Surface micropattern limits bacterial contamination

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Title:
Surface micropattern limits bacterial contamination
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English
Creator:
Mann, Ethan E.
Manna, Dipankar
Mettetal, Michael R.
May, Rhea M.
Dannemiller, Elisa M.
Chung, Kenneth K.
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BioMed Central (Antimicrobial Resistance & Infection Control)
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Abstract:
Background: Bacterial surface contamination contributes to transmission of nosocomial infections. Chemical cleansers used to control surface contamination are often toxic and incorrectly implemented. Additional non-toxic strategies should be combined with regular cleanings to mitigate risks of human error and further decrease rates of nosocomial infections. The Sharklet micropattern (MP), inspired by shark skin, is an effective tool for reducing bacterial load on surfaces without toxic additives. The studies presented here were carried out to investigate the MP surfaces capability to reduce colonization of methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) compared to smooth control surfaces. Methods: The MP and smooth surfaces produced in acrylic film were compared for remaining bacterial contamination and colonization following inoculation. Direct sampling of surfaces was carried out after inoculation by immersion, spray, and/or touch methods. Ultimately, a combination assay was developed to assess bacterial contamination after touch transfer inoculation combined with drying (persistence) to mimic common environmental contamination scenarios in the clinic or hospital environment. The combination transfer and persistence assay was then used to test antimicrobial copper beside the MP for the ability to reduce MSSA and MRSA challenge. Results: The MP reduced bacterial contamination with log reductions ranging from 87-99% (LR = 0.90-2.18; p < 0.05) compared to smooth control surfaces. The MP was more effective than the 99.9% pure copper alloy C11000 at reducing surface contamination of S. aureus (MSSA and MRSA) through transfer and persistence of bacteria. The MP reduced MSSA by as much as 97% (LR = 1.54; p < 0.01) and MRSA by as much as 94% (LR = 1.26; p < 0.005) compared to smooth controls. Antimicrobial copper had no significant effect on MSSA contamination, but reduced MRSA contamination by 80% (LR = 0.70; p < 0.005). Conclusion: The assays developed in this study mimic hospital environmental contamination events to demonstrate the performance of a MP to limit contamination under multiple conditions. Antimicrobial copper has been implemented in hospital room studies to evaluate its impact on nosocomial infections and a decrease in HAI rate was shown. Similar implementation of the MP has potential to reduce the incidence of HAIs although future clinical studies will be necessary to validate the MP’s true impact.
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Mann et al. Antimicrobial Resistance and Infection Control 2014, 3:28 http://www.aricjournal.com/content/3/1/28; Pages
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doi:10.1186/2047-2994-3-28 Cite this article as: Mann et al.: Surface micropattern limits bacterial contamination. Antimicrobial Resistance and Infection Control 2014 3:28.

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© 2014 Mann et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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Surfacemicropatternlimitsbacterial contaminationMann etal. Mann etal.AntimicrobialResistanceandInfectionControl 2014, 3 :28 http://www.aricjournal.com/content/3/1/28

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RESEARCHOpenAccessSurfacemicropatternlimitsbacterial contaminationEthanEMann1,DipankarManna1,MichaelRMettetal1,RheaMMay1,ElisaMDannemiller1,KennethKChung1, AnthonyBBrennan2andShravanthiTReddy1*AbstractBackground: Bacterialsurfacecontaminationcontributestotransmissionofnosocomialinfections.Chemical cleansersusedtocontrolsurfacecontaminationareoftentoxicandincorrectlyimplemented.Additionalnon-toxic strategiesshouldbecombinedwithregularcleaningstomitigaterisksofhumanerrorandfurtherdecreaseratesof nosocomialinfections.TheSharkletmicropattern(MP),inspiredbysharkskin,isaneffectivetoolforreducing bacterialloadonsurfaceswithouttoxicadditives.ThestudiespresentedherewerecarriedouttoinvestigatetheMP surfacescapabilitytoreducecolonizationofmethicillin-sensitive Staphylococcusaureus (MSSA)andmethicillin-resistant S.aureus (MRSA)comparedtosmoothcontrolsurfaces. Methods: TheMPandsmoothsurfacesproducedinacrylicfilmwerecomparedforremainingbacterialcontamination andcolonizationfollowinginoculation.Directsamplingofsurfaceswascarriedoutafterinoculationbyimmersion, spray,and/ortouchmethods.Ultimately,acombinationassaywasdevelopedtoassessbacterialcontaminationafter touchtransferinoculationcombinedwithdrying(persistence)tomimiccommonenvironmentalcontamination scenariosintheclinicorhospitalenvironment.Thecombinationtransferandpersistenceassaywasthenusedtotest antimicrobialcopperbesidetheMPfortheabilitytoreduceMSSAandMRSAchallenge. Results: TheMPreducedbacterialcontaminationwithlogreductionsrangingfrom87-99%(LR=0.90-2.18; p <0.05) comparedtosmoothcontrolsurfaces.TheMPwasmoreeffectivethanthe99.9%purecopperalloyC11000at reducingsurfacecontaminationof S.aureus (MSSAandMRSA)throughtransferandpersistenceofbacteria.TheMP reducedMSSAbyasmuchas97%(LR=1.54; p <0.01)andMRSAbyasmuchas94%(LR=1.26; p <0.005)compared tosmoothcontrols.AntimicrobialcopperhadnosignificanteffectonMSSAcontamination,butreducedMRSA contaminationby80%(LR=0.70; p <0.005). Conclusion: Theassaysdevelopedinthisstudymimichospitalenvironmentalcontaminationeventstodemonstrate theperformanceofaMPtolimitcontaminationundermultip leconditions.Antimicrobialcopperhasbeenimplemented inhospitalroomstudiestoevaluateitsimpactonnosocomia linfectionsandadecreaseinHAIratewasshown.Similar implementationoftheMPhaspotentialtoreducetheincidenceofHAIsalthoughfutureclinicalstudieswillbe necessarytovalidatetheMP ’ strueimpact.BackgroundEnvironmentalsurfacecontaminationprovidesapotentialreservoirforpathogenstopersistandcauseinfection insusceptiblepatients[1,2].Environmentalsurfacesnear patientssuchasbedrails,traytables,telephones,bedsidetables,patientchairs,andnursecallbuttonsare oftenheavilycontaminated[3-6].Methicillin-resistant Staphylococcusaureus (MRSA)andVancomycin-resistant Enterococcus (VRE)havebeenshowntosurviveon inanimatesurfacesforaminimumofafewweeksandin somecasesmonths[1,7-11].Pathogenscontaminatesurfacesthroughlargevolumesurfacesoaking(e.g.spills) ormicro-dropletaspirations(e.g.sneezes)andaretransferredsubsequentlytohealthcareworkers ’ handsand otherobjects(e.g.touchevents)[12-14].Recentevidenceconfirmsthatpatientsadmittedtoroomspreviouslyoccupiedbypatientsinfectedorcolonizedwith *Correspondence: sreddy@sharklet.com1SharkletTechnologies,Inc,12635E.MontviewBlvd,Suite160,Aurora, CO80045,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Mannetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated.Mann etal.AntimicrobialResistanceandInfectionControl 2014, 3 :28 http://www.aricjournal.com/content/3/1/28

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MRSAorVREhaveanincreasedriskofacquiringthe samepathogenasthepriorroomoccupants[15-20]. HealthcareinfectioncontrolguidelinesfromtheCentersforDiseaseControlandPrevention(CDC)now emphasizetheimportanceofcleaninganddisinfecting “ high-touchsurfaces ” andmonitoringtheseactivitiesto maintainasanitaryenvironmentinthehospital[21]. Thesedocumentsreflectanevolvingmindsetthatpatientareaenvironmentalcleanlinessinhealthcaresettingsplaysasignificantroleininfectionpreventionand control.Despitetheincreasedattentiontoenvironmentalhygiene,recentstudieshaveshownthatasfewas 40%ofnearpatientsurfacesarebeingcleanedinaccordancewithexistinghospitalpolicies[6,10,16].Surprisinglyfewtechnologicalsurfaceimprovementshavebeen implementedtoaddresstheproblemofcontaminated surfacesthatexistbetweenterminalcleanings[22]. Amongthefew,antimicrobialcopperhasrecentlybeen implementedasatechnologytopreventsurfacecontaminationbetweencleanings.Inonecoppertrial,patients whodevelopedHAIand/orcolonizationofMRSAand VREwassignificantlyreducedfrom0.123to0.071( p = 0.02)forpatientsthatresidedinICUroomswithcopper asopposedtoICUroomswithoutcopper[23].TheproportionofpatientsdevelopingHAIalonewasreduced from0.081to0.034( p =0.013)wherecopperwasused [23].Unfortunately,copperandantimicrobialsilverare expensivetoimplementandbothutilizekillmechanisms whichhavepotentialtoselectforresistantorganisms [24].Whiletheresultsofthecoppertrialsmayrequire furthervalidation[25,26],thedatafromthesestudiesindicatethatsustainedsurfacecontaminationreduction mayofferaclinicalbenefit. Amicropattern(MP)surfacewasevaluatedtoaddress theneedforimprovedsurfacetechnologytoresistbacterialcontamination.Specifically,previousstudiesshowthe SharkletMPtobethemosteffectiveamongorderedtopographies(pillars,channels,othergeometries)forinhibitingbioadhesion(Figure1)[27,28].TheMPreduces colonizationofavarietyofmarineorganismsandhuman pathogensinnutrient-richenvironments[27,29-34].The MPisaphysicalsurfacemodificationthatdoesnotintroducechemicaladditivesorantimicrobials;thereforethe bulkpropertiesofthematerialarenotaffectedbythe presenceofthetexturedsurface.Alternativesurfacemodificationsreportingtolimitbacterialcontamination existbutwerenottestedhere.Theyincludeexampleslike photo-activatingagents,polyethyleneglycol,anddiamond-likecarbonfilmsandwerereviewedrecentlyfor theirrolesincontaminationmitigation[35]. DemonstrationofMPefficacyrequirestestingdesignedtoassesstheremainingviablebacteriadirectly fromthesurfaceratherthanenumeratingbacteria remaininginsolutionsexposedtothesurfaceasdone inexistingstandardizedprotocolsforassessingantimicrobialsurfaces[36].Specificbacterialcontamination scenarioswereexaminedusingimmersion,spray,or touchtransferenceinoculationmethodswith,MSSA andMRSAtomimiccommoncontaminationscenarios. Ultimately,throughvalidationofindividualinoculation methods,acombinationofbothbacterialtransferand persistenceeventswerecombinedintoasingleassay. ThecombinationmethodwasusedtocomparethereductionofMSSAandMRSAontheMPtoantimicrobial copper.MethodsBacterialstrains,growthconditions,andinoculumsBacterialstrainsusedfortestingincludedmethicillinsensitive Staphylococcusaureus (MSSA;ATCC6538)or methicillin-resistant S.aureus (MRSA;ATCC43300),Each weregrowninashakingincubatorat37Cfor18 – 24hin trypticsoybroth(TSB)media(HardyDiagnostics,Santa Maria,CA).Priortoinoculation,strainsweresub-cultured intofreshTSBat1:100dilutionandgrownfor4h. Inoculumsuspensionswerepreparedbypelleting,resuspendingandadjustingthecellconcentrationofthe brothculturesinphosphatebufferedsaline(PBS;Hardy Diagnostics,SantaMaria,CA)usingaknownOD600/CFU ratio.Followingtheexperiment,bacterialinoculationsuspensionswereevaluatedforCFUs.Experimentswithinoculumswithin0.5logunitsofthetargetinoculumwere accepted.Bacterialchallengeonsurfaces TestsurfacematerialsFlatpolydimethylsiloxaneelastomer(PDMSe;Dow Corning,Midland,MI)oracrylicfilm(Flexcon,Spenser, MA),waseithercastagainstnickelshimsorembossed withaninverseSharklet ™ micropattern(MP)orsmooth Figure1 ScanningelectronmicrographofSharklet micro-patterned(MP)acrylicmaterial. Thescalebarinthe micrographrepresents20 m. Mann etal.AntimicrobialResistanceandInfectionControl 2014, 3 :28Page2of8 http://www.aricjournal.com/content/3/1/28

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surface(controls)bySharkletTechnologies,Inc(STI; Aurora,CO).TheinverseMPconsistsof2 mwide rectangularfeatureswithnonadjacentrepeatinglengths thatarerecessedintothesurfaceandarrangedinaperiodicdiamondpatternwith2 mspacingandafeature depthof3 m(Figure1).Copperfoil,a99.9%purealloy C11000(AlaskanCopperandBrassCompany,Seattle, WA),registeredasaUSEPAantimicrobialwaspurchasedforantimicrobialcoppertesting.Eachelastomer, plastic,orcopperfoilsamplewasfirmlyadheredtothe bottomofaPetridish,sterilizedfor10mwith95%ethanol,rinsed3timeswithdeionizedwaterandallowedto drypriortoeachexperiment.Ineachexperimental methoddiscussed,un-patternedsmoothsurfacesofthe identicalplasticmaterial,orsmoothacrylicforantimicrobialcoppercomparisons,wereusedasstandards toachievepercentreductioncalculations.SurfacetestingmethodsExperimentalmethodsusedtoevaluateattachmentand survivalwerebasedonareviewofpreviousstudiesassessingsurfacecontaminationorantimicrobialefficacy onsurfaces[1,5,7,8,36-41].Threeassaysweredeveloped thateachevaluatedvariedaspectsofreal-worldsurface contaminationscenarios.SprayinoculationassayToachieveevenbacterialloading,thesprayinoculation methodwasusedtotestMSSA.Suspensionsranging from1105to1107CFU/mlwerepreparedfromlogphasegrowthculturesinsterile1PBS.ACentral PneumaticProfessionalgravity-fedpaintsprayer(Harbor FreightTools,Camarillo,CA)wassterilizedbyspraying 50mlof95%ethanolthroughthedeviceandrinsedwith 100mlofsteriledeionizedwater.Usingsterile1PBS, theappropriatesprayconditionswereoptimizedtodepositbetween100 – 200 loffluidperdish.Perspray event,5 – 6platesweresecuredinthebiologicalsafety cabinetontesttuberacksangledatapproximately45 degrees.Thesprayerwasconnectedtocompressed nitrogentank(GeneralAir,Denver,CO)andwasloaded with50 – 100mlofpreparedbacterialsuspension.Test andcontrolsurfaceswerecutinto40mmradiussemicirclesandplacedside-by-sideinasinglePetridish.Experimentalplateswereweighedbeforeandafterspraying andthevolumeofdeliveredinoculumwascalculatedto ensurethesampleswerewithintheappropriatesprayinoculumrangeforenumerationthroughRODACsampling.RODACsamplingoccurreddirectlyfollowing dryingof30matambientconditionswithoutrinsing. Additionally,adisruptionanddilutionsamplingmethod, asdescribedbelowasapreviouslyoptimizedMSSA quantificationstandardprotocol,wasusedassupplementaryquantificationmethodstoconfirmtheability fortheRODACplatestorecovercellsfromthesurface aftersprayinoculation.ImmersioninoculationassayBacterialinoculumsofMSSAorMRSArangingfrom 1103to1104CFU/mlcompletelysubmergedthe testsamplesinthedishfor1hatroomtemperature (RT).Thebacterialsuspensionwasthenremovedand thedisheswererinsedwithsterile1PBS3times,for 10swhilerotatingat80rpm,toremovenon-attached cells.Afterdiscardingthefinalrinsate,surfaceswere driedunderambientconditionsfor1hthensampledfor viablebacteriausingRODACcontactplatesasdescribed below.TouchtransferenceinoculationassayMSSAandMRSAwereusedtoevaluatetheperformanceoftheMPinthisassay.Testandcontrolsurfaces werecutinto40mmradiussemi-circlesandplaced side-by-sideinasinglePetridish.Bacterialsuspensions (5ml)rangingfrom1105to1107CFU/mlwere usedtofloodsterilevelveteencloths(Bel-ArtProducts, Wayne,NJ)thatlinedthebottomofsterilePetridishes [42].Sterilevelveteenclothwasplacedonareplicaplatingtool(Bel-ArtProducts,Wayne,NJ)andinverted ontosaturatedvelveteen-containingbacterialinoculum for10sbeforebeingplacedontotestandcontrolsurfacesfora10scontacttime.Thetestsurfaceswerethen allowedtodryfor5 – 10sunderambientconditionsbeforebeingsampledusingRODACcontactplates,as describedbelow.CombinationtransferenceandpersistenceassaySuspensionsofMSSAandMRSAorganismsranging from1105to1107CFU/mlwereeachusedtochallengesmoothandtheMPacrylicfilms.Testandcontrol acrylicfilmwascutinto40mmradiussemi-circles andplacedside-by-sideinasinglePetridish.Touch transferenceinoculation(describedabove)wasusedto inoculatethechallengesurfaces.Reductionoftouch transferencewasmeasuredafter0mofdryingandreductionofpersistencewasmeasuredafter90mofdryingwasaddedincombinationwhileeachtimepointwas sampledusingRODACplates.Similarly,antimicrobial copperwassubjectedtochallengewithMSSAand MRSAusingthesamecombinationtransferenceand persistencetestinginhead-to-headcomparisonwiththe MPacrylicfilm.Samplingbacterialload RODACcontactagarFollowinginoculationandprocessingofeachsample surface,bacterialloadwasquantifiedusingRODACcontactagarplates.Ineachtest,perorganisminvestigated,Mann etal.AntimicrobialResistanceandInfectionControl 2014, 3 :28Page3of8 http://www.aricjournal.com/content/3/1/28

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theinoculationrangewasdeterminedexperimentallyas beingoptimalforyieldingacountablerangeofcolonies onRODACcontactplates(BBLPreparedRODACPlate, TrypticaseSoyAgarwithLecithinandPolysorbate80, BD,FranklinLakes,NJ),whichwereusedforcellrecoveryfromsamplesurfaces.Theagarcontactmethodwas usedtodirectlyquantifybacterialCFUtransferredfrom thesurfacetoa60mmdiameterRODACcontactplate forenumerationoftotalcolonycounts.TheRODAC contactplateswerepressedontoinoculatedsurfacesfor 5swhileavoidingairbubblesbetweenthesurfaceand RODACplate.TheRODACplateswerethenincubated for18 – 24hat37C.TheRODACplateswerephotographedandcountedusingmagnificationandImageJ colonycountingmethods.Theresultingcolonieswere enumerated,logtransformed,andrecordedaslogCFU/ RODAC.DisruptionanddilutionplatingSterilebiopsypunches(4mm;VWRInternational,Radnor,PA)wereusedtoobtainsamplesfrominoculated surfaces.Punchesweredroppedintoconicaltubes,each containing1mloffreshDey-Engley(DE)neutralization buffer(Sigma,St.Louis,MO).Thetubesweresonicated fortwominuteswith30svortexesbeforeandaftersonication[41].SerialdilutionoftheelutedbacteriainDE bufferwasthenplatedontoTSA,andtheplateswereincubatedfor18 – 24hat37C.Resultingcolonieswere enumerated,logtransformed,andrecordedaslogCFU/ ml.MicroscopyanalysisScanningelectronmicroscopywasusedtovisualizeMSSA duetoitsabilitytopotentiallyexistwithinthepatterned surface.Afterbacterialimmersion,twosamplesofeach theMPandtwosmoothsampleswereretainedforanalysiswithoutRODACexposure,whileanothertwosamplesofeachsurfacewerestampedwithaRODACplate. Eachsamplewassubsequentlyfixedwithosmiumtetroxidegas(ElectronMicroscopySciences,19150)for45m andthensubjectedtoadehydrationserieswith10mincubationsin25,50,75,andfinally95%ethanol(Decon LabsInc,KingofPrussia,PA),thenair-driedovernightat ambientconditions.Two8mmcirclesweretakennear thecenterofeachsamplewithabiopsypunch(Fisher, Waltham,MA),mounted,sputter-coatedwithgold,and analyzedviaSEM.Twoimagesweretakenpersmooth sampleandfourimagesweretakenpertheMPsamplefor qualitativeimageanalysis.DatareportingandstatisticalanalysisEachexperimentconsistedofatleastthreeexperimental replicatespersurfacetype(theMPandsmoothcontrol), generatingalogreductionvaluecalculatedfrompaired comparisonoftheMPandsmoothsurfacemeanlogcell densities.Eachsingleexperimentwasrepeatedatleast threetimestogenerateleastsquaresmeanlogreduction (LR)valuesandestablishstatisticalsignificance[43].The resultinglogreductionsweresubjectedtoasinglet-Test and,whenappropriate,anANOVAanalysiswitha Tukeytesttogeneratestatisticalsignificanceandgroupingacrosssamples,materials,andstraintypes.Smooth controlsurfacelogcelldensitieswerecomparedwhere appropriatetodetermineexperimentalvarianceforestablishingoptimumassayconditions.ResultsBacterialattachmenttosurfacesTheimmersionassaywithRODACrecoverywasusedto quantifybacterialattachmenttorepresentativeacrylic TheMPandsmoothsurfacesafterbeinginoculatedwith abacterialsuspension.MSSAandMRSAdemonstrated significantlyreducedattachmenttotheMPsurfaces comparedtosmoothcontrols,with99%(LR=2.18; p <0.001)and98%(LR=1.64; p <0.001)reductionsof eachoftheseorganisms,respectively(Figure2).BacterialpersistenceonsurfacesS.aureus (MSSA)wastestedforpersistenceontheMP withRODACrecoveryafterauniformsprayinoculation technique,mimickingacommonsurfacecontamination event.MSSAwasreducedby98%(LR=1.61; p <0.005) ontheMPcomparedtosmoothcontrols(Figure3A). TheMPreductionofMSSAcontaminationwasalso Figure2 Microbialattachment. MSSAandMRSAwereincubated insuspensiononsmooth(blackbars)ormicro-patterned(greybars) acrylicfilmfor1h.Afterrinsi ng3timesanddryingfor1hthe remainingviablebacteriaonthesurfaceswerequantified.Theplot representsaveragelogdensitiesandstandarderrorofthemean. Significancewasdeterminedusingasinglet-Testofthelog reductiondatapoints.Theaveragelogreductionvalueswerethen usedtocalculatethemedianpercentreductionvaluesindicated aboveeachcolumn. p< 0.005(***). Mann etal.AntimicrobialResistanceandInfectionControl 2014, 3 :28Page4of8 http://www.aricjournal.com/content/3/1/28

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visuallyapparentusingRODACrecovery(Figure3B). Sprayedsurfaceswerealsosampledusingdisruptionand dilutionplatingquantification.MSSAwassignificantlyreducedontheMPcomparedtosmoothsurface(Additional file1:TableS1)withthisrecoverymethod.Microscopy methodswereutilizedtoensurethattheRODACagar efficientlyremovedbacteriafromtheMPandsmooth surfaces.Scanningelectronmicroscopy(SEM)surface examinationindicatedthatbeforeRODACrecovery,the smoothsiliconesurfacehadextensivecontamination comparedtotheMPsurface(Additionalfile2:FigureS1A andB).AfterRODACrecovery,bothsurfaceswerewithoutvisiblecontamination(Additionalfile2:FigureS1C andD).Theseresultsareconsistentwithquantitativeresultsfromtheimmersionassay(Figure2).TransferandpersistenceofbacteriaonsurfacesBothMSSAandMRSAonasurfaceswereevaluated usingthecombinationtransferandpersistenceassaywith RODACrecoverymethods.TransferofMSSAontothe MPacrylicfilmwasreduced87%(LR=0.90; p <0.05) comparedtosmoothfilm(Figure4).MSSApersistenceon theMPwasfurtherreduced97%(LR=1.54; p <0.001) after90mofdrying(Figure4).ANOVAanalysisand TukeygroupingidentifiedthatreductionofMSSAafter 0m(transfer)groupedsignificantlydifferently( p <0.05) thanitsreductionsseenafter90mofdrying(survival). TheMPreducedMRSAcomparedtosmoothcontrolsby 91%(LR=1.04; p <0.005)after0mand94%(LR=1.26; p <0.005)after90mofdrying(Figure4). Antimicrobialcopper,whichismarketedforitsability toreduceenvironmentalcontamination[23,44],wasnot effectiveatreducingMSSAcontaminationcomparedto smoothacrylicfilmafter0mor90m.CopperdidreduceMRSAby80%(LR=0.70; p <0.002)after0mand 79%(LR=0.69; ns )after90mofdryingcomparedto smoothcontrols(Figure4).Importantly,theMPreductionsinMSSAcontaminationgroupedinstatistically higherlogreductiongroupsfromthatofantimicrobial copperusingTukeypost-testANOVAanalysis.These datademonstratedthattheMPwasmoreeffective thanantimicrobialcoppersurfacesinlimitingbacterialcontaminationtransferandsurvivalinthetouch transferassay. Figure3 Microbialpersistence. Smoothandmicro-patterned(MP) acrylicfilmswerechallengedwithasprayedinoculumanddriedfor 30m. A .)LogdensitiesofbacteriapresentonthesurfacesoftheMP comparedtosmoothcontrolsforMSSAareplottedwiththe associatedstandarderrorofthemean. B .)Arepresentativeimageof aRODACcontactplateafterMSSAsampling,theMPsurface(right) hasfewerbacteriacomparedtothesmoothsurface(left). p< 0.005 (***)n=3. Figure4 ComparisonofSharkletMPtoCopperantimicrobialsurface. MSSAandMRSAwereusedtochallengesmoothunpatternedfilm, theMPfilm,andcopperfoil(99.9%pure)usingatouchincidentwithtimepointssampledafter0and90mofdrying.Averagelogdensityvalues arepresentedforsmooth,theMP,andcoppersurfaces.ErrorbarsrepresenttheSEMfor3independentexperiments.Thepercentreduction valueswerecalculatedusingindividuallogreductionvaluescomparingeitherSharkletMPorcoppertosmoothcontrolsamples. p< 0.05(*), p< 0.01(**), p< 0.005(***). Mann etal.AntimicrobialResistanceandInfectionControl 2014, 3 :28Page5of8 http://www.aricjournal.com/content/3/1/28

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DiscussionTheMPconsistentlydemonstratedareductioninmicrobialattachment,transference,andsurvivalfollowing simulatedreal-worldinoculationmethods.WhiletheinitialphaseofthisstudyidentifiedtheMP ’ sabilitytolimit bacterialattachmentofMSSAandMRSA(Figure2), high-touchsurfacesintheenvironmentarecommonly contaminatedbytouchandsneeze-likeeventsandnot completeimmersion.Additionally,theMPhaspotential tolimittransferenceandpersistenceofbacteria,butthe immersionmethoddidnotallowevaluationofthose individualevents.Thereforemethodswerefurtherdevelopedtosimulatereal-worldinoculationevents.Spray inoculationallowedforuniformandreproducibleloadingofinoculumontosurfacestoevaluatebacterialpersistenceovertime(30 – 90m)andtouchtransference assayswereusedtomimicindirectbacterialspreadon high-touchsurfacesthroughtransferalonetherefore thoseassaysrequiredsamplingafter0m.Bacterialloads weresampledfromtheMPorsmoothsurfacesusing RODACcontactplates[5,45,46].TheRODACplates wereusedtoquantifyremainingbacterialloads,which provedtobeareproduciblemethodwhichisnot commonlyusedinstandardizedtestmethods[36,47,48]. ValidationofRODACsamplingefficacywasdonequalitativelyusingSEM(Additionalfile2:FigureS1)and quantitativelyusingpreviously-optimizedultra-sonication(Additionalfile1:TableS1)[34,41,49,50].These datasubstantiatetheuseofRODACcontactsamplingto testthebacterialloadpresentafterinoculationanddryingonboththeMPandsmoothsurfaces.Importantly, theMPdemonstratedreducedbacterialcontamination regardlessoftheinoculationandsamplingmethod. Interestingly,significantlysuperiorreductionsinbacterialloadofMSSAontheMPafterbothtransferand survivaltimepointscomparedtotransferalonewere identifiedusingTukeygroupinganalysis(Figure4; p <0.05).Thissuggeststhatindependentmechanisms arelimitingbacterialtransferaswellasbacterialsurvivalafterinteractionwiththeMPsurface.Thisreport isthefirsttodemonstratethatuseofamicrotopographycanresultinacceleratedlossofbacterialviabilitycomparedtoasmoothsurface(Additionalfile1: TableS1).Lossofbacterialviabilityfollowingreduced bacterialsurfaceinteractionisnotsurprisingsincemicrobialtransitiontoatolerantsessilephysiologyrelies heavilyonsurfaceadherence[51,52].TheMPwasengineeredandoptimizedtoachievespecificsurfaceenergies,whichreducebacterialinteractionandattachment comparedtoasmoothsurface[29,53].Therefore,the inabilityforbacteriatoefficientlyadheretotheMPis potentiallyresponsibleforthelossofbacterialviability inadditiontothelimitedinitialtransferofbacteriato theMP. Antimicrobialcopperhasbeenthemostpopularlyimplementedsurfacetechnologyabletodemonstrateareductioninbacterialcontaminationinbothlaboratory andclinicalenvironmentaltesting[23,44].Therefore,in thisstudy,antimicrobialcopperwascomparedtothe MP,andtheMPoutperformedantimicrobialcopperin reductionofbacterialtransferandsurvival.Copperwas ineffectiveinlimitingMSSA,whiletheMPreduced MSSAbyupto97%(p<0.05)whencomparedto smoothacryliccontrolsurfaces.Copperdemonstrated 80%(p<0.002)reductionofMRSAascomparedto94% (p<0.005)reductionwiththeMP(Figure4).ThefindingthattheMPwasmoreeffectiveatlimitingbacterial load90mafterinoculationisintriguing.WhiletheMP surfacelimitsinitialtransferduetosurfaceenergy changes[53,54]aswellasperceivedpersistenceoforganisms,thecoppersurfacesappeartoonlylimitpersistencebasedoncytotoxiceffectsoccurringafterlonger durations.ThefactthattheMPdoesnotrequirecytotoxiccompoundsorleachingchemicalstobeaneffectivealternativetotraditionalantimicrobialssuchas copperforlimitingbacterialcontaminationisadistinct advantage. Theimpactoftheseresultsishighlyrelevantgiventhe evidencelinkingsurfacecontaminationtonosocomial infections[5,11,55,56].Survivalof S.aureus (including MRSA)ondryinanimatesurfacescanrangefrom7days to7months[8].Theexistingandemergingsurfacedecontaminationandcleaningmethodologieswereclearly evaluatedinarecentreviewbyWeberandRutala[22]. Theydiscussedadvantagesanddisadvantagesofmany hygienepracticesandcontaminationresistantsurfaces includingtheMP.Unfortunately,theyfoundthatwhile educationandimprovedhygienepracticeswouldtheoreticallycontributetofewerHAIs,littlepositiveeffects havebeenobserved.Therefore,atechnologythatlimits contaminationregardlessofhumanerroriswarranted. Additionally,chemicalantimicrobialapplicationscan alsobeproblematictovulnerablepatientpopulations includingneonatesandyoungchildrenandareoften avoided.Wideimplementationofantimicrobialsurface technologieswithdirectkillmechanismsareconcerning duetotheirpotentialtoprovideselectivepressurefor resistantmicro-organisms.Heavymetalresistancehas alreadybeenidentifiedwithclinically-relevantbacterial speciesshowingresistancetosilver[24,57,58]andcopper[59].TheMSSAstraintestedinthisstudyexhibited tolerancetocoppersurfacesfor90mbutnottotheMP. Consideringthatantimicrobialcoppersurfaceshave beenshowntoreduceHAIratescombinedwithMRSA orVREcolonizationwhenimplementedinICUrooms [23]andtheMPoutperformedcopperwhentesting transferandsurvivalofMSSAandMRSA invitro ,this studysuggeststhattheMPmayhelpreduceinfectionMann etal.AntimicrobialResistanceandInfectionControl 2014, 3 :28Page6of8 http://www.aricjournal.com/content/3/1/28

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ratesandimprovepatientcare.Continuedtestingofthe MPsurfacesinclinicalsettingsshouldprovidefurther evidenceofthenatureandmagnitudeofthebenefitsto patients.ConclusionTheMPsurfaceisaneffectiveandattractivemethodto broadlyreducemicrobialcontaminationonsurfaceswithouttheuseofantimicrobialagents.Thestudiespresented hereclearlydemonstratethattheMPreducesmicrobial transferandwhencomparedtothesamematerialwithout theMPpresent.Whenadoptedintoreal-worlduse,applicationoftheMPontohigh-touchsurfacesinhospitalsor sharedpublicspacesisexpectedtolimitenvironmental contaminationofinfectiousmicroorganisms.Giventhat preliminaryclinicalevidenceexiststhatantimicrobialcopperimplementationinhospitalroomsdecreasesHAIrate, similarimplementationoftheMP,whichoutperformed copperinthetransferandpersistence invitro study,has potentialtoreducetheincidenceofHAIs.AdditionalfilesAdditionalfile1:TableS1. Quantificationofbacterialpersistence usingdilutionplating.MSSAwasaerosolizedontosmoothortheMP acrylicfilmandallowedtodryfor90m.8mmbiopsypuncheswere usedtocutfilmsamplestosuspendbacteriaanddilutionplate.Smooth andMPassociatedlogdensitieswithresultinglogreductionsare presentedalongwiththe p valueusingasinglepairedt-Test. Additionalfile2:FigureS1. MSSAcontaminationpersistencerecovery. 1107CFU/mLwaspreparedtoimmersesmoothandtheMPsurfaces. SamplesimmersedinasuspensionofMSSAwererinse3times,sampled, andthenpreparedforSEMimaging.SmoothsurfacebeforeRODAC (A) andSharkletMPbeforeRODACsampling (B) arepicturedadjacentto imagesafterRODACsampling (CandD) Competinginterests Allauthors,exceptABB,completedworkwhilebeingemployedbySharklet Technologies,Inc.ABBisapaidconsultantofSharkletTechnologies,Inc. Authors ’ contributions Allauthorshadsignificantcontributionstothesciencediscussed.EEM,DM, RMMettetal,RMMay,EMD,KKC,ABB,andSTRallcontributedtothedesign andobjectivesofexperimentalanalysis.EEM,DM,MRMettetal,andRMMay combinedtocarryouttesting.Allauthorsreadandapprovedthefinal manuscript. Acknowledgements TheauthorswouldliketorecognizethesupportofMelindaSogo,Trevor Hostetter,MichaelDrinker,BristiBasu,andCarolineFloresfortheirsupportin completingexperimenttasksandlaboratorysupport.Additionally,test materialdevelopmentandinnovationsupportprovidedbyMarkSpiecker, MiKaylaHenry,KelleyDoebler,andBryceStevenson.Testmaterialalso providedbyRyanStonebergandGurpreetChhiberat10Microstructures andBillSullivanandDaveConstantineatFLEXcon. Authordetails1SharkletTechnologies,Inc,12635E.MontviewBlvd,Suite160,Aurora, CO80045,USA.2DepartmentofMaterialsScienceandEngineering, UniversityofFlorida,Gainesville,FL,USA. 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ApplEnvironMicrobiol 2010, 76: 1341 – 1348.doi:10.1186/2047-2994-3-28 Citethisarticleas: Mann etal. : Surfacemicropatternlimitsbacterial contamination. AntimicrobialResistanceandInfectionControl 2014 3 :28.Mann etal.AntimicrobialResistanceandInfectionControl 2014, 3 :28Page8of8 http://www.aricjournal.com/content/3/1/28