Micro-patterned surfaces reduce bacterial colonization and biofilm formation in vitro: Potential for enhancing endotrach...

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Title:
Micro-patterned surfaces reduce bacterial colonization and biofilm formation in vitro: Potential for enhancing endotracheal tube designs
Physical Description:
Mixed Material
Language:
English
Creator:
May, Rhea M.
Hoffman, Matthew G.
Sogo, Melinda J.
Parker, Albert E.
O'Toole, George A.
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Springer (Clinical and Translational Medicine)
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Abstract:
Background: Ventilator-associated pneumonia (VAP) is a leading hospital acquired infection in intensive care units despite improved patient care practices and advancements in endotracheal tube (ETT) designs. The ETT provides a conduit for bacterial access to the lower respiratory tract and a substratum for biofilm formation, both of which lead to VAP. A novel microscopic ordered surface topography, the Sharklet micro-pattern, has been shown to decrease surface attachment of numerous microorganisms, and may provide an alternative strategy for VAP prevention if included on the surface of an ETT. To evaluate the feasibility of this micro-pattern for this application, the microbial range of performance was investigated in addition to biofilm studies with and without a mucin-rich medium to simulate the tracheal environment in vitro. Methods: The top five pathogens associated with ETT-related pneumonia, Methicillin-Resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Klebsiella pneumonia, Acinetobacter baumannii, and Escherichia coli, were evaluated for attachment to micro-patterned and un-patterned silicone surfaces in a short-term colonization assay. Two key pathogens, MRSA and Pseudomonas aeruginosa, were evaluated for biofilm formation in a nutrient rich broth for four days and minimal media for 24 hours, respectively, on each surface type. P. aeruginosa was further evaluated for biofilm formation on each surface type in a mucin-modified medium mimicking tracheal mucosal secretions. Results are reported as percent reductions and significance is based on t-tests and ANOVA models of log reductions. All experiments were replicated at least three times. Results: Micro-patterned surfaces demonstrated reductions in microbial colonization for a broad range of species, with up to 99.9% (p < 0.05) reduction compared to un-patterned controls. Biofilm formation was also reduced, with 67% (p = 0.12) and 52% (p = 0.05) reductions in MRSA and P. aeruginosa biofilm formation, respectively. Further, a 58% (p < 0.01) reduction was demonstrated on micro-patterned surfaces for P. aeruginosa biofilms under clinicallysimulated conditions when compared to un-patterned controls. Conclusions: This engineered micro-pattern reduces the colonization and biofilm formation of key VAP-associated pathogens in vitro. Future application of this micro-pattern on endotracheal tubes may prevent or prolong the onset of VAP without the need for antimicrobial agents. Keywords: Micro-pattern; Sharklet; VAP; Endotracheal tube; Biofilm inhibition
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May et al. Clinical and Translational Medicine 2014, 3:8 http://www.clintransmed.com/content/3/1/8
General Note:
doi:10.1186/2001-1326-3-8 Cite this article as: May et al.: Micro-patterned surfaces reduce bacterial colonization and biofilm formation in vitro: Potential for enhancing endotracheal tube designs. Clinical and Translational Medicine 2014 3:8.

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RESEARCHOpenAccessMicro-patternedsurfacesreducebacterial colonizationandbiofilmformation invitro : Potentialforenhancingendotrachealtube designsRheaMMay1,MatthewGHoffman1,MelindaJSogo1,AlbertEParker2,GeorgeAO ’ Toole3, AnthonyBBrennan4andShravanthiTReddy1*AbstractBackground: Ventilator-associatedpneumonia(VAP)isaleadinghospitalacquiredinfectioninintensivecareunits despiteimprovedpatientcarepracticesandadvancementsinendotrachealtube(ETT)designs.TheETTprovidesa conduitforbacterialaccesstothelowerrespiratorytractandasubstratumforbiofilmformation,bothofwhich leadtoVAP.Anovelmicroscopicorderedsurfacetopography,theSharkletmicro-pattern,hasbeenshownto decreasesurfaceattachmentofnumerousmicroorganisms,andmayprovideanalternativestrategyforVAP preventionifincludedonthesurfaceofanETT.Toevaluatethefeasibilityofthismicro-patternforthisapplication, themicrobialrangeofperformancewasinvestigatedinadditiontobiofilmstudieswithandwithoutamucin-rich mediumtosimulatethetrachealenvironment invitro Methods: ThetopfivepathogensassociatedwithETT-relatedpneumonia,Methicillin-Resistant Staphylococcus aureus (MRSA) ,Pseudomonasaeruginosa Klebsiellapneumonia,Acinetobacterbaumannii, and Escherichiacoli, were evaluatedforattachmenttomicro-patternedandun-patternedsiliconesurfacesinashort-termcolonizationassay. Twokeypathogens,MRSAand Pseudomonasaeruginosa, wereevaluatedforbiofilmformationinanutrientrich brothforfourdaysandminimalmediafor24hours,respectively,oneachsurfacetype. P.aeruginosa wasfurther evaluatedforbiofilmformationoneachsurfacetypeinamucin-modifiedmediummimickingtrachealmucosal secretions.Resultsarereportedaspercentreductionsandsignificanceisbasedon t -testsandANOVAmodelsoflog reductions.Allexperimentswerereplicatedatleastthreetimes. Results: Micro-patternedsurfacesdemonstratedreductionsinmicrobialcolonizationforabroadrangeofspecies, withupto99.9%( p <0.05)reductioncomparedtoun-patternedcontrols.Biofilmformationwasalsoreduced,with 67%( p =0.12)and52%( p =0.05)reductionsinMRSAand P.aeruginosa biofilmformation,respectively.Further,a 58%(p<0.01)reductionwasdemonstratedonmicro-patternedsurfacesfor P.aeruginosa biofilmsunderclinicallysimulatedconditionswhencomparedtoun-patternedcontrols. Conclusions: Thisengineeredmicro-patternreducesthecolonizationandbiofilmformationofkeyVAP-associated pathogens invitro .Futureapplicationofthismicro-patternonendotrachealtubesmaypreventorprolongthe onsetofVAPwithouttheneedforantimicrobialagents. Keywords: Micro-pattern;Sharklet;VAP;Endotrachealtube;Biofilminhibition *Correspondence: sreddy@sharklet.com1SharkletTechnologies,Inc.,Aurora,12635E.MontviewBlvd.Suite155, CO80045Aurora,CO,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Mayetal.;licenseeSpringer.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproduction inanymedium,providedtheoriginalworkisproperlycredited.May etal.ClinicalandTranslationalMedicine 2014, 3 :8 http://www.clintransmed.com/content/3/1/8

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BackgroundPneumoniaisthemostcommonhospital-acquiredinfection(HAI)inIntensiveCareUnits(ICUs) — comprising some27%oftheseinfectionsanddrivingmorethanhalfof theantibioticsprescription sintheICU[1,2].Ventilatorassociatedpneumonia(VAP)isaleadingHAIinICUs,accountingfor86%ofnosocomi alpneumoniacases[2,3]. TheannualU.S.hospitalcostofVAP-relatedtreatment isabout$1.5billionanddeathratesarebetween2560%[4,5].Approximately80%ofallVAPcasesare causedbytheESKAPEpathogens( Enterococcusfaecium, Staphylococcusaureus,Klebsiellapneumoniae,Acinetobacterbaumannii,Pseudomonasaeruginosa ,and Enterobacter species),whicharelargelyresponsibleforHAIs andaretheprimaryorganismsdemonstratingantimicrobialresistance,therebyescapingtheeffectsofantibacterial drugs[6,7].Currentepidemiologyshowsthat S.aureus,P. aeruginosa,K.pneumoniae,E.coli ,and A.baumannii cause28%,22%,10%,7%and7%,respectively,offirst episodeVAPandHAI-associatedpneumoniainfectionsworld-widemakingthemthetopfivecausative agentsoftheseinfections[ 7].Methicillin-resistant S.aureus (MRSA)and P.aeruginosa comprisethetoptwocausative organismsofVAPandareconsideredparticularlydevastatinglungpathogensastheycau sepersistentpneumoniainfections,areresistanttoanu mberofantimicrobials,and areassociatedwithahighattributablemortalityofpatients withVAP[7]. VAPisattributedtotheendotrachealtube(ETT)used duringventilation[1,2,5,8].Intubationdoesnotallow coughreflexesthatclearorganismsdescendingfromthe proximaloropharynxtothedistalbronchi[5],therefore thetubeprovidesbacteriaaccesstothelowerrespiratory tractviacontaminatedsecretions[9].Also,thesurfaces ofanETTprovidebacteriawithasubstratumthatpromotesmicrobialcolonizationandbiofilmformation [10-13].PathogensisolatedfromETTbiofilmshavebeen showntobethesamecausativeorganismsresponsible forthepatient ’ sVAP,whichassociatesthebiofilmpresenceontheETTwithinfection[14,15].Ithasbeen demonstratedthatthesebiofilmscandeveloponthe innerandoutersurfacesoftheETTinlessthan24hours ofpatientintubation[16],andithasbeendetermined thatmaturebiofilmpresenceratherthandurationofintubationisdirectlyrelatedtooccurrenceofVAP[17]. TreatmentsforVAPuniversallyincludetheuseofantimicrobialstocleartheinfection.PrescribingtheappropriateantimicrobialsattheonsetofVAPisincreasingly difficultbutextremelyimportant,asaninadequateinitial antimicrobialtherapyisassociatedwithincreasedmortality[2,18].Numerousfactorscontributetotreatment decisions( e.g. clinicalpresentation,localdrugresistance patterns,patientexposure)andthiscomplexityprevents thestandardizationofaVAPantimicrobialtreatment guideline[19,20].Broad-spectrumantimicrobialsare ofteninitiallyimplementedtoreducetheriskofpatient mortality.Unfortunately,thispathcontributestoincreasedcostandcomplicationsforthepatientaswellas heightenedriskforantimicrobialresistance[2,21].Itis alsowellknownthatthepresenceofantibioticsinduces robustbiofilmformationofarangeofpathogens,which mayfurthercontributetotheonsetofVAP[22-24]. CommonICUandVAPpathogendrugresistanceissteadilyrisingandoutpacingnewdrugdevelopment,leaving healthcareprofessionalsdefenselessagainstthesecommon infections. Topreventtheoveruseofanti-microbialtreatments, devicemodificationsandimprovedpatientcarepractices havebeenimplementedtoreducemicrobialaccesstothe lowerrespiratorytract.TheadditionofasubglottalsecretionportaswellasreducingthethicknessoftheETTcuff preventsmicroaspirationofsubglottalsecretionsintothe lungs.Thesedevicemodifications,inadditiontoelevation ofthepatient ’ shead,dailysedationinterruptions,andoral antiseptictreatments,haveshownsomebenefit[5],but therateofVAPremainssubstantial.Whilethereissome clinicalevidencethatsilver-coatedtubesreduceinfection rates[25,26],thecostremainsprohibitivelyhighforwidespreadadoption.Anovelsolutionthatwouldcontinueto limitbacterialpresenceinthelowerrespiratorytract, therebyimprovingpatientoutcomes,withoutpromoting antimicrobialresistanceiswarranted. Engineeredsurfacetopographies,particularlygeometries oforderedfeaturesdesignedwithuniqueroughnessproperties,elicitspecific,predictablebiologicalresponsesand havebeenshowntocontrolbio-adhesion[27-29].Studies haveshownthattheSharkletmicro-pattern(Figure1)is themosteffectiveamongorderedtopographies(pillars, channels,othergeometries)forinhibitingbio-adhesion [30].Thissharkskin-inspiredmicro-topographymay Figure1 ScanningelectronmicrographoftheSharklet micro-patterncastintosilicone. Thisisthemicro-patternusedin thisstudy(Scalebar=20 m). May etal.ClinicalandTranslationalMedicine 2014, 3 :8 Page2of9 http://www.clintransmed.com/content/3/1/8

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provideanalternativestrategyforVAPpreventionasit hasbeenshowntoinhibit E.coli and S.aureus biofilmformation invitro withouttheuseofantimicrobialagents [31,32].Theaimofthepresentstudywastodetermine theeffectivenessofthisorderedmicro-pattern,compared toun-patternedcontrolsurfaces,incombatingthetopfive VAPcausativepathogensbyassessingmicrobialcolonizationandbiofilmformationonbothsurfacetypesin simplifiedandclinicallysimulated invitro conditions.MethodsSamplefabricationMicro-patterned-surfacesamplesandun-patterned(control)surfacesampleswerecreatedasdescribedpreviously [31].Briefly,siliconwafermoldswerecreatedbytransferringtheSharkletpatterndesigntophotoresist-coatedsiliconwafersthroughphotolithographyandthefeatures weredeepreactiveionetchedtoadepthof3 mbefore beingcleanedandmethylatedpriortouse.Smooth,unetchedsiliconwaferswereusedtocreatetheun-patterned controls.DowCorningSilasticT-2poly-(dimethylsiloxane)elastomerfilmswerecastoffthesiliconwafermolds bymixingonepartbyweightcuringagentwithtenparts byweightresin,degassingundervacuum,pressingto 0.4mmthickwithglassplatesoverthesiliconwafer molds,andcuringfor1hourat65C.Eachflat0.4mm thicksiliconefilmwasthenpunchedinto12mmdiameter circularsamplesandtestedasdescribedbelow.Bacterialstrains,media,andgrowthconditionsAllclinicalisolatesanalyzedinthisstudywerepurchasedfromATCC(Table1).Thesestrainsinclude clinicallyisolatedstrainsofMRSA, P.aeruginosa K. pneumoniae A.baumannii and E.coli .Twoadditional strainsof P.aeruginosa werealsoassessedtoconfirm thevalidityoftheresultsacrossmultiplestrainswithina species:asecondclinicalisolateandalaboratorystrain. Thelaboratory P.aeruginosa strainPA14,carryinga bifA mutation wasincludedinthesestudiesasacomparative straincontrolduetoitsgeneticmodificationtooverproduceexopolysaccharideandthusformbiofilmsquickly andconsistently[33].Asinglecolonyofeachorganism, platedontrypticsoyagar(TSA;Criterion),wasusedtoinoculatetrypticsoybroth(TSB;Criterion)andgrownina shakingincubatorat37Cand280rpmovernight.ColonizationassayforthetopfiveVAP-associated pathogensMicro-patternedandun-patternedcontrolsampleswere suctionedtothebottomofasterilePetridisharound theouterperimeterofthedishusingethanol.Overnight microbialculturesweresub-culturedandgrowntoearly logphaseinTSBbeforecentrifugingaliquotsandresuspendingcellpelletswith1xphosphatebufferedsaline (PBS;CulGeneX).Sampleswereimmersedininoculum containing~107CFU/mLforincubationtimeof1to 4hoursstaticallyatroomtemperaturedependingonthe propensityofthespeciestocolonizethesurface;thecell densityonun-patternedcontrolswastargetedtoremain withinanaverageof2.5to6logs(Table1).Afterincubation,theinoculumsuspensionwasdecantedandsampleswererinsedthreetimesbyaddingsterile1xPBSto thedish,rotatingfor10secondsonanorbitalshakerset to80rpm,anddecantingtherinsate.Theouteredgeof eachsamplewasremovedtominimizethevariability causedbytheun-patternedsidewallsbyusingasterile 8mmbiopsypunch(VWRInternational);the8mm sampleswerethenasepticallyloadedintoa15mLconicaltubecontaining2mLsterileDey-Engleybroth(Sigma Aldrich).Attachedcellswer eremovedanddisaggregated fromeachsamplesurfacebyvortexingfor30seconds,sonicatingfor2minutesinabat hsonicator(Branson3510) andvortexinganadditional30seconds[34].Sampleswere enumeratedbya10-folddiluti onseries,platingeachdilutionontoTSA,andcountingthecolonyformingunits (CFU)aftersufficientgrowt hat37Covernight.EachCFU persampledatapointwaslog10transformedbeforestatisticalanalysis.Eachexperimentwasreplicatedatleastthree Table1StrainsandcolonizationmethodologySpeciesStrain Designation Inoculum concentration (CFU/mL) Inoculation duration (hours) Numberof replicatesper experiment Numberof experiments completed Colonizationon un-patterned controls (Averagelog10(CFU)across experiments) Colonizationon Micro-patterned samples (Averagelog10(CFU)across experiments) Repeatability ofcontrol colonization (repeatability standard deviation) MRSAATCC7006985E+0713or435.833.720.05 P.aeruginosa ATCC90275E+064432.781.220.53 P.aeruginosa ATCC101975E+064443.622.261.94 P.aeruginosa PA14 bifA1E+074433.961.720.44 E.coli ATCC7003365E+074483.511.210.57 K.pneumoniae ATCC277995E+074444.932.011.57 A.baumannii ATCC196061E+072432.740.880.32 May etal.ClinicalandTranslationalMedicine 2014, 3 :8 Page3of9 http://www.clintransmed.com/content/3/1/8

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timesforeachorganism;theexactnumberofexperiments conductedforeachorganismisshowninTable1.Allexperiments,exceptoneMRSAexperiment,containedfour samplespersurfacetype;thatMRSAexperimentcontained threesamplespersurfacetype(Table1).Statisticalanalysis tookplaceacrossthethreeormoreexperimentsforeach organismasdescribedbelow.BiofilmassaysMicro-patternedandun-patternedcontrolsampleswere suctionedtothebottomofasterilePetridisharound theouterperimeterofthedishusingethanol.ForMRSA biofilms:OvernightculturesofMRSA(ATCC700698) weredilutedintofreshTSBtoafinalbacterialconcentrationof106CFU/mLinTSB.Sampleswereimmersed with20mLofthebacterialsuspensionstaticallyat37C forfourdays,withgentlemediareplenishingeachday fromthecenterofthedish.For P.aeruginosa biofilms: Overnightculturesof P.aeruginosa ATCC9027and P. aeruginosa PA14 bifA weredilutedintominimalM63 mediasupplementedwith0.4%arginine(22mMKH2PO4, 40mMK2HPO4,15mM(NH4)2SO4,1mMMgSO4),or minimalM63mediasupplementedwith0.4%arginine, 2mg/mLporcinemucinthatcontainstheMucA/Bcomplexfoundinmucus(SigmaAldrich),and400 g/mLoxacillin(SigmaAldrich),toafinalbacterialconcentrationof 106CFU/mL[33,35-37].Sampleswereimmersedwith 20mLofthebacterialsuspensionstaticallyat37Cfor 24hours.Alldishesweregentlyrinsedtentimesthrough mediaexchangebyremoving10mLmediafromthecenterofthedishandadding10mLsterile1xPBS.Samples werefixedfor30minutesin2.5%gluteraldehyde(Electron MicroscopySciences)anddehydratedwithanethanoldehydrationexchangeseries(finalethanolconcentrationsof 25,50,75,and95%).Onetothreesampleswerestained withpropidiumiodideandimagestackswereobtainedin twotofivepre-selectedsitespersamplebyconfocallaser scanningmicroscopy(ZeissLSM510METAonAxiovert 200M).Semi-volumetricanalysiswasachievedbytotaling biofilmareacoverageinthex-yplaneforeachimage throughthez-stackusingImageJsoftwareafterthresholdingtheimageatthebrightestframeinthestack[38].Each biofilmareacoverage/imagedatapointwaslog10transformedbeforestatisticalan alysis.Allbiofilmexperimentswerecompletedintriplicate.StatisticalmethodsAlogreduction(LR)perexperimentwascalculatedby subtractingtheaveragelog10(micro-patterndatapoints) fromtheaveragelog10(un-patternedcontroldatapoints). Afterconfirmingthenormalityofthelogreductionsby residualandnormalprobabilityplots,themeanlogreductionwasinterpretedasthemedianpercentreduction withtheequation:1-10( Š LR).Statisticalsignificanceof thereductionsforthecolonizationandbiofilmassays wereassessedusinga1-sided t -testoflogreductions fromatleastthreeexperimentsperorganism.Estimates oftheamong-andwithin-experimentvarianceswere assessedusingANOVAofthelogtransformedcelldensitiesforeachun-patternedcontrolsampleandmicropatternedsample,witharandomeffectforexperiment. Therepeatabilitystandarddeviation(SD)oftheunpatternedsample,whichquantifiestherepeatabilityof themethodsbasedoncontrolvariances,isthesquare rootofthesumoftheamong-experimentvarianceand thewithin-experimentvariancedividedbythenumber ofreplicatesperexperiment[39].AllanalyseswereperformedusingthestatisticalsoftwareMiniTab16.ResultsColonizationassayforthetopfiveVAP-associated pathogensBacterialcolonizationonmicro-patternedandun-patterned surfaceswereevaluatedforthetopfiverespiratorypathogensassociatedwithVAP,includingclinicallyisolated strainsofMRSA, P.aeruginosa K.pneumoniae A. baumannii and E.coli .Twoadditionalstrainsof P. aeruginosa werealsoevaluatedtoconfirmthevalidityof theresultsacrossmultiplestrainswithinaspecies:a secondclinicalisolateandalaboratory,hyper-biofilm formingstrain.Themicro-patternedsurfacesignificantlyreducedthecolonizatio nofallspeciesandstrains comparedtoun-patternedcontrols,withmedianreductionsinCFUcountsrangingfrom95.6%to99.9%, p <0.05 whenevaluatedacrossmultipleexperiments(Figure2).To confirmtheconsistencyofthemethodsusedtoobtain theseresults,therepeatabilitySDofthelog10(CFU/control sample)wascalculatedacrossexperimentsforeachorganism(Table1).Exceptfor P.aeruginosa ATCC10197 and K.pneumoniae ,allun-patternedrepeatabilitySDs wereclosetoorbelow0.50log,ahistoricalbenchmark thatindicateshighlyreproduciblecolonizationoncontrolsacrossmultipleexperiments[40].Evenwiththeincreasedvariabilityobservedfor P.aeruginosa ATCC10197 and K.pneumoniae, themicro-patternedsurfacesmaintainedstrongstatisticallysignificantperformanceinthis assaytherebyindicatinggoodrepeatabilityofthelogreductionsforalloftheorganismstested(Figure2).BiofilmformationofMRSAand P.aeruginosa onthe micro-patternBiofilmsweregrownonmicro-patternedandunpatternedsurfacesforMRSAandtwostrainsof P.aeruginosa followedbyconfocalimagingandImageJanalysis toquantifythebacterialareacoverageforeachimagein thez-stacks.Themicro-patternedsurfaceshoweda67% (p=0.123)medianreductionofMRSAbiofilmvolumes grownoverafourdaydurationinaTSBmediawhenMay etal.ClinicalandTranslationalMedicine 2014, 3 :8 Page4of9 http://www.clintransmed.com/content/3/1/8

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comparedtoun-patternedcontrolsurfaces(Figure3). Similarobservationswereseenforthehyper-biofilm formingstrain P.aeruginosa PA14 bifA ,whereasignificant52%medianreductioninvolume(p=0.05)was observed(Figure3).Importantly,maturebiofilms[17] wereobtainedontheun-patternedcontrolsurfacesfor boththeMRSAand P.aeruginosa PA14 bifA strains, whereasbacterialcolonizationonthemicro-patterned surfaceswasmoredispersed(Figure3).Whenquantifying theresemblanceofthebiofilmvolumemeasurements onun-patternedcontrols,therepeatabilitySDsofthe log-transformedbiofilmcoveragedatawas0.081forthe PA14 bifA experimentsand0.681fortheMRSAexperiments.Whilethelevelofacceptablevariabilityofimage analysisdatahasnotbeenestablished,thesmallSDsfor PA14 bifA indicateshighconsistencyinbiofilmformationontheun-patternedcontrols.The P.aeruginosa ATCC9027straindidnotformasufficientquantityof biofilmunderthesenon-clinicalconditionstoaccurately quantifybiofilmvolumecoverage,asmostimagecoordinateshadnodepthfromwhichtoobtainimagestacks (Figure3).Biofilmreductionof P.aeruginosa inamucin-modified mediumThe P.aeruginosa clinicalisolateATCC9027didnotform biofilmsinthesimplifiedminimalmedium(Figure3),so additionalstudiesonthisstrainwereconductedina mediumthatwouldbothencouragebiofilmformation andsimulatetheclinicalenvironmentofthelungs.Thus, amucin-richminimalmediumsupplementedwithoxacillinwasusedtopromotebiofilmandprovidethemucin glycoproteinsthatmakeupthemaincomponentofthe mucuspresentinthelungs[22-24,35,36].Thismedium Figure2 Themicro-patternedsurfacesignificantlyreducesthe colonizationofthefivemicrobialspeciesmostfrequently associatedwithVAPwhencomparedtoun-patternedcontrols. Allmeanlogreductions(acrossatleastthreeexperiments)are statisticallysignificantlypositivebysingle t -test*p<0.05;**p 0.01. Errorbarsarethestandarderrorofthemeanlogreduction. Figure3 Themicro-patternreducesbiofilmformationoftwokeyVAP-relatedpathogens,MRSAand P.aeruginosa whencomparedto un-patternedcontrols. The P.aeruginosa (ATCC9027)straindidnotformrobustbiofilmsundertheseconditions,howevertheMRSAandthe P.aeruginosa PA14 bifAstrainsshowed67%( p =0.123)and52%( p =0.05)medianreductionsinbiofilmcomparedtocontrols,respectively. Arepresentativeimagepersurfacetypeforeachorganismwasselectedtoreflectquantitativeresults.Imagesobtainedbycompilingthestackof imagestakenthroughthebiofilm(Scalebar=20 m). May etal.ClinicalandTranslationalMedicine 2014, 3 :8 Page5of9 http://www.clintransmed.com/content/3/1/8

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changeresultedinenhancedbiofilmformationofthisclinicalisolateontheun-patternedcontrolsurfaces,whereas themicro-patternedsurfacesmaintainedaninhibitory effect,witha58%reductioncomparedtocontrols(p= 0.009)(Figure4).Therepeat abilitySDforbiofilmson controlsacrossthreeexperimentswaslowat0.11,suggestingthatthemethodologyusedintheseassaysobtainsrepeatablebiofilmgrowthandcoverage.DiscussionInnovativesolutionsforVAP-preventionviaETTdevice modificationswillbecriticalforimprovedpatientcare, asVAPaccountsfor86%ofhospital-acquiredpneumoniacasesinICUs[2,3].Recentstudieshaveshownthat biofilmdevelopmentonthesurfacesofanETTisa strongpredictorofVAP,withbiofilmsforminginaslittleas24hours.Currentdevicemodificationshavehad somesuccessinreducinginfectionrates,particularly cuffmodificationsthatlimitmicrobialaccesstothe lowerrespiratorytract.However,furtherlimitingmicrobialaccesstothelowerrespiratorytractiscrucialforreducingVAPratesinpatients.Thegoalofthisstudywas todeterminethefeasibilityofusinganovelmicropatternedsurfacetodecreasemicrobialcolonizationfor thetopfiveVAPpathogens,andbiofilmformationfor thetoptwo,undersimulatedETTenvironments. Itisknownthatsurfacecharacteristicsmodulatemicrobialinteractions – howeverthisisthefirststudythat hasshownperformanceofaspecific,orderedmicropatternagainstthecolonizationofthetopfiveorganisms thatareassociatedwithVAPandotherHAIs.Currentliteratureshowsthatthismicro-patternreduces S.aureus and E.coli biofilms[31,32],however P.aeruginosa ,anothercommonbiofilmformer,hadnotbeentested.This studyevaluatedthetoptwocausativeagentsofVAPin biofilmgrowthassaysandshowedareductionofboth MRSAand P.aeruginosa maturebiofilmvolumecoverageonmicro-patternedsurfaceswhencomparedtounpatternedcontrols.Beforethisstudy,itwasunknown whetherbiologicallyrelevantproteins,likeMucA/Bthat makeupthemucusfoundinthelungs[36],wouldcoat themicro-patternandinterferewithitscapabilitytoreducemicrobialcolonization.Therefore, P.aeruginosa was furtherchallengedinalungsecretion-likemucinmedium supplementedwithantibiotics,andthemicro-pattern demonstratedstatisticallysignificantbiofilmreduction evenunderthesemorechallengingconditions.Together, thesedatacontributetoourunderstandingoftherange ofbacterialinhibitionexhibitedbythisnovelmicropatternagainstseveralclinicallyrelevantpathogensand conditions. Asinvestigatedelsewhere,thetheoreticalbasisforthe topography ’ sfunctionisitsabilitytocreateastablenonwettingstatewhereairpocketsatkeypointsinthe microscopicfeaturesaremaintained,therebyinhibiting interactionofmicrobeswiththesurface[41].Specifically, potentialattachmentsitesthatwouldgenerallyinduce stableinteractionsofmicro-organismstothesurfaceare Figure4 Themicro-patternsignificantlyreduces P.aeruginosa (ATCC9027)biofilmformationinamucin-richenvironmentbyan averageof58%( p =0.009)overthreeseparateexperiments. Arepresentativeimagepersurfacetypefromeachexperimentwasselected randomlyasallimagesweresimilarwithineachexperiment.Imagesobtainedbycompilingthestackofimagestakenthroughthebiofilm (Scalebar=20 m). May etal.ClinicalandTranslationalMedicine 2014, 3 :8 Page6of9 http://www.clintransmed.com/content/3/1/8

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madeinaccessibletotheorganismsduetothenonwettingnatureofthetopography.Thisnon-wettingpropertyisenhancedbytheuseofspecificnanoforcegradients thatoccurbasedonthesizesofthemicroscopicfeatures, whichcreateanenergeticallyunstablesurfacetowhich themicro-organismsareunabletoformulatestableattachmentmodes[42]. Aspecificgrowthmediaformulationandantibiotics wereusedinthisstudytoachieveclinicallysimulated biofilmgrowth invitro. Robustandaccelerated P. aeruginosa biofilmgrowthwasobtainedbyusingan arginineminimalmediatoen couragemicrobialrespirationinthisbiofilmmodel[43],andtoinduceasessile lifestylethatpromotesbiofilmformation[44].Thenutrientrichmucinproteincomplex,inadditiontoantibiotics,havebeenshowntoenhancebiofilmformation [22,35,36]andbothwereincludedinthemediumtoinducerobustbiofilmformationandprovideasimulated clinicalenvironment[19].Thecombinedresultwasacceleratedbiofilmformationoncontrolsurfaceswithin 24hoursfor P.aeruginosa – agoalcommonlymetby otherbiofilmreactorsystems[45,46]. Thebiofilmspresentonun-patternedcontrolsurfaces inthisstudywerethree-dimensionalandmadeupof largecellularaggregates,whichwereincontrasttothe singlecellorsmallaggregatesobservedonthemicropatternedsurface.Giventhisobservation,implementation ofthemicro-patternontothesurfaceofanETTcould provideasynergisticeffecttogetherwiththehumanimmunesystemandantibiotictreatmentstoclearpotentially harmfulbacteriabeforetheyformabiofilmandcausean infection.Shouldsucharesultbedemonstratedinaclinicaltrial,itwouldultimatelyleadtodecreaseduseof antimicrobialtreatmentsandsilver-coatedETTs,thatmay contributetotheriseinantimicrobialresistanceobserved intheESKAPEpathogens. Futureworkwillincludedemonstratingtheefficacyof thismicro-patterninadvancedmulti-species invitro biofilmmodelswithair-flowandanimalVAPmodels[47-49] utilizingETTprototypeswiththemicro-patternonthe innerandoutertubesurfaces.Thesecomplexmodels wouldfurtherourunderstandingoftissuecompatibility andperformanceofthepatterninclinicallyrelevant environments,beforecondu ctingclinicaltrialsthat wouldevaluatepatientdrivenoutcomes.Sinceventilatorassociatedcomplications(VAC)areequallyimportantin prolongingpatientrecovery[50],futurestudieswillassess thecapabilityofthemicro-patterntoreducetubeocclusion,aninterestinghypothesisbasedonthegeneralantiwettingcapabilityofthemicro-patternedsurface[51,52]. Asthistechnologyhasnotyetbeenclinicallyevaluated, ourcurrentresultsdonotindicatewhatoutcomeswe canexpectforpatientswhouseamicro-patternedETT. Nonetheless,withfutureresearchwehopetodevelopa micro-patternedETTthatreducesmicrobialcolonization andbiofilmformationassociatedwithVAPaswellastube occlusionthatleadstoVAC.Oncedemonstratedtobeeffectiveinclinicalstudies,thismicro-patternedETT,combinedwithstate-of-theartdevicecuffdesignsandbundle practices,maysupplyclinicianswithanarsenaltodiminishVAPandVACrates.ConclusionsThemicro-patternsurfacemodificationinhibitstheattachmentofMRSA ,P.aeruginosa,K.pneumoniae,E. coli, and A.baumannii ,thetopfiveVAP-associated pathogens,byupto99.9%whencomparedtounpatternedcontrols.Themicro-patternalsoinhibitstheformationofMRSAand P.aeruginosa biofilms,thelatterof whichwasfurtherinvestigatedinacomplexmucin-rich mediummimickingthetrachealenvironment.Demonstrationofbiofilmreductionbythi smicro-patternedsurfacein arangeofconditionsandwitharangeofpathogenssuggest thatthemicro-patternimplementedontoETTsurfaces mayreduceVAPrateswithouttheuseofantimicrobial agents.Abbreviations HAI: Hospitalacquiredinfection;VAP:Ventilator-associatedpneumonia; ETT:Endotrachealtube;ICU:Intensivecareunit;MRSA:Methicillin-resistant Staphylococcusaureus ;TSA:Trypticsoyagar;TSB:Trypticsoybroth; PBS:Phosphatebufferedsaline;SD:Standarddeviation; VAC:Ventilator-associatedcomplications. Competinginterests RheaMay,MattHoffman,MelindaSogo,andShravanthiReddyare employeesofSharkletTechnologies,Inc.AlParkerandAnthonyBrennanare paidconsultantsforSharkletTechnologies,Inc. Authors ’ contributions RMparticipatedinthedesignofthestudy,carryingoutthecolonizationand biofilmassays,statisticalanalysis,dataanalysisandinterpretation,and draftingthemanuscript.MHparticipatedinthedesignofthestudyandin carryingoutthecolonizationassays.MSparticipatedindesigningand carryingoutthecolonizationandbiofilmassays.APparticipatedinstatistical analysis.GOandABparticipatedinthedesignofthestudyanddata interpretation.SRconceivedthestudy,participatedinitsdesignand coordination,datainterpretation,andhelpedtodraftthemanuscript. Allauthorsparticipatedinreviewingandeditingthemanuscript. Authors ’ information RM,MHandMSaremicrobiologyresearchersandSRistheDirectorof ResearchforSharkletTechnologies,Inc.(STI);GOisabiofilmexpertand professoratDartmouthMedicalSchoolandanun-paidcollaboratorwithSTI; APisastatisticianandprofessorfortheCenterofBiofilmEngineeringat MontanaStateUniversityandaconsultantforSTI;ABisamaterialsengineer andprofessorattheUniversityofFlorida,Gainesvilleandistheinventorof themicro-patterntechnologywherehecontinuestoconsultonthetechnology forSTI. Acknowledgements ThisstudywassupportedbyNIH/NHLBIPhaseISBIRfunding,grantnumber 1R43HL110444-01toSharkletTechnologies,Inc.,aswellasNIHgrant R01AI083256toG.A.O.ImagingexperimentswereperformedintheUniversityof ColoradoAnschutzMedicalCampusAdvanceLightMicroscopyCoresupported inpartbyNIH/NCRRColoradoCTSIGrantNumberUL1RR025780.Contentsare theauthors'soleresponsibilityanddonotnecessarilyrepresentofficialNIHviews.May etal.ClinicalandTranslationalMedicine 2014, 3 :8 Page7of9 http://www.clintransmed.com/content/3/1/8

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