Microbial Biofilms Sunita Panchawat * Department of Pharmaceutical Sciences, Mohanlal Sukhadia University, Udaipur (Rajasthan), India Email: Sunita_pharma2008@rediffmail.com Chapter 1 Current Research in Microbiology 1. Introduction and Historical Perspective Biofilm exhibit two types of growth mode i.e. planktonic cell and sessile aggregate. In biofilm (association of micro-organisms), cells stick to each other on a surface encased within matrix of extracellular polymeric substance produced by bacteria themselves [1]. A Dutch researcher, Antoni van Leeuwenhoek, for the first time observed ‘animalcule’ on surfaces of tooth by using a simple microscope and this was considered as the microbial biofilm discovery [2]. For marine microorganism i.e. bacterial growth and activity were substantially enhanced by the incorporation of a surface to which these microorganisms could attach is known as “bottle effect” observed by Heukelekian and Heller [3]. Zobell observed that the number of bacteria on surfaces was higher than in the surrounding medium [4]. Zo Bell introduced first about multicellular prokaryotic communities on submerged surfaces who stated the presence of adherent microbial associations in all natural environments [5,6]. The extensive physical and chemical analysis of bacterial biofilms did not begin until the late 1960s and early 1970s, when some of the investigators identified the extensiveness of bacterial biofilms. Scanning and transmission electron microscopy was used by Jones et al. to examine biofilms on trickling filters in a wastewater treatment plant and showed them to be composed of a variety of organisms (based on cell morphology). By using a specific polysaccharide-stain such as ruthenium red when coupled with osmium tetroxide fixative to show that the matrix material surrounding and enclosing cells in these biofilms was polysaccharide. In 1973 Characklis studied microbial slimes in industrial water systems and reported that they were not only adhering very closely but also highly resistant to disinfectants such as chlorine. Costerton et al. in 1978 gives a theory of biofilms based on observations of dental plaque and sessile communities in mountain streams that explain the mechanisms whereby microorganisms adhere to living and nonliving materials and the benefits arises by
27
Embed
Department of Pharmaceutical Sciences, Mohanlal Sukhadia ...openaccessebooks.com/current-research-in-microbiology/microbial-biofilms.pdfDepartment of Pharmaceutical Sciences, Mohanlal
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Microbial BiofilmsSunita Panchawat*
Department of Pharmaceutical Sciences, Mohanlal Sukhadia University, Udaipur (Rajasthan), India
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
2
www.openaccessebooks.com
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,
3
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].
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
5
CurrentResearchinMicrobiology
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.
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].
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
7
CurrentResearchinMicrobiology
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
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
9
CurrentResearchinMicrobiology
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].
Biofilmformationbeginswithplanktonic(free-swimming)bacteriawhichcanattachtoavarietyofsurfaces,fromwoods,metals,andplasticstolivingtissuesandstagnantwater.The cells are excreted a sugarymolecule called extracellular polymeric substance or EPShasastrand-likestructurethatholdsthecellstogetherandattachesthemtothesurfaceandcreatingamatrix.Thismatrixofcellsandstrandscanbequitecomplex:thecellsmayshare
10
CurrentResearchinMicrobiology
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].
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
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
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].
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].
[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
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].
17
CurrentResearchinMicrobiology
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]
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
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].
19.Hans-Curt Flemming, JostWingender,Ulrich Szewzyk, Peter Steinberg, ScottA, Rice and StaffanKjelleberg.“Biofilms:anemergentformofbacteriallife”.NatureReviewsMicrobiology.2016;14:563–575.
70. DechoAW. “Microbial exopolymer secretions in ocean environments: Their role(s) in food webs and marineprocesses”.OceanographyandMarineBiolologyAnnualReview.1990;28:73-153.
73. Garrett ES, Perlegas D,Wozniak DJ. “Negative control of flagellum synthesis in Pseudomonas aeruginosa ismodulatedbythealternativesigmafactorAlgT(AlgU)”.JournalofBacteriology.Dec1999;181(23):7401-7404.
74. Kokare CR, Chakraborty S, Khopade AN, Mahadik KR. “Biofilms; Importance and Applications”. Indian JBiotechnonogy.2009April;8:159-168.
124.Arnold JW andBaileyGW. “Surface finishes on stainless steel reduce bacterial attachment and early biofilmformation:scanningelectronandatomicforcemicroscopystudy”.PoultSci.2000;79:1839-1845.
129. BoschA, et al. “Characterization of Bordetella pertussis growing as biofilm by chemical analysis and FT-IRspectroscopy”.ApplMicrobCellPhysiol.2006;71:736-747.
148. Elsner C, Cavalcanti E, FerrazO,Di SarliA. Evaluation of the surface treatment effect on the anticorrosiveperformanceofpaintsystemsonsteelProgressinOrganicCoatings.2003Nov;48(1):50-62.