Systematic analysis of the ability of Nitric Oxide donors to dislodge biofilms formed by Salmonella enterica and Escheri...

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Title:
Systematic analysis of the ability of Nitric Oxide donors to dislodge biofilms formed by Salmonella enterica and Escherichia coli O157:H7
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Mixed Material
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English
Creator:
Massimiliano, Marvasi
Chen, Charles
Carrazana, Manuel
Durie, Ian A.
Teplitski, Max
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Springer (AMB Express, a Springer Open Journal)
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Abstract:
Biofilms in the industrial environment could be problematic. Encased in extracellular polymeric substances, pathogens within biofilms are significantly more resistant to chlorine and other disinfectants. Recent studies suggest that compounds capable of manipulating nitric oxide-mediated signaling in bacteria could induce dispersal of sessile bacteria and provide a foundation for novel approaches to controlling biofilms formed by some microorganisms. In this work, we compared the ability of five nitric oxide donors (molsidomine, MAHMA NONOate, diethylamine NONOate, diethylamine NONOate diethylammonium salt, spermine NONOate) to dislodge biofilms formed by non-typhoidal Salmonella enterica and pathogenic E. coli on plastic and stainless steel surfaces at different temperatures. All five nitric oxide donors induced significant (35-80%) dispersal of biofilms, however, the degree of dispersal and the optimal dispersal conditions varied. MAHMA NONOate and molsidomine were strong dispersants of the Salmonella biofilms formed on polystyrene. Importantly, molsidomine induced dispersal of up to 50% of the pre-formed Salmonella biofilm at 4°C, suggesting that it could be effective even under refrigerated conditions. Biofilms formed by E. coli O157:H7 were also significantly dispersed. Nitric oxide donor molecules were highly active within 6 hours of application. To better understand mode of action of these compounds, we identified Salmonella genomic region recA-hydN, deletion of which led to an insensitivity to the nitric oxide donors. Keywords: Biofilm control; Bacterial signaling; Food-borne pathogens; Nitric oxide
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Marvasi et al. AMB Express 2014, 4:42 http://www.amb-express.com/content/4/1/42; Pages 1-11
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doi:10.1186/s13568-014-0042-y Cite this article as: Marvasi et al.: Systematic analysis of the ability of Nitric Oxide donors to dislodge biofilms formed by Salmonella enterica and Escherichia coli O157:H7. AMB Express 2014 4:42.

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© 2014 Marvasi et al.; icensee Springer; licensee Springer. 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
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RESEARCHARTICLEOpenAccessSystematicanalysisoftheabilityofNitricOxide donorstodislodgebiofilmsformedby Salmonella enterica and Escherichiacoli O157:H7MassimilianoMarvasi1,CharlesChen2,ManuelCarrazana2,IanADurie1andMaxTeplitski1,3*AbstractBiofilmsintheindustrialenvironmentcouldbeproblematic.Encasedinextracellularpolymericsubstances, pathogenswithinbiofilmsaresignificantlymoreresistanttochlorineandotherdisinfectants.Recentstudiessuggest thatcompoundscapableofmanipulatingnitricoxide-mediatedsignalinginbacteriacouldinducedispersalofsessile bacteriaandprovideafoundationfornovelapproachestocontrollingbiofilmsformedbysomemicroorganisms.Inthis work,wecomparedtheabilityoffivenitricoxidedonors(molsidomine,MAHMANONOate,diethylamineNONOate, diethylamineNONOatediethylammoniumsalt,spermineNONOate)todislodgebiofilmsformedbynon-typhoidal Salmonellaenterica andpathogenic E.coli onplasticandstainlesssteelsurfacesatdifferenttemperatures.Allfivenitric oxidedonorsinducedsignificant(35-80%)dispersalofbiofilms,however,thedegreeofdispersalandtheoptimal dispersalconditionsvaried.MAHMANONOateandmolsidominewerestrongdispersantsofthe Salmonella biofilms formedonpolystyrene.Importantly,molsidomineinduceddispersalofupto50%ofthepre-formed Salmonella biofilm at4C,suggestingthatitcouldbeeffectiveevenunderrefrigeratedconditions.Biofilmsformedby E.coli O157:H7were alsosignificantlydispersed.Nitricoxidedonormoleculeswerehighlyactivewithin6hoursofapplication.Tobetter understandmodeofactionofthesecompounds,weidentified Salmonella genomicregion recA-hydN ,deletionof whichledtoaninsensitivitytothenitricoxidedonors. Keywords: Biofilmcontrol;Bacterialsignaling;Food-bornepathogens;NitricoxideIntroductionEverynaturalwetsurfaceisasubstrateformicrobialbiofilms.Thesesessilemulticellularmicrobialconsortiaare embeddedwithintheself-producedextracellularpolymeric substances(EPS)(Costertonetal.1987;Marvasietal. 2010;Solanoetal.2002;Wingenderetal.1999).Infood handlingfacilities,biofilmscouldbeparticularlyproblematic.Whilepathogensdonottypicallymakeupthebulkof thebiofilmsformedintheindustrialsettings, Salmonella spp,Listeriaspp, pathogenicstrainsof E.coli,Yersiniaspp, Pseudomonasspp,Shigellaspp,Staphylococcusspp, and Bacillusspp canbefoundinbiofilmsonvariouscontact surfaces(Blanpain-Avetetal.2011;ShiandZhu2009).Becausemicrobesinbiofilmsaresignificantlymoreresistant tochlorineandotherdisinfectants(ZhangandMah2008), thereisapressingneedtoidentifycompoundscapableof controllingbiofilmsbyothermeans. Adiscoveryofthenitricoxide ’ sfunctionininducing biofilmdispersalledtoconcertedeffortsontheidentificationofthecompoundscapableofdislodgingbiofilms (Landinietal.2010;McDougaldetal.2012).Severaldisinfectants,antibioticsandmessengermoleculeshavebeen studiedfortheirabilitytodispersebiofilms(Barraudetal. 2006;Barraudetal.2012;Barraudetal.2009a;Huynh etal.2012;McDougaldetal.2012).Nitricoxide(NO)gas andNOdonorsarecurrentlyusedclinically(RegevShoshanietal.2010).Inbacteriaandeukaryotes,nitric oxideisasignalingmolecule,activeatverylowconcentrations(Gaupelsetal.2011;Simontacchietal.2013).Atlow concentrations,itiseffectiveasabiofilmdispersant, functioningasamessengerratherthanagenericpoison (Barraudetal.2006;Barraudetal.2009a).Nitricoxide canbedeliveredtobiofilmsusingNOdonormolecules *Correspondence: maxtep@ufl.edu1SoilandWaterScienceDepartment,UniversityofFlorida-IFAS,Gainesville, FL,USA3CancerandGeneticsResearchCenter,GeneticsInstitute,2033MowryRoad, Gainesville,FL32611,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Marvasietal.;icenseeSpringer;licenseeSpringer.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.Marvasi etal.AMBExpress 2014, 4 :42 http://www.amb-express.com/content/4/1/42

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orimpregnatedontonanoparticles(Slombergetal. 2013;Wangetal.2005).TheapplicationofNOdonors hasthesameeffectondispersalofbiofilmsasthedirectadditionofnitricoxideandislesstoxicthantheapplicationofthenitricoxidegas(Barraudetal.2009a). Over105NOdonorshavebeencharacterized,butonly fewofthemhavebeentestedforcontrollingindustrialbiofilms(Wangetal.2005).Amongthese,activityofsodium nitroprussidehasbeenrecentlycharacterizedindetail.It canproduceafluxofnitricoxideof30pmolcm 2s 1,and thiscanefficientlyreducetheadhesionof Staphylococcus aureus Staphylococcusepidermidis ,and E.coli by96%, 48%,and88%,respectively(Charvilleetal.2008).Therefore,itappearsthatnitricoxidecouldhaveauniversal effectonthedispersalofbacterialbiofilmincluding bothGram-positiveandGram-negativebacteria(Xiong andLiu2010). Inthiswork,wetestedtheeffectivenessofNOdonors indispersalofbiofilmsformedbycommonfoodborne pathogens(non-typhoidal Salmonellaenterica andenterohaemorrhagic E.coli ,EHEC)onmaterialsthatare commoninthefoodindustry.Therationaleforthis studywasbasedonthereportsthatthesameNOdonorshavedifferentdispersionpotentialdependingon thebacterialstrain,temperatureandsurfaceproperties (Barraudetal.2006;Barraudetal.2009a;Charvilleetal. 2008;GilberthorpeandPoole2008).Becausetheultimategoaloftheseexperimentsistoidentifycommercially availablecompoundsforindustrialapplications,thefollowingcriteriawereusedtoselectcandidatecompounds:1)low/moderatetoxicity;2)havenomorethan 0.1%ofprobable,possibleorconfirmedhumancarcinogenicityaccordingtotheInternationalAgencyforResearch onCancer(IARC);3)low/moderatecost;4)commercially availability.Basedonthesecriteria,thefollowingcompoundswereselected:molsido mine(N-(ethoxycarbonyl)3-(4-morpholinyl)-sydnoneimine),MAHMANONOate (6-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1hexanamine),spermineNONOat e(N-[4-[1-(3-Aminopropyl)2-hydroxy-2-nitrosohydrazino]butyl]-1,3-propanediamine), diethylamineNONOatediethylammoniumsalt,anddiethylamineNONOatesodium. ThemechanismsbywhichNOeffectsthetransition fromsessilebiofilmorganismstofree-swimmingbacteriaarenotentirelyclear(Barraudetal.2006;Barraud etal.2009a). Microarraystudieshaverevealedthat P.aeruginosa genesinvolvedinadherencearedownregulateduponexposuretonitricoxide(Firovedetal.2004),andtheinvolvementofNOinregulatingbiofilmformationanddispersal in P.aeruginosa wasalsosupportedbyseveralstudies (Barraudetal.2006;Barraudetal.2009a;Darlingand Evans2003;VanAlstetal.2007).Severalgenesinvolved intheproductionandperceptionofthissignaling moleculehavebeencharacterizedin Pseudomonasaeruginosa PAO1.ThechemotaxisproteinBdlAisinvolvedin biofilmdispersionof P.aeruginosa :biofilmsformedbythe bdlA mutantdonotdetachwhenexposedtolowdosesof NOincontinuous-flowcultures(Barraudetal.2009a; PetrovaandSauer2012). P.aeruginosanirS and norCB encodeaNO2 reductaseandNOreductase.Amutation in nirS leadstoareducedbiofilmdispersion(Barraud etal.2006),whilebiofilmsformedbyaNOreductasedeficientstrain norCB didnotshifttotheplanktonic statewhenexposedtoendogenousnitricoxide.NohomologsofNirS,NorCBarefoundin Salmonella .However, whengrownanaerobicallywithnitrate, Salmonella is capableofgeneratingNOafternitriteaddition,likelyvia productsof fnr and hmp genes(GilberthorpeandPoole 2008).Becausethemechanismsofnitricoxide-mediated signalingin Salmonella appeartobedistinctfromthosein P.aeruginosa ,thisstudyalsoattemptedtoelucidategenes thatarepotentiallyinvolvedinthesesignalingpathways andcontributetobiofilmdispersalin Salmonella.MaterialsandmethodsBacterialstrainsandculturemediaEscherichiacoli EHECO157:H7ATCC43888, Salmonella enterica serotovarTyphimuriumATCC14028 sv.Braenderup 04E01347,Braenderup04E01556,Braenderup04E00783,sv. MontevideoLJH519,sv.JavianaATCCBAA-1593andsv. NewportC6.3(Noeletal.2010)wereusedinthisstudy. Whenacocktailof Salmonella strainswasused,itwas amixofequalvolumesofsixstrains:threestrainsofthe serovarBraenderup(04E01347,04E01556,04E00783),sv. MontevideoLJH519,Javiana(ATCCBAA-1593)and Newport(C6.3).pGFP-ON(astronglyfluorescentconstructcarryingGFPproteinexpressedfromthe Salmonella dppA promoter(Noeletal.2010))wastransformedinto thestrainsofinterestbyelectroporation. S. TyphimuriumA9isderivedfrom S. Typhimurium ATCC14028andlacksthegenomicregionbetween 2,974,854and2,990,668ntofNC_003197.1( recA through hydN ),whichwasreplacedwithakanamycin-resistance cassette.ItwasconstructedbysequentialDatsenkoand Wannermutagenesisasin(Santiviagoetal.2009). Allstrainsweremaintainedasfrozenglycerolstocks, andweresub-culturedintoLuriaBertanimediumwithappropriateantibiotics(50 gmL 1kanamycin,100 gmL 1ampicillin).NitricOxidedonorsAllwerepurchasedfromSigmaAldrich(St.Luois,MO, USA).Foreachcompound,1mmolL 1stocksolutions werepreparedinphosphate-bufferedsaline,pH7.3 (PBS,Fisher,Waltham,MA,USA)andaliquotswere storedat 80C.Fortheessays,serialdilutionswere alwayspreparedfreshinice-coldPBSjustbeforetheMarvasi etal.AMBExpress 2014, 4 :42 Page2of11 http://www.amb-express.com/content/4/1/42

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experimentsandusedwithin5minutesoftheirpreparation.Thebiofilmdispersionpotentialofthefivemoleculeswastestedonpolystyreneandpolypropylene.The abilityofmolsidominetodispersebiofilmswasalso testedonstainlesssteel.BiofilmformationanddispersalonplasticsOvernightLuriaBertaniculturesof Salmonella or E.coli strainswerediluted1:100inCFAmediumasdescribed previously(Teplitskietal.2006),and100 Lofthedilutedcultureswerealiquotedintowellsof96-wellpolypropyleneandpolystyreneplates(Fisher,Waltham,MA, USA).Plateswithbacteriawereincubatedfor24hours at37CinsideaZiplocbag.Uponcompletionoftheincubation,themediumwithplanktonicbacteriawasremovedandserialdilutionsofnitricoxidedonorsin PBS(in200 L)wereaddedtothewellswithbiofilms. Dispersalexperimentswereconductedat22Cor4C for6and24hours.Dispersalwasmeasuredbystaining theremainingbiofilmswith1%crystalvioletinethanol, asdescribedpreviously(Merrittetal.2005;O ’ Toole andKolter1998). Inparallel,tovalidatethestainingapproach,detachmentofcellsfrombiofilmswasalsomeasuredbydirectly monitoringtheincreaseoffluorescenceofplanktoniccells of S. Typhimurium14028pGFP-ONusingVictor-2multimodeplatereaderwitha485nm/535nmexcitation/emissionfilter(PerkinElmer,Waltham,MA,USA).LuminescencetestsEffectsofselectedNOdonorsonlightproductionbya constitutivelyluminescent Salmonella strainwerecharacterizedasindirectassessmentsoftoxicityofthecompounds.TwohundredmicrolitersofLuriaBertanibroth inoculatedwiththeovernight,1:50dilutedcultureof S.Typhimurium14028pTIM2442(harboringthe luxCDABE drivenbyastrongconstitutivephage promoter Alagelyetal.,2011)weregrowninblackpolystyrene plates(Corning,NewYork,USA)inpresenceofserialdilutionsofMolsidomine.MolsidominewasdilutedinPBS (9.89gL 1)(FisherScientific,Waltham,MA,USA)tofinal concentrationsof10 molL 1,10nmolL 1,and10pmolL 1. PBSwasusedasacontrol.Luminescenceof S. Typhimurium14028pTIM2442wasmeasuredovertimeusing Victor-2multimodeplatereader(PerkinElmer,Waltham, MA,USA).Eachexperimentincluded12replicas.BiofilmformationonstainlesssteelBiofilmswereformedonthestainlesssteelculturetube closures(FisherScientific,Waltham,MA,USA)essentiallyasdescribedaboveforplasticswiththefollowing modifications:onlydilutionsofmolsidominewereused, andonly24hoursofcontacttimewastested.Biofilm dispersalwastestedbymonitoringfluorescenceundera multimodemicroplatereaderequippedwitha485nm/ 535nmexcitation/emissionfilter(PerkinElmer,Waltham, MA,USA).AdditiveeffectofthedisinfectantSaniDate12.0with nitricoxidedonorsBiofilmsof S. TyphimuriumATCC14028weresetupas aboveusingovernightculturesofthepathogendiluted 1:100intheCFAmediuminwellsof96-wellpolypropyleneplates(Fisher,Waltham,MA,USA).Plateswithbacteriawereincubatedfor24hoursat37CinsideaZiploc bag.Uponcompletionoftheincubation,themedium withplanktonicbacteriawasremovedand10nmolL 1of MolsidomineorMAHMAnonoatewereaddedtothe wellswithbiofilms.Ascontrols,BPSalonewasused. Plateswereincubatedat22Cfor24hours.Uponcompletionoftheincubation,planktoniccellswereremoved, wellswerewashedtwicewithPBSand200 LofSaniDade 12.0(BioSafeSystem,Hartford,CT,USA)dilutedasper manufacturer ’ srecommendationsandwereloadedinto thewells.Thedisinfectantwasincubatedfor10minutes, aftertheincubationtime,biofilmdispersalwasmeasured bystainingtheremainingbiofilmswith1%crystalvioletin ethanol,asdescribedpreviously(Merrittetal.2005; O ’ TooleandKolter1998).12replicasforeachexperiment weredone.StatisticalanalysisThestatisticalsoftwareJMP(SAS)packagewasusedto infertheOne-wayANOVAanalysis(p<0.05).Tukey meansseparationanalysiswasinferredinordertogroup themeans.ResultsBiofilmformationondifferentplasticsMorerobustbiofilmswereformedby Salmonella and E.coli strainsonpolypropylenethanonpolystyrene (Additionalfile1:FigureS1).Onpolypropyleneand polystyrene, S.enterica svTyphimurium14028formed morebiofilmsundertheseconditionsthan E.coli O157:H7 (Additionalfile1:FigureS1).Biofilmformationisknown tovarydependingonthesurface,themediausedtodevelopbiofilms,aswellastheprotocoladopted(Kroupitski etal.2009;Teplitskietal.2006).BiofilmdispersalbymolsidomineMolsidominereleasesnitricoxideandformspolarmetabolitesrapidly;itshalf-lifeis1to2hoursinplasmaat pH7.4(Rosenkranzetal.1996).Intermsofbiofilmdispersal,molsidominewasthemostpotentmolecule.It waseffectiveindislodgingbiofilmsformedby S.enterica svTyphimurium14028,thecocktailofthesix Salmonella outbreakstrainsand E.coli O157:H7.Molsidomine wasparticularlyeffectiveonpolypropylene(Figure1).ItMarvasi etal.AMBExpress 2014, 4 :42 Page3of11 http://www.amb-express.com/content/4/1/42

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Figure1 Biofilmdispersalbymolsidomine. Exposuretomolsidominedosesrangingfrom10 Mto10pMinducedbiofilmdispersalasmeasured bystainingoftheattachedcellswithcrystalviolet. “ 0 ” indicatescontrol,anexistingbiofilmnottreatedwithmolsidomine. A. Biofilmformedby S.enterica sv.Typhimurium14028inwellsofamicrotiterplatebeforetreatmentwithmolsidomine.Attachedcellsarevisualizedwithcrystal violetstaining. B. Residualbiofilmaftertreatmentwith10nMofmolsidomine. C H. Inductionofbiofilmdispersalbymolsidomine.Biofilmswere pre-formedonpolystyrene.Contacttimeandtemperatureatwhichbiofilmswereexposedtothechemicalarelistedaboveeachpanel. Concentrationsofmolsidomineareindicatedon thex-axis.Errorbarsarestandarderrors. Marvasi etal.AMBExpress 2014, 4 :42 Page4of11 http://www.amb-express.com/content/4/1/42

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wasmosteffectiveat22C,inducingdispersalof~50% ofbiofilmsformedby Salmonella 14028andthecocktail ofsix Salmonella outbreakstrainsafterincubationfor 6hours(Figure1,p<0.0001).Upto75%ofbiofilmdispersalwasobservedaftera24-hrstreatmentof E.coli O157: H7biofilmswithmolsidomine(Figure1,p<0.0001). Biofilmdispersalbymolsidominewasalsoobservedon polystyrene(Additionalfile1:TableS1). Salmonella biofilmspreformedinpolystyrenewellsweredislodged after24hoursofcontacttimeatroomtemperature. Molsidominewasabletoinducesomedispersaleven whenbiofilmsweretreatedwiththecompoundat4C. Intriguingly,thestrongestdispersalwasobservedinresponsetothetreatmentwith10picomolarconcentrationsofmolsidomine(Figure1).Suchpotencyofthe compoundatverylowconcentrationsmakesitapotentiallyinterestingcandidateforcommercialapplications. Totestwhetherthedecreaseinthestainingoftheattachedcellswascausedbyanincreaseinthenumberof planktoniccells,biofilmsformedbyfluorescent Salmonella 14028pGFP-ONonpolypropyleneweretreated withmolsidomine.Fluorescenceoftheplanktonicand attachedcellswasmeasuredafter0,3and6hoursofexposuretothenitricoxidedonor.Theincreaseintotal fluorescenceofplanktoniccellsupontreatmentofbiofilms withmolsidominewasstatisticallysignificant(Additional file1:TableS1),reflectinganincreaseindetachmentatthe testedconcentrations.Similarly,molsidominetreatmentof the Salmonella biofilmsformedonstainlesssteelresulted in~0.3logincreaseinfluorescenceoftheplanktoniccells (datanotshown). Theabilityoftheconstitutivelyluminescent Salmonella constructdrivenbyaphage promotertoproduce lightinthepresenceofmolsidominewasusedasanindirectassessmentofthetoxicityofthecompound,and itsabilitytogenerallydisruptmetabolismorrespiration ofthebacteria.AsshowninAdditionalfile1:FigureS2, eventhoughmodestdecreaseinluminescencewasobservedinmolsidomine-treatedcultures,thereduction ofluminescencewasnotgreaterthan1-10%ofthe control.Dose-dependentbiofilmdispersalbydiethylamine NONOatediethylammoniumDiethylamineNONOatediethylammoniumrepresentsa classofmoleculeswhichspontaneouslydissociateina pH-dependentmanner,withahalf-lifeof16minutesat 22-25C,pH7.4toliberate1.5molesofNOpermoleof theparentalcompound(Keeferetal.1996;Maragos etal.1991).BiofilmdispersalinresponsetodiethylamineNONOatediethylammoniumwasdose-dependent (Figure2),howevertherelationshipwasinverse:low concentrationsofthenitricoxidewereassociatedwith higherbiofilmdispersion.Thestrongactivityinthe picomolarrangeissimilartowhatwasobservedwith molsidomine. DiethylamineNONOatediethylammoniumwasmost potentatdispersingbiofilmsonpolypropylenefollowing a6-hoursincubation.Astrongdispersalofbiofilms formedby Salmonella ATCC14028onpolypropylene wasobserved(Figure2).Biofilmsformedby E.coli O157:H7werealsodispersedbydiethylamineNONOate diethylammonium,butthedispersalwaslowerwhen comparedwith Salmonella .Thebiofilmformedbythe cocktailofsix Salmonella outbreakstrainsformedon polystyrenewasalsodispersed(Figure2andAdditional file1:TableS2andAdditionalfile1:TableS3).TheeffectofdiethylamineNONOatesodiumsalthydrate, MAHMANONOateandspermineNONOateonbiofilmsOfthetestedcompounds,diethylamineNONOatesodium saltwastheleasteffectivebiofilmdispersantfollowing24 and6hoursincubation.Thestrongestdispersionwasobservedinbiofilmsformedonpolypropylenebythecocktailof Salmonella strainsand Salmonella Typhimurium ATCC14028(Figure3).Upto50%ofreductionofbiofilm wasdetectedatroomtemperaturefor Salmonella 14028 andtheoutbreakstrainsonpolystyrene(Figure3).On Figure2 Theeffectofdiethylamin eNONOatediethylammonium onbiofilms. Remainingbiofilmsweremeasuredbycrystalviolet staining,whichwassolubilizedindilutedaceticacidandtheabsorbance at590nmwasmeasured.Concentrationsofthenitricoxidedonorare onthex-axis.Contacttimeandtemperatureatwhichpre-formed biofilmsweretreatedwithdiethylamineNONOatediethylammonium areindicatedaboveeachpanel. Marvasi etal.AMBExpress 2014, 4 :42 Page5of11 http://www.amb-express.com/content/4/1/42

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polystyrene,biofilmformationbythecocktailofthe Salmonella strainswasreducedbyupto30%when comparedwiththecontrol.DiethylamineNONOatewas alsoactiveindispersingbiofilmsformedby E.coli O157:H7, eventhoughthedispersionwasverylimited(~20%when comparedwiththecontrol)(Additionalfile1:TableS2and Additionalfile1:TableS3). TheeffectivenessofMAHMANONOate(whichhas half-lifeofminutes)wascomparedtothatofsperimne NONOate,whichhashalf-lifeofseveralhours.Thisaffects potencyandsustainabilityofthetreatment,forMAHMA NONOateshowingupto40timesthepotencyofspermine NONOate,butthelattershow ingactivatyforhours,comparedwithfewminutesf orMAHMANONOate(Wang etal.2005).MAHMANONOateisalsoanoptimaldispersantofpreformedbiofilmof Pseudomonasaerouginosa biofilm(Barnesetal.2013).Therefore,thetwomoleculeswere comparedwithin6hoursofincubationwith Salmonella and E.coli biofilms.AsexpectedMAHMANONOatewas amoreeffectivedispersantonboththematerials(Table1). However,spermineNONOatewasmainlyeffectiveatreleasingbiofilmsformedonpolypropylene(Table1). MAHMANONOatealsoappearstobeabroadlyactivemoleculeindispersingbiofilmformedbothon polypropyleneandpolystyrene(Figure4):itreleasedup to70%of E.coli biofilmspre-formedonpolystyreneat roomtemperatureaftera6-hourtreatment(Figure4).Biofilmsformedby S.enterica svTyphimuriumATCC14028 werealsodispersedbyMAHMANONOateatroom temperature,butcomparedtospermineNONOate,itwas moreeffectiveonpolystyrene(Figure4).Onpolypropylene,upto50%of S.enterica svTyphimuriumATCC14028 andthecocktailofsix Salmonella outbreakstrainsbiofilms weredispersedbyMAHMANONOatewhenincubated for24hoursatroomtemperature(Figure4).Biofilms formedby E.coli O157:H7werealsoeffectivelydispersed onpolypropylenebutlesswhencomparedwithpolystyrene (Figure4).EffectivenessofMAHMAnonoateindispersing Table1TheeffectofSpermineNONOateandMAHMA NONOateonpreformedbiofilmProbability>F (p=0.05)* MaterialStrainIncubation temperature Spermine NONOate MAHMA NONOate Polypropylene Salmonella 140284Cn.s.n.s. Polypropylene Salmonella 1402822Cn.s.0.0049 Polypropylene E.coli O157:H74C0.0273n.s. Polypropylene E.coli O157:H722C0.0486<.0001 Polypropylene Salmonella cocktail 4C0.00360.0185 Polypropylene Salmonella cocktail 22C0.0003<.0001 Polystyrene Salmonella 140284C0.04180.0229 Polystyrene Salmonella 1402822Cn.s.<.0001 Polystyrene E.coli O157:H74Cn.s.n.s. Polystyrene E.coli O157:H722Cn.s.n.s. Polystyrene Salmonella cocktail 4Cn.s.0.0184 Polystyrene Salmonella cocktail 22C0.0183<.0001*Statisticallysignificanteffects(p<0.05)oftheNOdonortreatmentswhen comparedwiththeuntreatedcontrol; “ n.s. ” notstatisticallysignificant biofilmdispersion. Figure3 Dispersalof Salmonella biofilmsbydiethylamineNONOatesodiumsalt. Biofilmswereformedovernightbyeither S.enterica sv. TyphimuriumATCC14028oracocktailofsix Salmonellaenterica strains(sv.Braenderup04E01347,Braenderup04E01556,Braenderup04E00783,sv. MontevideoLJH519,sv.JavianaATCCBAA-1593andsv.NewportC6.3)linkedtoproduce-associatedoutbreaksofgastroenteritis.Biofilmswere pre-formedoneitherpolystyrene(blackbars)orpolypropylene(greybars)priortothetreatmentwithdiethylamineNONOatesodiumsalt. Concentrationsofthenitricoxidedonorareonthex-axis.Contacttimeandtemperatureatwhichthetreatmenttookplaceareindicatedabove eachpanel.Residualbiofilmswerequantifiedbystainingwithcrystalviolet.Errorbarsarestandarderrors. Marvasi etal.AMBExpress 2014, 4 :42 Page6of11 http://www.amb-express.com/content/4/1/42

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biofilmswasfurtherincreasedbytreatmentwiththe microbiocideSaniDate(Figure5).TherecA-hydNgenomicregionisinvolvedinSalmonella biofilmdispersalInordertobetterunderstandhownitricoxidedonors inducedispersalof Salmonella biofilms,thegenomeof S. Typhimurium14028wasscannedforthehomologsof knowngenesinvolvedinnitricoxidesignaling.Amutant lackingthe~15kB Salmonellaenterica svTyphimurium ATCC14028genomicregionspanning15genes( recA hydN ) wasstudied(Figure6).Thisregionincludesputative NO-reductasemachinery: ygaA ,ananaerobicnitric oxidereductasetranscriptionalregulator;STM2840,an anaerobicnitricoxidereductaseflavorubredoxin; ygbD nitricoxidereductase; hypF ,hydrogenasematuration protein; hydN ,anelectrontransportproteinHydN. Proteinsencodedregioncanbeinvolvedintransferring electrontoNO,itsdetoxificationandgenerationofnitrousoxide.Consistentwiththepredictedfunctionsof thedeletedgenes,the recA hydN mutantformeda morerobustbiofilmonpolystyrenecomparedtothe wildtype,anddidnotrespondtothe24-hrstreatment withmolsidomine(Figure6).DiscussionControllingbiofilmsonsurfacesofclinicalandindustrial importancehasemergedasanimportantgoal.Whilean impressivetoolboxispotentiallyavailabletothoseaiming topreventmicrobesfromattachingtosurfacesandformingbiofilms(Campocciaetal.2013;Chenetal.2013),the approachesforcontrollingexis tingbiofilmsaresignificantly Figure4 BiofilmdispersalbyMAHMANONOate. Biofilmswereformedby E.coli O157:H7, Salmonella sv.Typhimurium14028andacocktailof thesix Salmonella strainslinkedtohumanproduce-relatedgastroenteritisoutbreaksonpolystyrene(blackbars)orpolypropylene(greybars)priorto thetreatmentwithMAHMANONOate.Contacttimesandtemperaturesatwhichbiofilmswereexposedtothenitricoxidedonorarelistedaboveeach panel.Concentrationsofthenitricoxidedonorareonthex-axis. Marvasi etal.AMBExpress 2014, 4 :42 Page7of11 http://www.amb-express.com/content/4/1/42

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morelimited.Aseminaldiscoverythatself-producednitric oxideactsasadispersalcuefor Pseudomonasaeruginosa biofilms(Barraudetal.2006) sparkedinterestinexploring theuseoftheNOgas,moleculesandnanoparticlescapable ofreleasingit(Landinietal.2010;McDougaldetal.2012). Variousnitricoxidegeneratingmoleculesandnanoparticlescanbeusedeffectivelytodislodgebiofilmsformed byGram-negative P.aeruginosa and E.coli ,Gram-Positive Staphylococcusaureus and S.epidermidis (Barraudetal. 2009b;CherayilandAntos2001;Slombergetal.2013) andpreventattachmentofzoosporesofthegreenalgae Ulva (Thompsonetal.2010).However,insomebacteria( Shewanellaoneidensis,Vibrioharveyi ),perception ofNOleadstoanincreasedbiofilmformation(Landini etal.2010;PlateandMarletta2012).Therefore,even thoughNOisasignalemployedbyadiversityoforganisms,responsestoitarenotuniversallyconserved.Becauseofthesebiologicaldifferencesintheconsequences ofNOdetection,itisimportanttoestablishhowandto whatextentcommerciallyavailablenitricoxidedonors Figure6 Involvementofthe SalmonellarecA-hydN genomicregioninthenitricoxide-mediatedsignaling. Salmonella sv.TyphimuriumA9 (mutantlackinga15kBfragmentspanning recA-hydN genes)formedmoreabundantbiofilms,whichwerenotresponsivetotreatmentwithnitric oxidedonormolsidomine.Detachmentofthewildtypebiofilmsinresponsetomolsidomine (A) .LackofresponsetomolsidominebytheA9 mutant (B) .AppearanceofthebiofilmsformedbytheA9mutantandthewildtype (C) .GenomiccontextoftheA9deletion (D) .Thickarrows areannotatedORF.Geneswithputativefunctionsinnitricoxidesignalingareshadedgrey. Figure5 AdditiveeffectofthemicrobiocideSaniDateandnanomolarconcentrationsofMolsidomineandMAHMAnonotate. Barsrepresent thestandarderror.Differentlettersrepresentsignificantdifferentmeans(p=0.05). Marvasi etal.AMBExpress 2014, 4 :42 Page8of11 http://www.amb-express.com/content/4/1/42

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canbeusedforcontrollingbiofilmsformedbyimportant foodbornebacterialpathogens,knowntopersistasbiofilmsintheindustrialfacilities. Withthisstudy,wefocusedontheeffectsofoff-theshelfNOdonorsonthebiofilmsformedbysevenstrains of S.enterica andpathogenic E.coli onsurfacesthat mimicthosefoundinfoodprocessingfacilities.Thefive compoundsselectedforthesetestsbasedontheirlow potentialtoxicity,commercialavailabilityandpredicted potencydemonstratedvaryinglevelsofspecificityand efficacy.Ofthecompoundstested,molsidomineand diethylamineNONOatediethylammoniumarethemost promisingastheyarecapableofdislodgingbiofilmsformed by S .Typhimurium14028,acocktailofsix Salmonella strainsisolatedfromoutbreaksand E.coli O157:H7under mostofthetestedconditions.Formostofthecompounds tested,theirhighestactivityappearstobeinthepicomolar range,suggestingthattheirapplicationscouldbefurtheroptimizedfortheeconomicalandeffectiveindustrialapplications.Theactivityofthecompoundsinthe nano-andpicomolarrangesfurthersuggeststhatNO actsasapotentcue,consistentwithpreviousreports. Lowbiofilm-dispersingactivityofthenitricoxidedonorsathigherconcentrationsis,perhaps,notsurprisingconsideringthatNOcanbebactericidalata concentrationofmgL 1(McDougaldetal.2012;Miller etal.2009),andthemethodsemployedinthisstudy wouldnotnecessarilydistinguishbetweenliveanddead bacteriawithinanexistingbiofilm. Interestingly,somecompounds(molsidomineordiethylamineNONOatediethylammonium)wereeffectiveat dispersingbiofilmsunderrefrigeratedconditions(4C). Temperatureisanimportantfactoraffectingthedissociationconstant.NONOate(s)areverysensitiveto temperature:a1Cchangefrom37Ccanresultsinan approximate13%changeinNOrelease(Ramamurthiand Lewis1997).Therefore,itisreasonabletohypothesize thatwhenbiofilmsareexposedtothenitricoxidedonors atlowertemperatures,theyexperiencedthetreatmentfor alongerperiodoftime,andthisincreasedtheirpotency. Theimplicationsofthisobservationforindustrialapplicationsarepotentiallyexciting:theabilityofthenitricoxide donorstodispersebiofilmsat4Cmakesthemgoodcandidatesforcleaningrefrigeratedsurfaces,commoninthe foodindustryandevenremovingpathogenbiofilmsfrom refrigeratedfoods.While Salmonella and E.coli are thoughttobemetabolicallyinactiveundertheseconditions,theirpopulationsinbiofilmsformedonrefrigerated foodsremainrelativelysteadyoveranextendedperiodof time(Kroupitskietal.2009).Furthermore,acomplement of Salmonella genesthatweredifferentiallyexpressedduringbiofilmformationoncutlettuceduringcoldstorage hasbeenidentified(Kroupitskietal.2013),suggestingthat eventhoughthecellsofthesehumanpathogensarenot dividing,physiologicalprocessesandgeneexpressionstill takeplaceunderrefrigeratedconditions.Itwouldbeof greatinteresttodeterminewhetherthesespecificgenes inducedinbiofilmsonfoodsat4-8Ccouldbesubject tomanipulationbyNO.Morebroadly,itremainstobe determinedwhichofthe Salmonella genesinvolvedin biofilmformation(Hamiltonetal.2009;Teplitskietal. 2006)aresubjecttoregulationbynitricoxide,and – conversely – whichoftheknown Salmonella genesresponsivetonitricoxidecontributetotheNO-mediated biofilmdispersal(HenardandVazquez-Torres2011; Karlinseyetal.2012;Richardsonetal.2011).AdditionalfileAdditionalfile1:FigureS1. Biofilmformationondifferentplasticsby Salmonella and E.coli O157:H7.Biofilmswereestablishedonpolypropylene andpolystyrenesurfacesinCFAmediumfor24hrsandstainedwith1% crystalvioletandthenwashed.Theabsorbedcrystalvioletandthe biofilmweredissolvedin33%aceticacidandA590wasmeasuredwith aspectrophotometer.Errorbarsrepresentstandarderror.Significantdifferent meansaredisplayedwithdifferentletters. FigureS2. Luminescenceof Salmonella 14028pTIM2442uponexposuretomolsidomine.General metabolicstateofthecellswasassessedusingtheredox-coupled FMNH2/Luciferaseproducedbya S. TyphimuriumATCC14028strain harboringhighcopynumberplasmidinwhichthe luxCDABE operonis underthephage promoter(pTIM2442).Concentrationsofmolsidomineto whichculturesof Salmonella 14028pTIM2442wereexposedarelistedon thefigure.Errorbarsrepresentthestandarderrorof12replicas. TableS1. Fluorescenceofthe Salmonella cellsdetachedbymolsidominetreatment Measurementof Salmonella 14028planktoniccellsdetachedfrom preformedbiofilmduringtreatmentwithmolsidomine.polypropylene, *Statisticallysignificanteffects(p<0.05)ofmolsidominetreatment. TableS2. ThecompletesetofexperimentsontheeffectoftheNO donorsonpreformedbiofilmafter24hoursofcontacttime.*Statistically significanteffects(p<0.05)oftheNOdonortreatments.n.s.,notstatistically significantbiofilmdispersion.MOL,Molsidomine;M1555,MAHMA NONOate;S150,SPERMINENONOate;D5431,diethylamineNONOate diethylammoniumsalt;D184,diethylamineNONOatesodium;S8432 sulfoNONOatedisodiumsalt. TableS3. Thecompletesetofexperiments ontheeffectoftheNOdonorsonpreformedbiofilmafter6hoursof contacttime.*Statisticallysignificanteffects(p<0.05)oftheNOdonor treatments.n.s.,notstatisticallysignificantbiofilmdispersion.MOL, Molsidomine;M1555,MAHMANONOate;S150,SPERMINENONOate; D5431,diethylamineNONOatediethylammoniumsalt;D184,diethylamine NONOatesodium;S8432sulfoNONOatedisodiumsalt. Competinginterests ThisresearchwassupportedbyfundingprovidedbytheFloridaTomato Committee,Grant#106486andbytheUC-DavisCenterforProduceSafety, Grant#2014-308. Authors ’ contributions MM,MTconceivedexperiments;MM,CC,MC,IADconductedexperiments; MManalyzeddata;MM,MTwrotethemanuscript.Allauthorsreadand approvedthefinalmanuscript. Acknowledgements WearegratefultoM.McClellandforprovidingA9mutant. Authordetails1SoilandWaterScienceDepartment,UniversityofFlorida-IFAS,Gainesville, FL,USA.2MicrobiologyandCellScienceDepartment,Universityof Florida-IFAS,Gainesville,FL,USA.3CancerandGeneticsResearchCenter, GeneticsInstitute,2033MowryRoad,Gainesville,FL32611,USA.Marvasi etal.AMBExpress 2014, 4 :42 Page9of11 http://www.amb-express.com/content/4/1/42

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! Supplementary File Article title: Systematic analysis of the ability of Nitric Oxide donors to dislodge biofilms formed by Salmonella enterica and Escherichia coli O157:H7 Journal Name: AMB Express Authors and Affiliations: Massimiliano Marvasi 1 Charles Chen 2 Manuel Carrazana 2 Ian A. Durie 1 and Max Teplitski 1* 1 Soil and Water Science Department, University of Florida IFAS 2 Microbiology and Cell Science Department, University of Florida IFAS *Corresponding author. Email address: maxtep@ufl.edu ; phone: (352) 273 8189 Mailing address: Cancer and Genetics Research Center, 2033 Mowry Road, Genetics Institute, Gainesville, FL, USA 32611

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! # Supplemental Material Figure S1 Biofilm formation on different plasti cs by Salmonella and E. coli O157:H7. Biofilms were established on polypropylene and polystyrene surfaces in CFA medium for 24 hrs and stained with 1 % crystal violet and then washed T he absorbed crystal violet and the biofilm were dissolved in 33% acetic acid and A590 was measured with a spectrophotometer. Error b ars represent standard error. Significant different means are displayed with different letters.

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\057 \130 \120 \114 \121 \110 \126 \106 \110 \121 \106 \110 \003 \122 \111 \003 \066 \104 \117 \120 \122 \121 \110 \117 \117 \104 \003 \024 \027 \023 \025 \033 \003 \123 \067 \054 \060 \025 \027 \027 \025 \003 \104 \111 \127 \110 \125 \003 \120 \122 \117 \126 \114 \107 \122 \120 \114 \121 \110 \003 \127 \125 \110 \104 \127 \120 \110 \121 \127 \003 \024 \023 \031 \024 \023 \030 \024 \023 \027 \023 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \030 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \024 \023 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \024 \030 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \025 \023 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \025 \030 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \003 \046 \122 \121 \127 \125 \122 \117 \003 \013 \063 \045 \066 \014 \060 \122 \117 \126 \114 \107 \122 \120 \114 \121 \110 \003 \024 \023 \227 \060 \060 \122 \117 \126 \114 \107 \122 \120 \114 \121 \110 \003 \024 \023 \121 \060 \060 \122 \117 \126 \114 \107 \122 \120 \114 \121 \110 \003 \024 \023 \123 \060

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! $ Supplemental Material Figure S2. Luminescence of Salmonella 14028 p TIM2442 upon exposure to molsidomine. General metabolic state of the cells was assessed using the redox coupled FMNH 2 /Luciferase produced by a S. Typhimurium ATCC14028 strain harboring high copy number plasmid in which the l uxCDABE operon is under the phage promoter (pTIM2442) Concentrations of molsidomine to which cultures of Salmonella 14028 pTIM2442 were exposed are listed on the figure. Error bars represent the standard error of 12 replicas

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Supplementary Table S 1 Fluorescence of the Salmonella cells detached by molsidomine treatment Measurement of Salmonella 14028 planktonic cells detached from preformed biofilm during treatment with molsidomine polypropylene Conc Log (Fl u o rescence 485nm/535nm ) P< 0.05 0 hours 3 hours 6 hours Biofilms formed on polypropylene 0 4.335 0.065 n.a. 4.450 0.177 0.5492 10 M 4.307 0.067 4.846 0.054 4.466 0.080 <.0001 10 nM 4.275 0.055 4.780 0.050 4.753 0.075 <.0001 10 pM 4.355 0.064 4.815 0.125 5.002 0.144 0.0024 Statistically significant effects (p<0.05) of molsidomine treatment

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Supplementary Table S2 The complete set of experiments on the effect of the NO donors on preformed biofilm after 24 hours of contact time. NOD Material Strain Incubation temperature Prob>F (p=0.05) D184 Polypropylene Salmonella 14028 4¡C < 0 .0001 D184 Polypropylene Salmonella 14028 22 ¡C n.s. D184 Polypropylene E.coli O157 :H7 4¡C n.s. D184 Polypropylene E.coli O157 :H7 22 ¡C n.s. D184 Polypropylene Salmonella cocktail 4¡C n.s. D184 Polypropylene Salmonella cocktail 22 ¡C < 0 .0001 D5431 Polypropylene Salmonella 14028 4¡C < 0 .0001 D5431 Polypropylene Salmonella 14028 22 ¡C < 0 .0001 D5431 Polypropylene E.coli O157 :H7 4¡C 0.0004 D5431 Polypropylene E.coli O157 :H7 22 ¡C < 0 .0001 D5431 Polypropylene Salmonella cocktail 4¡C < 0 .0001 D5431 Polypropylene Salmonella cocktail 22 ¡C < 0 .0001 M1555 Polypropylene Salmonella 14028 4¡C n.s. M1555 Polypropylene Salmonella 14028 22 ¡C 0.0049 M1555 Polypropylene E.coli O157 :H7 4¡C n.s. M1555 Polypropylene E.coli O157 :H7 22 ¡C < 0 .0001 M1555 Polypropylene Salmonella cocktail 4¡C 0.0185 M1555 Polypropylene Salmonella cocktail 22 ¡C < 0 .0001 MOL Polypropylene Salmonella 14028 4¡C < 0 .0001 MOL Polypropylene Salmonella 14028 22 ¡C < 0 .0001 MOL Polypropylene E.coli O157 :H7 4¡C < 0 .0001 MOL Polypropylene E.coli O157 :H7 22 ¡C n.s. MOL Polypropylene Salmonella cocktail 4¡C 0.0001 MOL Polypropylene Salmonella cocktail 22 ¡C < 0 .0001 D184 Polystyrene Salmonella 14028 4¡C n.s. D184 Polystyrene Salmonella 14028 22 ¡C n.s. D184 Polystyrene E.coli O157 :H7 4¡C n.s. D184 Polystyrene E.coli O157 :H7 22 ¡C n.s. D184 Polystyrene Salmonella cocktail 4¡C < 0 .0001 D184 Polystyrene Salmonella cocktail 22 ¡C < 0 .0001 D184 Polystyrene Salmonella 14028 4¡C < 0 .0001 D5431 Polystyrene Salmonella 14028 22 ¡C 0.0006 D5431 Polystyrene E.coli O157 :H7 4¡C 0.0001 D5431 Polystyrene E.coli O157 :H7 22 ¡C n.s. D5431 Polystyrene Salmonella cocktail 4¡C < 0 .0001 D5431 Polystyrene Salmonella cocktail 22 ¡C < 0 .0001 M1555 Polystyrene Salmonella 14028 4¡C 0.0229 M1555 Polystyrene Salmonella 14028 22 ¡C < 0 .0001 M1555 Polystyrene E.coli O157 :H7 4¡C n.s. M1555 Polystyrene E.coli O157 :H7 22 ¡C n.s. M1555 Polystyrene Salmonella cocktail 4¡C < 0 .0001

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! Statistically significant effects (p<0.05) of the NO donor treatment s. n.s., not statistically significant biofilm dispersion. MOL, Molsidomine ; M1555 MAHMA NONOate ; S150 SPERMINE NONOate ; D5431 diethylamin e NONOate diethylammonium salt; D184 diethylamine NONOate sodium ; S8432 sulfo NONOate disodium salt. M1555 Polystyrene Salmonella cocktail 22 ¡C < 0 .0001 MOL Polystyrene Salmonella 14028 4¡C 0.0465 MOL Polystyrene Salmonella 14028 22 ¡C 0.0078 MOL Polystyrene E.coli O157 :H7 4¡C n.s. MOL Polystyrene E.coli O157 :H7 22 ¡C n.s. MOL Polystyrene Salmonella cocktail 4¡C < 0 .0001 MOL Polystyrene Salmonella cocktail 22 ¡C < 0 .0001

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Supplementary Table S3 The complete set of experiments on the effect of the NO donors on preformed biofilm after 6 hours of contact time. NO donor Material Strain Incubation temperature Prob>F D184 Polypropylene Salmonella 14028 4¡C < 0 .0001 D184 Polypropylene Salmonella 14028 22 ¡C n.s. D184 Polypropylene E.coli O157 :H7 4¡C "#$# D184 Polypropylene E.coli O157 :H7 22 ¡C 0.0551 D184 Polypropylene Salmonella cocktail 4¡C "#$# D184 Polypropylene Salmonella cocktail 22 ¡C < 0 .0001 D5431 Polypropylene Salmonella 14028 4¡C < 0 .0001 D5431 Polypropylene Salmonella 14028 22 ¡C < 0 .0001 D5431 Polypropylene E.coli O157 :H7 4¡C 0.0004 D5431 Polypropylene E.coli O157 :H7 22 ¡C < 0 .0001 D5431 Polypropylene Salmonella cocktail 4¡C < 0 .0001 D5431 Polypropylene Salmonella cocktail 22 ¡C < 0 .0001 M1555 Polypropylene Salmonella 14028 4¡C "#$# M1555 Polypropylene Salmonella 14028 22 ¡C 0.0049 M1555 Polypropylene E.coli O157 :H7 4¡C "#$# M1555 Polypropylene E.coli O157 :H7 22 ¡C < 0 .0001 M1555 Polypropylene Salmonella cocktail 4¡C "#$# M1555 Polypropylene Salmonella cocktail 22 ¡C < 0 .0001 MOL Polypropylene Salmonella 14028 4¡C < 0 .0001 MOL Polypropylene Salmonella 14028 22 ¡C < 0 .0001 MOL Polypropylene E.coli O157 :H7 4¡C < 0 .0001 MOL Polypropylene E.coli O157 :H7 22 ¡C "#$# MOL Polypropylene Salmonella cocktail 4¡C 0.0001 MOL Polypropylene Salmonella cocktail 22 ¡C < 0 .0001 S150 Polypropylene Salmonella 14028 4¡C n.s. S150 Polypropylene Salmonella 14028 22 ¡C n.s. S150 Polypropylene E.coli O157 :H7 4¡C 0.0273 S150 Polypropylene E.coli O157 :H7 22 ¡C 0.0486 S150 Polypropylene Salmonella cocktail 4¡C 0.0036 S150 Polypropylene Salmonella cocktail 22 ¡C 0.0003 D184 Polystyrene Salmonella 14028 4¡C "#$# D184 Polystyrene Salmonella 14028 22 ¡C "#$# D184 Polystyrene E.coli O157 :H7 4¡C "#$# D184 Polystyrene E.coli O157 :H7 22 ¡C "#$# D184 Polystyrene Salmonella cocktail 4¡C < 0 .0001 D184 Polystyrene Salmonella cocktail 22 ¡C < 0 .0001 D5431 Polystyrene Salmonella 14028 4¡C < 0 .0001 D5431 Polystyrene Salmonella 14028 22 ¡C 0.0006 D5431 Polystyrene E.coli O157 :H7 4¡C 0.0001 D5431 Polystyrene E.coli O157 :H7 22 ¡C n.s.

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D5431 Polystyrene Salmonella cocktail 4¡C < 0 .0001 D5431 Polystyrene Salmonella cocktail 22 ¡C < 0 .0001 M1555 Polystyrene Salmonella 14028 4¡C 0.0229 M1555 Polystyrene Salmonella 14028 22 ¡C < 0 .0001 M1555 Polystyrene E.coli O157 :H7 4¡C n.s. M1555 Polystyrene E.coli O157 :H7 22 ¡C n.s. M1555 Polystyrene Salmonella cocktail 4¡C 0.0184 M1555 Polystyrene Salmonella cocktail 22 ¡C < 0 .0001 MOL Polystyrene Salmonella 14028 4¡C < 0 .0001 MOL Polystyrene Salmonella 14028 22 ¡C 0.0078 MOL Polystyrene E.coli O157 :H7 4¡C n.s. MOL Polystyrene E.coli O157 :H7 22 ¡C n.s. MOL Polystyrene Salmonella cocktail 4¡C < 0 .0001 MOL Polystyrene Salmonella cocktail 22 ¡C < 0 .0001 S150 Polystyrene Salmonella 14028 4¡C 0.0418 S150 Polystyrene Salmonella 14028 22 ¡C n.s. S150 Polystyrene E.coli O157 :H7 4¡C n.s. S150 Polystyrene E.coli O157 :H7 22 ¡C n.s. S150 Polystyrene Salmonella cocktail 4¡C n.s. S150 Polystyrene Salmonella cocktail 22 ¡C 0.0183 Statistically significant effects (p<0.05) of the NO donor treatment s. n.s., not statistically significant biofilm dispersion. MOL, Molsidomine ; M1555 MAHMA NONOate ; S150 SPERMINE NONOate ; D5431 diethylamin e NONOate diethylammonium salt; D184 diethylamine NONOate sodium ; S8432 sulfo NONOate disodium salt.