University of Groningen
Coupled adhesion of bacteria to surfaces
Skogvold, Rebecca van der Westen
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2018
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Skogvold, R. V. D. W. (2018). Coupled adhesion of bacteria to surfaces. Rijksuniversiteit Groningen.
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C H A P T E R
6
GENERALDISCUSSION
Inthisthesis,focusisontheviscoelasticnatureoftheadhesivebondbetween(bio)colloids andsubstratumsurfaces,ormorespecificallyhowenvironmentalconditions(i.e.ionicstrengthand surfacehydrophobicity)influencethebondcharacteristics.Severalinstrumentswereusedtothisend. The quartz crystal microbalance (QCM), measures the changes in frequency and dissipation upon (bio)colloidaladhesiontodeterminetheviscoelasticpropertiesofthebond.Avectornetworkanalyzer basedQCM(VNA),whichholdstheabilitytochangethedrivingvoltageoftheresonancecrystal,was usedtoexaminewhetherbondscanberupturedandatwhatforce.Lastly,totalinternalreflection microscopy (TIRM) was employed for analysis of nanoͲscopic vibrations exhibited by bacteria with differentsurfaceappendages,inwhichtheelasticityofthebondisrelatedtothevibrations.
(Bio)colloidaladhesionisatopicthathasgainedalargeinterestoverthepastdecades1–3due
tothefactthatgainingknowledgeontheforcesresponsibleforadhesionandevenmoresoforparticle detachment from substratum surfaces is crucial in many biomedical, industrial and environmental processes. Several models4,5 have been developed to determine the bond strength between (bioͲ
)colloidsandsubstratumsurfaces.Todate,themostcommonphysicoͲchemicalapproachesemployed tostudy(bioͲ)colloidaladhesiontosubstratumsurfacesaresurfacethermodynamics6,7and(extended)
Derjaguin, Landau, Verwey and Overbeek (DLVO)Ͳtypes of analyses.8,9 Surface thermodynamics
comprisethecalculationoffreeenergiescalculatedbymeansofcontactanglesofsurfacesinorderto obtain interfacial free energies. These interfacial energies predict the outcome of adhesion. DLVO analysesincludeLifshitzͲVanderWaals,electrostaticandacidͲbaseinteractions.
(Bio)colloidaladhesionanalysisbymeansofQCMͲDwasexploredinchapter2.Thepositive frequencyshiftsaswellasthedissipationsshiftsretrievedfromtheexperimentswererelatedtothe viscoelasticity of the (bio)colloidalͲsubstratum bond according to a coupled resonator model.10 All
quantitativepropertiesoftheadhesivebondreportedinchapter2havebeenobtainedbyfittingthe springconstant(k),dragͲforce(ʇ),andthemassoftheparticle(mp).Thecoupledresonatormodelwas firstexploredbyDybwad10,11inthe1950stoexplainthepositivefrequencyshiftsexhibitedbycolloidal particles,anditwasconcludedthatcolloidaladhesionisacouplingbetweentwooscillatorsthrough verysmallcontactpoints,thusthefrequencyisnotonlydependentonthedegreeofadsorbedmass butalsoonthestiffnessofthebondbetweentheadsorbedmassandsurface.Chapter2focusesmostly onthepossibilitytoobtaintheviscoelasticparametersfor(bioͲ)colloidaladhesion,whichupuntilnow wasnotpossiblebymeansofQCMͲD,andinthepastonlythespringconstantcouldbedetermined.12 Goodqualitiesofthefitswereconfirmedbythelowrootmeansquaredeviation(RMSD).Valuesfork weremuchhigherforthebacteriaindicativeofmuchstiffercontactpoints,whichisnotunexpected since bacteria, especially those with surface tethers form stiff contact points followed by bond maturation,13causingalesserimpactfromthedragcoefficient.Oppositelyitwasobservedforsilica
particles which showed little influence from k but a large influence from ʇ. The masses calculated matchedverywelltheexpectedvalueforbacteria,albeitinthecaseofsilicaparticlestherewasaslight overͲestimation. This overͲestimation could be explained by reasoning that since it is known that viscoelasticityinfluencesthefrequencyshifts,thisfurtherinfluencesthemass.Morespecifically,the resonancefrequency(f)isgivenby14 ݂ ൌ ଶగଵ ට [1] inwhichitkisthespringconstantandmisthemass.Furthermore,itcanbeseenthatiftheresonance frequencydecreases,themassincreases,anddependingonhowthe(bio)colloidsadhereandinfluence thefrequencyshifts,itshowsthatthemassisaffectedsimultaneously.Alsowhatshouldbetakeninto considerationisthefactthatemployingthecoupledresonatormodel,the(bio)colloidsfunctionas resonatorsthemselves,andperhapsinthecaseforsilicaparticlestheydonotalwaysadherethrough specific contact points but instead move along with the movement of the crystal, leading to more negativefrequencyshiftsandthereforeinfluencethemassescalculated.
Chapter 3 also focused on the quantitative analyses of the physicalͲchemical bond characteristicsobtainedbymeansofthecoupledresonatormodel.Inadditionacomparisonwasmade basedonphenomenologicalKelvinͲVoigtorMaxwellmodelsandthepossiblerolewhichpolydispersity might have in the analysis of the QCMͲD response to adhesion of a fibrillated and nonͲfibrillated streptococcalstrainandabioticpolystyreneparticles.Itwasshownthatpolydispersityonlymadea differenceforthefittingofaMaxwellmodel.Moreover,itwasnotpossibleatthistimetoconclude whichmodelgavethebestoutputparameters,insteadthestudyshowedthatdependentonwhich model was used, different bond characteristics were obtained. The Maxwell model showed more emphasisontheelasticresponsethantheKelvinͲVoigtmodel,meaningsincespringanddashpotare putinseries,theelasticresponseintheMaxwellmodelactsindependentlyofdamping.Oppositely,in theKelvinͲVoigtmodel,thespringisplacedinparallelwiththedashpotandcontinuouslyopposesits responsethroughthedashpot.Moreover,itshouldalsobenotedthatwithregardsto(bio)colloidal adhesionitperhapsisnotsufficienttomodelthecontactpointsaseitheroneKelvinͲVoigtelementor oneMaxwellelementbutinsteadaseriesofeachoftheseelements.IfaMaxwellelementisputinto seriesthefirstinitialspringanddashpotwouldfunctionasthespringandtheremainingspringsand dashpotsinserieswouldthenfunctionasadashpotasawhole,basicallythesameprincipleasina KelvinͲVoigtmodel.Compared,ifaKelvinͲVoigtmodelwasputintoaseriestheinitialspringwouldbe themostdominantone,andremainingspringswouldthenhavelittleornoinfluence.Theseareall conceptsneededtobeexplored,albeitthisthesisisafirststepintoquantitativelyobtainingphysicoͲ
chemicalbondparametersfromtheQCMwithregardsto(bio)colloidaladhesion,andgaininginsight intothefactorswhichinfluenceinitialbacterialadhesion.Thetwofirstexperimentalchaptersofthis thesis(chapter2andchapter3)focusedonobtainingbondpropertiesfor(bio)colloidaladhesion.Yet afewthingshaveremainedunexplored.Adhesionofbacteriatodifferentsubstratumsurfaceswas donebyemployinganadhesionbuffer,yetthisdoesnotconstitutearealenvironmentforbacteria, becausethebacteriaemployedwereaccordinglynotphysiologicallyactiveinabsenceofnutrients.In ordertohavethemphysiologicallyactive,adhesionshouldbeperformedinagrowthmediumspecific forthesebacteria.Oneproblemarisesinthiscase,sincethenutrientspresentinthemediumwould also adsorb to the QCMͲD crystal surface thereby giving rise to molecular adsorption rather than colloidaladhesion.Inapreviousstudy15itwasshownthatbacteriaproducingextracellularpolymeric substances(EPS)adsorbdirectlyonthecrystal,accordingtotheconventionalmassͲloadingtheory16 ratherthanviathecoupledresonancetheoryasdescribedaboveforcolloids.Inordertomakeupfor thatOlssonetal.15adheredEPSproducingbacteriainoneexperimentandnonͲEPSproducingbacteria inanother.FromtheobtainedchangesinfrequencyanddissipationtheycouldsubtractQCMͲDsignals fromeachotherandobtainthecorrectchangesinfrequencyanddissipation.Inasimilarmanner,to accountforphysiologicallyactivebacteriainanutrientͲrichsolution,itcouldbeenvisagedtoadhere bacteriafrommediumbymeansofQCMͲDtoobtainthechangesinfrequencyanddissipation,andin anextseriesofexperimentsallowbacteriatoadherefromabufferoronlyadsorptionof medium components. By subtracting the changes in frequency and dissipation in different experiments, changes in frequency and dissipation for bacteria adhering in their natural environment, including effectsoftheirphysiologicalactivitycanbeobtained. Inaprecedingstudyitwasinvestigatedthatforbacteriathevibrationalamplitudesordistance variationsparalleltoasurfacedecreasedwithincreasingadhesionforcesactingperpendiculartothe surface.5Meaningthatbyinvestigatingthevibrationalamplitudesof(bio)colloidswouldalsocreatea deeperunderstandingofthebindingmechanismsandhowtheseareinfluenced.Moreoverthestudy confirmedthatfor(bio)colloidstheadhesiontoasubstratumsurfaceinvolvedmultiplebindingtethers whichdetachedandreͲattachedtoasurface,leadingtoanirreversibleadhesion.Inspiredbythisnew knowledgeonhowbacteriaadhereandmoveparalleltoagivensubstratumsurface,knowledgeon howtheymoveperpendiculartoasurfacewaspursued.Therefore,inchapter4,bymeansofTIRM having a penetration depth of approximately 200 nm, it was studied how two different strains of streptococci interact with a substratum surface or more specifically, how their outermost surface wouldinteractwithasubstratumsurface.Inchapter4twotypesofsubstratumsurfaces,hydrophobic andhydrophilicwereemployedatvariousionicstrengthstocontrolthelevelofbacterialadhesion. Bacteriacontainingfibrillarsurfacetethersadheredtoasubstratumsurfaceinanirreversiblefashion bytetherͲcouplingtothesurface.Bacteria,knownashavingamore“bald”surface(nodemonstrable
fibrillarsurfacetethers),adhereddifferently;theyabidebyDLVOͲtypesofanalysis,wherethebacteria reside in a secondary energy minimum. Accordingly, a distinction between “tetherͲcoupled” and “floating”adhesionwasmade,whichpresentsanewwaytolookathowbacteriaadheretoasurface. Although novel insight is obtained on the adhesion mechanisms, several questions arise when analyzing the vibrational amplitudes for the fibrillar and nonͲfibrillar streptococcal strains perpendicular to the surface. Most notably is the fact that the largest population of the adhering fibrillarbacteriaonlymovearound5Ͳ10nmawayfromthesurfaceirrespectiveofionicstrengthofthe bufferandsubstratumsurfacehydrophobicity.Thisisoppositetowhathasbeenfoundforbacteria through parallel movement, moving around 100Ͳ200 nm over a certain time period and what was confirmed by in silico modeling.5,12 Accordingly the question arises, what makes parallel versus
perpendicularmovement/measurementofadhesionsodifferent.Itisnotexpectedthatbacteriawill moveasmuchintheperpendiculardirectionespeciallynotwhentetherͲboundascomparedtoina parallel direction, since bacteria move over a larger distance parallel to a surface than secondary minimum or tetherͲextension will allow, till they have found a highͲaffinity spot where they will permanently attach.17 It has been postulated that the penetration depth plays a vital role in TIRM
monitoringtheperpendicularBrownianmotiondisplayedby(bio)colloids,mostlybecauseTIRMhasa small penetration depth and therefore when monitoring the adhesion it is no longer longͲrange LifshitzͲVanderWaalsforcesthatareinplaybutinsteadshorterͲrangedattractiveforces.Thuswhat isthenobservedinTIRMarethebacteriathatarestronglyadheringascomparedtomonitoringwith a bright field camera. Although this is a valid point, it may not be sufficient to explain the smaller vibrational amplitudes obtained in chapter 4. As calculated by Sjollema et al.,5 one way to more
preciselydeterminehowbacteriamoveaccordingtoasurfaceisbycalculatingtheirmeansquared
perpendiculardisplacementasfunctionoftime.
Figure 1. Mean Squared perpendicular Displacement (MSD) as a function of time for two different
bacterialstrains,S.salivariusHBC12andS.salivariusHB7.Thecoloredlinesarefortheadhesionat57 mMandthedottedlinesfortheadhesionat0.57mM. InFigure1itcanbeseenthatforS.salivariusHB7,thevariationsovertimearearound10nm, confirmingthatforfibrillarbacteriamovementperpendiculartothesurfaceisverylimited.Oddly,it seemsthatfortheadhesioninahighionicbuffersolution(57mM)adheringbacteriakeepmoving muchmore,andnotreallyreachingaplateau.Thiscouldbeduetothecompressionoffibrils,making themactlikemoreunsteadysprings,thereby causing aconstantmovement ofthebacteria.ForS.
salivariusHBC12,theMSDisquitestable,afterthefirst5saplateauisreached.Thevariationsover time are around 30 nm which is also what has been deduced from calculating the vibrational amplitude.
Since chapter 4 gave insight into more detail about the physicoͲchemical properties of two differentstrainsofbacteria,combiningthisknowledgewiththephysicoͲchemicalpropertiesobtained fromtheQCMͲD,explorationintomoredetailregarding(bio)colloidalbindingispivotal. Inchapter5avectornetworkanalyzerͲbasedQCMwasemployedwhereitwaspossibletovary thedrivingvoltagesfrom0.01Vto0.4V.Moreover,itwaspossibletocalculatetheoscillationforce exertedontheadhering(bio)colloidsofferingthepossibilitytomeasuretheforceneededtoprevent (bio)colloidaladhesionortocausedetachmentfromdifferentcrystalsurfaces.Unfortunatelywithin the range of oscillation forces possible with the instrument, it was not feasible to observe any detachmentfromthesurfaceforthefibrillarstreptococcalstrain.Thiswasnotentirelyunexpected since the bacteria are let to adhere for an hour before applying an oscillation force, leading to a prolongedresidencetimeonthesurfacecausingastrongerbond.18Althoughashorteradhesiontime
could increase the chances of fibrillar bacteria to detach, it might not be sufficient to cause detachment.OtherfactorssuchascrystalresonancefrequenciesaswellasQCMqualityfactorsshould ^͘ƐĂůŝǀĂƌŝƵƐ HBC12 57mM ^͘ƐĂůŝǀĂƌŝƵƐ HB7 0.57mM 57mM0.57mM
6
beincreased.19Itwasbelieved,bychangingtheviscosityofthesystem,i.e.subjectingthe(bioͲ)colloids
to a glycerol solution, causing faster dampening of the oscillation amplitudes, the Q factors might increase,therebyincreasingtheoscillationforce(seeEq.2) ܨ௦ିൌ ଵఘோయሺொሻమబమ ହඥሺொሻమାସோమ [2] ThiswasevaluatedforthehydrophilicSAMͲcoatedcrystalswheretheglycerolconcentration waschangedfrom10%to50%,andunfortunatelynoincreaseinQwasobserved(seeTable1),but instead a systematic decrease. This possibly indicates that the glycerol adjacent to the oscillating crystalmovesalongwiththecrystal.
Table1Qualityfactors,QfortheVNAͲbasedQCMforhydrophilicSAMͲcoatedcrystalsatsixdifferent
overtones submerged in different glycerol concentrations. Data represent averages ± standard deviationsovertriplicateexperimentswithseparatecrystals.
Overtone (n)
HydrophilicSAM
50%glycerol 20%glycerol 10%glycerol
1 1174±10 2416±40 2830±22 3 2051±19 4298±157 4972±13 5 2713±56 5171±278 6353±32 7 3062±178 6511±51 7733±17 9 3465±326 7423±17 8686±67 11 3864±273 8547±54 9103±211 Nextstepwouldbetochangethecrystaldimensionssuchthattheirresonancefrequencies(f0) wouldbehigher.Anotherinterestingpartinregardstopreventingadhesionorcausingdetachmentof (bio)colloidsfromasurfacewouldbetoevaluatethesizeorthesizedistributionofbacteria.Most bacteria have radii of around 500 nm, but rodͲshaped bacteria have different dimensions. This differenceindimensionwouldbeinterestingtoevaluatewiththeVNAͲbasedQCM,sinceEq.2also
statesthattheradius(R)playsacrucialrole.UseofrodͲshapedcoliformbacteriasuchasEscherichia
coliwouldofferinsightintotheroleofshapeonadhesionofbacteriatosubstratumsurfaces.
CONCLUSION
ThisthesisdemonstratesthatanalysisofthebacteriumͲsubstratuminterfacewithregardsto their viscoelastic properties offers novel and pivotal insight into the mechanisms governing initial bacterialadhesion.Moreover,theuseofseveraltypesofinstrumentsinthisthesishashelpedgain insight into the bond properties of (bio)colloids to different types of substratum surfaces, which strengthens the understanding of the adhesive bond stiffness as well as their perseverance of adhesion.Coupledadhesionof(bio)colloidstosurfacesiscomplexandthereforeneedstobeexplored further,preferablyassessedwithmultiplenonͲinvasivetechniques.
REFERENCES
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Summary
S U M M A R Y
SUMMARY
Bacterialadhesionoccursonessentiallyallnatural,aswellmanͲmadesurfacesandposesa majorconcerninengineeringandmedicine.Controllinginitialbacterialadhesionisofkeyimportance to prevent later problems after bacteria have grown into e.g. pathogenic biofilms on biomaterials implantsanddevicesthatposeathreattohumanhealth.
Chapter1givesabriefoverviewofbacterialadhesionphenomena,aswellasofthemethods
employed in this thesis to study adhesion. Methods are explained and the significance to study bacteriumͲsubstratumbondsareexplained,givingrisetotheaimofthisthesis,whichistogaininsight intohowtheviscoelasticityofthebondbetween(bio)colloidsandasubstratumsurfacedependon environmentalconditions,likeionicstrengthofthesurroundingfluid,substratumhydrophobicityand absenceorpresenceofbindingtethersonabacterialcellsurface. Thebondbetweena(bio)colloidandasubstratumsurfaceisnotrigidaspreviouslythought, butinstead,iscurrentlyconsideredtobeviscoelastic.
Based on this consideration, chapter 2 focuses on the quantitative derivation of the viscoelasticbondparametersusingQCMͲDonbacteriaandsilicaparticlesadheringtoQCMͲDcrystal surfaces.Thequartzcrystalmicrobalancewithdissipation(QCMͲD)hasbecomeapowerfultoolfor studying the bond viscoelasticity of biotic and abiotic colloidal particles adhering to substratum surfaces. A windowͲequipped QCMͲD allows highͲthroughput analysis of the average bond viscoelasticity,measuringover106particlessimultaneouslyinonesingleexperiment.Othertechniques
require laborious analyses of individual particles. In the protocol described in chapter 2, the quantitative derivation of the springͲconstant and dragͲcoefficient of the bond between adhering colloidalparticlesandsubstratumsurfacesusingQCMͲDisexplainedforbacteriaandsilicaparticles, using the particleͲmass derived for validation. Bond viscoelasticity is calculated using a coupled resonatormodel,payingspecialattentiontotheprotocolformathematicalfittingneededtoobtain reliablequantitativeoutput.Knowledgeoftheviscoelasticityofthebondbetweencolloidalparticles andsubstratumsurfacesfacilitatesdevelopmentofnewstrategiestodetachadheringparticlesfrom orretainthemonasurface. Next,inchapter3thebondbetweentwohydrophilicstreptococcalstrainswith91nmlong andwithoutfibrillarsurfaceappendagesandmicronͲsizedhydrophobicpolystyreneparticlesonQCMͲ Dcrystalsurfaceswithdifferenthydrophobicities,wereanalyzedemployingbothaKelvinͲVoigtanda Maxwell model. A Poisson distribution was implemented in order to determine possible virtues of includingpolydispersitywhenfittingmodelparameterstothedata.Qualityofthefitsdidnotindicate whetheraKelvinͲVoigtorMaxwellmodelispreferentialandonlypolydispersityinspringͲconstants improvedthefitforpolystyreneparticles.KelvinͲVoigtandMaxwellmodelsbothyieldedhigherspring constantsforthebaldstreptococcusthanforthefibrillatedone.Inbothmodels,thedragcoefficients
increased for the bald streptococcus with the ratio of electronͲdonating over electronͲaccepting parametersofthecrystalsurface,whileforthefibrillatedstrainthedragcoefficientwassimilaronall crystalsurfaces.Combinedwiththepropensityoffibrillatedstreptococcitobindtothesensorcrystal asacoupledresonatorabovethecrystalsurface,thissuggeststhatthedragexperiencedbyresonatorͲ coupled,hydrophilicparticlesismoreinfluencedbytheviscosityofthebulkwaterthanbyinterfacial wateradjacenttothecrystalsurface.HydrophilicparticlesthatlacksurfacetethersaremassͲcoupled justabovethecrystalsurfaceandaccordinglyprobeadragduethethinlayerofinterfacialwaterthat is differently structured on hydrophobic and hydrophilic surfaces. Hydrophobic particles without surfacetethersarealsomassͲcoupled,buttheirdragcoefficientdecreaseswhentheratioofelectronͲ donating over electronͲaccepting parameters increases, suggesting that hydrophobic particles experiencelessdragbystructuredwateradjacenttoasurface.
Chapters 4 and 5 further advance a new model recently proposed (Sjollema J. et al.,
DetachmentandsuccessivereͲattachmentofmultiple,reversiblyͲbindingtethersresultinirreversible bacterialadhesiontosurfaces.ScientificReports,2017,7:4369)whichdescribesbacterialadhesionas
aresultoftetherͲbindingtoasurface,basedonTotalInternalReflectionMicroscopy(TIRM)andQCM, respectively.Currentmodelsforbacterialadhesiontosubstratumsurfacesallincludeuncertaintywith respect to the (ir)reversibility of adhesion. In a model, based on vibrations exhibited by adhering bacteria parallel to a surface, adhesion was described as a result of reversible binding of multiple bacterialtethersthatdetachfromandsuccessivelyreͲattachtoasurface,eventuallymakingbacterial adhesion irreversible. Here, we use TIRM to determine whether adhering bacteria also exhibit variationsovertimeintheirperpendiculardistanceabovesurfaces.Streptococciwithfibrillarsurface tethers showed perpendicular vibrations with amplitudes of around 5 nm, regardless of surface hydrophobicity.Adhering,nonͲfibrillatedstreptococcivibratedwithamplitudesaround20nmabove ahydrophobicsurface.Amplitudesdidnotdependonionicstrengthforeitherstrain.Calculationsof bacterialenergiesfromtheirdistancesabovethesurfacesusingtheBoltzmanequationshowedthat bacteriawithfibrillartethersvibratedasaharmonicoscillator.Theenergyofbacteriawithoutfibrillar tethers varied with distance in a comparable fashion as the DLVO (Derjaguin, Landau, Verwey and Overbeek)Ͳinteractionenergy.Distancevariationsabovethesurfaceovertimeofbacteriawithfibrillar tethersaresuggestedtobegovernedbytheharmonicoscillations,allowedbyelasticityofthetethers, piercingthroughthepotentialenergybarrier.Bacteriawithoutfibrillartethers“float”aboveasurface inthesecondaryenergyminimum,withtheirperpendiculardisplacementrestrictedbytheirthermal energy and the width of the secondary minimum. The distinction between “tetherͲcoupled” and “floating”adhesionisnew,andmayhaveimplicationsforbacterialdetachmentstrategies.
Forces required to detach adhering bacteria from substratum surfaces are not trivial to measureandhenceinchapter5itwasattemptedtousetheQCMtothisend.Variationofthedriving
voltage and crystal oscillation amplitude in QCM has seldom been explored but offers interesting possibilities to measure (bio)colloidal particle adhesion forces and characteristics of the bond, as achievedduringdifferentdevelopmenthistories.Tothisend,webuiltamodifiedQCM,referredtoas avectornetworkanalyzer(VNA)whichallowstovarytheQCMdrivingvoltagesfrom0.01Vto0.4V. QualityfactorsQoftheVNAͲbasedQCM,usingthesamecrystalsandchamberastheQͲsense,were foundcomparablewiththoseoftheQͲsenseQCMͲDandwereusedtocalculatetheoscillationforces exertedonthe(bio)colloidalparticlesduringadhesiontoanoscillatingcrystalintheVNAͲbasedQCM. Forcestoprevent(bio)colloidalparticleadhesionrangedfrom0.2pNto30pN,whileforcesrequired todetachadheringparticleswerehigherandrangedfrom2pNto30pN.Althoughtheseforcesare ordersofmagnitudesmaller than generallyderived fromatomic forcemicroscopy,theyareofthe same order of magnitude as obtained using optical tweezers and flow displacement systems. This negatesoftenvoicedcriticismonQCMdatathatitshighoscillationfrequencyinfluences(bio)colloidal particleadhesion.However,bondcharacteristics,derivedinacoupledresonatormodelbasedonthe routineQCMoutputanduseofaphenomenologicalKelvinͲVoigtmodel,variedwiththeoscillation forces applied during adhesion. Importantly, this chapter adds a simple, and easy to interpret instrumenttomeasure(bio)colloidaladhesionforces,thatcoincidegenerallywellwithforcevalues obtainedusingopticaltweezersandflowdisplacementsystems,butnotwithforcesobtainedusing atomicforcemicroscopy.Therewith,thechapterhelpssolvethequestionwhetherbacterialadhesion forcesareinthenNͲrange(atomicforcemicroscopy)orpNͲrange(QCM,opticaltweezersandflow displacementsystems). Inchapter6,thegeneraldiscussionofthisthesis,criticalpointsarehighlightedwithrespect to the different chapters, including suggestions for future experiments like carrying out QCM experiments with bacteria in growth medium. These critical points raised in combination with the thesis as a whole, may help strengthening current understanding of the adhesive bond between (bio)colloidandsubstratumsurfaces.
6
Samenvatting
S A M E N V A T T I N G
SAMENVATTING
Bacteriëlehechtingkomtveelvooropnatuurlijkeenartificiëleoppervlakkenenveroorzaakt een groot probleem binnen talrijke industriële en geneeskundige processen. Het beperken van de initiëlehechtingvanbacteriënisvancruciaalbelanginhetvoorkomenvandezeproblemen,wanneer bacteriënzijngegroeidtoteenpathogenebiofilm,zoalsopbiomedischeimplantatenenapparatuur.
Hoofdstuk1geefteenkortoverzichtoverbacteriëlehechtingsmechanismenende
methoden die in deze thesis gebruikt zijn om hechting te bestuderen. De methoden worden onderbouwdenhetbelangvandebacterieͲsubstraatinteractiewordtuiteengezet,welkeleidentot de doelstelling van deze thesis, namelijk het verkrijgen van inzicht in de viscoͲelastische interactie tussen(bio)colloïdaledeeltjeseneenoppervlakonderverschillendeionsterktenenopmaterialenmet verschillendehydrofobiciteit.
Hoofdstuk2richtzichopdekwantificeringvanviscoͲelastischebindingendoormiddelvan
QCMͲD analyse van gehechte bacteriën en silicadeeltjes. Een QCMͲD meetkamer met een inkijk venster,maakteenanalysevandegemiddeldeviscoͲelasticiteitvandebindingenmethogeefficiëntie mogelijk, door het meten van meerdere miljoenen deeltjes tegelijkertijd in een enkel experiment. Andere technieken vereisen veel tijd doordat ze individuele deeltjes bemeten. In het protocol beschreven in hoofdstuk 2 wordt de kwantitatieve afleiding van de veerconstante en de frictiecoëfficiënt gebruikt om de binding tussen gehechte colloïdale deeltjes en oppervlakken, gebruikmakendvandeQCMͲD,besproken.Deafgeleidemassavandedeeltjeswordthierbijgebruikt ter validatie. Berekeningen zijn gebaseerd op het resonantie model voor twee gekoppelde resonatoren.
Vervolgens wordt in hoofdstuk 3 de binding tussen twee hydrofiele streptokokken met en zonder91nmlangefibrillenenhydrofobepolystyreendeeltjesopQCMͲDkristaloppervlakkenmet verschillende hydrofobiciteiten bestudeerd, middels een KelvinͲVoigt en een Maxwell model voor gekoppelderesonatoren.EenPoissonverdelingwerdgeïmplementeerdomdemogelijkeinvloedvan polydispersiteit te onderzoeken. De kwaliteit van de fit was voor het KelvinͲVoigt en het Maxwell modelgelijk,terwijlalleenhettoevoegenvanpolydispersiteitindeveerconstantevoordepolystyreen deeltjestoteenbeterfitleidde.ZowelinhetKelvinͲVoigtmodelalsookinhetMaxwellmodel,hadden de kale streptokokken een hogere veerconstante dan de gefibrilleerde variant. In beide modellen verhoogdedefrictiecoëfficiëntvandekalestreptokokkenwanneerderatiovanelektronͲdonerende enelektronͲaccepterendeparametersvanhetkristaloppervlakhogerwas,terwijlbijdegefibrilleerde streptokokkendefrictiecoëfficiëntopallekristaloppervlakkengelijkwas.Ditsuggereertdatdefrictie ervaren door de gefibrilleerde streptokokken meer beïnvloed wordt door de viscositeit van het omgevingswaterdandoordegebondenwaterlaagaanhetkristaloppervlak.Hydrofielestreptokokken, zonderfibrillenwordenvlakbovenhetkristaloppervlakgebondenenvoelendaardoordefrictievan
de dunne laag water in het raakvlak, welke een verschillende structuur heeft op hydrofobe en hydrofieleoppervlakken.
Indehoofdstukken4en5wordteennieuwmodelvoorbacteriëlehechtingverderuitgewerkt (SjollemaJ.etal.,DetachmentandsuccessivereͲattachmentofmultiple,reversiblyͲbindingtethers result in irreversible bacterial adhesion to surfaces. Scientific Reports, 2017, 7:4369) en beschrijft bacteriëlehechtingaanhetoppervlakalsgevolgvan“ankerverbindingen”zoalsfibrillen,gebaseerdop TotaleInterneReflectieMicroscopie(TIRM)enQCM.Huidigemodellenvoorbacteriehechtingaande oppervlakkenvansubstratenbevattenonzekerheidvoorwatbetreftvande(on)omkeerbaarheidvan de hechting. In een model gebaseerd op de vibraties van hechtende bacteriën parallel aan het oppervlak, kan deze hechting omschreven worden als het resultaat van reversibele bindingen van meerderebacteriëleankerverbindingen,diekunnenlosrakenenooksuccesvolopnieuwhechtenmaar nooitallemaaltegelijkloslatenvaneenoppervlak,watuiteindelijkleidttotonomkeerbarebacteriële hechting.HierhebbenweTIRMgebruiktomtebepalenofdehechtendebacteriënookvariatieslaten zieninhunafstandloodrechtboveneenoppervlak.Streptokokkenmetfibrillenalsankerverbindingen latenloodrechtevibratieszienmetamplitudenvanongeveer5nm,ongeachtdehydrofobiciteitvan hetoppervlak.Hechtendekalestreptokokkenvibreerdenmetamplitudenvanongeveer20nmboven eenhydrofooboppervlak.Deamplitudenwarennietafhankelijkvandeionsterkte.Berekeningenvan de bacteriële hechtingsenergie gebaseerd op de afstand tot het oppervlak middels de Boltzman vergelijking,toondenaandatbacteriënmetfibrillenalsankerverbindingopeenzelfdemaniervibreren alseenharmonischeoscillator.Dehechtingsenergievanbacteriënzonderfibrillenvarieerdemetde afstand op een vergelijkbare manier als beschreven in DLVO (Derjaguin, Landau, Verwey and Overbeek)Ͳinteractieenergie.Erwordtgesuggereerddatdeverschilleninafstandbovenhetoppervlak indetijdvoorbacteriënmetfibrillenalsankerverbindingwordengereguleerddoordeharmonische oscillaties,eeneigenschapmogelijkdoordeelasticiteitvandeankerverbindingen.Bacteriënzonder ankerverbindingen zweven als het ware boven het oppervlak in het secundaire DLVO energie minimum, waarbij hun loodrechte verplaatsing beperkt wordt door de thermische energie van de bacterie en de breedte van het secundaire minimum. Het onderscheid tussen “gekoppelde” en “zwevende” hechting is nieuw en kan belangrijke gevolgen hebben voor bacteriële verwijderingsstrategieën.
De krachten benodigd om gehechte bacteriën te verwijderen van oppervlakken zijn niet gemakkelijktemetenenomdieredenwerdinhoofdstuk5deQCMgebruiktomditprobleemaante pakken.DevariatieinhetaandrijvingsvoltageendeamplitudevandekristaloscillatieinQCMisnog maar zelden onderzocht, maar biedt interessante mogelijkheden voor het meten van de hechtingskrachtenvan(bio)colloïdaledeeltjesendekarakteristiekenvandeverbindingen.Hiervoor werdereengemodificeerdeQCMgebouwd(devectornetwerkanalysator(VNA)),welkehetmogelijk
maakthetaandrijvingsvoltagetevariërentussen0.01Ven0.4V.DekwaliteitsparametersQvande VNAͲgebaseerdeQCM,gebruikmakendvanhetzelfdekristalenmeetkameralsindeQͲsense,waren vergelijkbaarmetdievandeQͲsenseQCMͲDenwerdengebruiktvoordeberekeningvandeoscillatie krachtenuitgeoefendopde(bio)colloïdaledeeltjesgedurendehechtingaaneenoscillerendkristalin de VNAͲgebaseerde QCM. Krachten benodigd om de hechting van (bio)colloïdale deeltjes te voorkomen varieerde van 0.2 pN tot 30 pN, terwijl krachten benodigd om gehechte deeltjes te verwijderenhogerwaren,variërendvan2pNtot30pN.Alhoeweldezekrachtenordenvangrootte lageruitvallendangemetenmetatomairekrachtmicroscopie,zijnzeindezelfdeordevangrootteals gemetenwordenmetoptischepincettenofstroomkamersvoorbacteriëlehechting.Ditweerlegtde vaak gehoorde kritiek op QCM gegevens dat de hoge oscillatie frequentie de (bio)colloïdale deeltjeshechtingbeïnvloedt.Debindingskarakteristieken,afgeleidineengekoppeldresonantiemodel gebaseerdopdestandaardQCMoutputenhetgebruikvaneenfenomenologischKelvinͲVoigtmodel, varieerden met de oscillatiekrachten toegepast gedurende de hechting. Dit hoofdstuk voegt een simpeleengemakkelijkteinterpretereninstrumenttoeaandemethodendiegebruiktwordenomde hechtingskrachten van (bio)colloïdale deeltjes aan oppervlakken te meten, en geeft resultaten die goed overeenkomen met de krachten gemeten met optische pincetten en stroomkamers voor bacteriële hechting. Daarmee helpt dit hoofdstuk de vraag te beantwoorden of bacteriële hechtingskrachten zich in het nNͲbereik (atomaire krachtmicroscopie) of in het pNͲbereik (QCM, optischepincettenenstroomkamersvoorbacteriëlehechting)bevinden.
In hoofdstuk6,dealgemenediscussie vandezethesis, worden belangrijke kanttekeningen geplaatstmetbetrekkingtotdeverschillendehoofdstukken,waarondersuggestiesvoortoekomstige experimentenzoalshetuitvoerenvanQCMproevenmetbacteriëningroeimedium.
Acknowledgements
ACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS
InthecourseofthepastfiveyearsofmelivinginGroningenIhavehadthepleasuretomeet so many wonderful people, many of which have become very close friends of mine. When I first embarkedonmyjourneytoGroningenIdidnotexpectmylifetochangeinsomanyways.Ihave learnedthatevenwhenyouarechallengedwiththebiggestobstacleofyourlifeyoujusthavetolook aroundandtherearehelpinghandseverywhere.ForthatIamforevergrateful! Sowheredoesonestartwhenwritinganacknowledgment,whomistobementionedfirst, whatarethenorms?!And(yes,Istartedasentencewith“and”,Icandothat,Iamadoctornow!),how doesonenotforgettomentionsomeone?!Butjusttoclarify,theorderofpeoplementionedsays nothingaboutimportance;youareallspecialtome. Soheregoes, DearHenk,IamforevergratefulforyourguidancethroughoutmyPhD,andevenmoresoin thelastfewmonths.Wehavenotalwaysseeneyetoeye,andIapparentlyalwaysmadeyoudizzywith mytablesandgraphs.Inspiteofthat,wehavesharedmanyinterestingscientificaswellaspolitical discussions,fromwhichIhavelearnedagreatdeal.Iwishyouallthebestonallyourfutureendeavors, andwherevertheymaytakeyou,youwillbeaninspiration! Henny,dearHenny,Ihavesomanythingstosaytoyou(onlygood,obviously!),butsolittle spacetosayitin.Youareanamazingprofessor,scientist,lecturer,andmentor!Iamtrulygratefulfor alltheconversations,bothscientificandpersonalwehaveshared,bothatwork,atconferencesand duringourroadtriptoDenmark.Youhavebeenagreatinspirationtome! DearPrashant,firstof,thankyouforwantingtotakeonanotherScandinavianstudent,despite thefactthatIhadlessofabeardthanthepreviousone!Iamthankfulfortheopportunityyouhave givenme,andappreciateallyourhelpduringmyPhD. Tothereadingcommittee,Prof.FrederikHook,Prof.MarkvanLoosdrechtandProf.YijinRen, Iamgratefulthatyouagreedonbeingpartofmyreadingcommittee.Thankyou! Jelmer,youhavebeenagreathelpwithmythesistheselastfewmonths,andIamgrateful thatyoualwaystookyourtimetohelpmeout,nomatterhowbusyyouwere.Thenagain,Iwouldnot leaveyourofficeuntilyoudidhelpme,soyouwereprobablyforced.Eitherway,Iamthankful.
Hans Kaper, thank you for teaching me how to use the VNAͲbased QCM, I know it was
sometimesfrustrating,butIamveryappreciativeofthetimeyoutooktofixitwhenneeded. Robert,howdoIstartthankyou?!Westartedcollaborationmorethantwoyearsago,and sincethenyouhaveevolvedfrombeingacolleaguetoafriend.Youhavealwaysbeensopatientwith meandIamthankfulforthat.Youtaughtmesomuchaboutmicroscopeobjectives,andmicroscopes thatIfeelIcanstartworkingforNikon!Thankyouforwhoyouare! ACKNOWLEDGMENTS
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To the awesome team of technicians at the BME, Gezinda, Willy, Betsy, Melissa, Willem, Minie,Rene,Jelly,Ed,andMarja,youhaveallbeensohelpfulthroughallmyyearsatthedepartment, andIhavesharedmanygoodtimesworkingwithyouinthelab,thankyousomuchforthat!Ed,an extrathankyouforallyoursupportandcomfortingwordsthroughoutmyPhD,Iwillneverforgetit! Joop,aspecialthankyoutoyouaswellforalwayshelpingme,nomattertheissueathand,Icannot thankyouenough. Ina,wordscannotexpresshowmuchIhavelovedallofourconversationsinyouroffice.You havealwaysbeensosupportive,kindandhelpfultome.Thankyouforeverything! Wya,thankyouforalwaysbrighteningupthedaywithyourwitandsarcasm.
Patrick, thank you for always being willing to listen to me every time I passed your office, havingbothpersonalandscientificquestions!Iadmireyouasapersonaswellasascientistandasa supervisor. TomyfellowPhDcolleaguesatBME(oldandnew!),somanynewstudentshavearrivedat ourdepartmentandmadeitamulticulturalenvironment.IsometimesIfeellikeIaminairportwith allthedifferentculturesaround,butthisonlymakesourdepartmentextraspecial.Iwillnottryto nameallofyou,sinceIamworriedIwillforgetone,sothiswayismywayofthankingallofyou!Good lucktoeveryoneofyou! Brandon,aspecialthankyouforwelcomingmeintothedepartmentwhenIfirstmovedto Groningen.Yourdoorwasalwaysopenforachatoradvice,thankyousomuchforthat!Iamsure whereverweareinthefuturewewillalwaysstayintouch. HansDeRaedt(andEdderkop!),IhavenowordstotellyouhowmuchIappreciateyoucoming intomylife.Westartedoffascollaborators(wellmorespecifically,Ineededyourscientifichelp),but this collaboration turned into a friendship! I would share all aspects of my personal life as well as academiclifewithyou,andyouwouldalwayslistenandgivemeadvice,mostlywithadoseofhumor attached.IamforevergratefulthatIhadthechancetomeetsomeonelikeyou,andIhopethatno matterwherewebothareinthefuture;wewillstillstayintouch!Mysincerestaffectionstoyouand Kristel! Vera,nooffensetoanyone,butyoudogetaprivatethankyousinceyouandIarepractically oneoftheeldestonesinthedepartment(IdonotmeanageͲwisebuttimewise,justtobeclear!).I haveenjoyedourmanylaughsandprivateaswellasscientificconversations,thankyouforthat! Rene,notsurehowIwouldstarttothankyouforbeingmycolleagueattheBME,allIcansay is,Ihaveenjoyedeveryconversationwehaveeverhad(althoughIamtheonewhoprobablydidmost ofthetalking),andthedepartmentwouldnothavebeenthesameifyouhadneverstartedworking there.Sothankyouforallthediscussionsandrantswehavesharedoverthelastfiveyears.Andas youwouldsaytomenow:Leukverhaal,lekkerkort! ACKNOWLEDGMENTS
Sara,mysecondandbestofficemate,aswellasmydearestfriend!Ihavemissedyousomuch sinceyouleftthedepartment,butluckilywealwaysmanagedtostayintouchandbepartofeach other’slives.Imissyoutremendously,butIknowthatinthefuturewewillhavemoretimetogether! Youaresomuchmorethanafriendtome,youaremyfamily! Jesse,youweremyfirstfriendattheBME,myfirstfriendinGroningen,andafterfiveyearsI amproudtosaythatyouarestillmyfriend!YouwereamentortomeinthebeginningofmyPhDand youalwayssupportedmewhenIsometimesfoundithardtobeawayfromhome.Youareanincredible womanandIthankwhateverhigherpowerthereis,forsendingyouintomylife!You,MaxandPieter holdaspecialplaceinmyheart. Barbara,allIcansayishowluckyIamtoknowsomeoneasspecialasyou!Iamproudtocall youmyfriend!Youareextraordinaryandsogratefulthatourpathshavecrossed!Ithinkperhapswe canthankJanforthat!SothankyouJanforBarbaraaswellasbeinganawesomeofficemate! Gulcan,mysearbebisim!Youcametoworkatthedepartment,anditwasadorationatfirst sight!Youbecamesuchaclosefriendofmine,andIamsohappytohavemetsomeonelikeyou!I hopeourpathswillsooncrossagain!Lotsofloveforyou,butalsoformybabyRuzgar. Katia,mysister,myfriend,myparanimf!WheredoIbegin…Youaresuchanamazingperson andwithoutyoumylifewouldnotbefull!Wehaveknowneachotherforyears,butsincethemoment wemetwehadthisinstantconnection,andyouunderstoodme!Ihavewatchedyougrowasawoman as well as becoming an entrepreneur with your own company, and what you have accomplished alreadyisamazing!Wewillalwaysbefriends! Corka,mydeardearCorka,whatawoman!Youcameintomylifealittlelessthantwoyears ago,andwhatanimpactyouhavealreadymade.Veryfewpeoplehavethateffectonme,butyou did/do!HowIadmireyou!Iamgratefultocallyoumyfriend,andIwillalwaysbehere,justtheway thatIknowyouwillalwaysbethereforme! Laetitia,(alsofellowPhDsurvivoraswellasparanimf).Thankyousomuchforbeinghereto supportmeonmybigday.Youalwaysmanagetostaycoolinwhateversituationyouarein,andI admireyouforthat!Moreover,havingyoubymysideonthisdaywillkeepmecalmandcollected,not becauseyouarecalm,butbecauseIamscaredyouwillotherwisebemeantome.No,jokeaside,you aretrulyanamazingpersonandIamsoverygratefultohavehadthechancetomeetsomeonelike you! Sonia,Julia,Geraldine,Aline,Seargeoh,Ivy,howIjustadoreyouguys!HowcanIbesolucky tobeenrichedwithamazingpeoplelikeyouinmylife!Ihaveenjoyedeverymomentspentwithyou andlookforwardtospendingmanymore.Youarejustextraordinary! Susi,dearSusi,mywalkingpartnerincrime!Whatagreatpleasureitistohaveyouinmylife. Wehavecrossedpathssomanytimesbeforewebecameclosefriends…Iguesswearelikeagood ACKNOWLEDGMENTS
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cheese,weneededsomeagingtime!Ihaveenjoyedsomanyconversationswithyou,andwantto thankyouforthatfromthebottomofmyheart.
HenkandWilma,Iamsogratefulthatyouweremy“neighbors”thefirstfewyearsofmeliving
inGroningen.Icouldalwayscountonyourhelporjustfunsmalltalks.Youfeellikefamilytome! To my family in Rotterdam; tante Ton, Ome Ruud, Petra, Martin and Marc, your support throughoutmyPhD,aswellasprivately,hasmadeitpossibleformetogetthroughitall.Youarethe bestfamilymembersonecouldaskfor,andIamforevergratefultocallyouMYfamily! Brigitte,therearenowordstoexpresshowgratefulIamtohavemetyouandyourfamily(Jop, Tilly,Michelle,andthekids)!Fromdayoneyouhavealwaysofferedmesupportandhugswhenever needed.YouareremarkableandIhopewewillalwaysbeineachother’slives,nomatterwherelife takesus! Wearenowventuringtowardscolderweather,thatisright;wearegoingtoScandinavia… Anja,mydearfriend,mysoulsister,mypartnerincrime!Youaresoamazing!HowcanIexplain whatyoumeantome!Youhavebeenoneofmyclosestfriendsfor12years,andthereisnotaday thathasgonebywherewe,onewayoranother,nothavebeenincontactwitheachother.Youknow everythingaboutmeandviceversa,soIhavedecidedthatweHAVEtostayfriendsforever,youknow toomuchaboutmetoliveotherwise!Cannotwaitforwhatthefutureholdsforus.
Pernille, mah! The first time we met we were just kids; I do not think the two of us ever imagined thatafterso manyyearswe wouldstillbeas close assisters!We haveshared moments togetherthattakesalifetimetofindsomeonethatwillunderstandyouthewayyouunderstandme! Twopeasinapod,itsoundsoclichébutinourcaseitistrue!YouareanamazingwomanandIamso proudandthankfultocallyoumyfriend! Sarah(bae!),mysisterfromanothermister!Iknewyouwouldbeoneofmyclosestfriends fromtheveryfirsttimeIsawyou!Withyourbeautifulpersonality,yourkindspirit,yourpatience,your charisma,Ijustknewyouhadtobepartofmylife!Nowitsoundslikeyouaremytherapist,butwhat Imeantosayisthatyouhavetaughtmehowtobeme,andhowtoaccepthelpfrompeoplearound me,ifeverneeded!Youarejustextraordinary!Thankyouforbeingmyfriend,mysister! SarahMaass,Idonotevenrememberwhenwebecamefriends,allofasuddenyoujustwere oneofmyclosestfriends,andithasstayedthatwayeversince!Wehavesharedsomanyamazing moments,andIamsogratefultohaveyouinmylife!Nomatterwhereweareintheworld,Iknow thatwewillalwaysstayconnected! Henriette(Lose),youcameintomylifewhenIwas13,youwereabitolder(notgoingtosay howoldexactly,aladyneverrevealsherage!),youstartedworkingformymotherinhercompany, andallIcanrememberthinkingis:Wow,IwishIcanbeasamazingasHenriettewhenIgrowup!And guesswhat?!Thatdidnothappen,soIamcurrentlyworkingonaPlanB.Jokeaside,Henrietteyou ACKNOWLEDGMENTS
havealwaysbeenpartofmylifeandyouhaveshowedmewhatastrongwomanissupposedtobe like!IadmireyougreatlyandIthankmymotherforbringingyouintomylife! Pappa,asyougetolder,youcometorealizewhoyourparentsreallyare.Yourealizethatthey arenotjustannoyingcreaturesputonthisearthtotellyouwhatyoucanandcannotdo(although theywillkeepdoingthatuntilyouareatleast50,Ithink).IhavecometorealizethatthemanIcallmy fatherisoneTHEmostfantasticpeopleIhaveevermet!Pappa,youaresmart,patient,understanding, diplomatic,funny,andhandsome(Ihavetosaythat,peoplesayIlooklikeyou!)Icannotexpressthe loveIhaveforyou,andwithoutyouandyoursupportIwouldnothavemadeitthisfarinlife!Afather likeyouiswhateverydaughterdreamsof! Mama,mysoulmate,mybestfriend,myeverything!IloveyouinwaysthatonlyyouandIwill everknowandunderstand!Ioweyoueverything,youhavebeenmybiggestfansincethedayIwas born,youhavesupportedmeinsomanywaysandyouneveraskedforanythinginreturn!Iamwhat Iambecauseofyou(soifanyonedoesnotlikeme,Iwillblameyou,justsoyouknow).Writingthis partwasthehardestpartandIwaswaitingarounduntillastminutetodothis.Iknowyouareunable toreadthis,butIneededtogetthisoffmychest,mostlybecauseyoudeservemorethananyoneto bementionedinthisthesis!Iloveyouandonedaywewillmeetagain(noworries,Iwillbringyoua copy;Iknowyouwouldhavelovedtosee,readandholdmythesis).Foreveryours!Ikhouvanjou mama,vooraltijd!
