptic soy broth (TSB) at vested at 6-hour intervals following inoculation until 24
371C. The green fluorescent protein (GFP)-expressing hours,andanalyzedbyepifluorescenceandSEM.Totest
WoundRepReg(2011)19400 410 (cid:2)c 2011bytheWoundHealingSociety 401
Animalmodelforbiofilmimpairedwoundhealing Gurjalaetal.
for the presence of bacteria in the wound bed, bacterial werealsoanalyzedusinglaserscanningconfocalmicroscopy
countsandlaserscanningconfocalmicroscopywerecon- (Zeiss LSM 510 META, Carl Zeiss Microimaging, LLC,
ductedonPODs4,6,8,10,and12.Inaddition,SEMwas Thornwood,NY)tovisualizeGFP-labeledS.aureus.
performed POD 12 to visually confirm biofilm formation
within the wound bed. For the wound healing experi-
SEM
ments,woundswereharvestedatPOD12forhistological
analysisusinghematoxylin&eosin(H&E)staining.RNA Todeterminethestructureofbiofilmsformedinrabbitear
extractionandreversetranscriptionquantitativepolymer- wounds, wound samples were mounted by double-sided
asechainreaction(qPCR)wascarriedoutonPOD6and tapetospecimenstubs,andexamined‘‘insitu’’byvariable
POD 12 on wounds to determine the cytokine profiles as pressureSEM.Theprocedurebypassesthestepsforsam-
theresultofthehostinflammatoryresponse. ple preparation used in conventional SEM and therefore
minimizes the introduction of artifacts, providing an ex-
cellent preservation of sample integrity. Imaging of the
Viablebacterialcountsusingdropplatemethod
sampleswasaccomplishedbytheuseofaCarlZeiss1450
For viable bacterial counts, wound beds were biopsied VP scanning electron microscope (Carl Zeiss Microima-
with 10mm punches and using No. 15 scalpels (Becton ging,LLC)operatedatthegaschamberpressureof70Pa
Dickinson AcuteCare, Franklin Lakes, NJ) the wound with the specimen cooling stage set at (cid:4)251C and the
beds were excised and tissue samples collected into sepa- scanningvoltagesetat10kV.
rate MagNA Lyser Green Beads tubes (Roche Diagnos-
tics, Indianapolis, IN) each containing 1mL of PBS. All
TotalmRNAextractionandreverse-transcriptionqPCR
samples were homogenized at 5,000rpm for 30 seconds
(MagNA Lyser, Roche Diagnostics), and then sonicated WoundswereharvestedformRNAextractionandsubse-
(MicrosonUltrasonicCellDisrupter,HeatSystems-Ultra- quent cDNA conversion as part of reverse transcription
sonics Inc., Farmingdale, NY) for 2 minutes at 5–8W to qPCR.Thedermallayerontheventralsideoftheearwas
break up any bacterial aggregates that were present in removed and the wound bed and circumferential areas
woundsamples.Timeintervalandpowersettingusedfor (rings) extending from the wound bed were punched out
the sonication were preempirically determined to ensure and immediately snap-frozen in liquid nitrogen. Wound
thatbacterialviabilitywasmaintainedintheprocess. samples were homogenized using a Mini-bead beater-8
Theresultingsolutionswereseriallydilutedandplated equipment(BiospecProductsInc.,Bartlesville,OK)using
on both Blood Agar (Hardy Diagnostics, Santa Maria, zirconiabeads(2.0mmdiameter,BiospecProductsInc.)in
CA) and Lipovitellin Salt Mannitol Agar (selective me- the presence of Trizol Reagent (Sigma-Aldrich). Total
dium for S. aureus) (Hardy Diagnostics) and incubated RNAwasisolatedaccordingtothemanufacturer’sproto-
overnightat371C.CFUsweredeterminedbythestandard col.ContaminatinggenomicDNAduringRNAprepara-
colonycountingmethod. tionwasremovedusingtheTurboDNA-freekit(Ambion,
Austin, TX). Five mg of total RNA was used to prepare
cDNA using superscript II (Invitrogen) with 100ng of
Histologicalanalysis
randomprimers(Invitrogen).
Wounds were excised with a 10mm punch and bisected at For quantitative analysis of the expression level of
theirlargestdiameterforH&Estaining.Tissueswerefixed mRNAs,realtimeqPCRanalysesusingSYBRgreen1were
in formalin, embedded in paraffin, cut into 4mm sections, performed utilizing an ABI prism 7000 sequence detection
andstainedtobeanalyzedunderalightmicroscope.Slides system(AppliedBiosystems,FosterCity,CA).PCRprimers
wereexaminedforquantificationofepithelialandgranula- weredesignedusingthePrimer3program(http://frodo.wi.
tiongap,andtotalgranulationareausingadigitalanalysis mit.edu/). Expression of each gene was normalized to the
system (NIS-Elements Basic Research, Nikon Instech Co., levelofglyceraldehyde3-phosphatedehydrogenase(GAD-
Kanagawa, Japan) as described previously.12 Two blinded PH),thehousekeepinggene,togetDC.The2DDCt method
t
and independent observers evaluated all histological sec- wasusedtocalculategeneexpressionofinterleukin(IL)-1b
tions. The results of both examiners were averaged. Slides andtumornecrosisfactor-a(TNF-a)ofcontrol,biofilm-in-
wereomittedifresultsdiffered > 30%amongexaminers. fectedwounds,andwoundsunderactiveinfection.Expres-
sion of genes was detected by PCR with the following
Fluorescentstainingandlaserscanningconfocal oligonucleotides:IL-1b(50-CCACAGTGGCAATGAAAA
TG-30and50-AGAAAGTTCTCAGGCCGTCA-30),TNF-
microscopy
a(50-CCAGATGGTCACCCTCAGAT-30 and50-TGTTC
Woundsamplesforfluorescencestainingwereembeddedin TGAGAGGCGTGATTG-30), GADPH (50-AGGTCATC
O.C.T. Compound (Sakura Finetek, Torrance, CA), snap- CACGACCACTTC-30 and 50-GTGAGTTTCCCGTTCA
frozeninliquidnitrogen,andstoredat(cid:4)801Cuntiltimeof GCTC-30).
cryosectioning. Ten micrometer sections of biofilm-infected
woundtissueswerepreparedwiththeuseofacryostat.Sec-
Statisticalanalysis
tions for the biofilm time course experiment were stained
with concanavalin-A conjugated to Texas Red (Invitrogen, Dataarepresentedingraphicalformasmean(cid:5)standard
Carlsbad,CA)toidentifythepresenceofexopolysaccharides errors and were analyzed using the Student’s t-test (two-
richinmannoseandglucosethatarecommonlythebasisof tailedandunpaired)tocomparecontrols,activeinfection,
biofilm extracellular matrix (ECM), and with 40-6-diami- andbiofilm woundsamples.Thelevel ofsignificancewas
dino-2-phenylindole to visualize host cells. Section slides setatp < 0.05.
402 WoundRepReg(2011)19400 410 (cid:2)c 2011bytheWoundHealingSociety
Gurjalaetal. Animalmodelforbiofilmimpairedwoundhealing
Figure1. Time-courseofbiofilmdevelopmentin6-hourintervalsfrominoculationtotheformationofmaturebiofilm.Staphylococ-
cusaureusestablishesamaturebiofilminrabbitdermalulcerswithin24hoursasreviewedbyscanningelectronmicroscopy(SEM)
(A–D)andfluorescencelightmicroscopy(E–H).(A)and(E)showthatat6hourspostinfectionbacteria(green)sparselycoverthe
woundsurface(blue).At12hourspostinfectionS.aureusspreadsmoreevenlyoverthewoundsurface(BandF).(C)and(G)show
thatby18hourspostinfectionS.aureusassumesearlybiofilmmorphology.Notethechangeofarchitectureofthebiofilmasre-
vealedbySEM.By24hourspostinfection,amaturebiofilmofS.aureusisformedwithbacteriaencasedwithinexopolysaccharide
matrix(red)(DandH)(magnification:A–D,(cid:6)200;E–H,(cid:6)1,000).
RESULTS confocal microscopy was also performed at these time
points,showingthepresenceofGFP-labeledS.aureusat
To determine the kinetics of biofilm formation in vivo, a
POD 6 (Figure 3A) and POD 12 (Figure 3B) within the
time-course study using the rabbit biofilm wound model
wound bed, clearly separated from the underlying, intact
wasperformed.TheresultsshowedthatS.aureusformeda
earcartilage.Inaddition,SEMwasperformedatPOD12,
mature biofilm within 24 hours in wounds after inocula-
showing the presence of individual bacteria within a well
tion (Figure 1). A spatially organized three-dimensional
biofilm structure began to emerge 18 hours postinocula-
tionasrevealedbySEM(Figure1C).At24hourspostin-
oculation the bacteria on the wound became more
organized showing a complex architecture with dense
ECM(Figure1D).Stainingofmannoseandglucosemoi-
etieswithinthewoundbyTexasRed-labeledconcanavalin
Aalsorevealedincreasingexopolysaccharidesaroundthe
bacteriasuchthatby24hourstheyarecompletelyencased
byacarbohydrate-richECMasshownbyepifluorescence
microscopy(Figure1GandH).
As part of model testing and validation, biofilm devel-
opmentandviabilitywasverifiedthroughseveraldifferent
techniques. Viable bacterial counts, measured at multiple
timepointsfollowinginoculation,showedthepersistence
oflivebacteriawithinthewoundbedsofbiofilmanimals Figure2. Viable bacterial count measurements from biofilm-
(Figure2).Inparticular,followinginoculationonPOD3 infected wounds at successive time points following inocula-
with approximately 106CFUs of planktonic bacteria, an tiononPOD3.Inoculationwithapproximately106colony-form-
initial proliferation on POD 4 up to 107 is followed by ing units (CFUs) of Staphylococcus aureus is followed by an
topical antibiotic and absorptive, antimicrobial dressing initialproliferationofbacteriabyPOD4,followedbytopicalan-
placement, resulting in a significant (p < 0.05) decreased tibiotic and antimicrobial, absorbent gauze placement to de-
numberofviablebacteriaatPOD6.Thisdecreasecanbe velop and maintain a biofilm-dominant wound. Note an initial
attributed to the elimination of free-floating planktonic significantdecreaseinviablebacteriabetweenPOD4andPOD
bacteria, creating a biofilm-dominant wound. However, 6, most likely representing the elimination of free-floating
betweenPOD6andPOD12,biofilmwoundsmaintaina planktonicbacteriabytheaforementionedtreatmentmethods.
consistent level of viable bacteria averaging between 105 SuccessivebacterialcountmeasurementatPODs6,8,10,and
and106CFUsperinfectedwound,verifyingthatwounds 12showviablebacteriathataremaintainedataconsistentlevel
maintainlivebiofilmbacteriathroughoutthetime-course over time, averaging between 105 and 106CFUs per wound,
of our animal model. This consistency models the representative of the ‘‘steady-state’’ that is often seen be-
presumed ‘‘steady-state’’ that develops between chronic tween wound biofilms and their respective hosts (np<0.05)
wound biofilms and their respective hosts. To correlate (n58–15wounds/time-point). POD, postoperative day. Error
with bacterial count measurements, laser scanning barssignifystandarderrorofmeans.
WoundRepReg(2011)19400 410 (cid:2)c 2011bytheWoundHealingSociety 403
Animalmodelforbiofilmimpairedwoundhealing Gurjalaetal.
Figure3. Laser scanning confocal
microscopy of biofilm wounds in-
fected with green fluorescent pro-
tein-labeled Staphylococcus aureus.
ImagingdoneatPOD6andPOD12
validatesthepresenceofviablebac-
teriawithinbiofilmwoundsoverthe
course of time used in our model.
Notetheclusteringofbacteriawithin
thewoundbed(green)withunderly-
ingintactcartilageoftheear(arrow)
(magnification(cid:6)20). POD, postoper-
ativeday.
developed, encasing biofilm matrix architecture that cov-
eredthewoundbedsurface(Figure4)
Despite the spectrum of infection that exists between
planktonic- and biofilm-phase bacteria, our model has
been developed to create a distinct infection phenotype
characterized by a predominance of biofilm-phase bacte-
ria.Toshowthismodelcapability,viablebacterialcounts
were performed from biofilm-dominant and planktonic-
dominant,i.e.,activeinfection,woundsatPOD6(Figure
5A).Thisanalysisrevealednosignificantdifferenceinvi-
able bacteria despite differences in how the wounds were
treated. At this same time-point, host inflammatory
response to these two types of infections was quantified
using real-time qPCR (Figure 5B and C). Biofilm infec-
tion elicited a significantly lower-grade host response
(p < 0.05), measured through IL-1b and TNF-a expres-
sion, than active infection wounds, indicatinga true phe-
notypic difference in the bacteria–host response between
thesetwotypesofinfectiondespiteasimilarbacterialbur-
den. In addition, when the expression of these inflamma-
tory mediators in biofilm wounds was measured at POD
12, there is evidence of a maintained low-grade host re-
sponse, with minimal decrease in the level of TNF-a ex-
pression and a continued high level of IL-1b expression.
(Figure6)Toensurethatourmodelcreatedonlylocalized
wound infections, thus preventing the activation and in-
fluence of systemic inflammatory mediators, successively
largercircumferentialringsoftissuewereanalyzedoutside
of the wound bed. As seen in Figure 7, expression of in-
flammatorymediatorsquicklytaperstosignificantlylower
levels in both biofilm and active infection wounds
(p < 0.05),verifyingthatourmodelmaintainsalocalized Figure4. Scanning electron microscopy of Staphylococcus
woundinfection.Furthermore,noanimalsineithergroup aureusbiofilm-infectedwoundsonPOD12showingdeveloped
showedsignsofsystemicinfectionandmaintainednormal biofilm architecture. (A) Intricate extracellular matrix encases
body weights throughout the experimental period (data individualbacterialcells (arrow)in dense,lattice-like structure
not shown). These data not only distinguish and charac- (magnification (cid:3)(cid:6)1000).(B)Highermagnificationviewofindi-
terize the biofilm wound phenotype, which triggers and vidualS.aureuswithsurroundingextracellularmatrixmaterial
maintains a low-grade host inflammatory response, but adjacent to bare wound surface (arrow) (magnification (cid:3)(cid:6)
also validate the sensitivity of our model to distinguish 4000).POD,postoperativeday.
404 WoundRepReg(2011)19400 410 (cid:2)c 2011bytheWoundHealingSociety
Gurjalaetal. Animalmodelforbiofilmimpairedwoundhealing
Figure5. Demonstration of pheno-
typic differences between biofilm-
and planktonic-dominant (i.e., active
infection) wounds. (A) At POD 6,
thereisnosignificantdifferenceinvi-
able bacterial counts between un-
treated active infection (AI) wounds
and treated biofilm-dominant (BF)
wounds (n515wounds/group). (B)
and (C) despite similar bacterial bur-
dens,biofilmwoundstriggerasignifi-
cantlylower-gradehostinflammatory
response,measuredthroughinterleu-
kin(IL)-1bandtumornecrosisfactor-a
(TNF-a)woundbed(WB)expression,
as compared with active infection
wounds. This validates a phenotypic
differencebetweenthetwoinfection
groups in their interaction with the
host (np<0.05 as compared with
CTRL; nnp<0.05 as compared with
BF)(n56wounds/group).POD, post-
operativeday.Errorbarssignifystan-
darderrorofmeans.
betweenthesedifferentwoundphenotypesdespitesimilar planktonic bacteria damage the cartilage, while biofilm
levelsofinitialbacterialburden. histology shows cartilage integrity. Large amounts of de-
Having established and validated our wound biofilm brisintheactiveinfectionwerepresentrepresentinganin-
model,weaimedtoassesstheeffectsofS.aureusbiofilm creasedinfluxofwhitebloodcells(neutrophils)abovethe
on quantitative wound healing parameters histologically. woundbed.Thedebriswasalsofoundinthebiofilm,but
WoundswereharvestedonPOD12,withgrossevidenceof significantly reduced, confirming a decreased inflamma-
minimal wound healing in both biofilm- and planktonic- tory response histologically. Through quantitative image
dominant(activeinfection)woundsascomparedwithcon- analysis, measurement of epithelial gap (Figure 10A),
trol (Figure 8). Also note the increased level of purulent granulationtissuegap(Figure10B),andnewgranulation
exudateoveractiveinfectionwounds(Figure8B)ascom- tissue (Figure 10C) showed significant differences across
pared with the so-called ‘‘film’’ overlying biofilm wounds all analyzed parameters between control and infected
(Figure 8C), consistent with the differences seen in host groups (p < 0.05), verifying that our model is capable of
inflammatory response. Looking histologically, S. aureus simulating the impairment of wound-healing seen in bio-
in the form of active or biofilm infections impaired cuta- film-infectedwounds.
neouswoundhealing(Figure9).Infectedwoundsshowed
delayedreepithelializationoverthewoundbedandthick-
DISCUSSION
eningoftheepidermallayerattheleadingedge(Figure9B
and C). Differences between active infection and biofilm Predating any bacterial biofilm theories, chronic wounds
arealsoseeninthecartilageanddebrisaroundthewound ofalltypeshavesharedacommoncharacteristic:apersis-
bed. In the active infection, highly virulent and invasive tent inflammatory state, marked by a destructive milieu
Figure6. Maintenance of a low-grade
inflammatory response secondary to
biofilm wound infection over time. (A)
Expression of tumor necrosis factor-a
(TNF-a) is maintained between POD 6
andPOD12atverysimilarlevels.(B)In-
terleukin(IL)-1bexpressionisdecreased
butremainsmarkedlyelevatedatPOD
12, validating the idea that biofilm trig-
gersalow-grade,chronicinflammatory
response, which can be simulated
through this model (n56wounds/
group). POD, postoperative day. Error
barssignifystandarderrorofmeans.
WoundRepReg(2011)19400 410 (cid:2)c 2011bytheWoundHealingSociety 405
Animalmodelforbiofilmimpairedwoundhealing Gurjalaetal.
Figure7. Comparison of inflamma-
tory profiles from control and in-
fected wounds, showing highly
localized infections at POD 6. Note
significantlyhigherlevelofinflamma-
torymediatorexpressionforbothtu-
mornecrosisfactor-a(TNF-a)(A)and
interleukin (IL)-1b (B) within wound
beds of infected wounds as com-
pared with successive outer rings
(ring 1: concentric ring of tissue
2mmdistaltothewoundedge;ring
2:concentricringoftissue2mmdistaltotheedgeofring1)(np<0.05ascomparedwithwoundbed)(n56wounds/group).POD,
postoperativeday.Errorbarssignifystandarderrorofmeans.
and failure of the wounds to progress normally into the wound microbiology was conventionally conceptualized
proliferative and remodeling phases of wound healing.13 as contamination, colonization, and infection by plank-
Clinically, chronic wounds are observed to languish in a tonic bacteria, it is now clear that biofilm phase bacteria
friable, highly exudative, and oftentimes ‘‘slimy’’ condi- playaprominentroleinthespectrumofclinicalwounds.16
tion,mostdisplayingnoevidenceofplanktonicorfrankly This spectrum includes both infections that are predomi-
purulent bacterial infection, some responding tofrequent nantlyintheplanktonic-orbiofilm-phases,althoughitis
debridementandwoundcare,andothersremainingrecal- clearthatamixtureofbacterialphenotypescan,andtend
citranttowoundhealinginterventions.14 to, exist within a given wound. There is likely a balance
Bacterial biofilms offer a powerful and parsimonious betweenhostvulneraryfactors/defensesandbacterialpro-
explanation for both the cause and persistence of the in- liferationinitsvariousforms,thepredominanceofwhich
flammatoryarrestofchronicwounds.Thelogicalconnec- influences wound outcome. Bacteria in a wound may be
tion between bacteria and chronic wounds is intuitively either overcome or cleared by host defenses; if bacteria
clear,butwaslikelydelayedbymedicine’shistoricreliance persist asbiofilms,theymaybe heldincheckbythehost
on planktonic culture techniques, which fail to recover defenses,yetcreateastateofchroniclow-gradeinflamma-
biofilm phase bacteria, especially from chronic wound tion.Giventhisexpandedviewofwoundmicrobiology,it
samples.Indeed,asbiofilmshaverecentlybeenfoundus- iscriticalthatanyinvivomodelofbiofilmsimulateboth
ingmoresophisticatedtechniquestobepresentinpressure itsabilitytoimpaircutaneouswoundhealinganditsmain-
sores,venousstasisulcers,anddiabeticfootulcers—tradi- tenance of a low-grade, chronic inflammatory host re-
tionally regarded as having distinct etiologies—all three sponse that is distinct from the response to a more
chronicwoundarchetypesmayinfactbelinkedbyasingle ‘‘active’’planktonic-dominantinfection.
contributing factor. This factor has high potential as a Recentworkhastakenimportantstepstowardachiev-
therapeutic target, as elimination of bacterial biofilms inganinvivobiofilmmodel,withRashidetal.17showing
alone could be instrumental in shifting the balance be- thatcutaneousPseudomonasaeruginosabiofilmcanbeex-
tween inhibitory and vulnerary factors toward a healing perimentally grown in thermally induced mouse wounds.
phenotype. In addition to the classic tenets of hypoxia, Using their porcine model, Davis and colleagues inocu-
ischemia-reperfusion,andintrinsichostdisease,then,bac- lated S. aureus onto partial thickness cutaneous wounds,
terialbiofilmsholdconsiderablepromiseasafourthmajor andassessedforpresenceofbiofilmafter48hours.Multi-
pillarofimpairedwoundhealing.15 plemodesofimagingwereabletoconfirmpresenceofliv-
Theadditionofbiofilmtheorytowoundshasincreased ing biofilm in these wounds.18 In 2009, our own group
our understanding of bacteria–host interaction. Whereas published the infected splinted mouse model, in which
Figure8. Photographs of control
(uninfected)andinfectedwoundsat
POD12.Control(A),activeinfection
(B), and biofilm infection (C). Note
theabsenceofpurulenceanddebris
inbiofilmwoundsascomparedwith
active infection wounds, instead
showinga‘‘film’’ofbacteriaspread
across the wound. Also note the
clear difference in extent of wound
epithelializationbetweencontroland
infected wounds. POD, postopera-
tiveday.
406 WoundRepReg(2011)19400 410 (cid:2)c 2011bytheWoundHealingSociety
Gurjalaetal. Animalmodelforbiofilmimpairedwoundhealing
wounds, this removal of dermis more closely models the
dermal-lossseeninhumanchronicwounds.Additionally,
themajorityofhumanwoundshealthroughepithelializa-
tionandgranulation,incontrasttothecontracture-based
healingseeninmice.19Theunderlyingcartilageoftherab-
bit ear serves as a natural splint, preventing healing by
contracture,andthusallowingforaccuratequantification
ofepithelialandgranulationtissueformationfromthepe-
riphery of the wound. Previous work with the rabbit ear
dermal ulcer model has also showed that hypertrophic
scarringwithintherabbitearissimilartothatseeninhu-
mans grossly, histologically, and in its response to scar
treatmentssuchassteroidinjection.31,32Moreover,multi-
pleidenticalwoundscanbemadeinoneanimalwithcon-
tralateralcontrols,creatingbothastandardizedandhigh-
throughputwoundmodel.Inoculationofwoundsisdone
onpostwoundingday3usingculturemedium-grownbac-
teria with a measured size of inoculum of approximately
106CFUsofbacteria.Incontrasttootherpublishedmod-
els where preformed in vitro biofilm is directly applied
to wounds, we believe that inoculation with planktonic,
free-floating bacteria more closely represents the seeding
mechanismofhumanchronicwounds,withthewoundbed
itselfplayingacriticalroleinthetransformationofbacteria
into the biofilm state.33,34 Furthermore, as suggested by
our presented data,inoculationin this manner triggers the
formationofbiofilm-phasebacteriawithin24hours.
In another significant departure from other published
animalmodels,followinginvivoproliferationoftheinoc-
ulated bacteria, including formation of bacterial biofilm,
our wounds are treated with topical antibiotic. As ex-
Figure9. Woundhealinghistology.Hematoxylin&eosinstain- pected, this reduces the presence of active, planktonic-
ingofPOD12rabbitearwoundsfromcontrol(A),activeinfec- phase bacteria, but also by definition leaves behind what
tion(B),andbiofilminfection(C)groups.Thewoundedgewas should be predominantly biofilm-phase bacteria, which
determinedbytheappearanceofanickinthecartilagemade are more resistant to antimicrobial challenge due to a
during wounding. Cartilage nick (arrow), epidermal thickening protective ECM. We then utilize a combination of an
(arrowhead),cartilagedegradation(D),anddebrisfromwhite occlusive dressing (Tegaderms) with an underlying anti-
blood cell influx (W) are labeled (magnification, (cid:6)20 [A– microbial (polyhexamethylene biguanide) absorptive
C]).POD,postoperativeday. gauze pad (AMD Telfas). This form of wound coverage
helps maintain the predominance of biofilm-phase bacte-
openwoundswereinoculatedwitheitherbiofilm-ornon- ria in two ways. First the antimicrobial impregnation of
biofilm–forming bacteria, and treated with a biofilm in- thegauzehelpslimit proliferationofplanktonicbacteria.
hibiting peptide; data from this paper suggested delayed Second,itisclearthatactivelyinfectedwoundsarepredis-
epithelializationofthesewounds,andthatthisdelaywasa posed to the formation of purulent exudates. Previously
biofilm-dependent effect.19 Most recently Zhao et al.20 published models have utilized fluid impermeable, occlu-
have developed an infected wound model in db/db mice, sivedressingsfortheirinfectedwoundswithoutanunder-
applyingpreformedP.aeruginosabiofilmtofull-thickness lying absorptive gauze pad.19,20 Clinical observations, as
punch wounds, and found a marked reduction in a gross wellasourownpilotstudies,demonstratethatplacement
measure (percent healing) of infected vs. noninfected ofocclusivedressingsoverthisdevelopingfluidcollection
wounds. createsaseromawithinthedeadspacebeneaththedress-
Despitetheadvancesinvivoanimalmodeling,currently ing,representinganidealculturemediumforproliferation
availablewoundbiofilmmodelspossesscertainlimitations of planktonic-phase bacteria. In this setting a mixed
and shortcomings. In trying to address these deficiencies, planktonic-biofilminfectioncanbecomeapredominantly
we have developed the aforementioned rabbit ear wound planktonic, purulent infection, more similar to a superfi-
biofilmmodel,whichwebelieveisthemostaccuraterep- cial abscess than the wound surface biofilms seen in
resentation of biofilm-infected human chronic wounds to chronic wounds. Therefore, the use of absorptive gauze
date. Our model utilizes an adaptation of the rabbit der- helps to minimize seroma formation, which when com-
mal ulcer model,21 an FDA-recognized model of wound bined with previously administered antibiotics, creates a
healingthathasbeenutilizedbyourlabandothersfor20 ‘‘steady-state’’ predominantly biofilm-phase infection by
years.22–30 In this model, full-thickness, circular punch- POD6.Finally,wealsoperformfrequentdressingchanges
woundsaremade inthe earsofNew ZealandWhite rab- beforewoundharvest,modelingthecommonclinicalman-
bits down to cartilage, affording a number of important agement of chronic wounds. In summary, our in vivo
advantages. For example, in contrast to partial-thickness wound biofilm model is the most accurate and consistent
WoundRepReg(2011)19400 410 (cid:2)c 2011bytheWoundHealingSociety 407
Animalmodelforbiofilmimpairedwoundhealing Gurjalaetal.
Figure10. Quantification of healing
parameters for uninfected (control),
active-,andbiofilm-infectedwounds.
(A)Epithelialgap,(B)granulationgap,
and (C) granulation tissue area. In-
fected wounds have significantly
lesshealingacrossallmeasuredpa-
rameters when measure to control
wounds, verifying in particular that
biofilm does impair cutaneous
woundhealingandthatthisresultis
effectivelysimulatedwiththismodel
(np<0.05ascomparedwithcontrol
wounds) (n58–12wounds/group).
Error bars signify standard error of
means.
simulation of biofilm-infected human chronic wounds to islocalizedtothewoundbed,similartoahumanchronic
date,whichwevalidatethroughthedatapresentedinthis wound that elicits little systemic inflammatory effects.
manuscript. Taking the clinical applicability of our model further, we
Development of a model requires systemic testing and investigated the impairment of wound healing related to
verificationbeforeitcanbeusedforexperimentationand biofilm, an increasingly recognized phenomenon in non-
analysis.Wehavepresentedanextensivesetofdatadem- healingwounds.Grossly,thereisacleardifferenceinboth
onstrating this validation. Using epifluorescence and the hostinflammatoryresponseand extentof healingbe-
SEM,wehaveshownthatinoculationwithplanktonicS. tween infected and control wounds, whichis further sup-
aureus establishes mature biofilm in cutaneous wounds, ported by histological analysis. In fact, quantitative
with marked speed and under no special conditions. To analysisyieldsstatisticallysignificantdifferencesinmulti-
ensurethatourmodelmaintainsviablebiofilm-phasebac- ple histological wound healing parameters between unin-
teriaoverthecourseofourprotocoltimeline,wemeasured fected and biofilm-infected wounds. Interestingly, vs.
viablebacterialcountsatvarioustimepointspostwound- granulation tissue formation, we found maximal impair-
ingand-inoculation.Althoughourdevelopmentofabio- ment to be in reepithelialization of biofilm-infected
film-predominant infection through antimicrobial wounds. This observation agrees well with the frequent
techniquesdoesdecreaseviablebacterialcountsfromtheir clinicalobservationofrecedingofepitheliumorcomplete
postinoculationvalues,theconsistentlevelofbacteriaseen failure ofreepithelialization(even intheface ofadequate
between POD 6 and POD 12 reveals a trend that models granulationtissue)inhumanwounds.Thesefindingssug-
the ‘‘steady-state’’ or chronic nature of biofilm, and di- gestthattheprimarymechanismofbiofilmimpairmentof
rectly correlates to the static nature of human chronic woundhealingmaybethroughinhibitionofepithelializa-
wounds. Corroborating this bacterial count analysis, we tion. Although our data only represents part of the con-
usedlaserscanningconfocalmicroscopyandSEMtover- tinued investigation into biofilm, we submit that our
ify the physical presence of viable bacteria within the model provides a powerful in vivo methodology for
woundbedandtheexistenceofcomplexbiofilmstructure woundbiofilmstudythatiscapableofquantitative,repro-
morphologicallyatPOD12.Theseexperimentsclearlyin- ducible,andsensitivedataanalysisthroughbothimaging
dicate that our model is capable of developing consistent andnonimagingmodalities.
woundbiofilmsthatprovideafoundationforfurtherbio- Although we have ensured biofilm phase infection and
filmcharacterizationandexperimentation. mirrored key inflammatory aspects of chronic wounds in
Wethenaimedtoassessthecapabilitiesandsensitivity thismodel,ourassessmentofwoundhealingisstilloccur-
of our model, utilizing quantitative analysis of host in- ringwithinanacutetimeframe.Furtherworkwillbenec-
flammatory gene expression to distinguish between our essary to investigate the long-term effects of our biofilm
developed biofilm wounds and untreated, planktonic- protocol.Moreover,althoughmorphologybasedonSEM
dominant,activeinfectionwounds.Correlatingwithclin- is the current gold standard for identifying bacterial bio-
icalsuspicion,wehaveshownthatthereisadistinctphe- films, it would have been ideal to confirm biofilm pheno-
notypic difference between biofilm and active infection typeinmorespecificways.Unfortunately,visualizationby
wounds. In particular, despite similar bacterial burdens, fluorescence is currently limited to using nonspecific car-
thehostwoundbedshowsauniqueinflammatorymedia- bohydrate markers as concanavalin A, as no specific
torprofileinresponsetothebiofilmphenotype.Addition- markers for biofilm yet exist. In addition, our work thus
ally,weverifiedthatthisuniqueprofile,characterizedbya farhasbeenlimitedtoasinglespeciesofbacteria,namely
low-gradeinflammatoryresponse,ismaintainedovertime S. aureus. Organisms such as P. aeruginosa and various
withinbiofilm-infectedwoundsandthatusingourmodelit anaerobic bacteria are also very common within chronic
408 WoundRepReg(2011)19400 410 (cid:2)c 2011bytheWoundHealingSociety