Department of Pharmaceutical Sciences, Mohanlal Sukhadia...

Microbial BiofilmsSunita Panchawat*

Department of Pharmaceutical Sciences, Mohanlal Sukhadia University, Udaipur (Rajasthan), India

Email: [email protected]

Chapter 1

Current Research inMicrobiology

1. Introduction and Historical Perspective

Biofilmexhibit twotypesofgrowthmodei.e.planktoniccellandsessileaggregate.Inbiofilm(associationofmicro-organisms),cellssticktoeachotheronasurfaceencasedwithinmatrix of extracellular polymeric substance produced by bacteria themselves [1].ADutchresearcher,AntonivanLeeuwenhoek,forthefirsttimeobserved‘animalcule’onsurfacesoftoothbyusingasimplemicroscopeandthiswasconsideredasthemicrobialbiofilmdiscovery[2].Formarinemicroorganismi.e.bacterialgrowthandactivityweresubstantiallyenhancedby the incorporationofa surface towhich thesemicroorganismscouldattach isknownas“bottleeffect”observedbyHeukelekianandHeller [3].Zobellobservedthatthenumberofbacteriaonsurfaceswashigherthaninthesurroundingmedium[4].ZoBellintroducedfirstaboutmulticellularprokaryoticcommunitiesonsubmergedsurfaceswhostatedthepresenceofadherentmicrobialassociationsinallnaturalenvironments[5,6].

Theextensivephysicalandchemicalanalysisofbacterialbiofilmsdidnotbeginuntilthelate1960sandearly1970s,whensomeoftheinvestigatorsidentifiedtheextensivenessofbacterialbiofilms.ScanningandtransmissionelectronmicroscopywasusedbyJoneset al. toexaminebiofilmsontricklingfiltersinawastewatertreatmentplantandshowedthemtobecomposedofavarietyoforganisms(basedoncellmorphology).Byusingaspecificpolysaccharide-stain such as ruthenium red when coupled with osmium tetroxide fixativeto show that the matrix material surrounding and enclosing cells in these biofilms waspolysaccharide.In1973Characklis studiedmicrobialslimesinindustrialwatersystemsandreportedthattheywerenotonlyadheringverycloselybutalsohighlyresistanttodisinfectantssuchaschlorine.Costertonet al. in1978givesa theoryofbiofilmsbasedonobservationsofdentalplaqueandsessilecommunitiesinmountainstreamsthatexplainthemechanismswherebymicroorganismsadheretolivingandnonlivingmaterialsandthebenefitsarisesby

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CurrentResearchinMicrobiologyPanchawatS

thisecologicniche[7-9].

Costerton and Geesey specified that glycocalyx acted as an ionic exchangematrix,trappingnutrientsthatweretransportedintocellsbyhighlyefficientpermeases[10].In1981glycocalyx was characterised as a hydrated polyanionic polysaccharide matrix which isproducedbypolymerasesthatisattachedtothelipopolysaccharidecomponentofthebacterialcellwall. Biofilmproductionofglycocalyxinaqueousenvironmentisprevalentwithorganicandinorganicnutrientsbeingconcentratedatthesolid/liquidinterface.Theglycocalyxprovidesa physical/chemical barrier, offers partial protection against antibacterial agents [11]. Thestructuresofdifferentbiofilmshavedistinctfeaturesbecauseitformsunderdiverseconditionsandcomposedofsingleormultiplespecies.Thestudyrelatedtobiophysical,structuralandchemicalpropertiesofbiofilmhaveledtoausefulbasicconceptof“biofilmmodel”[12].

The important advances of the development andbehavior of biofilmsweremade in1998,whenmoleculargeneticsapproachescombinedwithconfocallaserscanningmicroscopy(CLSM). Traditionally, microbiologists have performed physiological experiments withmicroorganisms grown in liquid monocultures where the cells are “free swimming” orplanktonic[13].Itisnowwidelyacceptedthat99%ofallmicro-organismsattachtoasurfaceandgrowasa bioflim.Animportantsurvivalstrategyformicro-organismsinthehealthcareenvironment is thegrowthof biofilmmode.According to theCenters forDiseaseControlandPrevention,theassociationofbiofilmsisapproximately65%ofall healthcare-associatedinfections.Thus,theirpresenceinmedicaldevices, chronicwounds and surgicalsiteinfectionsisofgrowingconcern[14].

Figure 1:HistoricalDevelopmentofBiofilm

2. Definition

Manynovel,organiccompoundshavebeendevelopedinlastfewyearsthatarereleasedintotheenvironment.Thesecompoundsincludeheavymetals,poly-aromatichydrocarbons,polychlorinated biphenyls, pesticides, chemical fertilizers, detergents, paints, disinfectants,

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CurrentResearchinMicrobiology

lubricants, antibiotics and nanoparticles. Many of them are toxic to humans and otherorganisms.Managing the harmful effects of these pollutants is a challenge to sustainabledevelopmentglobally.Usingbiofilmsinbioremediationcanallownewtechnologiestoremainenvironmentallysustainableifintegratedmethodsarecorrectlydevelopedandapplied[15]. Biofilms vary greatly in structure and composition from one environmental condition toanothersothattheyarenoteasilydefined.Microbialbiofilmsareextremelycomplexmicrobialecosystemsconsistingofmicroorganismsattachedtoasurfaceandembeddedinanorganicpolymermatrixofmicrobialorigin.Non-cellularmaterialssuchasmineralcrystals,corrosionparticles,andclayorsiltparticles,bloodcomponentsmayalsobefoundinthebiofilmmatrix.Thereforebiofilmmaybedefinedas“microbialcellsimmobilizedinamatrixofextracellularpolymersactingasan independentfunctioningecosystem,homeostaticallyregulated”[16]. Biofilmisacommunityofbacteriathatattachtoasurfacebyexcretingasticky,sugarysubstancethatencompassesthebacteriainamatrix. Bacteria,fungiandprotistsarethemicroorganismsthatformbiofilms.Biofilmsarecomplexsystemsthataresometimescomparedtomulticellularorganisms.Biofilmshavebeenfoundgrowingonmineralsandmetals.Theyhavebeenfoundunderwater,undergroundandabovetheground.Theycanalsogrowonplanttissuesandanimaltissues, implantedmedicaldevicessuchascathetersandpacemakersetc [17,18].Bacterialbiofilmscanbeconsideredtobeanemergentformofbacteriallife,inwhichcommunallifeiscompletelydifferentfrombacteriathatliveasfree-livingcells[19].Biofilmsmayformonlivingornon-livingsurfacesandcanbeprevalentinnatural,industrialandhospitalsettings[1,20].Themicrobialcellsgrowsonbiofilmarephysiologicallydistinctfromplanktoniccellsofthesameorganism,which,bycontrast,aresingle-cellsthatmayfloatorswiminaliquidmedium[21].ThemorphologicalstructuresofbiofilmareshowninFigure 2[22].

Figure 2ThemorphologicalsimilarityinthestructureofaP. aeruginosa biofilmandaMyxococcus fruitingbodyisevidentinthesetop-downphotographs.Bothorganismsformdistinctaggregatesofcellsthatarewellseparatedfromtheirneighbors.Left:8-h-oldbiofilmofP. aeruginosa grownonPVCplasticat400_magnification.Right:FruitingbodiesofMyxococcus xanthus after6honstarvationagarplatesat5_magnification.Microcoloniesandfruitingbodiesareindicatedbyarrows.

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3. Classification of Biofilms [23].

3.1 On basis of its location:

a. Supragingival-Presentcoronaltothegingivalmargin

b. Subgingival-Presentapicaltothegingivalmargin

3.2. On basis of pathogenicity

a. Cariogenic-Generallyacidogenicandgram-positive

b. Periopathogenic-Mostlybasophilicandgram-negative

4. Composition of Biofilm

Abiofilmcomprisesanysyntrophicconsortiumofmicroorganismsinwhichcellssticktoeachotherandalso toasurface.Theseadherentcellsbecomeembeddedwithinaslimyextracellularmatrixthatiscomposedofextracellularpolymericsubstances(EPS).ThecellswithinthebiofilmproducetheEPScomponents,whicharetypicallyapolymericconglomerationofextracellularpolysaccharides,proteins,lipidsandDNA[1,24,25].Theyhavebeendescribed(metaphorically) as “cities for microbes” because they have three-dimensional structureand represent a community lifestyle formicroorganisms [26,27].TheEPShas a complexbiochemical composition, comprising predominantly carbohydrates and proteins, althoughlipidsandextracellularDNA(eDNA)havealsobeenidentified[28],alongwithexogenousinorganicororganicsubstanceswhichmaybecomeentrappedwithintheEPS,forexample,ironormanganese[29].EPSprimarilycomposedofpolysaccharidesandmayvaryinchemicalandphysicalproperties.FortheEPSgram-negativebacteria,someofthesepolysaccharidesareneutralorpolyanionic.Thepresenceofuronicacids(suchasD-glucuronic,D-galacturonic,andmannuronicacids)orketal-linkedpryruvatesconferstheanionicpropertyofEPS[30]. Thispropertyallowsassociationofdivalentcationssuchascalciumandmagnesium,whichhavebeenshowntocross-linkwiththepolymerstrandsandprovidegreaterbindingforceinadevelopedbiofilm[31].AbiofilmisanimmobilemicrobialcommunitycomposedofcellsimmersedinamatrixofEPSattachedtoasubstratumorinterface.Essentiallythematrixisofmicrobialoriginandthecellsencasedinthismatrixpresentamodifiedphenotype,especiallywithregardtogrowthrateandgenetranscription[32].Thetermofslime wasusedtodefinetheglycocalixproducedbythestronglyadherentstrainsofStaphylococcus epidermidis isolatedfromtheinfectedsurfaceofmedicalimplants[33,34].

Biofilms are group or micro-organisms in which microbes produced extracellularpolymericsubstances(EPS)suchasproteinsincludingenzymes,DNA,polysaccharidesandRNAandinadditiontothesecomponentswater(upto97%)isthemajorpartofbiofilmwhich

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is responsible for theflowofnutrients inside thematrixofbiofilm.Thecomplexstructureof biofilm consists of twomain components i.e.water channel (for transport of nutrients)anddenselypackedcells(aregionhavingnoprominentporesinit)[35].Thecomponentsofbiofilms(Table1)havethecapacitytomakeitresistantagainstvariousenvironmentalfactorsandsignify thebiofilm integrity [36,37].Thechemicalcompositionofbiofilm is shown inTable 1.

5.Role and Importance of Biofilm in Different Field

5.1 In Medical Field

Microorganismsareabletoadheretovarioussurfacesandtoformathree-dimensionalstructureknownasbiofilm.Bacteriaembeddedinthebiofilmcanescapeandformwellknownplanktoniccells(freeflowingbacteriainsuspension),thatareonlyapartofthebacteriallifecycle.Bacteriaalsoadheretomedicaldevicessuchascatheters,eitherurinaryorintravenous,artificialheartvalves,orthopedicimplantsthatcausesdevice-relatedinfectionslikecystitis,catheter-related sepsis, endocarditis etc.Once a biofilmhas been establishedon a surface,thebacteriaholdinsidearelessexposedtothehost’simmuneresponseandlesssusceptibletoantibiotics.Asanimportantcauseofnosocomialinfectionsthebiofilmmustremaininthecentreofthemicrobiologist’sattention[38].

ThefourthleadingcauseofdeathintheUnitedStatesisnosocomialinfections(infectionsacquiredatahospital).About65%oftheseinfectionsareduetobiofilmsonimplantedmedicaldevices[39].BiofilmsdifferfromaninfectionofplanktonicbacteriaisduetotheEPSmatrixof the biofilm,which is important in cell adhesion and aggregation.ThisEPSmatrix alsohinders the normal functions of antibodies and the phagocytic cells of the host’s immunesystem[40].Anotherkeyfactorthatmakesbiofilmsparticularlydifficultinmedicalsituationsistheirheightenedresistancetoantibiotics.Therearethreeproposedmethods[41]:

a. Theantibiotic isdeactivated faster than it candiffuseandalsonot able topenetrate thesurfacelayersofthebiofilm.

b.Thedifferentchemicalenvironmentsofbiofilmcanaffecttheactionoftheantibiotic.Thecauseofnon-growingstateofbacteriaislowlevelofnutrientsinthelowerlayersofbiofilm.

Table 1: ChemicalCompositionofBiofilms

S. No. Components Percentage

1 Microbialcells 2-5%

2 Polysaccharides 1-2%

3 DNAandRNA <1-2%

4 Proteinsincludingenzymes <1-2%

5 Water Upto97%

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c.About1%ofthepopulationmayexhibitaphenotypicstate(whichpersistsundercontinuedexposuretoanantibiotic),evenwhenthebiofilmistoothintoinhibitdiffusionoftheantibioticorofnutrients.

Becauseoftheseproperties,cells(existinbiofilms)canbe1000timesmoreresistantto antimicrobial agents than the same cells in planktonic form.Cells at the surface of thebiofilmcaninfectthehostwhendetachfromthebiofilmmatrix.Therefore,biofilmscanactasareservoirofprotectedbacteria(oninsertedmedicaldevices)oftenpersistsuntiltheremovaloftheinfecteddevices[42,43].TogetridcompletelyfromtheInfectionsassociatedwiththebiofilmgrowtharechallengingtaskduetothefactmaturebiofilmsdisplaytolerancetowardsantibioticsand the immune response.The rapidlygrowing industry forbiomedicaldevicesandtissueengineeringrelatedproductsisalreadyat$180billionperyearworldwide.Theseindustries continue to suffer frommicrobial colonization [44,45].VariousmicroorganismsdevelopedonmedicaldevicesareshowninTable 2.

5.2. In Industry

Biofilmformedwhenbacteriaareabletoattachtoandcolonizeenvironmentalsurfaceswhichallowtheorganismstopersistintheenvironmentandresistdesiccation,UVlightandtreatment with antimicrobials and sanitizing agents. Biofilms are formed when microbesattach toa solidsupportand toeachotherbyextracellularpolymericsubstances (EPS)onawidevarietyofsurfaces includingmetal,plastic, rockand livingordead tissue.Bacteriacanbeseveralordersofmagnitudeinbiofilmwhichismoreresistanttoantimicrobialsthantheir planktonic forms [46]. Inmarine and other aquatic environments algae, diatoms andbacteriathatareabletoattachandformbiofilmsonships’hullsandbecomeresistanttothedifferentantifoulingpaints(developedtopreventtheinitialcolonization)resultsinincreasedfluidfrictionalresistanceandfuelconsumption.Inthefoodindustry,contaminationoffoodprocessingand/orfoodcontactequipmentoftenleadstopost-processcontaminationandreducetheshelflifeofproducts[47].

Table 2:Microorganismsassociatedwithbiofilmdevelopedonmedicaldevices

S. No. Microorganism Medical Devices

1. Psudomonas aeuginosa Artificialhipprosthesis,Centralvenouscatheter,Urinarycatheter

2. Candida albicans Artificialhipprosthesis,Centralvenouscatheter,Prostheticheartvalves,Intra-uterinedevices

3. Staphylococcus aureus Artificialhipprosthesis,Centralvenouscatheter,Prostheticheartvalves,Intra-uterinedevices

4. Enterococcus Spp. Artificialhipprosthesis,Urinarycatheter,Prostheticheartvalves

5. Klebsiella pneumoniae Centralvenouscatheter,Urinarycatheter

6. C o a g u l a s e - n e g a t i v e staphylococci

Central venous catheter, Urinary catheter, Intra-uterine devices,Prostheticheartvalves

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Biofilms can also be utilized for useful purposes. Sewage treatment plants includeasecondarytreatmentstageinwhichwastewaterpassesoverbiofilmsgrownonfilterswhichextract and digest organic compounds. In such condition of biofilms, bacteria are mainlyresponsibleforremovaloforganicmatter,whileprotozoaandrotifersaremainlyresponsibleforremovalofsuspendedsolids,includingpathogensandothermicroorganisms.Slowsandfiltersdependsonbiofilmdevelopmenttofiltersurfacewaterfromlake,springorriversourcesfordrinkingpurposes.Toeliminatepetroleumoilfromcontaminatedoceansormarinesystems,biofilmscanbehelpfulbythehydrocarbondegradingactivitiesofmicrobialcommunities[48].Biofilmsareusedtogenerateelectricityfromavarietyofstartingmaterials,includingcomplexorganicwasteandrenewablebiomassintheformofinmicrobialfuelcells(MFCs).Biofilmsarealsousedtoenhancethemetaldissolutioninbioleachingindustry[49-52].

5.3. In Food industry

Biofilmformationisadynamicprocessinwhichvariousmechanismsareinvolvedintheirattachmentandgrowth.Biofilmshavebeenamatterofinterestinthecontextoffoodhygiene.Ifthemicroorganismsfromfood-contactsurfacesarenotcompletelyremoved,theymayleadtoformbiofilmwhichincreasesthebiotransferpotential[53].Biofilmsarecomplexmicrobialecosystemsformedbyoneormorespeciesimmersedinanextracellularmatrixofdifferentcompositionswhichdependsonthefoodmanufacturingconditionsandthecolonizingspecies[54].TheformationofBiofilmsinfoodindustryenvironmentsisveryfast.Thefirsttwostepsare;a)theconditioningofthematerialssurfacesb)thereversiblebindingofthecellstothatsurface.Thebindingbecomesirreversiblethatcausesdevelopmentofmicrobialcolonies.Finally,thetridimensionalstructureofbiofilmisformed,andthiscomplexecosystemisreadyfordispersion[55-57].Theextracellularmatrixismainlycomposedofpolysaccharides,suchascellulose,proteinsorexogenousDNAand itcanbefixed tohardsurfacessuchas foodindustry equipment, transport, dispensing and storage surfaces, soil, etc. or to biologicalstructuresviz.vegetables,meat,bones,fruits.Theextracellularmatrixisresponsibleforthestrongpersistenceofthesebiofilmsinthefoodindustry.Thisgeneratescomplexgradientswithrespecttonutrientsandoxygendiffusion,containsextracellularenzymesusedfornutritionalpurposes.Thesecomplexgradientsallowforthetransferofcellcommunicationmolecules,andprotecttheembeddedcellsagainsttoxiccompounds[58].

The biofilm layer is foundon themesocarp inherently formedby various yeast andlacticacidbacteria.ThesebacteriaandyeaststrainsplayaroleinthefermentationofoliveandalsotheybecomeadominantfloraonthefruitwhichpreventstheolivefrommicrobialspoilageoriginatedbyGramnegativebacteria. In this regard, thequalityandsafetyof thetableoliveandalsothetasteandflavourofthelastproducthasbeendeterminedbybiofilmformingmicroorganismsfoundonthemesocarpofthefruit.Biofilmformingabilityisadesiredpropertyoffermentedfruitproducts[59].Beneficialeffectofthebiofilmformationisaboutthe

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yeaststrainsusedcommonlyinthefoodindustry.SomeyeastspecieshavingbiotechnologicalrelevancesuchasSaccharomyces cerevisiae mightregulatetheQStype.IntheQSmechanismoftheyeaststrains,aromaticalcoholsarethemostobservedsignalmoleculeswhichcanresultinmodificationandimprovementofindustrialprocesses[60].Themicrobialinteractionshaveanimportanceforfoodindustry.Fermentation,brewingandcheeseripeningaresomeareaswheremicrobialinteractionshavebeenobserved.Mixtureoffungi,yeastandbacterialspeciesplayaledroleintheproductionofwine,fromripeningofgrapesinvineyardstowinebottling.Thegrowthofsomebacterialspecies,suchasLeucobacter sp.orBrevibacterium aurantiacum,significantlyreliesonthepresenceoftheyeast[61].

5.4 In aquaculture

Aquacultureisdefinedastheproductionofaquaticplantsandanimalsandthisisafastestgrowingfoodindustry(FAO-FisheriesandAquacultureDepartment2012).Aquaculturehasexpanded12‐foldwithanannualgrowthrateof8.8%andthisdatawasobservedduringthelast30years.In2010,ithasreachedatotalvolumeof60milliontonnesperyear.Amajorshareofglobalaquacultureproductioniscoveredbyfreshwaterfish(56.4%),mostnotablybycarpcultureinChina(16milliontonnes).Approximately38%ofthetotalaquacultureproductionisfrommarineaquaculture[62].Infishculture,presentlythemostcommonformisfloatingnetcages,whichcontainslargeamountsoffishatminimalcosts.Tankandpondculturesaremoreexpensive;howevertheyareeasiertoaccessandarethusthebestchoiceforlabor‐intensiveculturessuchaslarvae,juvenilesandbroodstock.Are-circulatingaquaculturesystemareafurtherdevelopmentofpondor tankculturesandisarelativelynewculturetechniquethatpresupposedtheavailabilityofdurabletechnicalequipmentaswellasbiologicalandtechnicalknowledge originating from wastewater treatment research [63].Microbial community ofbiofilmoccursinblocksof20-60uinwaterandsediment,harvestablebymanyplanktonicfishlikesilvercarp,rohu,catla,mulletsandmilkfish.Themicrobialcommunityflourishesusingorganicandmineralfractionsoforganicmanureassourceofenergyandnutrients.Fishesareabletoharvesttheseorganismsdirectlyinsignificantquantities.Themicrobialfilmcoatingthatisrelativelyindigestiblesubstrateofthedetritusanditisdigestedwhilethesubstrateitselfpassesthroughthefishgutwhichthengetre-colonizedbymicrobesandre-harvestedbyfish[64].Numerousstudieshaveshownthatbiofilmcanbeareservoirforpotentiallypathogenicbacteriainfreshwateraquaculture[65,66].

6. Functions of Biofilm in Microbial Communities

6.1 Environmental protection

ExtracellularPolymericSubstances(EPS)playsdifferentrolesinstructureandfunctionof biofilm communities. EPS act as an anion exchanger to prevent the access of certainantimicrobialagentsintothebiofilm.Itrestrictsthediffusionofcompounds fromsurroundings

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into the biofilm. Antibiotics that are hydrophilic and positively charged such as amino-glycosidesshowmorepronouncedattractiontowardsthiseffect.EPShasalsobeenreportedtosequestermetalions,cationsandtoxinsthatprovideprotectionfromvarietyofenvironmentalstressessuchaspH-shift,UVradiation,osmoticshockanddesiccation[34,67-69].

6.2 Availability of nutrients

Theeffectivemeansofexchangingnutrientandmetabolitesiswaterchannel.Aqueousphaseenhancestheavailabilityofnutrientandalsoremovesthepotentiallytoxicmetabolites.Fermentivebacteriaproduceacidsandalcoholsinitiatedbytheprocessofcatabolism,whicharethenutilizedassubstratebyacetogenicbacteria.Biofilmsprovidesanidealenvironmentfor the establishment of syntrophic relationship.Syntrophism is a symbiosis inwhich twometabolicallydistinctbacteriadependsoneachothertoutilizecertainsubstratestypicallyforenergyrequirements[70,71].

6.3. Acquisition of new genetic trait

Acquisitionofnewgenetictraitgiveschancestothemicrobialcommunitiestotranscribethenecessarygamestobecometheactivememberofbiofilmcommunities.TheproductionofalginatewhichinvolvesthetranscriptionofalgCgeneisincreasedapproximatelyfourfoldinbiofilmassociatedcellsascomparedtoplanktoniccells[12,72].

6.4. Penetration of antimicrobial agent

Diffusionistherate limitingsteptoinactivatethebiofilmformingmicrobialcommunitybyantimicrobialagents.EPSactsasdiffusionbarrierforthesemoleculesthatinfluencestherateoftransportofthereactionofantimicrobialagentswiththematrixmaterial.Advantagesofbiofilmgrowthtowardsthemicrobialcommunityare:

a.AsthegrowthisrestrictedalltheenergyisusedupbythebacteriainmakingtheEPSthatwillgiveprotectiontothemicrobialcommunity[73].

b.Asthegrowthisrestricted,bacteriawillremainindormantstagesthatwillgiveprotectiontothemicrobialcommunityagainstantibiotics(mostoftheantibioticsareactiveagainstthegrowthphaseofthebacteria)[74].

7. Formation of Biofilm

Biofilmformationbeginswithplanktonic(free-swimming)bacteriawhichcanattachtoavarietyofsurfaces,fromwoods,metals,andplasticstolivingtissuesandstagnantwater.The cells are excreted a sugarymolecule called extracellular polymeric substance or EPShasastrand-likestructurethatholdsthecellstogetherandattachesthemtothesurfaceandcreatingamatrix.Thismatrixofcellsandstrandscanbequitecomplex:thecellsmayshare

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geneticmaterialandhaveorganizedstructure.Abiofilmcanbeasthinasasinglecellorasthickasseveralinchesdependontheconditionsoftheenvironment.Biofilmsbecomematureandthickensastheygrowsanddevelop.Inthepresenceofsufficientwaterandnutrients,thebiofilmwilldevelopuntilsmallportionsdetachandfloattoanothersurfaceandcolonize[75].Complexprocessofbiofilmformationinvolvesseveraldistinctphasesstartwithadsorptionontothetoothsurfaceofaconditioningfilmderivedfrombacterialandhostmoleculesformstootheruptionortoothcleaning.Thisadsorptionprocessisfollowedbypassivetransportofbacteriamediatedbyweaklong-rangeforcesofattraction.Covalentandhydrogenbondscreatestrong,short-rangeforcesthatresultinirreversibleattachment.Theprimarycolonizersformabiofilmbyauto-aggregation(attractionbetweensamespecies)andco-aggregation(attractionbetween different species). Co-aggregation results in a functional organization of plaquebacteriaandformationofdifferentmorphologicalstructuressuchasCorncobsandRosettes.Themicroenvironmentnowchangesfromaerobic/capnophilictofacultativeanaerobic.Theattachedbacteriamultiplyandsecreteanextracellularmatrix(EPS),whichresultsinamixed-populationofmaturebiofilm.Organizationtakesplacewithinbiofilmafteroneday.Formationofaclimaxcommunitytakesplaceduringtransmissionthatoccursfromothersites,leadingtoincorporationofnewmembersintothebiofilm.Thethicknessoftheplaqueincreasesslowlywithtime,increasingto20to30μmafterthreedays[76].

Theformationofabiofilmbeginswiththeattachmentoffree-floatingmicroorganisms(Planktonic) to a surface [77]. The first colonist bacteria of a biofilm may adhere to thesurface initiallyby theweakvanderWaals forcesandhydrophobiceffects. If theyarenotimmediately separated from the surface, they can anchor themselves more permanentlyusingcelladhesionstructuressuchaspili. Hydrophobicityaffectstheabilityofbacteriatoformbiofilms.With increasedhydrophobicitybacteriahave reduced repulsionbetween thesubstratumandthebacterium.Bacteriawithincreasedhydrophobicityhavereducedrepulsionbetweenthesubstratumandthebacterium.Motilebacteriacanrecognizesurfacesandaggregatetogethereasilythannon-motilebacteria.Bacteriacellsareabletocommunicateusingquorumsensing(QS)productssuchasN-acylhomoserinelactone(AHL)duringsurfacecolonizationprocess.Bacterialbiofilmsenclosespolysaccharidematricesthatalsocontainmaterialfromthesurroundingenvironment[78].Biofilmsaretheproductofamicrobialdevelopmentalprocess.ThediagramofbiofilmformationisshowninFigure3[79].

Theprocessissummarizedbyfivemajorstagesofbiofilmdevelopment[80]:

1.Initial/reversibleattachment(bindingof1stcolonist)

2.Irreversibleattachment(theyanchorthemselvesusingpili)

3.MaturationI(intercommunicationthroughquorumsensing)

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4.MaturationII/Development(finalstageofmodification)

5.Dispersion(essentialstageforbiofilmformationandlifecycle)

8. Chracterization/Evaluation of Biofilm

Mostcommonlyusedmethodsofbiofilmcharacterizationarequantitativecharacterizationandqualitativecharacterization.

8.1 Quantitative Characterization Methods

Biofilmdynamicsandcomplexarchitecturecreateschallengesforbasicmeasurementsregarding the number of viable cells, mass accumulation, biofilm morphology, and othercriticalproperties.Thesechallengesarenotinthemeasurementsthemselvesbutinthelackofstandardizedprotocolsforcharacterizationanduniformtrainingavailabilityforindividuals.Oneofthemostbasicandmostcommonlyacquiredtypesofbacterialmeasurements,whetherinplanktonicorbiofilmcultures is thedeterminationofhowmuch ispresent.Avarietyofdirectandindirectmethodshavebeenusedtoquantifycellsinbiofilms[81].

8.1.1. Direct Quantification Methods

Direct countingmethodspermit enumerationof cells that canbecultured, includingplatecounts,microscopiccellcounts,Coultercellcounting,flowcytometry,andfluorescencemicroscopy.Directmethodsforbiofilmquantificationarethosethatrelyondirectobservationfor quantification of the desired parameter (number of cells, total biofilm volume, etc.).Imagingandautomatedcellcountingarethemostcommonmethodsofbiofilmquantification.Furthermore,theuseofstainsorfluorescentmarkers,inordertomoreaccuratelyidentifycellsofinterestanddistinguishfromculturedebris,allowforeasierandincreasedaccuracyofcellcountinganddatainterpretation.Imagingmethods,includinglightandconfocalmicroscopyprovide manual platforms to count cells and determine total biofilm volume. Instruments

Figure 3: BiofilmFormation

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incorporatingflow,suchasautomatedcellcountersandflowcytometers,providemechanizedmethods.Differentdirectmethodsforthecharacterizationofbiofilmare[82]:

8.1.1.1. Plate Count Method (colony forming units/ml or CFUs)

Thismethodisusedforthedeterminationofviablecellnumbersby akaCFU/mlassayoraerobicplatecount[83-86]. Thisassayisusedtoseparatetheindividualcellsonanagarplateandgrowcolonies fromcells, thereforedifferentiatesandquantifies living fromdeadcellswithoutuseofdyesorinstrumentation.Thefirststepofthisprocedurestartswithaliquidplanktoniccultureoramaturebiofilmwhichissuspendedandhomogenizedinliquidmediumvia scraping, vortexing or sonicating.The platingmethod involves the aseptic removal ofaliquotsofthesuspendedbiofilm,followedbyserialdilutionandplatingontonutrientagar.After24-72hours(whenincubationiscomplete)coloniesarecountedontheplatesandthenumberofcellspermilliliter(cfu/mL)arecalculatedusingthemeancolonycounts.Duringtheprocessitisimportanttonotetheincubationtimeandkeepituniformtoexpandeachculturebythesameamount.Itisadvisabletohaveanexperimentcontrolwithnotreatment[85].Opticaldensity(OD)canbemeasuredpriortoplatingtoobtainacalibrationcurveusedtocorrelatecellnumberandabsorbanceinpureculturebyenumerationmethod.Therebyabsorbanceofasampleofunknowncellnumbercanthenbemeasuredtodeterminethecellconcentration[87,88].TheCFUtechniquecanbeperformedbytrainedindividualinlaboratoryscaleanddoesnotrequirehighlyspecializedadvancedequipment.However,thistechniqueistimeandlaborintensive,sometimesrequiredaystoperformenoughreplicatestoobtainreproducibleresults.Thistechniqueisalsovulnerabletocountingerrorespeciallywhenthegivennumberofcoloniesishighand/orthecountisdonemanually[89].

8.1.1.2. Flow-based Cell Counting

Inthismethodcellsinliquidcultureflowthroughnarrowaperturesandaremeasuredastheypass.Coultercountingandflowcytometrybothrequirehomogenizedandsuspendedbiofilm in liquid cultures.TheCoultermethod involves passing of charged particles in anelectrolytesolutionthroughanaperture(partofanelectricalcircuit).FlowcytometrygivesmoreinformationaboutcellsduringmeasurementwhileCoultercountersarelessexpensive[90,91].Thevoltagepulsesarethencountedoveraperiodoftimeandcorrelatedwithcellnumber.Thistechniqueisverysimplebutcannotdifferentiateliveanddeadcells[92].Inflowcytometertechnique,cellsflowthroughanarrowopening(topassthroughsinglefile).Alaserisrequredtodetectthecellsastheypassviascattering,absorbanceorintrinsicandextrinsicfluorescencemeasurements.Themajoradvantagesofthismethodarethespeed,simplicityandaccuracyassociatedwithmeasurements.Additionalinformationaboutthecellsalsogatheredby using this method including the cell dimensions, surface properties metabolic activityandthedifferentiationstateofthecellswithendogenousfluorescenttags(suchasGFP).The

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maindisadvantageofthismethodisthecostoftheinstrumentapproximatebetween$50,000-100,000[93].

8.1.1.3. Light and Fluorescence Microscopy

Biofilm3Dcharacterizationandcellcountingcanbedonebyusingseveralmicroscopymethodsrangingfromsimplelightmicroscopytoconfocallaserscanningmicroscopy(CLSM)[81].

Compound light and fluorescence microscopes

Smallstructuresofbacterialcellscanbevisualizedbyacompoundlightmicroscope.Resolutionofbacterialcells(2-8μminlength)requirestotalmagnificationof200xorgreater.Use of Contrast enhancement methods such as phase contrast or differential interferencecontrast(DIC)canimprovetotalqualityoftheimages.Fluorescencemicroscopyenlargestheopticalcapabilitiesoflightmicroscopytointrinsicoraddedfluorescentlightemission[94].

Confocal laser scanning microscopy

Confocallaserscanningmicroscopy(CLSM)produceshigh-resolution,sharpimagesof biofilms in three dimensions [97-100].The area of focus is scanned across the sampletoproducehigh-resolution2-D“slices”atvariousheights that areassembled toproduceafinal3Dimage.Confocalmicroscopycanutilizesingleormultipleexcitationlaserstoviewmultiplefluorescentmarkerssimultaneously.Theseinstrumentsalsorequireexperiencedandhighlytrainedusersforaccuratemeasurementandanalysis[95].

Fluorescent dyes and proteins

Intrinsic biomolecules, such as NADH and NAD(P)H or chlorophyll which havefluorescentpropertiescanbeusedinfluorescencemicroscopy.Fluorescentdyesandproteinsareusedtointroducefluorescenceintoasample.Fluorescentdyesarefluorescentmolecules(knownasfluorophores)absorbsandemitslightwhileincorporatedinthebiologicalstructure.The emitted light is detected to analyze biofilm features, such as spatial cellular viability,shapeand function [96].Someexamplesoffluorescentdyes areDAPI (4’,6-Diamidino-2-phenylindole dilactate), lipophilic dyes such as FM 4-64, SYTO 9 and Propidium Iodide(PI)[97].Greenfluorescentprotein(GFP),enhancedgreenfluorescentprotein(EGFP)[98],CyanFluorescentProtein(CFP)andYellowFluorescentProtein(YFP)aretheexamplesoffluorescentprotein[99].

8.1.2. Indirect Quantification Methods

Thegrowthofbiofilm(quantityofbiofilm)canbedetermined indirectlyusingaproxymarkersuchasdrymass,totalproteincontent,DNA,RNA,polysaccharidesormetabolites.

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Alltheindirectquantificationmethodsinvolvebasicassumptionthatthesubstanceorpropertytobequantifiedcorrelatestothenumberofcellsoramountofprotein/DNA/mass[100].

8.1.2.1. Dry Mass Measurement

Drymass(massperunitarea)orbiofilmdensityisawidelyusedmarkerforquickgrowthquantification.Thebiofilmtogetherwithgrowthsubstrateisplacedinanovenataconstanttemperature(dependsonsubstrateheattolerancecapacity)untilthewaterisremovedandaconstantweightisachievedtofindthedrymass.Ifthesubstrateisheatsensitive,thebiofilmcanbescrapedfromthesurfacethensuspendedinphysiologicalsalineafterthatprecipitatedwithcoldethanolandprecipitatesarecollectedforanalysis.Aftercompletedryingthesampleisweighed, the biomass is scraped from the substrate and then substrate isweighed.Drybiomassiscalculatedas thedifferenceinweightbetweenbiomassonthesubstrateandthesubstratewithnobiomass[101,102].

8.1.2.2. Total Organic Carbon (TOC) Quantification

Totalorganiccarbon(TOC)isanindirectmeasurementoftheamountofcarboninasampleassociatedwithorganiccompoundsorcarboncompoundsderivedfromlivingthingssuchasproteins,lipids,ureaetc.Thisisopposedtoelementalcarbon(EC)suchasgraphiteorcoal,andinorganiccarbon(IC)consistingofsimplecompoundsincludingsimplecarbonoxides(COandCO2),carbonates,carbides,andcyanides[103].TOCmeasurementisgenerallyusedtodeterminethequalityofenvironmentalwaterandfortestingofinstrumentcleanlinessusedinthepharmaceuticalindustry.Thismethodisalsousedinthequantificationofbiofilmaccumulation[104,105].TheTOCquantificationofbiofilmsfollowsatwo-stepprocessinwhichtotalcarbon(TC)andICaremeasuredandTOCiscalculatedbythedifferencebetweenthesetwovalues(TOC=TC–IC).Theexactmethodisdeterminedusinginstrumentssuchas theOceanic International CarbonAnalyzer,Analytik JenaMulti N/C 2100S, or aUICincorporatedModelCM5012CO2coulometer[106,107].

8.1.2.3. Crystal violet assay

The primary component and commonly used dye for gram staining (identificationandvisualizationofbacteria)iscrystalviolet,abasictri-anilinedyewhichiscellmembranepermeable[108].Forbothgrampositiveandnegativecells,thecrystalvioletisusedandthedyewillfreelypassfromthecellduringthede-decolorizationstepallowingforthequantificationofcrystalvioletviaspectroscopy.Thisquantificationhasprovenextremelyusefulasacellestimateforbiofilmgrowth[109,110].

8.1.2.4. Tetrazolium salt

Tetrazoliumsaltsaremostwidelyusedinbiologyformonitoringmetabolismin vitro

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[111].AvarietyofsaltssuccessfullyutilizedforbiofilmevaluationwhichallowforquantificationandvisualizationofcellularviabilityandmetabolismwiththehelpofUV-Visandfluorescencespectroscopy.Thetetrazoliumsaltisdilutedintoaphysiologicallyrelevantsolution,suchasmediaorsaline,andthebiofilmisallowedtoincubatefor1-3hoursatculturetemperatureorroomtemperatureandcellularviabilityisdetectedbyvisualorfluorescentspectrometersormicroscopes[112,113].Thereductioncanresultinwatersolubleorwaterinsolubleformazan,water soluble formazans solubilize in the treatmentbufferused for real-timeevaluationofcellularviabilityandmetabolism[114,115].Water-insolubleformazancrystallizesandtrappedwithinthecellmembrane,crystalscanbeevaluatedviaflowcytometryandmicroscopy[116].SomeexamplesofcommonlyusedtetrazoliumsaltsaregiveninTable 3.

8.1.2.5. ATP bioluminescence test

ATPbioluminescence isawell-establishedmicrobial testused todetect thepresenceof microbial contamination on surfaces in food and biomedical communities. Adenosinetriphosphate(ATP)isanucleosidetriphosphatewhichactsastheprimaryenergysourceinallorganisms,soitisusedasaprimemarkerforviability.Intheprocessofbioluminescenceorganismsconvertchemicalenergytolightandtheamountoflightcanbeusedinferbiofilmviabilityandbiomass.Thisassayisveryreliable,canbeperformedquickly,andonlyrequiresaluminometerforanalysis.TheassayishighlyaccurateatlowATPlevels[117-119].

8.1.2.6. Total protein determination

Proteincontenthasbeenfoundtocorrelatewiththenumberofcellsinbiofilms.Totalproteincontentdeterminationiswidelyacceptedmethodtodetectthegrowthofbiofilm[107].In thisprocess thebiofilmsare removed from their substrate andhomogenized in a liquidsuspensionandthecellsarelysed.Someprotocolsrequireincubation(at55°C)inthepresenceofastrongbaseordetergentsolutionandproteinprecipitateswithtrichloroaceticacid(TCA).Thislysismadeproteasefreeinthepresenceofproteasesenzymethatbreakdownproteins.After lysis, the protein content canbemeasuredby color change (eg.CoomassieBrilliantBlueG-250dye),andcolourchangeresultfromthedye-proteininteraction.ThechangeinabsorbanceofthecoloredspeciesataparticularwavelengthisproportionaltotheconcentrationofproteinbytheBeers-Lambertlaw.Bradford,Lowry,andbicinchoninicacid(BCA)aresome

Table 3. Commonlyusedtetrazoliumsaltsusedforin vitro studyofbiofilms

S. No. Name

1. 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazoliumbromide(MTT)

2. 5-Cyano-2,3-di-(p-tolyl)tetrazoliumchloride(CTC)

3. 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazoliumchloride(INT)

4. 2,3,5-TriphenylTetrazoliumChloride(TTC)

5. (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide)(XTT)

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establishedmethodsusedfortotalproteindetermination[120].

8.1.2.7. Quartz crystal microbalance

Quartz crystalmicrobalance (QCMs) is used for the nondestructivemeasurement ofbiofilmaccumulation.TheinstrumentconsistsofasmalldiscofAstatine(AT)-cutsinglecrystalquartzthatisdrivenattheresonantfrequencybyanappliedoscillatingpotentialdifference.ThediscmaybecoatedbyGold(Au)orSiliconOxide(SiO2)andservesasthegrowthsubstrate.Inthisstudy,adirectcorrelationbetweenwetmassofthefilmandQCMfrequencyshiftisshown,givingaquantitativemeasureofmassfromtheQCMdevice.Themajoradvantageofthistechniqueisthemonitoringofmassaccumulationtong/cm2accuracyinreal-timewithoutsacrificingthesampleandallowsfortheinvestigationwithmultipleanalytictechniques[121-123].

8.2.Qualitative Characterization Methods

The characteristics which are helpful in the qualitative determination of biofilmare imaging the physiological biofilm surface, structure evaluation of surface roughness,morphology,spatialorganization,andinteractionofthebiofilmwiththeenvironment.Surfacestructureanalysisisdonebylightandfluorescentmicroscopy,ScanningElectronMicroscopy(SEM)methodsthroughhighresolutionimaging.

8.2.1. Scanning Electron Microscopy (SEM)

SEMisusedforhighresolutionmagnifiedimageofsurfacetopography.ThemagnificationrangeofSEMisabout10-500,000timeswhichmakesthistechniqueinvaluableintheanalysisof microscopic structures and biofilm morphology. SEM utilizes a concentrated beam ofelectrons to observe a sample through a number of electromagnetic lenses [124, 125].Anadvantageofelectronmicroscopyistheeasyavailabilityoftandemspectroscopictechniquesforquantitativeelementalanalysisandthehighresolutionofthesurfaceimagescanrevealdetailsaboutbiofilmstructureandtopography.SEManalysiscannotbeperformedonlivingsamplesandtestingisdoneunderhighvacuum,extensivepreparationisrequiredpriortotheanalysisofbiologicalsamples[126].

8.2.2. Alternative Qualitative Characterization Methods

Alternative methods used for qualitative characterization of biofilm growth arescanningelectrochemicalmicroscopy(SECM)[127],Infrared(IR)andRamanspectroscopiccharacterization[128],SurfaceEnhancedRamanSpectroscopy(SERS)[129],Smallanglex-rayscattering(SAXS)[130],SurfacePlasmonResonanceimaging(SPRi)andElectrochemicalSurfacePlasmonResonance(EC-SPR)[131].

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9. Management of Biofilms

Theimportancefromapublichealthperspectiveistheroleofbiofilminantimicrobialdrugresistance,posesaseriousthreattothePharmaceuticalindustries.Thereforepreventionofbiofilmformationisrecommendedratherthantreatment[132].Biofilmformationcanbepreventedbysignalingmoleculesthatblocktheattachmentofbacterialcellstosubstratesurface[133] andbychemical reactions thatprevent synthesisofpolymers in extracellularmatrix[134]. Substancesthatblockcommunicationbetweenbacteriacanpreventbiofilmformationorstimulateitsdispersion[135,136].Biofilmdispersioncanbeinducedbytheuseofenzymesthatbreakdownpolymersinextracellularmatrix[137].

Treatmentofperiodontalbiofilms- In these treatment individual considerationsmustbetakencareof.Biofilmcontrolisfundamentaltothemaintenanceoforalhealthandtothepreventionofdentalcariesgingivitisandperiodontitis[138].

9.1. Possible strategies to control oral biofilms [138]

Inhibitionofbacterialcolonization•

Inhibitionofbacterialgrowthandmetabolism•

Disruptionofestablishedplaque•

Modificationofplaquebiochemistry•

Alterationofplaqueecology•

9.2. Clinical approaches

a. Mechanical plaque control [139]

Toothbrushes•

Manual•

Electrical•

Interdentalcleaningaids/brushes•

Woodenandrubbertips•

Dentalfloss•

Oralirrigationdevices•

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b.Chemical plaque control [140]

Enzymes(Mucinase,Dehydratedpancrease,Lactoperoxidasehypothiocyanate)•

Antibiotics(Penicillin,Vancomycin,Erythromycin)•

Phenols(Thymol,Delmopenol)•

Quaternary Ammonium Compounds (Benzalkonium chloride, Cetylpyridinium•chloride)

Bisbiguanides(Chlorhexidine,Alexidine)•

Bispyridines(Octenidine)•

MetallicSalts(Zinc,Tin,Copper)•

Aminoalchohols(Octapenol,Decapenol)•

Herbalextracts(Sanguinarine)•

Surfactant(Sodiumlaurylsulfate)•

10.Application of Biofilm

Specific applications of bound bioactivemolecules to surfaces (biofilm) in differentsectorsorscientificdisciplinesaredescribedbelow;

10.1. Food industry application

In food processing industry antimicrobial polymers (active packaging) can be usedto improve the safetyof food [141]. Immobilized lysozyme, glucoseoxidase and chitosanhavebeenusedaspackagingfilms.Thesepackagingtechnologiesplayanimportantroleinextendingshelf-lifeoffoodsandreducetheriskofgrowthofpathogenicmicroorganisms[142].Material/compoundsproposedandtestedforantimicrobialactivityinfoodpackagingincludesorganic acids, antibacterial peptides and fungicides [143-145]. Triclosan containing foodcontactsurfacessuchasincludecuttingboardsanddishclothseffectivelyreducesthebacterialcontamination. Enzyme immobilization reduced the overall bioactivity after denaturation[146].Whensurfacemodificationstrategiesareappliedtoobtainantibacterialfoodprocessingsurfaces,theycanhelpreducebiofoulingandcross-contamination[147].TheeffectivenessofcoatingSSwithanticorrosionundercoatpaintwasreportedinvariousstudies[148].Biofilmformationinfoodmaybeavoidedbyequipmentdesign,temperaturecontrolandbyreductionofwaterandnutrients.Effectivecleaning(alkalicompounds)isthemainfocustocontrolthegrowthofbiofilm.Thesanitizersusedinfoodindustryarehalogens,acids,peroxygensand

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quaternaryammoniumcompounds(cationicsurfactantsanitizers)[132].

10.2. Biomedical application

Modifiedmaterialsarenotrecommendedforthemedicalpurposebecauseifthesubstanceswill leachout itmaycausecytotoxicity [149].Ametallicmaterialwhich is implanted intohumanbodyreleasemetalionsmaycausevarioushealthproblemsduetometalaccumulationinorgans,allergyandcarcinoma[150-152].Biocompatibilityisthemostimportantpropertythatmust involve in amodified abiotic surface. Biocompatibility can be divided into twokinds,oneisthebulkpropertyofthebiomaterialandotherisitssurfaceproperty.Therigidityofmodified implantsmustmatchwith that of the adjacent tissueotherwisehyperplasia orabsorptionofthetissuewilloccurresultinginfailureofimplantation[153].

11. A Future Prospectus for Research

Thebiofilmisviscoelasticinnaturewhichisuniversalbutwhenexposedtodifferentenvironment,hydrodynamicconditionswillchangethestructure,compositionandphysicalproperties of theirmatrix. Biofilm science is highly exciting research area because it is amixtureofbiology,microbiology,biotechnology,biophysic,chemistryandmuchmore[154].Researchonmicrobialbiofilmsopensmanyfrontswithspecialattentiononelucidationofthegenesexpressedbybiofilm-associatedorganisms,evaluationofcontrolstrategiestocontrolorpreventbiofilmcolonizationofmedicaldevicesanddevelopmentofnewmethodsforassessingor evaluating the efficacyof these treatments.The focused research area shouldbeon theroleofbiofilmsinantimicrobialresistance,biofilmsasareservoirforpathogenicorganismsandtheparticipationofbiofilmsinchronicdiseases.Asthepharmaceuticalandhealth-careindustries embrace this approach, novel strategies for biofilm prevention and control willdefinitelyemergeinfuture.Thekeytosuccessmaydependuponacompleteunderstandingofwhatmakesthebiofilmphenotypesodifferentfromtheplanktonicphenotype[155].

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