University of Groningen Coupled adhesion of bacteria to surfaces Skogvold, Rebecca van der Westen

University of Groningen

Coupled adhesion of bacteria to surfaces

Skogvold, Rebecca van der Westen

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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

4

Floating- and Tether- coupled Adhesion of

Bacteria to Hydrophobic and Hydrophilic

Surfaces

This chapter is published with permission from American chemical society:

Rebecca van der Westen, Jelmer Sjollema, Robert Molenaar, Prashant K. Sharma, Henny C. van der Mei and Henk J. Busscher

ABSTRACT Modelsforbacterialadhesiontosubstratumsurfacesallincludeuncertaintywithrespectto the(ir)reversibilityofadhesion.Inamodel,basedonvibrationsexhibitedbyadheringbacteriaparallel toasurface,adhesionwasdescribedasaresultofreversiblebindingofmultiplebacterialtethersthat detachfromandsuccessivelyreͲattachtoasurface,eventuallymakingbacterialadhesionirreversible. Here,weuseTotalInternalReflectionMicroscopytodeterminewhetheradheringbacteriaalsoexhibit 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.

 

INTRODUCTION Bacterialadhesionoccurstomanydifferentsurfacesinawidevarietyofenvironments,andis eitherdesirablesuchasinmanybioreactorsystems,soilremediationortointestinalsurfacesinthe humanbodyoris,amongstothers,thecauseofsevereinfections,foodspoilageormicrobiallyͲinduced corrosion.1_{Accordingly,thereisanongoingquesttomodelbacterialadhesion.Thisquestisontheone} handledbybiochemiststryingtodiscovermoreandmorespecificligandͲreceptorsystemsfacilitating bacterialadhesiontosurfaces,whileontheotherhandphysicoͲchemistsattempttodesigngenerally validmodelsthatexplainandpredictadhesionofbacteriatosurfacesbytreatinglivingorganismsas (bio)colloidalparticles.2,3_

 The twomostcommonphysicoͲchemicalapproachesused to modelbacterialadhesionare surfacethermodynamic4,5_{and(extended)DLVO(Derjaguin,Landau,VerweyandOverbeek)Ͳtypesof} analyses.6,7_{Applicationofsurfacethermodynamicsinvolvesthemeasurementofcontactangleswith} differentliquids,followedbycalculationandcomparisonofthefreeenergiesofthesubstratumand bacterialcellsurfacestoyieldaninterfacialfreeenergyofadhesion.Negativevaluesfortheinterfacial freeenergyofadhesionareassumedtobepredictiveforbacterialadhesiontooccur,butthishasnever becomeagenerallyvalidobservation,possiblybecausethesurfacethermodynamicrequirementof reversibilityisseldomornevermetandtheinterfacebetweenabacteriumandasubstratumsurface is a dynamic one, changing over time.8,9_{ DLVOͲtypes of analyses calculate the interaction energy}

between a (bio)colloidal particle and a substratum surface as a function of distance between the particle and the substratum surface. Under most relevant conditions, electrostatic interactions in bacterial adhesion are repulsive, that together with attractive LifshitzͲVan der Waals forces yield a secondaryinteractionminimumatadistanceofaround20–50nm10_{fromthesubstratumsurface,a}

potential energy barrier that impedes close approach and a deep primary minimum close to the surfacethatcanonlybereachedonceaparticlehasovercomethepotentialenergybarrier.AlsoDLVOͲ typeapproacheshaveneveracquiredageneralvalidityacrossdifferentbacterialstrainsandspecies. An important reason for this is, that bacterial cell surfaces can possess a wide variety of surface appendagesofdifferentlength,width,compositionandsurfacedensitythathavebeensuggestedto be able to pierce the potential energy barrier and tether a bacterium to a surface. Moreover, possessionoftetherswithlengthsthatmayrangeuptoseveralmicrometers,9_{makesitimpossibleto} adequatelydefinetheinteractiondistanceinDLVOͲtypeanalysesofbacterialadhesionasitcreatesa multiscaleroughnessonthebacterialcellsurface.11_{Severalofthetroublesomeissuesinvolvedinthe} applicationofsurfacethermodynamicandDLVOͲtypesofanalysesofbacterialadhesionhavebeen addressedinanewmodelofbacterialadhesiondescribingirreversibleadhesionofbacteriaasaresult ofthereversiblebindingofmultipletethersthatdetachfromandsuccessivelyreͲattachtoasurface,

withtheirreversibleadsorptionofhighͲmolecularͲweightproteinstosurfaces,mediatedbymultiple, reversiblyͲbinding molecular segments and was confirmed by in silico modeling of the keyͲ observations underlying the model: 1) forceͲdistance curves in single probe bacterial atomic force microscopy showing detachment events indicative of multiple binding tethers, 2) nanoscopic displacementsofbacteriawithrelativelylongautocorrelationtimesuptoseveralseconds,inabsence ofmacroscopicdisplacement,3)nanoscopicvibrationalamplitudesofadheringbacteriaparalleltoa surfacedecreasingwithincreasingadhesionͲforces,and4)increasesinmeanͲsquaredͲdisplacements overprolongedtimeperiodsaccordingtotɲ_{with0<ɲ<<1,indicativeofconfineddisplacement.}9_ Theroleofadhesionforcesactingperpendiculartoasubstratumsurfacemayseempuzzling inamodelthatisbasedonparalleldisplacementsofadheringbacteriaoverthesurface.However,in

silico modeling suggested that tether adhesion forces merely dictate the frequency with which individualtethersdetach.Thisleavesthequestionopenastowhetherthedistanceofanadhering bacteriumaboveasubstratumsurfacealsovariesovertime,similarasitspositiononasubstratum surface,andwhethertetherͲbindingplaysaroleheretoo.

In order to answer this question, this paper aims to determine whether adhering bacteria exhibitvariationsovertimeintheirdistanceperpendiculartosubstratumsurfaces,andifso,whether thesevariationsdifferfortwostreptococcalstrainswithandwithout91nmlongfibrillar,tethers.Total InternalReflectionMicroscopy(TIRM)12–14_{willbeusedtodeterminethevariationsindistanceabove}

different substratum surfaces over time, employing hydrophobic and hydrophilic glass surfaces as substrata.Distancevariationswillberelatedwiththeshapeandwidthofthesecondaryinteraction minimumanditsdistancefromthesubstratumsurfaceinaDLVOͲtypeofanalysis.

EXPERIMENTALSECTION 

BacterialStrains,CultureConditionsandHarvesting.StreptococcussalivariusHB7,possessing

91 nm long fibrillar tethers and its isogenic mutant HBC12, considered bald without demonstrable surfacetethers,15_{wereemployedinthisstudy(seeFigure1).BothS.salivariusstrainsarenegatively} charged,yetdifferinsurfacehydrophobicitywithS.salivariusHBC12,beingslightlymorehydrophilic thanS.salivariusHB7.16_{BothS.salivariusstrainswerepreͲculturedin10mLofToddHewittBroth} (THB,OXOID,Basingstoke,UK),understaticconditions.PreͲculturesweregrownfor24hat37°C.After 24h,preͲcultureswereinoculatedinto200mLofTHB,andmaintainedunderidenticalconditionsfor another16h.Streptococciwereharvestedbycentrifugationat5000gfor5minat10°C,subsequently washedthreetimesin100mLadhesionbufferhavinganionicstrengthof57mM(50mMpotassium chloride,2mMpotassiumphosphate,and1mMcalciumchloride,pH6.8)orionicstrengthof0.57 mM(10timesdilutedadhesionbuffer).Followingthis,bacteriaweresonicatedonice3timesfor10s at30W(VibraCellModel375;SonicsandMaterialsInc.,Danbury,CT)tobreakbacterialchainsand

4

obtainsinglebacteria.Finally,bacteriawerereͲsuspendedinadhesionbuffertoafinalconcentration of3x108_{bacteriapermL,asdeterminedusingaBürkerͲTürkcountingchamber.}   Figure1Transmissionelectronmicrographsofnegativelystained(1%methylaminetungstate)sections ofS.salivariusHB7(a)andS.salivariusHBC12(b).Thebardenotes100nm.AdaptedfromVanderMei etal.withpermissionfromthepublisher,SpringerNature.15_ 

Preparation of Substratum Surfaces. Glass microscope slides (15 mm x 15 mm; Thermo

Scientific, 38116 Braunschweig, Germany) were used as substrata. Prior to each experiment, glass surfaces were cleaned by 10 min sonication at 100 W (Bransonic 2510E, Danbury, USA) in 2% Hellmanex (Hellma GmbH & CO., Müllheim, Germany), 99% ethanol and finally in ultrapure water (specific resistance > 18 Mɏ cm). Next, glass surfaces were treated with UV/ozone, yielding a hydrophilicsurfacewhileforthepreparationofhydrophobicsurfaces,glassslideswerethoroughly dried after waterͲwashing in an oven at 80°C, followed by silanization in 0.05% (w/v) dimethyldichlorosilane(DDSSigmaAldrich)in99%ethanolforapproximately15min.

Total Internal Reflection Microscopy. Adhesion of streptococci onto uncoated and  DDSͲ

coatedglasssurfaceswasestablishedfromadhesionbuffer(0.57mMor57mM)atroomtemperature. Tothisend,astreptococcalsuspensionwasintroducedintothecircularlyͲshapedflowchamber(14 mmdiameterand0.35mminheight)oftheinstrumentusingaperistalticpumpataflowrateof300 ʅLperminduring60min.Nextthechamberwasperfusedwithbuffer,afterwhichTIRMlightscattering wasmeasured.TIRMwasperformedonanobjectiveͲbasedtotalinternalreflectionfluorescence(TIRF) microscope (Nikon, Eclipse Ti with TIRF module, Tokyo, Japan), equipped with a high numerical aperture objective (Olympus, PLANOͲAPO 100×, 1.45, Tokyo, Japan) illuminated by a 488 nm laser (MellesGriot,DynamicLaser,SaltLakeCity,Utah,U.S.A.)laterallyfocusedonthebackfocalplane.To avoidoverͲexposurebythereflectedlaserbeam,aspatialfilterwasemployedtoblockthereflected beaminthebackfocalplanewithoutimageinterference.Thefiltercubecontainedonlya488dichroic mirror. Scattering light was captured on an electron multiplying, chargeͲcoupled device camera (Andor,ixonDUͲ885BVAndor,Dublin,Ireland).Imagesizewascroppedto512x512pixelresolutions toachieveaframerateof33framessͲ1_{over2000frames.Priortoeachexperiment,theTIRMangle}

was observed as 2 diffraction limited spots, separated by the bacterial diameter, recognized as a cometͲorbitshape.17_ Foreachadheringsinglebacterium,therelativezͲdisplacement,zt,attimetwithrespecttotheclosest distanceencountered,wascalculatedaccordingto  ݖ௧ൌ െ݈݊ ቀ ூ೟ ூ೘ೌೣቁ ݀௣[1] 

withIttheintensityofthescatteredlightintheevanescentfieldattimet,Imaxthemaximumintensity

belongingtothedistanceofclosestbacterialapproachanddpthepenetrationdepthoftheevanescent

wave(185nm).Absolutedistancescouldnotbeobtained,becausetheevanescentwaveintensitywas notconstantovertheentirefieldofview.AsaresultthemaximumintensityImaxwasdifferentforeach

bacteriumoverthefieldofview.Thevibrationalamplitudeȴzwascalculatedasthestandarddeviation ofallztͲdisplacementsovertheexperimentaltime.AllTIRMexperimentswerecarriedoutintriplicates

onuncoatedandDDSͲcoatedglasssurfacesusingdifferentbacterialculturesforeachexperiment.

Contact Angle Measurements. Contact angles were measured on the uncoated and DDSͲ

coated glass surfaces with water, formamide, and methyleneiodide. Three 0.5 μL droplets of each liquidwererandomlypositionedonone microscopeslide,employingthreeslidesforoneseriesof measurementswitheachoftheabovethreeliquids.Imagesofthedropletswererecordedbyacamera about5safterplacingadropletonacoverslipssurfaceandthedropletcontourdigitizedaftergreyͲ valuethresholding,afterwhichcontactangleswerecalculatedfromthedigitizedcontoursusinghomeͲ madesoftware.Contactanglesonbacteriaweremeasuredbypreparingmacroscopicbacteriallawns onmembranefilters.Bacteriallawnsweremadebysuspendingbacteriatoaconcentrationof3x108 mLͲ1_{inwater,followedbydepositiononacelluloseacetatemembranefilter(porediameter0.45μm)} placedonafrittedglasssupportbyfiltrationofthesuspension.Atleastthreeseparatefilters,from three different cultures were used for each bacterial strain tested. Strains deposited similarly in a smoothandevenlayer.Thefilterswiththeirdepositedbacteriallawn,wereplacedonametalsample discandallowedtoairdryfor30to90min,18_{inordertoobtainrelativelystable,soͲcalled“plateau”} contactangles,indicativeofbacteriainahydratedstatebutwithoutfreewaterinbetween.Contact anglesweresubsequentlymeasuredasdescribedabove.Thecontactangelspresented,representthe averagesfromthreeexperimentswithseparatepreparedsurfacesaswellasbacterialcultures. Next,contactanglesoneachsurfacewereconvertedtoaLifshitzͲVanderWaals(ɶLW_)andacidͲ

base (ɶAB_{) surface free energy component, while the acidͲbase component was split up into an}

electronͲdonating(ɶͲ_{)andanelectronͲaccepting(ɶ}+_{)parameteraccordingto}



[2]



in which ɶ denotes the surface free energy and/or its components and parameters of the various liquidsusedorthesolidsurfacetobeanalyzed,whileɽrepresentsthecontactangleofthedifferent liquids.19_{ Surface free energy components and parameters of the liquids used can be found in}

SupportingInformationTableS1.

 Bacterial and Substratum Zeta Potentials. To determine the zeta potentials of the two

bacterial strains, particulate microelectrophoresis (Zetasizer nanoͲZS, Malvern Instruments, Worcestershire,UK)wascarriedoutatlowandhighionicstrength(0.57mMand57mM,respectively) atpH6.8.20_{Streamingpotentialmeasurementswereemployedtodeterminethezetapotentialsof}

uncoatedandDDSͲcoatedglasssurfaces.Tothisend,glassslideswithandwithoutDDSͲcoatingwere mounted in a homemade parallel plate flow chamber, separated by a 100 μm Teflon spacer.21_{ A}

platinumelectrodewaslocatedoneachsideofthechamber.Thestreamingpotentialsweremeasured atpressuresrangingfrom50to400mbar,andeachpressurewasappliedfor10sinbothdirections. Followingthis,thezetapotentialswerecalculatedbylinearregression,i.e.linearleastsquaresfitting ofthestreamingpotentials.

DLVO Theory. The DLVO theory, describes (bio)colloidal particle adhesion as a result of

attractive LifshitzͲVan der Waals and attractive or repulsive electrostatic forces. Accordingly, the interactionenergybetweenacolloidalparticleandasubstratumcanbeexpressedasafunctionof theirseparationdistance(d)as



ܩ்ை்_{ሺ݀ሻ ൌ  ܩ}௅ௐ_{ሺ݀ሻ ൅  ܩ}ா௅_{ሺ݀ሻ[3]}



inwhichGTOT_,GLW_,andGEL_{representthetotal,LifshitzͲVanderWaals,andelectrostaticinteraction}

energies,respectively.TheLifshitzͲVanderWaalsinteractionbetweenasphericalcolloidalparticleand aplanarsurfaceisgivenby  ܩ௅ௐ_{ሺ݀ሻ ൌ  െ}஺ ଺ቂ ௔ ௗ൅ ௔ ௗାଶ௔൅ ݈݊ ቀ ௗ ௗାଶ௔ቁቃ[4] 













»» » » » ¼ º « « « « « ¬ ª    » » » » ¼ º « « « « ¬ ª » » » » ¼ º « « « « ¬ ª         2 . cos 1 2 . cos 1 2 . cos 1 RGLGH PHWK\OHQHL RGLGH PHWK\OHQHL IRUPDPLGH IRUPDPLGH ZDWHU ZDWHU /: RGLGH PHWK\OHQHL RGLGH PHWK\OHQHL /: RGLGH PHWK\OHQHL IRUPDPLGH IRUPDPLGH /: IRUPDPLGH ZDWHU ZDWHU /: ZDWHU J T J T J T J J J J J J J J J J J J

inwhichAistheHamakerconstant,aistheradiusofthecolloidalparticle.19_{TheHamakerconstant}

was derived from the LifshitzͲVan der Waals component of interfacial free energy of adhesion accordingto



ܣ ൌ  െͳʹߨ݀଴ଶ߂ܩ஺ௗ௛௅ௐ       [5]



in which d0 is the distance of closest approach (0.157 nm).22–24 Analogously, the electrostatic

interactioncanbecalculatedusingmeasuredzetapotentialsaccordingto  ܩா௅_{ሺ݀ሻ ൌ ߨߝܽ൫ߞ} ௕ଶ൅ ߞ௦ଶ൯ ൜ଶ఍_఍್ା఍ೞ ್మା఍ೞమŽ ଵାୣ୶୮ሺି఑ௗሻ ଵିୣ୶୮ሺି఑ௗሻ൅ ݈݊ሾͳ െ ‡š’ሺെߢ݀ሻሿሽ[6] 

inwhichɸreferstothedielectricpermittivityofthemediumɺbandɺsreferstothezetapotentialsof

thebacteriumandsubstratumsurface,respectively.1/ʃistheDebyeͲHückellength,givenby  ߢ ൌ  ቂ ௘మ ఌ௞ಳ்σ ݖ௜ ௜݊௜ቃ ½        _{[7]} 

in which e corresponds to the electron charge and kB is the Boltzman constant, T is the absolute

temperature,ziisthevalenceofionspresentandniisthenumberofionsperunitvolume.

StatisticalAnalysis.Allexperimentswerecarriedoutintriplicateswithseparatelyprepared

bacterial cultures as well as different surfaces, and all data are presented as means ± standard deviations.ResultswerecomparedpairͲwiseforthetwodifferentstrainsofbacteriafortheeffectsof ionicstrengthbyusingaStudent’stͲtest.p<0.05wasconsideredtobestatisticallysignificant.  RESULTS  Table1summarizesthecontactanglesmeasuredwithdifferentliquidsonbothsubstratum surfacesandbacterialstrains.Onthebasisofthewatercontactangles,uncoatedandDDSͲcoatedglass surfacescanbeclassifiedashydrophilicandhydrophobic,respectively.AlthoughS.salivariusHB7was morehydrophobicthanS.salivariusHBC12,bothbacterialstrainscanbeclassifiedashydrophilic.   

4

Table1ContactangleswithdifferentliquidsforuncoatedandDDSͲcoatedglasssurfacesaswellasfor

S.salivariusHB7andS.salivariusHBC12.Datarepresentaverageswithstandarddeviationsoverthree

droplets on three different glass surfaces of each type and bacterial lawns prepared from three differentbacterialcultures.  Surface/ Bacterialstrain ɽǁĂƚĞƌ (degrees) ɽĨŽƌŵĂŵŝĚĞ (degrees) ɽŵĞƚŚLJůĞŶĞŝŽĚŝĚĞ (degrees) Glass 23.3±1.5 19.3±2.3 52.3±6.4 DDSͲcoatedglass 97.0±1.7 74.3±6.7 63.3±3.8 ^͘ƐĂůŝǀĂƌŝƵƐHB7 34.3±4.6 12.3±2.5 24.0±6.0 ^͘ƐĂůŝǀĂƌŝƵƐHBC12 21.6±3.6 24.7±4.9 38.0±16.2  

Surface free energy components and parameters, calculated from contact angles with the threedifferentliquidsweresubsequentlycompiledinTable2,mostnotablyshowingasmallacidͲbase component for hydrophobic DDSͲcoated glass, due to both small electronͲdonating and accepting parameters. In addition, the ratio of electronͲdonating over electronͲaccepting parameters varied betweenthetwosubstratumsurfaces,indicativeofdifferentstructuringofwatermoleculesnearby thesurface.19,25_{BothstreptococcalstrainshadsimilaracidͲbasecomponents,butS.salivariusHBC12} hadamuchhigherelectronͲdonatingsurfacefreeenergyparameterthanS.salivariusHB7,resulting indifferentratiosbetweentheirelectronͲdonatingandelectronͲacceptingparameters.Zetapotentials ofthedifferentsurfaces,alsocompiledinTable2,werenegativeforallsurfacesatbothlowandhigh ionicstrength,whilebeingsignificantlymorenegativeinthelowionicthaninthehighionicstrength buffer.   

Table2Surfacefreeenergycomponentsandparameterstogetherwithzetapotentialsforuncoated

andDDSͲcoatedglasssurfacesaswellasforS.salivariusHBC12andS.salivariusHB7,respectively. Data regarding surface energetics represent averages with standard deviations over three measurementsonthreedifferentglasssurfacesofeachtypeandbacteriallawnspreparedfromthree differentbacterialcultures.Bacterialzetapotentialsareaverageswithstandarddeviationsoverthree experiments with different bacterial cultures, while substratum zeta potentials are averages with standarddeviationsoverthreestreamingpotentialmeasurementswithdifferentuncoatedandDDSͲ coatedsurfaces.  Surfacefreeenergy componentsandparameters (mJmͲ2_) Glass DDSͲcoated glass ^͘ƐĂůŝǀĂƌŝƵƐ HBC12 ^͘ƐĂůŝǀĂƌŝƵƐ HB7 ɶ 54.9±1.1 27.7±1.0 51.5±2.6 57.0±1.2 ɶLW_{ 34.0±3.0 26.8±1.3 40.1±7.9 46.3±2.1} ɶAB_{ 21.1±3.8 0.4±0.4 11.5±8.8 10.6±3.2} ɶͲ_{ 48.0±0.8 2.1±1.8 51.8±4.9 35.1±10.2} ɶ+_{ 2.3±0.9 0.4±0.7 0.9±0.9 1.0±0.8} ɶͲ_/ɶ+_{ 20.6±0.9 4.1±3.2 59.7±5.5 35.1±13.5} Zetapotentials (mV) Glass DDSͲcoated glass ^͘ƐĂůŝǀĂƌŝƵƐ HBC12 ^͘ƐĂůŝǀĂƌŝƵƐ HB7 0.57mM Ͳ84.9±0.4 Ͳ55.2±1.4 Ͳ20.0±0.4 Ͳ16.9±2.4 57mM Ͳ39.5±0.6 Ͳ27.8±0.0 Ͳ8.2±0.2 Ͳ9.1±2.5  ThesurfacefreeenergycomponentsfromTable2canbeusedtogetherwiththeircounterparts forwater(seeSupportinginformationTableS1)tocalculatetheLifshitzͲVanderWaalsinterfacialfree energy of adhesion (Eq. 4) and subsequently using Eq. 5, to calculate the Hamaker constant for bacterialinteractionwithuncoatedorDDSͲcoatedglassinanaqueoussuspension.Sincetheconcept of interaction distance in the DLVO approach loses its meaning when the bacterial cell surface possessesamultiscaleroughness,11_{suchasduetofibrillarsurfacetethersinS.salivariusHB7,these}

calculationswereonlymadeforS.salivariusHBC12,yieldingHamakerconstantsof3.8x10Ͳ21_Jand1.6

x10Ͳ21_{JagainstglassandDDSͲcoatedglass,respectively.DLVOinteractionenergiesversusdistancefor} S. salivarius HBC12 for glass and DDSͲcoated glass were subsequently calculated inserting these

HamakerconstantsandthezetapotentialsfromTable2,intoEqs.3,4and6,assumingabacterial radiusof500nm,26_{andaDebyeͲHückellength1/ʃ}_{forthetwoionicstrengths(0.57mMand57mM)}

of1.3x10Ͳ8_m_and1.3x10Ͳ9_{m,respectively.}



Figure2InteractionenergiesbetweenS.salivariusHBC12anduncoatedorDDSͲcoatedglasssurfaces

indifferentionicstrengthsuspensions,asafunctionoftheirsurfaceͲtoͲsurfaceseparationdistance. Insets represent part of the interaction energy curve at a different scale to better visualize the secondaryminimum.

Atlowionicstrength(Figure2),thesecondaryminimumwasextremelyshallowwithadepth of0.5and0.25kTforuncoatedandDDSͲcoatedglass,respectivelyandlocatedapproximately140to 150nmawayfromthesubstratumsurface,respectively.Duetothedecreaseofelectrostaticrepulsion, the secondary minimum at 57 mM ionic strength was much deeper than in 0.57 mM suspensions (Figure2)andamountedaround5kTand3kTforglassandDDSͲcoatedglass,respectively,whilebeing locatedapproximately15nmfromthesurface.Thepotentialenergybarrieratcloseapproachmaybe consideredtoohighforabacteriumtoovercomeandadhereintheprimaryminimumasawholeinall cases.27_{DuetotherelativelystrongelectronͲdonatingandelectronͲacceptingparametersofglassas} comparedwithDDSͲcoatedglass,bothstrainswillalsoexperiencelargemonoͲpolarrepulsionatclose approach,thatwillbefarlessorabsentonhydrophobic,DDSͲcoatedglassthanonhydrophilicglass (seealsoTable2).ForS.salivariusHBC12onglassmonoͲpolarrepulsionȴGAB_(d 0)amounts+30.4mJ

mͲ2_{,turningintoattraction (Ͳ10.6 mJ m}Ͳ2_{)onDDSͲcoatedglass, butsince thisisat thedistanceof}

closestapproachd0,itisnotreflectedintheinteractionenergiespresentedinFigure2accordingto Ͳ10 Ͳ8 Ͳ6 Ͳ4 Ͳ2 0 2 4 6 8 10 0 50 100 150 200 250 G (k T) Distance(nm) Ͳ6 Ͳ4 Ͳ2 0 2 0 50 100 150 G (kT) Distance(nm) _Ͳ10 Ͳ8 Ͳ6 Ͳ4 Ͳ2 0 2 4 6 8 10 0 50 100 150 200 250 Distance(nm) Ͳ4 Ͳ2 0 2 0 50 100 G (k T) Distance(nm) 57mM _57mM Glass DDScoatedglass Ͳ10 Ͳ8 Ͳ6 Ͳ4 Ͳ2 0 2 4 6 8 10 0 50 100 150 200 250 G (k T) Ͳ1 0 1 110 160 210 G (k T) Distance(nm) 0.57mM Ͳ10 Ͳ8 Ͳ6 Ͳ4 Ͳ2 0 2 4 6 8 10 0 50 100 150 200 250 Ͳ1 0 1 110 160 210 G (k T) Distance(nm) 0.57mM

As a first step in the TIRM measurements, the chamber was perfused with a bacterial suspensionandthenumberofadheringstreptococcienumerated,assummarizedinFigure3.

S.salivariusHB7withitsfibrillartethersadheredinapproximatelyequalnumberstouncoatedand

DDSͲcoatedglass,regardlessofionicstrength(11x106_cmͲ2_and13x106_cmͲ2_{).S.salivariusHBC12}

demonstratednomicroscopicallyenumerable numberstothe uncoatedglasssurface,but onDDSͲ coatedglassenumerablenumberswereobserved,amounting2x106_cmͲ2_and4x106_cmͲ2_{forthelow}

andhighionicstrengthsuspension,respectively. 



Figure3NumbersofadheringS.salivariusHB7andS.salivariusHBC12onglassandDDSͲcoatedglass

surfaces at ionic strengths of 0.57 mM and 57 mM. Note that on uncoated glass, the number of adheringS.salivariusHBC12wastoolowformicroscopicenumeration.



 Vibrational amplitudes, ȴz, of S. salivarius HB7 adhering on a hydrophilic, uncoated glass surface(Figure4)wererelativelysmall,around5nmirrespectiveofionicstrength,whileS.salivarius HBC12adheredintoolownumbersforTIRMexperiments.S.salivariusHB7alsoexhibitedarelatively small vibrational amplitude ȴz of around 5 nm on hydrophobic, DDSͲcoated glass, albeit here the vibrationalamplitudewasslightlyhigheratlowionicstrength(notstatisticallysignificant;p>0.05, Student’stͲtest)thanathighionicstrength.Strikingly,S.salivariusHBC12demonstratedmuchhigher vibrationalamplitudesȴzonhydrophobic,DDSͲcoatedglassthanasobservedforS.salivariusHBC7. In addition, these vibrational amplitudes decreased slightly towards high ionic strength (not statisticallysignificant;p>0.05,Student’stͲtest).  0 5 10 15 20 0.57 57 Number of ba ct er ia (10 6cm Ͳ2) Ionicstrength(mM) Glass DDScoatedglass 0 5 10 15 20 0.57 57 Ionicstrength(mM) ^͘ƐĂůŝǀĂƌŝƵƐ HB7 ^͘ƐĂůŝǀĂƌŝƵƐ HBC12

4

Figure4VibrationalamplitudesȴzaboveglassorDDSͲcoatedglasssurfacesforS.salivariusHB7and

S. salivarius HBC12 in low and high ionic strength buffers, obtained using TIRM. Note that on

hydrophilicglass,thenumberofadheringS.salivariusHBC12wastoolowforTIRMmeasurements. Data represent averages over three separate experiments with error bars indicating the standard deviationsoverthreedifferentbacterialcultures.



DISCUSSION

Using TIRM, the variations in distance over time from a substratum surface to which they adhered,weremeasuredfortwostrainsofS.salivariuswithandwithoutfibrillarsurfacetethers.The strainwithfibrillartethersshowedvibrationalamplitudesofaround5nm,regardlessofionicstrength orsubstratumhydrophobicity.Thestrainwithoutfibrillartethersdidnotadhereinsufficientnumbers toderivevibrationalamplitudesonhydrophilic,uncoatedglass,duetounfavorablethermodynamic conditions(interfacialfreeenergyofadhesion27,28_{calculatedfromthedatainTable2:+26.5mJm}Ͳ2_ duetostrongmonoͲpolarrepulsion).Oppositely,onhydrophobic,DDSͲcoatedglass(interfacialfree energyofadhesion:Ͳ12.3mJmͲ2_{inabsenceofstrongmonopolarrepulsion)thenonͲfibrillatedstrain}

adhered reasonably well and vibrated perpendicularly above the surface with a fivefoldͲhigher amplitudearound25nm,regardlessofionicstrength.Previously,TIRMhasbeenusedtoanalyzethe changeinseparationdistanceofthesestreptococcalstrainsadheringforonly5mintoasubstratum surfaceuponreducingionicstrength.29_{Whentheionicstrengthwasreducedfrom57mMto5.7mM,}

thedistancebetweenthebacterialcellofS.salivariusHB7andthesubstratumincreasedfrom45nm to 90 nm, which suggests that fibrils change from a compressed, sideͲon conformation to a fully stretched state. This conclusion was later confirmed by QCMͲD (Quartz Crystal Microbalance with Dissipation)experiments30_{suggestingcollapseofstreptococcalfibrillartetherswithinseveralminutes}

aftercontactwithasubstratumsurface.Thevibrationalamplitudeofthefibrillatedstrainobserved here(5nm)isnotonlymuchsmallerthanthedistanceatwhichabacteriumadheresabovethesurface andunaffectedbyionicstrength,butalsomuchsmallerthanthefibrillarlengthorthedistanceabove the surface measured before, probably because in our measurements 60 min of adhesion were

0 10 20 30 40 50 0.57 57 ȴ z (nm ) Ionicstrength(mM) Glass DDScoatedglass 0 10 20 30 40 50 0.57 57 Ionicstrength(mM) ^͘ƐĂůŝǀĂƌŝƵƐ HB7 ^͘ƐĂůŝǀĂƌŝƵƐ HBC12 HB7 HBC12 Strain Fibrillength(nm) 91 < 5

allowed before measurements, causing tether collapse over time under influence of the adhesion forcesarisingfromthesubstratumsurface.

The perpendicular, vibrational amplitudes of adhering S. salivarius HBC12 without fibrillar surfacetetherscanberelatedwiththeDLVOinteractionfreeenergycurvesbutduetolownumbers of adhering bacteria resulting from combined monoͲpolar and low ionic strength electrostatic repulsionsonlyathighionicstrength.Underhighionicstrengthconditions,thereisaclearsecondary minimum (Figure 2). Accounting for a thermal energy of 1.5 kT for a bacterium,30_{ this allows a}

bacterium adhering in the secondary minimum to float and move away from and towards the substratum surface. This floating behavior is constrained by the width of the secondary minimum, whilebacteriaremaintoadhereatanaveragedistanceabovethesurfacedictatedbytheabsolute secondaryinteractionminimum.Thewidthofthesecondaryinteractionminimumat1.5kTabovethe absoluteminimumamountsaround15to20nm,whichcoincideswiththevariationsindistance(ȴz) abovethesurfaceobservedusingTIRM(Figure4).Perpendicular,vibrationalamplitudesofS.salivarius HBC12, adhering in a “floating” mode are much larger than of strain S. salivarius HB7, possessing fibrillartethers(Figure4).Tethercouplingtothesurfaceclearlyrestrictsthevibrationalamplitudes.

InordertoobtainfurtherevidenceforafloatingortetherͲcoupledmodeofbacterialadhesion, the energy of adhering bacteria as a function of their distance above a substratum surface can be compared with the distantͲdependent DLVO interaction energy (see Figure 2). The probability of a bacteriumbeinglocatedatacertainpositionzaboveasurfacefollowsfromthefrequencyhistogram of bacterial positions around its equilibrium, P(ztͲ<zt>), which was related to the Boltzmann

distribution32_{accordingto}  ܲሺݖ௧െ ۃݖ௧ۄሻ ൌ ܣ כ ݁ݔ݌ ቀെீሺ௭೟ିۃ௭೟ ۄሻ ௞ಳ் ቁ[8]  inwhichAisanormalizationconstant,G(ztͲ<zt>)theinteractionenergyatapositionrelativetothe equilibriumzͲposition,<zt>,ofabacteriumǤ TheinteractionenergycannowreadilybecalculatedexpressedinkTunitsaccordingto  ீሺ௭೟ିۃ௭೟ۄሻ ௞ಳ் ൌ െ Ž‘‰ሺܲሺݖ௧െ ۃݖ௧ۄሻሻ ൅ Ž‘‰ሺܣሻ[9] 

Neglecting log (A) as a constant that merely defines the absolute energy level, the distance dependenceoftheinteractionenergyfollowsdirectlyfromthevibrationamplitudesandassociated probabilitiesthatapositionabovethesurfaceisoccupied.ShapeͲwise,theinteractionenergiesofS.

salivariusHB7(Figure5)arehighlysymmetricalandparabolicatbothionicstrengthsandcanbewell fittedtoaharmonicoscillatormodel32_{accordingto}  ܩሺݖ௧െ ۃݖ௧ۄሻ ൌ ଵ ଶ݇ כ ሺݖ௧െ ۃݖ௧ۄሻ ଶ_{    [10]}        inwhichkisthespringconstantofthetether,thatcanonaveragebecalculatedtobe2.5x10Ͳ5_NmͲ1 regardlessofionicstrengths.Thusitcanbeconcludedthatstreptococciwithfibrillarsurfacetethers coupledirectlytoasubstratumsurface,whichrequirespiercingoftheDLVOpotentialenergybarrier bythetethers,whichhasbeensuggestedbefore33_{butneverbackedͲupwithexperimentalevidence.}  

Figure 5 Comparison of the distance dependence of the interaction energy (calculated from

perpendicularvibrationamplitudesabovethesurface)betweenadheringS.salivariusHB7withfibrillar surfacetethersandhydrophobic,DDSͲcoatedglasssurfaceattwoionicstrengths(0.57mMand57 mM)withthedistancedependencecalculatedaccordingtoaharmonicoscillatormodel.Blacklines representthecalculatedinteractionenergyasafunctionoftherelativedisplacement(ztͲ<zt>),and

the red dotted lines represent their fitting to a harmonic oscillator model. The figure refers to 15 individualbacteria,eachrepresentedbyonepairofblackandreddottedlines,i.e.fits.

  

 ThedistancedependenceoftheinteractionenergycalculatedfromvibrationamplitudesforS.

salivariusHBC12iscompletelydifferentthanforS.salivariusHB7(compareFigures5and6).Most notably,itsdistancedependenceisnotsymmetricalaroundanequilibriumdistanceandtherewithnot according to a harmonic oscillator model but resembling the asymmetry of the DLVO secondary potentialenergyminimum(compareFigures2and6).ThisconfirmsabsenceoftetherͲcouplinganda modeofadhesionthatweproposetocall“floatingͲadhesion”abovethesurface.



Ͳ Ͳ Ͳ Ͳ

 Figure6Thedistancedependenceoftheinteractionenergy(calculatedfromperpendicularvibration

amplitudesabovethesurface)betweenadheringS.salivariusHBC12withoutfibrillarsurfacetethers and hydrophobic, DDSͲcoated glass surface at two ionic strengths (0.57 and 57 mM), showing an asymmetrical distribution around their equilibrium position. Black lines represent the calculated interactionenergyasafunctionoftherelativedisplacement(ztͲ<zt>).Thefigurerefersto15individual

bacteria,eachrepresentedbyoneline. 

 In summary, we have provided experimental evidence for the existence of two modes of bacterial adhesion, as schematically summarized in Figure 7. Bacteria with fibrillar surface tethers adheretoasubstratumsurfaceinanirreversiblefashionbytetherͲcouplingtothesurface,i.e.piercing ofthetetherthroughthepotentialenergybarrier(Figure7a).Distancevariationsabovethesurface overtimearegovernedbytheharmonicoscillationsallowedbythespring.Bacteriawithoutfibrillar surfacetethersadhereinthesecondaryenergyminimumwiththeirperpendiculardisplacementover timerestrictedbythewidthofthesecondaryminimumat1.5kTabovetheminimumitself(Figure7b). Thedistinctionbetween“tetherͲcoupled”and“floating”adhesionisnew,andmayhaveimplications for bacterial detachment strategies, since detachment of tetherͲcoupled bacteria may involve disruptingthebondofmultipletetherswithasurfacewhile“floating”adhesionwillbedisruptedby decreasingthedepthofthesecondaryinteractionminimum,whichisrelativelyeasye.g.bychanging prevailingionicstrengthconditionsorslightͲrinsingofthesubstratawithadheringbacteria(datanot shown).(FordetailsondetachmentforcesinvolvedinslightͲrinsingseeGomezͲSuarezetal.34_) 

0.57mM

Ͳ

Ͳ

Ͳ

Ͳ

57mM

4

  Figure7SchematicsoftetherͲcoupledadhesionofabacterium(a)andfloatingadhesion(b).Notethat tetherͲcoupledadhesionrequirespiercingoftheelastictether(indicatedasaredspring)throughthe DLVOͲpotentialenergybarrier.   SupportingInformation Surfacefreeenergyparametersandcomponentsofwater,formamide,methyleneiodideused forcontactanglemeasurements.  ACKNOWLEDGEMENTS ThisstudywasentirelyfundedbyUMCG,Groningen,TheNetherlands.HJBisalsodirectorof aconsultingcompanySASABV.Theauthorsdeclarenopotentialconflictsofinterestwithrespectto authorshipand/orpublicationofthisarticle.Opinionsandassertionscontainedhereinarethoseofthe authorsandarenotconstruedasnecessarilyrepresentingviewsofthefundingorganizationortheir respectiveemployer(s).    

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SUPPORTINGINFORMATION

TableS1Surfacefreeenergyparametersandcomponentsofthethreeliquidsusedforcontactangle

measurements.ɶͲ_andɶ+_{aretheelectronͲdonatingandelectronͲacceptingparameters,whileɶ}AB_and

ɶLW_{aretheacidͲbaseandLifshitzͲVanderWaalscomponents,respectively.Alldatain(mJm}Ͳ2_)and

takenfromVanOssetal.32_

Parametersand components

Water Formamide Methyleneiodide

ɶͲ _{25.5 39.6 <0.1} ɶ+ _{25.5 2.3 <0.1} ɶAB _{51.0 19.0 0.0} ɶLW_{ 21.8 39.0 50.8}