Plasticity of Candida albicans Biofilms · protocols for new ones. ... culture conditions, and...

Plasticity of Candida albicans Biofilms

David R Soll Karla J Daniels

Developmental Studies Hybridoma Bank Department of Biology The University of Iowa Iowa City Iowa USA

SUMMARY 565INTRODUCTION 565DEFINING A C ALBICANS BIOFILM LESSONS FROM BACTERIA 566THE BEGINNINGS OF CANDIDA ALBICANS BIOFILM RESEARCH 568THE DOUGLAS MODEL A GOOD STARTING POINT 568BIOFILM DISPERSAL 571OTHER MODELS 571METHODS FOR MEASURING BIOFILMS MAY BE A SOURCE OF VARIABILITY 575STRAIN VARIABILITY 577VARIABILITY DUE TO THE ORIGINAL PLANKTONIC CELL INOCULUM 578VARIABILITY DUE TO FLOW ROTATION OR ROCKING 579VARIABILITY DUE TO DIFFERENCES IN DEVELOPMENTAL TIMING 581EFFECTS OF MEDIA ON BIOFILM DEVELOPMENT 581VARIABILITY DUE TO THE MTL CONFIGURATION 582WHITE-OPAQUE SWITCHING MATING AND BIOFILM FORMATION 583ARCHITECTURE OF MTL-HOMOZYGOUS BIOFILMS AND THE ROLE OF PHEROMONE 583PERMEABILITY PENETRABILITY AND DRUG SUSCEPTIBILITY OF ALTERNATIVE BIOFILMS 584ldquoSEXUAL BIOFILMSrdquo LESSONS FROM BACTERIA 585FACILITATION OF MATING BY SEXUAL BIOFILMS 585IMPACT OF ENVIRONMENTAL CONDITIONS ON MTL-HOMOZYGOUS BIOFILMS 585STRAIN VARIABILITY OF MTL-HOMOZYGOUS BIOFILMS 586CONCLUDING REMARKS 586ACKNOWLEDGMENTS 587REFERENCES 587

SUMMARY

Candida albicans the most pervasive fungal pathogen that colo-nizes humans forms biofilms that are architecturally complexThey consist of a basal yeast cell polylayer and an upper region ofhyphae encapsulated in extracellular matrix However biofilmsformed in vitro vary as a result of the different conditions em-ployed in models the methods used to assess biofilm formationstrain differences and in a most dramatic fashion the configura-tion of the mating type locus (MTL) Therefore integrating datafrom different studies can lead to problems of interpretation ifsuch variability is not taken into account Here we review theconditions and factors that cause biofilm variation with the goalof engendering awareness that more attention must be paid to thestrains employed the methods used to assess biofilm develop-ment every aspect of the model employed and the configurationof the MTL locus We end by posing a set of questions that may beasked in comparing the results of different studies and developingprotocols for new ones This review should engender the notionthat not all biofilms are created equal

INTRODUCTION

Microbial biofilms represent the first and perhaps most suc-cessful attempt by organisms to employ multicellularity to

succeed in an aquatic environment (1ndash6) This life form still re-mains a dominant component of the biota in some estimatesaccounting for over half of the earthrsquos present biomass (7 8) Over3 billion years ago the photosynthetic cyanobacteria (blue-greenalgae) in the oceans converted the early toxic atmosphere to one

that better supported modern terrestrial life (9ndash11) The cyano-bacteria formed biofilms that incorporated sediment generatingstromatolites (12 13) structures that can still be seen on thebeaches in Shark Bay Australia (14ndash17) These most importantand ancient prokaryotes also have a lesson to teach us about theevolution and adaptability of biofilms Today these organisms canalso inhabit terrestrial niches in the form of biofilms wreakinghavoc by etching both ancient stone monuments and modernstone buildings (18ndash20) And bacteria including those that colo-nize humans continue to live in aquatic environments primarilyas biofilms (4 21ndash24) As is the case for bacteria the fungi includ-ing those that are pathogenic have also developed the capacity toform biofilms The major yeast pathogen Candida albicans thefocus of this review can form biofilms on a variety of host tissuesdentures teeth catheters and other biomedical implants (25ndash32)Depending on the configuration of the mating type locus (MTL)it can also form alternative biofilms one exhibiting traits consis-tent with a ldquopathogenicrdquo biofilm and the other exhibiting traitsconsistent with a ldquosexualrdquo biofilm (33 34)

But before considering the plasticity of the biofilms formed byC albicans the meaning of the term ldquobiofilmrdquo must be considered

Published 1 June 2016

Citation Soll DR Daniels KJ 2016 Plasticity of Candida albicans biofilms MicrobiolMol Biol Rev 80565ndash595 doi101128MMBR00068-15

Address correspondence to David R Soll david-solluiowaedu

Copyright copy 2016 American Society for Microbiology All Rights Reserved

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September 2016 Volume 80 Number 3 mmbrasmorg 565Microbiology and Molecular Biology Reviews

on May 20 2020 by guest

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Can the term as applied to prokaryotic communities that rimbodies of water clog pipes form on the surface of your petrsquos waterbowl and obstruct the respiratory tracts of cystic fibrosis patientshave the same meaning in regard to the C albicans communitiesthat coat dentures and form plaque on teeth spread along thewalls of intestines and line the walls of catheters Is the meaning ofthe term so general that it can apply to any population of micro-organisms on a substratum including a colony of cells on nutrientagar Or does the term ldquobiofilmrdquo have specific functional and ar-chitectural connotations that apply to all species both prokaryoticand eukaryotic In delving into the literature on C albicans bio-films formed in vitro and relating it to that on the more intensivelystudied bacterial biofilms it became apparent that the definitionof the term had indeed evolved into something more than simplya film or colony of microorganisms supported by a substratum inan aqueous environment It connoted a community of interactingcells anchored tightly to a substratum made up of multiple cellphenotypes embedded in a self-generated extracellular matrix(ECM) It connoted a population in a self-established microenvi-ronment with specialized functional and architectural character-istics and a mechanism for dispersal And as is the case for allcommunities there appeared to be the potential for a significantdegree of plasticity depending upon the genotype and differentenvironmental pressures It became clear that there is a lack ofappreciation of the variability of C albicans biofilms due to straindifferences and the plasticity of these biofilms resulting from dif-ferent conditions employed in vitro Attempting to integrate datafrom different studies that employed different strains differentculture conditions and different quantitative methods of assess-ment can indeed lead to problems of interpretation without con-sidering the possibility that the biofilm preparations might differphysiologically and developmentally There can also be a majorproblem in interpreting the body of knowledge that has emergedfor bacterial biofilms formed in vitro (35ndash37) The problems for Calbicans are particularly poignant when attempts are made to gen-erate general models of biofilm development signal transductionpathways regulating biofilm formation and drug resistance Theproblem is further exacerbated for C albicans by the discoverythat depending upon the configuration of the mating type locus(MTL) (a versus aa or ) cells form biofilms that have dis-tinctly different functional characteristics (38 39) In reviewingthe literature it became clear that researchers rarely test whetherthe strain they employ is representative and rarely assess the con-figuration of the MTL locus For C albicans the latter omissionhas not had a profound effect on most studies since 90 to95 of strains are MTL heterozygous (a) (40ndash42) Howeverfor the closely related species C dubliniensis which shares mostof the developmental processes exhibited by C albicans (43ndash46)the omission is indeed worrisome given that one-third of all nat-ural strains are MTL homozygous (44)

The goal of this review is to consider the variability and plas-ticity of C albicans biofilms formed in in vitro models with theintent of engendering awareness that they are developmentallycomplex and depending on the strain and culture conditions notcreated equal This review does not cover in detail the literature onthe signal transduction pathways that regulate gene expressionduring biofilm formation the genes that are differentially regu-lated or the molecular mechanisms regulating matrix forma-tion or drug resistance unless that information pertains to thespecific aim set forth The reader is directed to a number of

excellent reviews on these subjects published in the past sev-eral years (47ndash55)

DEFINING A C ALBICANS BIOFILM LESSONS FROMBACTERIA

Considering what a C albicans biofilm represents may best beaccomplished by first reviewing bacterial biofilms given that de-tailed studies of their formation in vitro appeared in the literatureapproximately 2 decades prior to studies of C albicans biofilmsThe first report of the cellular nature of a microbial biofilm wasthat of Antonie van Leeuwenhoek in 1684 when he described tothe Royal Society of England the variety of microorganisms indental plaques which he examined with his newly developed mi-croscope (56 57) However not until the 1940s did researchersrealize that the bacterial slime covering surfaces in aquatic ecosys-tems represented more than disorganized detritus Rather theywere complex and organized communities of cells (58 59) In1978 J W Costerton and his coworkers (60 61) published thefirst formal description of a bacterial biofilm in researching howbacteria adhered to surfaces in aquatic systems They defined abiofilm as a matrix-encapsulated population of microorganismsadhering to a surface in a nutrient-sufficient environment Subse-quently they and others described the fundamental differencesbetween bacteria in biofilms and bacteria that are free-living(planktonic) (62ndash64) They noted (60) that over 99 of the bac-terial biomass in the natural aquatic ecosystems they first studiedwere in the form of biofilm communities on wet or submergedsurfaces rather than free and independent in suspension Becausethe bacteria include so many species one general biofilm descrip-tion cannot encompass the variety of their forms HoweverCosterton and others extracted a number of generally commonsteps in the formation of bacterial biofilms in vitro as well asseveral shared attributes (21 65ndash67) which are outlined briefly inFig 1 As will become evident in this review many of the steps ofbacterial biofilm formation and the attributes of these biofilmshave proven relevant to the emerging descriptions of C albicansbiofilm formation in vitro The first step in generating a bacterialbiofilm in vitro is to grow cells planktonically for inoculation intothe biofilm model The concentration of cells in the inoculum canbe adjusted to generate an initial monolayer on the substratum ofthe model employed The second step is to add the inoculum ofplanktonic cells to an adherent substratum to facilitate adhesionStrong adherence is necessary for anchoring especially when thereare fluid flow and mechanical shear forces that could release singlecells not firmly attached to the substratum during initial biofilmdevelopment It should be noted that although most in vitro mod-els include a solid substratum for adherence cells in natural set-tings may also converge at air-liquid or liquid-liquid interfaceswhere differences in density hydrophobicity and water structurecan result in a discrete physical interface that can be assessed bycell surface receptors (68 69) Cells may also use each other assurfaces through cohesion to form suspended aggregates or floc-cules exhibiting properties of a biofilm on a substratum (70ndash73)In vitro adherence to a substrate is basic to the establishment of ageographically confined population Bacterial adhesion has beenseparated into two phases primary reversible adhesion some-times referred to as ldquodockingrdquo and secondary irreversible adhe-sion sometimes referred to as anchoring or ldquolockingrdquo (74ndash77)(Fig 1) For biofilm formation in vivo a question arises as towhether one cell a number of independent cells or a fragment of

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566 mmbrasmorg September 2016 Volume 80 Number 3Microbiology and Molecular Biology Reviews


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a previous biofilm is responsible for dissemination ie the initi-ation of a new biofilm in another location (60 78 79) In all threescenarios however the first step must still be adhesion to thesubstratum In the process of adhesion an adhesin or anotherform of a mechanoreceptor activates signal transduction path-ways that effect the developmental transition from a planktonicstate to a sessile immobilized state (67 80 81) This transition canbe associated with a change in cellular phenotype For instancewhen motile bacteria such as Escherichia coli adhere to a substra-tum (33 82 83) motility ceases cell multiplication continues(84ndash87) and early biofilm-related genes are upregulated includ-ing those that encode additional adhesins (88ndash90) AlthoughKaratan and Watnick (91) suggested that bacteria at this point canform either an adherent monolayer biofilm or a multilayer bio-film the former is not sufficient to encompass some of the maincharacteristics of a complex biofilm most notably the formationof ECM and a three-dimensional controlled microenvironmentthat facilitates biofilm growth nutrient acquisition control of gasand pH and cell-cell signaling (92ndash97) It therefore may not belegitimate to consider an adhering cell monolayer lacking ECM abona fide biofilm Once cells adhere to a surface and begin tomultiply they remain aggregated both through adherence to thesubstratum and through coherence (cell-cell adhesion) (91 98)Coherence may be facilitated by several mechanisms includingdirect cell-cell adhesion the formation of an encapsulating ECMthat mechanically traps cells in place or the formation of an ECMthat acts as glue (99ndash101) The adhering cells then multiply in thez axis to form a tightly packed cohering basal polylayer of cellsthat further anchors the biofilm (Fig 1) It must be realized at this

point that the final architecture of the in vitro biofilm depends onthe original density of adhering cells Low-density inocula thatresult in sparse independent cells on the substratum may formpatches of cells or independent cell mounds while high-densityinocula may form uniform carpets of cells that result in a uniformbiofilm across the substratum While bacteria multiply they de-posit ECM The final thickness of the mature biofilm appears to bethe result of a variety of possible pressures These include the massthat can be anchored effectively to a substrate relative to shearforce (102) the penetrability of nutrients (103) gas exchange(104) the concentrations of secreted enzymes for extracellulardigestion of nutrients and for matrix modification (105) and invivo the topography of the substratum to which the biofilm ad-heres (106ndash108) Intercellular signaling develops gradually andcontrols the size of the final biofilm (109) and it may also play arole in the development of late biofilm architecture As the biofilmevolves it undergoes cellular differentiation and multicellularmorphogenesis In select bacteria there is stratification of cellularphenotypes and the development of an often complex multicellu-lar architecture (110ndash113) In Pseudomonas aeruginosa cells dif-ferentiate from a round to an elongated phenotype (113) and theyexhibit changes in physiology gene transcription and proteinsynthesis (81 114ndash116) One of the most dramatic phenotypictransitions is that of E coli (80 86) and P aeruginosa (81 117118) Both are motile when planktonic but become nonmotileduring adherence and as noted the flagellar motor can play adirect and early role in the initial adhesion event (119ndash122) As Paeruginosa biofilms grow the cells remain nonmotile but at ma-turity mushroom-like pillars separated by water channels formwithin the biofilms which contain motile cells that exit in theprocess of dispersal (81 118 123 124) Not only the size of thebiofilm but also multicellular morphogenesis is regulated by quo-rum sensing (125ndash127) Cell death which plays a major role invertebrate development most notably in limb formation (128129) may also play a role (130ndash132) In the case of Bacillus subtiliswhich makes wrinkled colonies on agar areas of localized celldeath underlie areas of buckling

During bacterial biofilm growth and maturation cells con-tinue to deposit an extracellular matrix which plays roles in cellcohesion (106 133 134) gas exchange (104) drug resistance (21135ndash137) defense against penetration by white blood cells (138ndash142) and the maintenance of architecture and biofilm rigidity(106 143 144) The ECM is hydrated and therefore is composedprimarily of water The matrix that supports the gel is composedof structural proteins and polysaccharides and contains extracel-lular DNA (eDNA) (145ndash151) Recently McCrate et al (152)demonstrated that the ECM of E coli biofilms contains celluloseand curli proteins with the latter forming fibrous amyloid bodiesStudies combining electron microscopy biochemistry and solid-state nuclear magnetic resonance (NMR) spectroscopy have led tothe perception that the major component of ECM is in fact protein(153ndash156) Studies have also demonstrated that the protein-cellu-lose architecture is responsible for the viscoelasticity of the ECM(154 155) It is likely that the composition and architecture of theECM change with developmental time In the complex matrix of abacterial biofilm a microenvironment is established that has beenshown to contain secreted enzymes for catalyzing the polysaccha-ride linkages and modifications that are basic to matrix architec-ture and function (106 144 157 158) and secreted enzymes in-volved in the extracellular digestion of nutrients from the host

FIG 1 General steps in the formation of a bacterial biofilm The general de-velopmental program provides us with a contextual framework for defining aC albicans biofilm since it includes a number of analogous steps




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environment or from the medium in an in vitro model (21 159160) The role of eDNA is unclear but experiments suggest that itindeed plays a role in biofilm formation in vitro (151 161) TheECM contributes to the establishment of a microenvironmentthat facilitates cell-cell communication through signaling (109162 163) Finally the mature bacterial biofilm must maintainitself rejuvenate and release cells or biofilm fragments for disper-sal (67 106 164) Bacterial biofilms have also been shown to fa-cilitate conjugation the bacterial version of mating through thetransfer of DNA between cells through direct cell-cell contact(165ndash167) As noted many of the developmental steps and traitsof bacterial biofilm formation in vitro have analogs in the forma-tion of a complex C albicans biofilm in vitro Formation of abacterial biofilm therefore provides a contextual framework fordefining a C albicans biofilm as well as assessing variability andplasticity

THE BEGINNINGS OF CANDIDA ALBICANS BIOFILMRESEARCH

It took more than 15 years after Costerton et alrsquos pioneering workon bacterial biofilms (60) for medical mycologists to recognize thepossible relevance of biofilm formation in the life history of Calbicans and to develop an in vitro model (168) Even the compre-hensive review and bibliography of C albicans literature by FrankC Odds in his second edition of the book Candida and Candido-sis a Review and Bibliography published in 1988 failed to haveldquobiofilmrdquo as a topic in the table of contents or index (169) Thismonumental work which included a review of 5796 publicationsdid however include a review of the literature on the adherence ofC albicans to host and synthetic surfaces the first step in biofilmformation The importance of the subject was underscored by itsplacement as the first comprehensively covered subject in thesection ldquoDeterminants of Virulence of Candidardquo in chapter 26ldquoPathogenesis of Candidosisrdquo in Frank Oddsrsquo book (169) In thisreview which covered the literature up through 1986 the range ofbiological surfaces to which C albicans adhered sometimes selec-tively was extensive These surfaces included buccal epithelia

vaginal epithelia cervical epithelia skin cells urinary epitheliagastrointestinal epithelia endothelia and even spermatozoa(169) Synthetic surfaces included catheters dentures and contactlenses (169) Possibly one of the first images of a biofilm formed bya Candida species was published in 1984 by Marrie and Costerton(24) Candida parapsilosis was identified in a sample from a cath-eter wall Yeast cells and extensive matrix were observed in thebiofilm but no hyphae were present suggesting that it repre-sented the smooth switch phenotype of this species (170) One ofthe first reports of the association of C albicans with a biofilm wasin a study by Ahearn and colleagues over 25 years ago (171) Theyassessed the efficacy of disinfectants and air drying on the clearingof biofilms from contact lens cases They noted that C albicansaccounted for only a small fraction of the cells of the predomi-nately bacterial biofilm Like the report of Ahearn and coworkers(171) there were several additional reports in the early 1990s of Calbicans in mixed biofilms forming on a variety of materials in-cluding silicone rubber prostheses (172) impression materials(173) catheters (174) and dentures (175ndash177) However it wasHawser and Douglas (168) who developed the first in vitro modelof C albicans biofilm formation in which the biofilm was formedby and composed solely of that species The development and useof an in vitro model composed exclusively of C albicans were thefirst steps in understanding how this species forms biofilms Themodel was adapted from one developed in the late 1980s byProsser et al (178) for the formation of E coli biofilms in vitroHowever during the 5 years following the landmark paper byHawser and Douglas (168) the number of published studies wasfewer than 10 per year on average (Fig 2) Medical mycologiststhen realized the importance of biofilm formation in C albicanscolonization and the number of published studies increased in anexponential fashion such that by the year 2015 the number ofpapers per year approached 300 (Fig 2)

THE DOUGLAS MODEL A GOOD STARTING POINT

As we shall see the original Douglas model continues to be usedbroadly with lab-specific variations because it supports the for-

FIG 2 The approximate number of papers published per year on Candida albicans biofilms began to increase at a nearly exponential rate after the developmentof the first in vitro model by Hawser and Douglas from 1994 to 1998

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mation of a mature biofilm that is robust thick architecturallycomplex and phenotypically and genotypically homogeneous atleast initially In the initial protocol of Hawser and Douglas from1994 (168) yeast-phase cells were first grown overnight in suspen-sion (ie planktonically) in yeast nitrogen base (YNB) medium(Fig 3A step 1) The concentration of the planktonic cell culturewas adjusted for optimum biofilm formation by measuring theoptical density at 520 nm (OD520) and then aliquots were inocu-lated into YNB medium in the wells of a microtiter plate (Fig 3Astep 2) The composition of YNB medium which usually containsglucose as the major carbon source is presented in Table 1 A disccut from catheter material that had been treated with serum wasinserted at the bottom of each plastic well of the multiwell plasticculture plate The planktonic cell inoculum was incubated in thewells for 60 min at 37degC in air without mixing (ie static withoutrotation or rocking) to facilitate adhesion to the disc (Fig 3A step3) Nonadhering cells were then gently removed and fresh YNBmedium added (Fig 3A step 4) In the original model cultureswere incubated statically for up to 72 additional hours (168) In asubsequent report (179) cultures were continuously mixed byrotation for 47 additional hours (Fig 3A steps 5 and 6) Aspects ofthis model were changed over time by other research groups Theplastic bottoms of the wells of multiwell tissue culture dishesrather than serum-treated catheter discs were employed as sub-strata for purposes of expediency since tissue culture plasticproved to be adhesive (180ndash182) YNB medium was also replacedwith different media to support planktonic growth and biofilmformation (38) In Table 1 the compositions of a number of me-dia used to develop biofilms are presented demonstrating thevariety of media and the potential for biofilm variation

The general developmental program of biofilm formation inthe Douglas model in this case using an adaptation developed byDaniels et al (38) is diagrammed in Fig 3B This variation isemployed because the architecture of the biofilm formed has beenassessed over time in detail (38 45 183 184) In this version of theDouglas model planktonic cells are grown to stationary phase insLeersquos medium to obtain a majority of unbudded yeast-phase cellsin stationary phase sLeersquos medium is Leersquos medium (185) supple-mented with zinc and arginine according to the method of Bedelland Soll (186) (Table 1) Since biofilm formation occurs morerobustly in RPMI 1640 medium buffered with morpholinepro-panesulfonic acid [RPMI 1640 (MOPS)] (Table 1) this mediumwas used by Daniels et al in the actual biofilm model in the mi-crotiter wells (38) The substratum in this model is a disc cut fromsilicone elastomer a catheter material and the incubation condi-tions after adhesion include air at 37degC with gentle rocking (30degdeflection 18 cyclesmin) (38 183 184) The cells that adhere tothe catheter disc between 0 and 15 h of static incubation form arelatively uniform contiguous monolayer if the cell concentrationis adjusted correctly [eg 1 107 cells in 2 ml of RPMI 1640(MOPS) medium added to a well containing a 156-mm-diametersilicone elastomer disc] In Fig 4A a differential interference con-trast (DIC) micrograph is presented of the yeast cell monolayerformed after 15 h (Fig 3B) A nearly confluent uniform sheet ofadherent cells is apparent In Fig 4B fixed cells are stained withthe DNA dye Syto 9 (Life Technologies Inc) to visualize nuclei InFig 4C a higher-magnification DIC image is presented of live cellsin the monolayer which reveals the uniform unbudded yeast-phase phenotype The cells in the monolayer then multiply in theyeast phase over the next 4 to 6 h forming a basal yeast cell poly-

layer that is approximately six cells thick (20 m) (Fig 3B)Between 6 and 8 h germ tubes (incipient hyphae) emerge at thedorsal surface of the yeast cell polylayer (Fig 3B) During thesubsequent 40 h the germ tubes grow in the z axis forming truehyphae (Fig 3B) Elongation in this model is oriented verticallyslowing with time as the biofilm matures (184) In Fig 4D a sideview is presented of a projection image of 500 scans obtained byconfocal laser scanning microscopy (CLSM) of a 48-h biofilm ofstrain SC5314 The image was processed with Imaris 3D imageprocessing and analysis software (Bitplane Zurich Switzerland)(45) to accentuate the vertical orientation of the hyphae in themature biofilm in this model In Fig 4E a scanning electron mi-crograph (SEM) is presented from the top of a collapsed 48-hbiofilm prepared such that the extracellular matrix was removed(note the homogeneity of hyphae in the top-view image) (45) InFig 4F an SEM is presented from the top of a collapsed 48-hbiofilm prepared to preserve the ECM (179) Note how the hyphaeare covered by the dehydrated ECM In Fig 4G a time series ispresented of top views and side views of CLSM projection imagesof biofilm formation through 24 h Note that the hyphae appearpunctate in the 16- and 24-h top-view images since they are ori-ented vertically (Fig 4G) The individual hyphae composing theupper biofilm layer each consists of a linear sequence of highlyelongated cellular compartments separated by septa Each hyphagrows apically and each of the cell compartments in the lineararray preceding the dividing apical compartment contains one ormore large vacuoles a nucleus and minimal particulate cyto-plasm These true hyphae are distinct from pseudohyphae in thatthey lack indentations at septal sites (187) In this model hyphaecontinue to grow vertically and apically for approximately 40 h(38 183 184) Very little branching or lateral yeast cell formationoccurs (Fig 4D) In other media such as Spider medium yeast cellformation and branching occur (183) in both cases from thedistal end of internal hyphal compartments just below the septaljunction (169)

Hawser and Douglas (168) showed in their original model thatgrowth (assessed by dry weight) protein synthesis ([3H]leucineincorporation) and metabolic activity (measured by the reduc-tion of tetrazolium salts) in static cultures in YNB medium at 37degCin air is approximately 75 complete after 24 h of incubation andover 90 complete by 48 h of incubation The kinetics of theseparameters (168) correlate with the gradual decrease in the rate ofbiofilm growth over time in the modified model of Daniels et al(184) (Fig 4G) Hawser and Douglas (168) found that in YNBmedium these kinetics were similar whether 50 mM glucose or500 mM galactose was used as the carbon source but the finalvalues at 48 h for all three parameters for biofilms formed inmedium containing galactose were twice those for biofilmsformed in YNB medium containing glucose Hawser et al (179)subsequently found that rotating (20 rpm) the biofilm culture tomix the medium increased the final dry weight There was noeffect on the reduction of tetrazolium salts but as discussed in alater section of this review this assay has been found to be anexpedient but sometimes problematic measure of biofilm growthand development Using SEM Hawser et al (179) reported thatlow rates of rotation during biofilm formation also increased ECMdeposition Daniels et al (183) in their modified version of theDouglas model showed by CLSM of calcofluor-stained prepara-tions that the upper portion (upper 70 to 80) of a 48-h biofilmwas composed of vertically oriented hyphae equidistant from each




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FIG 3 The Douglas model established by Hawser and Douglas (168) has served as the basis for a number of models with variations in one or more conditions(A) Procedure used by Hawser et al (179) (B) Representation of the cellular and architectural development of a C albicans biofilm using a variation of theDouglas model in which sLeersquos medium was used to grow cells planktonically RPMI 1640 (MOPS) medium was used to support biofilm development and thecultures were rocked to mix the media (38) In panel B cells in the yeast-phase cell monolayer and polylayer are represented by spheres germ tubes arerepresented by tubes hyphae are represented by vertical tubes and ECM is represented by gray fibers

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other presumably separated from one another by the encapsulat-ing ECM

BIOFILM DISPERSAL

Because the model of biofilm development described in Fig 3B islimited to cell phenotype and architecture there are many aspectsof biofilm formation that are not included One such aspect isdispersal which may have an architectural component The firstexperimental demonstration of this process was by Uppuluri andLoacutepez-Ribot (188 189) Using a flow model described in a latersection of this review they found that yeast-phase cells were re-leased from a developing biofilm These cells exhibited severaltraits that were enhanced compared to those of planktonic cellsincluding increased pathogenicity in a mouse model of systemicinfection (188) They found that between 1 and 8 h of biofilmdevelopment the rate of yeast-phase cells released into the me-dium increased by 1 order of magnitude The rate plateaued atapproximately 24 h (188) Nobile et al (190) subsequently dem-onstrated in a variation of the Douglas model using Spider me-dium that the rate of accumulation of budding yeast cells in themedium decreased between 24 and 60 h It was not clear whetherthe yeast-phase cells accumulating in the medium originated inthe adhesive yeast cell basal layer were released from the hyphalupper layer or were released from yeast cell pockets within thehyphal upper region of the developing biofilm The lowest rate ofrelease was observed in RPMI 1640 (MOPS) medium which isconsistent with the observation that biofilms formed in this me-dium contain vertically oriented hyphae with few lateral yeast cells

(45 183 184) This is therefore consistent with the suggestion thatdispersed cells may be formed by hyphae in media that facilitatelateral yeast cell formation There appears to be no time compo-nent for release of yeast-phase cells that can be correlated with anarchitectural transition in the developmental program of biofilmdevelopment so dispersal was not incorporated into the temporalscheme shown in Fig 3B

OTHER MODELS

A majority of the procedures that research groups have used togenerate biofilms in vitro are based on the Douglas model How-ever there are several models including those incorporating flowand those performed in an in vivo setting that are markedly dif-ferent Andes and coworkers (192) and Ghannoum and cowork-ers (191) developed in vivo intravenous catheter models and An-des and coworkers (187) developed an in vivo urinary cathetermodel In the original intravenous model developed by Andes etal (31 192) (Fig 5A) a polyethylene tube was inserted into thejugular vein of a rat conditioned by back flow (without the addi-tion of C albicans for 24 h) and flushed It was then inoculatedwith C albicans cells obtained from colonies grown on Sab-ouraudrsquos dextrose agar Upon inoculation the catheter was lockedand incubated for up to 72 h At the termination of the experi-ment the catheter was flushed with heparinized saline to removeblood and nonadhering cells The catheter was then assessed forbiofilm formation and the bloodstream and kidneys of the ratwere assessed for fungal load The biofilm in the catheter wasvisualized on the lumen surface by SEM (192) which entails fix-

TABLE 1 Compositions of media used to grow planktonic cultures and to support biofilm formation in Candida albicans biofilm models

Mediuma Carbon source (per liter) Remaining contents (per liter)

YNB medium 9 g dextrose 2 g biotin 400 g calcium pantothenate 2 g folic acid 2000 g inositol 400 g niacin 200 gp-aminobenzoic acid 400 g pyridoxine hydrochloride 200 g riboflavin 400 g thiaminehydrochloride 500 g boric acid 40 g copper sulfate 40 g potassium iodide 200 g ferricchloride 400 g manganese sulfate 200 g sodium molybdate 400 g zinc sulfate 1 g potassiumphosphate monobasic 500 mg magnesium sulfate 100 mg sodium chloride 100 mg calcium chloride10 mg L-histidine-HCl 20 mg methionine 20 mg tryptophan 2

YPD 20 g dextrose 20 g Bacto peptone 10 g yeast extractb

sLeersquos medium 125 g dextrose 5 g NaCl 5 g ammonium sulfate 25 g potassium phosphate dibasic 13 g L-leucine 1 g L-lysine 05 gL-alanine 05 g L-phenylalanine 05 g L-proline 05 g L-threonine 02 g magnesium sulfate 01 gL-methionine 70 mg L-ornithine 70 mg L-arginine 1 mg D-biotin 01 mM zinc sulfate

Spider medium 10 g mannitold 10 g nutrient brothc 2 g K2HPO4

SD medium 20 g dextrose 145 g yeast nitrogen base without amino acids and ammonium sulfate 5 g ammonium sulfate 50 mguracil 110 mg L-tryptophan 180 mg L-leucine 90 mg L-histidine 30 mg L-methionine

Sabouraudrsquos medium 20 g glucose 10 g Bacto peptoneRPMI 1640 (MOPS)

mediume

2 g dextrose 01 g calcium nitratemiddot4H2O 48 mg magnesium sulfate 04 g potassium chloride 2 g sodium bicarbonate6 g sodium chloride 08 g sodium phosphate dibasic 02 g L-arginine 50 mg L-asparagine 20 mgL-aspartic acid 652 mg L-cystinemiddot2HCl 20 mg L-glutamic acid 10 mg glycine 15 mg L-histidine 20mg hydroxy-L-proline 50 mg L-isoleucine 50 mg L-leucine 40 mg L-lysinemiddotHCl 15 mg L-methionine15 mg L-phenylalanine 20 mg L-proline 30 mg L-serine 20 mg L-threonine 5 mg L-tryptophan 28mg L-tyrosinemiddot2Namiddot2H2O 20 mg L-valine 02 mg D-biotin 3 mg choline chloride 1 mg folic acid 35mg myo-inositol 1 mg niacinamide 1 mg p-aminobenzoic acid 025 mg D-pantothenic acid 1 mgpyridoxinemiddotHCl 02 mg riboflavin 1 mg thiaminemiddotHCl 5 mg vitamin B12 1 mg glutathione (reduced)53 mg phenol redmiddotNa 03 g L-glutamine 35 g MOPS

a YNB yeast nitrogen base defined YPD yeast extract-peptone-dextrose undefined Spider medium undefined SD medium synthetic defined medium defined Sabouraudrsquosmedium undefined RPMI 1640 (MOPS) defined sLeersquos medium Leersquos medium (185) supplemented with arginine biotin and zinc according to the method of Bedell and Soll(186) definedb Bacto peptone is produced by peptic digestion of animal tissue Yeast extract is made by extracting the yeast cell content from the cell wall It is undefinedc Nutrient broth is composed of the following per liter 1 g beef extract 2 g yeast extract 5 g peptone and 5 g NaCl It is undefinedd Note that Spider medium is the only medium that contains mannitol a sugar alcohol rather than dextrose as a carbon source Mannitol is produced by plants algae fungi andselect bacteria but is not produced in abundance by humanse RPMI 1640 medium without MOPS is referred to as RPMI 1640 and that with MOPS is referred to as RPMI 1640 (MOPS)




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ation critical point dehydration and coating with palladium andgold SEM analysis revealed that the biofilms that formed con-tained yeast-phase cells pseudohyphae and hyphae embedded ina dense ECM (192) Hyphae dominated the outer region of thebiofilm but the actual architecture was difficult to assess in part

because the hyphae and matrix were collapsed and matted pre-sumably as a result of SEM fixation as was the case in SEMs ofmature biofilms presented by Hawser et al (179) for the Douglasmodel (Fig 4F) Metabolically active cells in the biofilm were as-sessed by use of the fungal viability stain FUN-1 and the polysac-

FIG 4 Microscopic images of yeast-phase cells and nuclei in a relatively confluent monolayer the hyphal upper layer and both top and side images of confocalprojections of biofilms forming in the Douglas model using planktonic cells grown in sLeersquos medium RPMI 1640 (MOPS) medium for biofilm formation anda silicone elastomer disc as the substrate (A) Differential interference contrast (DIC) microscopy image of an adhering yeast-phase cell monolayer after 90 minof incubation of planktonic cells in the model well (B) Nuclear staining with Syto 9 imaged by fluorescence microscopy (C) Increased magnification of themonolayer formed after 90 min imaged by DIC microscopy (D) Side view of a projection image of 500 CLSM scans of a 48-h C albicans biofilm stained withcalcofluor (E) SEM image of a 48-h C albicans biofilm prepared using methods that removed the ECM from a collapsed biofilm (F) SEM image of a 48-h Calbicans biofilm prepared using methods that left the collapsed ECM adhering to the hyphae and substratum (Republished from reference 179 with permissionof the Society for General Microbiology permission conveyed through Copyright Clearance Center Inc) (G) Top and side views of projection images eachcomposed of 500 CLSM scans of developing biofilms prepared using a variation of the Douglas model at 0 4 16 and 24 h

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FIG 5 Catheter denture and subcutaneous models developed for C albicans biofilm formation Panels cite references 192 (A) 203 (B) 211 (C) 210 (D) and208 (E)




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charide content was determined by staining with the lectin conca-navalin A (ConA) which identifies -D-mannosyl- and -D-glucosyl-containing molecules Polysaccharide localizationappeared to increase in the direction of the catheter lumen Pock-ets of matrix were also localized close to the catheter wall Thearchitectural differences between the biofilm formed in this modeland those formed in the Douglas model were considered by theauthors (192) but were difficult to assess although it is safe to saythat both biofilms contained the same general cellular phenotypesand ECM The intravenous catheter model developed by Andesand coworkers (Fig 5A) (192) has proven extremely valuable inmolecular experiments and appears to be excellent for investigat-ing a major medical problem ie catheter-based C albicans in-fections The model has been used successfully to assess the role ofadhesion molecules (193) drug susceptibility (194) gene expres-sion (195 196) and select mutants (193 197ndash202) This modelhas been used almost exclusively by the Andes laboratory at theUniversity of Wisconsin in a number of cases in collaborativestudies and has not been adopted generally probably as a re-sult of the expertise required rather than its efficacy This ob-servation is in no way meant to detract from the value of thismodel Schinabeck et al (191) developed an intravenous cath-eter model in the jugular vein of a rabbit The catheter wasflushed with saline every day and removed at 7 days SEMsrevealed dense matrix and the contours of yeast-phase cells butthere were no signs of hyphae or pseudohyphae This model hasalso not been adopted generally for the same reasons noted forthe model of Andes et al (36) but it also appears to be anexcellent one for studying catheter infections

A urinary catheter model also developed by Andes and co-workers (203) and diagrammed in Fig 5B involved inserting asilicone catheter into the urethra of an immunosuppressed rattreated with antibiotics (Fig 5B) The catheter was conditioned bydraining urine from the bladder prior to inoculation of yeast-phase cells suspended in yeast extract-peptone-dextrose (YPD)medium (Table 1) After 2 h the inoculum with nonadhering cellswas removed the rat returned to the cage and urine collectedthroughout the experimental time course At 24 and 72 h cathe-ters were analyzed by SEM (187) Because of fixation staining anddehydration in the SEM procedure the architecture of the biofilmformed on the catheter wall is difficult to compare with thatformed in the Douglas model but it is clear in the published im-ages that the upper region of the biofilm was composed mainly ofhyphae and matrix Viable cells were quantitated in the collectedurine and in the biofilm population adhering to the catheter Bio-films reached confluence at 48 h but the C albicans burden in theurine increased through 72 h indicating continued releaseandor multiplication of released cells The urinary cathetermodel warrants in vitro comparisons in which the medium inthe Douglas model is replaced with synthetic urine (SU) me-dium (204) or fresh filtered donor urine Using the XTT[23-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxianilide] reduction assay Uppuluri et al (204) demon-strated that biofilms formed in SU medium were as metabolicallyactive as those grown in RPMI 1640 medium although in SUmedium the final concentration of cells was lower the hyphaewere shorter the biofilm architecture was less complex and theexpression of select filamentation-associated genes (EFG1 ALS3and HWP1) was reduced As is the case for the intravenous cath-eter model the urinary catheter model developed by Andes and

coworkers (188 203 205 206) is extremely effective for studyingurinary catheter infections In a subcutaneous model van Dijckand coworkers (207 208) seeded pieces of catheter with implantedC albicans yeast cells subcutaneously in the back of a mouse (Fig5E) After 6 days the catheters could be removed and analyzedCells formed a dense biofilm composed of yeast cells hyphae andECM on the inner tube surface (208) Alternatively biofilmscould be assessed by using strains expressing a luciferase geneattached to the cell wall and noninvasive bioluminescence imag-ing (207) The technology for this preparation appears to be rela-tively simple and should be useful in in vivo studies of drug sus-ceptibility

Models have also been developed in which biofilms are formedon an acrylic denture placed on the palate of a rat In 1978 Olsenand Bondevik (209) first used an inserted acrylic plate fitted to thepalate of a rat to study Candida stomatitis but in that study thebiofilm that formed on the plate was not assessed In 2010 Nett etal (210) developed a denture model using temporary dentalacrylic deposited directly onto the palate of immunosuppressedrats treated with antibiotics (Fig 5D) The denture was securedwith dental wire between molars In this model a spacer wasplaced against the hard palate prior to depositing acrylic resinAfter the spacer was removed a 1-mm-wide void between thepalatal and acrylic surfaces was created that was filled with a sus-pension of C albicans yeast-phase cells After 72 h of incubationNett et al (210) found that a mixed-species biofilm developed onthe acrylic surface composed of one-third C albicans cells andtwo-thirds intestinal bacteria In controls in which there was noinoculum biofilms formed that were composed of up to one-fourth indigenous C albicans cells and three-fourths intestinalbacteria SEM analyses revealed budding yeast cells and hyphaebut no consistent architecture Another rat denture model (Fig5C) was developed by Lee and colleagues (211 212) A denturewas made from a dental impression cast for each individual ratemploying techniques similar to the prosthodontic proceduresused for humans The custom-fit removable denture containedan embedded metal rod secured to the anterior hard palate bymagnets within the fixed denture which was in turn secured byorthodontic wires to the rear molars Unlike the model by Nett etal (210) the rats in this study were immunocompetent and werenot treated with antibiotics C albicans was introduced into thetight interface between palate and denture as a paste of centrifugedC albicans yeast-phase cells The paste was applied directly to themucosal surface prior to placement of the denture In this modelbiofilms formed on both the denture surface and the palate (211)Biofilms containing yeast-phase cells hyphae and ECM formedon the denture after approximately 4 weeks postinoculation andon palates after 6 weeks even though colonization of the generaloral region by C albicans remained constant after 7 days No mix-ture of C albicans and bacteria was observed in the biofilms pos-sibly as a result of the tight fit of the denture to the palate Thebiofilms formed on the palate and the denture as viewed by SEMappeared similar to those formed in vitro using the Douglas modelwith human saliva-conditioned acrylic (211 213) Hence in thatstudy (211) the in vitro and in vivo models were architecturallysimilar The reason for comparability may have been due to theflow dynamics in the area between denture and palate which waslow Models in which C albicans was inoculated directly into theoral cavity or under the tongue of a rodent in the absence of den-tures appeared to involve primarily invasion of hyphae at the ven-

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tral surface of the tongue and thus appear to be better as models ofmucosal invasion (214ndash217) However small biofilms did appearto form in the crevices between papillae on the tongue surface

Recently Srinivasan et al (104 218ndash220) developed a uniquein vitro model which they referred to as a ldquonano-biofilmrdquo con-sisting of C albicans cells embedded in rat tail type I collagenalginate hydrogels In the model the nano-biofilms were printedonto microarray slides SEM images presented by Srinivasan et al(219 220) revealed that the seeded yeast-phase cells within thechips formed hyphae in all directions but an organized biofilmwas not evident presumably because of the structural impedi-ments of the synthetic matrix Hence this model may moreappropriately be useful for invasion rather than biofilm stud-ies Several additional in vitro biofilm models distinct from theDouglas paradigm have been developed that introduce flowacross the biofilm during development They are described in alater section of this review Suffice it to say that the variety ofmodels especially those with catheters and between denturesand palate introduce conditions quite distinct from those inthe Douglas model

METHODS FOR MEASURING BIOFILMS MAY BE A SOURCEOF VARIABILITY

Even if studies in different laboratories employ similar modelsdifferent methods of assessment have the potential to generatedifferent results and interpretations Therefore before consider-ing the variability of C albicans biofilms resulting from the use ofdifferent strains and different model conditions in vitro the vari-ability inherent in the different methods of assessment must beaddressed Hawser et al (168 179) originally evaluated biofilmformation by measuring three parameters dry weight the incor-poration of [3H]leucine into total acid-perceptible protein andthe reduction of tetrazolium salts to formazan These and subse-quent studies also routinely used SEM but primarily for assessingcellular phenotype and matrix deposition and usually for onlyone strain Assessing dry weights of 48-h mature biofilms isstraightforward and highly reproducible (45 179) Dry weighthowever provides no information regarding cellular phenotypearchitecture or the contribution of the ECM Indeed Pujol et al(45) showed that biofilms formed by a number of C dubliniensisstrains exhibited very different architectures but still attained sim-ilar dry weights If used in conjunction with other assessmentmethods however dry weight remains a fundamental parameterof biofilm growth Assessing [3H]leucine incorporation is repro-ducible (168) and provides a measure of the rate of protein syn-thesis However when used to assess a 48-h biofilm it representsan assessment of the rate of translation during biofilm mainte-nance not that during biofilm growth and development It is alsonot an expedient method and is institutionally restricted becauseof the use of radioactivity Tetrazolium salt reduction has been themost common assay primarily for expediency in analyses of theeffects of antifungal drugs on biofilm development This assaymeasures respiratory or metabolic activity The reduction of MTT[3-(45-dimethyl-2-thiazolyl)-25-diphenyl-2H-tetrazolium bro-mide] or XTT both of which are water-soluble tetrazolium saltsby NADH-dependent oxidoreductase in the mitochondria and inspecialized vesicles produces formazan which can be quantitatedcolorimetrically (221 222) The assay was developed originally toassess the level of oxidative metabolism (223) and has provenuseful for assessing cell death (181 224 225) However tetrazo-

lium salt reduction is an indirect method for measuring the extentof biofilm growth especially for mature biofilms for several rea-sons First stationary-phase yeast cells in the basal layer of a bio-film and the more proximal compartments of a hypha are notundergoing cell division These compartments may therefore befar less active metabolically than the most apical compartment ofhyphae Second such a measure does not reflect biofilm massvolume thickness cellular differentiation or architecture ormost importantly the extent of matrix deposition Its expediencyfor assessing drug susceptibility in 96-well plates has been demon-strated and its reproducibility verified (226) but its usefulness incomparing extents of biofilm development is suspect In a briefbut revealing side-by-side comparison using the XTT reductionassay and dry weight Kuhn et al (227) found that while someisolates of C albicans from dentures catheters blood bronchialtubes urine the vagina and skin formed biofilms with similarlyhigh XTT reduction values and dry weights others formed bio-films with relatively high dry weights but relatively low XTT re-duction values and vice versa In an independent study Li et al(228) found that the variation between biofilms formed by differ-ent strains obtained from the oral cavity the environment and thevaginal canal was greater for crystal violet (CV) absorption thanfor XTT reduction CV targets primarily mucopolysaccharidesand amyloids (229) which may vary dramatically as a result ofdifferences in matrix deposition In an excellent analysis of theparameters that can affect XTT reduction methods for antifungalsusceptibility studies Nett et al (230) provided a list of problemsand possible reasons for those problems They also providedmethods for optimization However these precautions do notalter the inherent problem of the assay namely that it mea-sures mitochondrion- and vesicle-based NADH-dependent oxi-doreductase activity (231) which probably differs between themultiple phenotypes in a biofilm between different phases of thebudding cycle and between the proximal and apical compart-ments of hyphae during elongation Moreover measuring XTT orMTT reduction at 48 h after formation of the biofilm is over 90complete and the rate of growth has decreased over 10-fold frompeak values (168) does not seem to be a very direct or accuratemeasure of biofilm growth and development To make things evenmore complicated Liu et al (231) reported that MTT is mem-brane impermeant and they concluded that the tetrazolium saltmay have to be taken up into living cells by endocytosis The MTTand XTT reduction assays may therefore be at least in part ameasure of uptake not cell metabolism

One of the most important traits for assessing biofilm de-velopment is architecture As noted above SEM (168 181 192232 233) and to a lesser extent CLSM have been used to assesscellular phenotype extracellular matrix relative growth andarchitecture (181 213 232 234) However both methods havelimitations and artifacts and can lead to variability dependingupon how they are performed There have been two proceduresused for SEM analysis of Candida albicans If biofilms are fixedwith glutaraldehyde in cacodylate buffer flash frozen and thenfreeze-dried (179 235) one preserves the collapsed ECM butloses resolution of the hyphal and yeast morphologies (Fig 4F)If biofilms are fixed and dehydrated (45) hyphae and yeast cellsare imaged but the ECM is lost (Fig 4E) As noted by Little etal (236) for bacterial biofilms solvent replacement of waterfrom a biofilm removes matrix components In addition




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whether the SEM preparation is frozen or dehydrated afterfixation native architecture is lost

CLSM (237ndash239) can provide information on internal archi-tecture cellular phenotype and biofilm thickness In contrast tothe SEM procedure there is no dehydration step after fixationwith paraformaldehyde or other fixatives and thus more of thenative architecture is preserved (240) CLSM involves stainingrather than surface shadowing and the collection of hundreds ofoptical sections of a biofilm through the z axis provides informa-tion about different depths Staining artifacts however can arisein CLSM For instance when a 48-h fixed biofilm is overlaid witha solution of calcofluor which targets cellulose and chitin (241)staining is most intense in the upper portion of the biofilm pre-sumably because of disproportionate binding of the dye to hyphaeat the top of the biofilm during dye penetration (45 183 213)This artifact is evident in side views of projection images of a stackof CLSM scans such as those presented in Fig 4D and G CLSM ishowever effective at comparing the thicknesses of biofilms (183)and with advanced processing software it can be used to image thearchitecture of intact hyphae in side views of projection images(45) (Fig 4D) An additional problem arises when CLSM is usedto view the ECM with dyes such as calcofluor which binds tochitin and cellulose (242) These ECM polysaccharides are alsolocalized in the hyphal wall In the case of calcofluor hyphae stainfar more intensely than the diffuse ECM One must thereforelower the laser intensity to view hyphae by CLSM at the expense ofviewing the ECM If intensity is increased the ECM can be visu-alized at the expense of resolving distinct hyphae because hyphalwalls stain so much more intensely (39 243) Light attenuation isless problematic when multiphoton CLSM is applied (97)

Finally transcription profiling is evolving as a major methodfor assessing biofilm development Profiling can be performed byassessing genome-wide transcription by using microarrays (195200 244ndash248) NanoString nCounter technology (249 250) andRNA sequencing (RNA-Seq) (197 200) or by using quantitativereverse transcription-PCR (RT-PCR) or Northern blot analysis ofa smaller select group of genes (251) Such comparisons are oftenmade between biofilm preparations and planktonic cultures Un-fortunately assessing the accuracy reproducibility and concur-rence of these technologies has been hampered by the variety ofgenome-wide transcription platforms employed and the differingconditions used to prepare biofilms such as different media sub-

strata times of analysis and fluid flow or mixing methods (Table2) In two general reviews of the reliability and reproducibility ofchip technology it is noted that reproducibility can be attained forabundant transcripts but that this is not always the case for low-abundance transcripts (252 253) Canales et al (252) noted thatthe latter problem is especially true for measurements on differentplatforms (252) As global analyses of gene expression segue fromDNA chips to RNA-Seq new challenges arise This is best exem-plified by the elegant study of Nobile et al (200) who used bothtechnologies Using a mutant library they developed a model of atranscriptional network controlling biofilm formation in which1000 target genes were regulated by six key transcription factorsIn that study they pooled data from a DNA chip analysis and anRNA-Seq analysis to determine genes differentially expressed dur-ing biofilm development rather than selecting genes that inter-sected in the two studies (ie genes upregulated or downregulatedin the same way by both methods of assessment) This was donefor inclusiveness However the question related to our focus inthis review is how comparable the results of the two methods weregiven that the preparations were generated in the same study andusing the same model for comparing planktonic and biofilm geneexpression levels The threshold employed for a significant differ-ence between planktonic and biofilm expression levels for a genewas 2-fold (ie 2-fold) Of the 2235 genes that were identifiedas up- or downregulated by both platforms or by one or the other1792 were actually assessed by both platforms Of the latter genes405 (23) were found to be regulated similarly by both methods(see the supplemental material in reference 200) which is a sur-prisingly low level of correspondence The study by Nobile et al(200) was followed by an additional one by the same group usingthe same model and the same microarray platform but not includ-ing an RNA-Seq analysis (245) In the supplemental material ofthe expanded study (245) comparison of the transcription pro-files of planktonic (log-phase) cells and biofilms identified 238genes that were upregulated or downregulated 2-fold and werealso identified as similarly regulated in the study of Nobile et al(200) However 845 additional genes were found to be differen-tially regulated 2-fold that were not assessed in the Nobile et al(200) study In the Nobile et al (200) study 577 genes were iden-tified as up- or downregulated 2-fold that were not identified tobe regulated similarly in the Fox et al (245) study Finally 31 geneswere oppositely regulated (upregulation versus downregulation

TABLE 2 Review of the models substrates media assay times and assays employed for transcriptional profiling of Candida albicans biofilms

Authors yr (reference) Model Substrate Mediuma Assay time(s) (h) Platform (source)

Garciacutea-Saacutenchez et al 2004 (246) Flow Thermanox YNB medium 48ndash72 Microarray (Eurogentec)Murillo et al 2005 (247) Douglas Tissue culture plastic Hamrsquos F-12 medium 05ndash65 Microarray (Affymetrix)Yeater et al 2007 (248) Douglas Silicone elastomer

denture acrylicYNB medium 6 12 48 Microarray (in-house)

Sellam et al 2009 (292) Flow Silicone elastomer YPD 1 3 6 Microarray (BiotechnologyResearch Institute)

Nett et al 2009 (195) Venouscatheter

Catheter wall Blood 12 24 Microarray (BiotechnologyResearch Institute)

Bonhomme et al 2011 (244) Flow Thermanox SD medium 24 32 40 Microarray (Eurogentec)Nobile et al 2012 (200) Douglas Tissue culture plastic Spider medium 48 Microarray (Agilent Technology)

RNA-SeqDesai et al 2013 (197) Douglas Tissue culture plastic Spider medium 48 RNA-Seq (Illumina GA2 solid system)Fox et al 2015 (245) Douglas Tissue culture plastic Spider medium 8 24 48 Microarray (Agilent Technology)a The compositions of the media are given in Table 1

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or downregulation versus upregulation) 2-fold in the two stud-ies Some of these differences certainly arose between measure-ments very close to but not attaining the significance thresholdBut even so if such differences arise between sequential studies inthe same laboratory in which the same methods are used to obtainpreparations and the same microarray platforms are used for as-sessment one wonders what differences would be found espe-cially for low-abundance transcripts between studies in differentlaboratories using different conditions and different platformsGenome-wide transcription profiling does reveal differences inselect abundant genes many of them reproducible betweenlaboratories and when limited to such genes it provides apowerful tool for assessing the progression and maturation ofbiofilm formation It would be extremely helpful in these stud-ies if the abundances of transcripts especially for those notshowing congruence were noted In addition a threshold of2-fold means that a gene must be expressed at a level onlytwice that of a planktonic culture or half that of a planktonicculture to be considered differentially regulated in a biofilmThe reliability and reproducibility of genome-wide profilingfor a particular platform and the congruence between plat-forms need to be addressed The use of this technology be-comes even more problematic when phenotypic heterogeneityis considered An and Parsek (254) point out in the last line oftheir discussion on the use of DNA microarray technology ingenome-wide assessments of transcription in bacterial bio-films that ldquothe real payoff might be when we are able to profiledistinct spatial or functional subpopulation in the commu-nityrdquo This also holds true for C albicans biofilms

STRAIN VARIABILITY

The complexity of C albicans biofilm formation in both space andtime and the problems that arise in measuring its progress providea contextual framework for assessing genetic and conditional fac-tors responsible for biofilm variability The first of these factors isstrain variability Do all strains form biofilms similarly This ques-tion is fundamental to biofilm studies dealing with gene expres-sion and mutational defects or drug resistance since a majority ofthese studies employ one strain or mutants derived from a singleparent strain Single-strain studies assume that the strain em-ployed is representative of all strains a majority of strains or theaverage strain Hawser and Douglas (168) performed the firststudy of strain variability They analyzed 15 strains but two weremutants one that could not form hyphae and one that could notform yeast-phase cells Both mutant strains were derived from thesame parent strain Their results are summarized for the 13 natu-ral strains (ie nonmutant strains) in their collection along withthe results of other studies of strain variability in Table 3 Theextremes of the ranges for the three traits (dry weight [3H]leucineincorporation and MTT reduction) analyzed by Hawser andDouglas (168) differed by 21- 23- and 32-fold respectively forcultures in medium containing 50 mM glucose and by 23- 38-and 32-fold respectively for cultures in medium containing 500mM galactose (Table 3) The standard deviations (as percentagesof the means) were 24 24 and 21 respectively for biofilmsformed in YNB medium plus glucose and 18 32 and 28respectively for biofilms formed in YNB medium plus galactose(Table 3) It should be noted that the strains used by Hawser andDouglas (168) and by all but one of the other studies reviewed forTable 3 were not assessed for the configuration of the mating type

locus which affects the size and functional characteristics of bio-films (39) and the susceptibility of planktonic cells to antifungals(41) As noted however it is reasonably safe to assume that arandom collection of strains as in the study by Hawser and Doug-las (168) will usually contain no more than 10 MTL-homozy-gous isolates (40 255ndash257) In a subsequent study of variability byKuhn et al (26) employing the Douglas model biofilm formationparameters were compared among 10 strains by measuring XTTreduction and dry weights of 48-h biofilms Isolates varied in rel-ative XTT reduction values (A492) which were between 065 and124 and in relative dry weights which were between 055 and130 mg (Table 3) For both measurements the extremes variedapproximately 2-fold The standard deviations (as percentages ofthe means) were 29 and 30 respectively (Table 3) These vari-ability measurements were close to those obtained by Hawser andDouglas (168) (Table 3) As previously noted Kuhn et al (26)found that there was poor correspondence of the relative rankingsin the collection for individual strains between XTT reduction anddry weight measurements

In 2003 Li et al (228) compared 115 isolates representing thefour major clades of C albicans They used a variation of theDouglas model developed by Ramage et al (181) that employedthe tissue culture plastic surface of the wells of 96-well platesrather than a catheter disc as the substratum The collection wasseparated into isolates from the oral cavity the vaginal canal andthe environment Li et al (228) measured XTT reduction activityand CV staining Just as Kuhn et al (26) reported there was poorcorrespondence of relative rankings between the XTT and CVmeasurements for individual strains within a clade Li et al (228)found that the means standard deviations and ranges for XTTreduction and CV staining were statistically indistinguishable be-tween clades The means standard deviations for XTT reduc-tion for 16 oral 31 environmental and 37 vaginal isolates were0077 0052 0079 0025 and 0063 0020 respectively Thestandard deviations of XTT reduction values (as percentages ofthe means) were 62 32 and 32 respectively which are muchhigher than the standard deviations in both the Hawser and Doug-las (168) and Kuhn et al (26) studies (Table 3) The standarddeviations (as percentages of the means) for crystal violet stainingwere even higher ie 159 113 and 57 respectively sug-gesting a problem in the reliability of the latter measurement giventhe parallel results for XTT reduction Jain et al (258) used thesame variation of the Douglas model as well as the XTT reductionassay to compare biofilm formation levels among 63 isolates fromsaliva in RPMI medium or artificial urine The standard devia-tions (as percentages of the means) for XTT reduction measure-ments were 9 for RPMI 1640 medium and 20 for urine (Table3) The value for RPMI 1640 medium was lower than those ofHawser and Douglas (168) Jain et al (258) did not provide indi-vidual strain data for C albicans so the range and extremes couldnot be computed Villar-Vidal et al (259) used the same modelbut sampled the biofilms at 24 h rather than 48 h as well as thesame XTT reduction measurement to analyze variability among16 oral isolates and 12 blood isolates The means standard de-viations for XTT reduction were 10 04 and 09 04 respec-tively (Table 3) The ranges were 03 to 13 for oral isolates and 03to 15 for blood isolates representing 4- and 5-fold differencesbetween extremes (Table 3) The standard deviations (as percent-ages of the means) were 40 and 41 for oral and blood isolatesrespectively or approximately double the values obtained in the




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Hawser and Douglas (168) Kuhn et al (26) and Jain et al (258)studies Finally Pujol et al (45) performed an architectural com-parison of three strains that had been typed a for the MTL con-figuration namely P37039 P37037 and SC5314 They found ahigh level of uniformity in thickness (78 5 m 76 2 m and74 3 m respectively) They also found similar basal yeast cellpolylayers hyphal upper layers vertical orientations of hyphaeand densities of the ECM However the sample size was too smallfor valid comparisons with the previous studies reviewed hereThey also presented thickness measurements for six aa and six strains which had similarly low variability (Table 3) Thedifferences in extremes were 12-fold and 11-fold for the aa and strains respectively However the variability of dry weightsfor a collection of 10 a strains containing 5 aa and 5 cells was similar to that obtained by Hawser and Douglas for astrains (Table 3)

Assessing variability between strains may therefore be influ-enced by a scientistrsquos technique or the assessment parametersthemselves as well as the variability due to differences betweenstrains The data at hand however suggest that variabilityamong strains can be as great as 2-fold or more and this mustbe considered in comparing data from different studies espe-

cially those that employ only one test strain For such studies itwould be prudent to consider whether the strain employed isrepresentative (ie near the mean) of a collection of naturalstrains at least for basic parameters such as dry weight phe-notypic composition and architecture SC5314 the strain thatis parental to a number of derivative strains used for mutantanalyses appears to be quite normal in the formation of bio-films in vitro (183 184) However that does not mean that allof the engineered derivatives used to select mutants form bio-films normally Therefore a parental strain that is a derivativeof SC5314 or another natural strain not the original naturalstrain must serve as the immediate control in comparisons ofbiofilm formation and it should be compared to the originalnatural strain such as SC5314

VARIABILITY DUE TO THE ORIGINAL PLANKTONIC CELLINOCULUM

In the original experiments performed by Hawser and Douglas(168) SEM images of cultures in YNB plus 500 mM galactoserevealed that after a 1-h period of incubation of planktonic cells inthe wells of the multiwell plate the adhering population was farfrom confluent It is not clear if this was due to too dilute an

TABLE 3 Strain variability in studies in which collections of three or more isolates were analyzed in parallela

Authors yr(reference)

Mediumd

(sugar) Substrate No of strains Assay Mean SDSD as of mean Range

Fold differenceof extremes

Hawser and Douglas1994 (168)

YNB medium(glucose)

Catheter 13 Dry wt (mg) 082 020 24 052ndash110 21[3H]leucine

incorporation4476 1058 24 2550ndash5886 23

MTT A540 052 011 21 038ndash066 32YNB medium

(galactose)Catheter 13 Dry wt (mg) 176 032 18 051ndash199 23

[3H]leucineincorporation

8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)

YNB medium Catheter 10 Relative dry wt 082 023 29 055ndash130 24Relative XTT 102 030 30 065ndash124 19

Li et al 2003 (228) YNB medium TCP 16 (oral) XTT A490 0077 0052 62 0053ndash0329 62CV A570 0209 0424 159 0050ndash2179 434

31 (environmental) XTT A490 0079 0025 32 055ndash0158 29CV A570 0217 0246 113 0087ndash1032 119

37 (vaginal) XTT A490 0063 0020 32 0049ndash0157 32CV A570 0143 0081 57 0043ndash0526 122

Jain et al 2007 (258) RPMI 1640e TCP 63 Relative XTT 055 005 9 mdash mdashUrine TCP 63 Relative XTT 100 020 20 mdash mdash

Villar-Vidal et al2011 (259)b

RPMI 1640e Catheter 16 (oral) XTT A492 101 040 40 030ndash133 44Urine 12 (blood) XTT A492 090 037 41 030ndash150 50

Pujol et al2015 (45)c

RPMI 1640e Catheter 3 (a) Thickness (mm) 76 2 3 74ndash78 11Catheter 6 (aa) Thickness (mm) 56 3 5 51ndash58 12Catheter 6 () Thickness (mm) 58 2 3 56ndash61 11Catheter 10 (a ) Dry wt (mg) 075 013 17 048ndash098 20

a TCP tissue culture plastic MTT A540 MTT reduction as measured by the absorbance at 540 nm relative dry weight dry weight relative to the wild type or the mean relativeXTT XTT measure relative to the wild type or the mean CV A570 crystal violet staining as measured by the absorbance at 570 nm XTT A490 or A492 XTT reduction as measured at490 nm or 492 nm mdash data not provided for calculationb The only study in which sampling occurred at 24 h not 48 hc The only study performed at 28degC not 37degC because of white cell induction at temperatures of 35degCd Sugar contents for the different studies were as follows for the study of Hawser and Douglas (168) 50 mM glucose or 500 mM galactose for the study of Kuhn et al (26) 50 mMglucose for the study of Jain et al (257) 11 mM glucose for the study of Li et al (227) 50 mM glucose for the study of Villar-Vidal et al (258) 11 mM glucose and for the studyof Pujol et al (45) 11 mM glucosee Buffered with MOPS

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inoculum or to a loss of cells during preparation for SEM If ex-periments are performed to assess adhesiveness as a function ofinoculum concentration by light microscopy conditions can bedefined empirically to obtain a uniform monolayer of yeast cellsafter the 60- to 90-min incubation period as in Fig 4A and B It isalso evident in the SEMs provided by Hawser and Douglas (168)that the original planktonic cells used for inoculation were in dif-ferent stages of the budding cycle This could have been the resultof heterogeneity of the yeast cell cycle in the planktonic cellpopulation grown overnight Growing cells planktonically indifferent media can lead to differences in the heterogeneity ofthe budding yeast cell cycle in late log phase or stationaryphase This in turn can result in phenotypic heterogeneity ofthe initial adhering cell population in the biofilm model Cellscan be in different phases of the yeast cell cycle (G1 S G2 andM) heterogeneously undergoing changes in evagination budformation intracellular architecture cell separation DNAreplication and nuclear division (260ndash262) Cells can also beundergoing mitochondrial duplication and migration hetero-geneously (263) Heterogeneity will have an impact on the syn-chrony of cell division at the onset of the formation of a basalyeast cell polylayer This in turn might affect measurements ofearly biofilm development Attaining synchrony was of para-mount importance more than 4 decades ago in studies relatedto the yeast cell cycle and cellular physiology (264ndash266) as wellas to the bud-hypha transition in C albicans (267)

Almost all planktonic preparations of C albicans are grown inthe yeast phase overnight in culture tubes or flasks that are rapidlyrotated Cell density is then commonly measured by determiningthe optical density Optical density is not however a direct mea-sure of cell number but rather of mass (268) and it must benormalized empirically to the cell concentration The OD may beinfluenced by the heterogeneity of the budding cycle adhesionand flocculation and by differences in strain-specific cell sizeThe use of a hemocytometer for verification of concentrationmay be a bit more time-consuming than measuring the opticaldensity but it provides researchers with two benefits an accu-rate count of cell number and a visual estimate of the hetero-geneity of the budding yeast cell cycle including the presenceof pseudohyphae and hyphae in the preparation For white andopaque cells in MTL-homozygous cultures it provides a mea-sure of phenotypic homogeneity The concentration can thenbe adjusted by dilution and the adjusted planktonic suspensioninoculated into test wells of the in vitro biofilm model And ifheterogeneity is a problem another medium strain or prepa-ration can be selected

Phenotypic homogeneity of the planktonic cell preparationcan be a concern especially for measurements during the earlystages of biofilm formation Cell cycle-regulated genes have beenidentified in Saccharomyces cerevisiae (269) including modulationof histone messengers first reported in 1981 (270) For C albicansKusch et al (271) used two-dimensional (2D) electrophoresis tocompare proteins between log- and stationary-phase cells Dra-matic differences were observed in proteins involved in all aspectsof metabolism Unfortunately the different media used to cultureC albicans planktonically result in quite different levels of cellcycle heterogeneity in stationary phase In Fig 6 representativefields of cells and the proportions of unbudded yeast-phase cellsare presented for the a strain SC5314 cultured planktonically at26degC for 24 and 48 h with rotation in a variety of media that have

been used for planktonic cell growth The compositions of thesemedia are presented in Table 1 At 24 h cultures in YNB YPDsLeersquos Spider SD and RPMI 1640 (with or without MOPS) mediaconsisted of over 70 unbudded yeast-phase cells (Fig 6A to Fand H) At 48 h however only planktonic preparations in YPDand sLeersquos media contained over 70 unbudded yeast-phase cellsie 93 and 96 respectively (Fig 6B and D) suggesting thatheterogeneity may increase as planktonic cultures become sta-tionary There are multiple reasons for why it may be advanta-geous to obtain a relatively uniform population of unbudded yeastcells in stationary phase for the initial planktonic inoculum of abiofilm model First cell cycle synchrony at the onset of a biofilmexperiment allows one to interpret measurements as representa-tive of a single cell phenotype Second cells in one growth phasesuch as stationary phase provide a single physiology The differ-ences in cell physiology between log- and stationary-phase cellshave been described primarily for bacteria (272ndash274) In additionthe susceptibilities to antifungal agents of planktonic cells of Scerevisiae differ between log- and stationary-phase cells (275) asdo the proteomes (271) Third the presence of a significant num-ber of pseudohyphae or hyphae adds a second dimension to phe-notypic heterogeneity Therefore documenting that a majority ofcells in the planktonic cell inoculum have entered stationaryphase are unbudded and of uniform size and are uniformly in theyeast phase should be considered in the development of in vitromodels of biofilm formation

VARIABILITY DUE TO FLOW ROTATION OR ROCKING

In their original report Hawser and Douglas (168) did not mixtheir cultures by rotation or rocking but in a subsequent report(179) they rotated cultures at 30 rpm which increased biofilmthickness Mixing can affect O2 and CO2 tension in the biofilmmicroenvironment as well as increase accessibility to nutrients Itcan also cause shear forces that affect mechanoreceptors Suchreceptors have been demonstrated to exist on the surfaces of hy-phae and function in thigmotropism (276) Whether these recep-tors play a role in the architecture of the upper hyphal region of abiofilm is not known However if the amount of mixing (ie viarotation shaking rocking or continuous flow) is too great cellarchitecture uniformity along the substratum matrix depositionand synchrony of the developmental program may be affectedTherefore mixing or flow conditions must be optimized empiri-cally for each in vitro model of biofilm formation Because there isthe reasonable perception that biofilms are challenged with con-stant flow in vivo several researchers have incorporated flow intotheir models For bacterial biofilms two major flow models havebeen employed a flow cell (277) and a rotating disc reactor (278279) The flow cell is constructed from polycarbonate or polym-ethyl methacrylate and contains three open tubular chambersThe chambers are covered by a glass coverslip (the substratum forthe biofilm) Medium enters through an inlet port and exitsthrough an outlet port (277) The rotating disc reactor contains astir disc to generate flow across 18 removable polycarbonate chipswith adherent cells (278) To generate flow across a C albicansbiofilm Loacutepez-Ribot and colleagues (188 280) developed a modelin which a strip of serum-coated silicone elastomer was first incu-bated with cells in a conical tube to promote adherence The stripwas then placed in a tube with inlet and outlet ports and bufferedRPMI 1640 medium was continuously dripped down the strip Ina second model (281) discs seeded with cells were placed in mul-




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tiple wells bored into an acrylic block through which medium waspumped In both cases SEM images revealed that robust biofilmsformed Garciacutea-Saacutenchez et al (246) developed a model in which aseeded Thermanox plastic coverslip was placed in a test tube thatcontained a narrower tube that released medium and air Mediumwas continuously removed through an outlet port at the top Theturnover rate was 06 mlmin for a 40-ml chamber or 15 of thevolume of the medium per minute The diagram of the modelprovided in the study (246) suggests that bubbles of air may haveflowed across at least the upper portion of the biofilm SEM im-ages of wild-type biofilms formed in this flow model revealedpatches on the substratum containing mixtures of tangled hyphaepseudohyphae and budding cells Some of the budding cells wereunusually large and possessed at least six bud scars Results ob-

tained with the different flow chambers may be difficult to com-pare given the differences in design In developing flow chambersfor bacteria Crusz et al (277) emphasized the importance of min-imizing bubbles since they were shown to disrupt biofilm devel-opment More importantly in most studies of C albicans biofilmsin which medium is either mixed or moved by flow it is difficult tocompare in vitro shear forces with those on the surfaces of hostniches where C albicans biofilms form In addition there havebeen no systematic studies of the effects of transient changes inflow on biofilm formation Such changes do occur in several hostniches For instance although there is normally very little flowacross biofilms that form between dentures and palate periodicmovement may cause turbulence and disruption Moreover den-tures are usually washed frequently rather than retained in place

FIG 6 Comparison of budding cell phenotypes of planktonic cultures of C albicans strain SC5314 grown in culture tubes shaken for 24 and 48 h using eightdifferent media to obtain planktonic yeast-phase cell cultures for in vitro C albicans biofilm studies Budding cells were scored as cells with buds exhibiting adiameter of less than two-thirds that of the mother cell and still attached to the mother cell The percent unbudded cells in culture is presented in the upper leftcorner of each panel Between 500 and 1100 cells were scored for each preparation Arrows point to examples of budding cells Compositions of the media canbe found in Table 1

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for 72 h (210) or up to 8 weeks (212) When an individual is awakethe flow rate from the salivary gland is approximately 07 mlminor 42 mlh (282ndash284) but it decreases dramatically during sleep(285) Swallowing causes agitation and rapid movement of thebolus of saliva to the esophagus (286) and chewing causes ex-treme turbulence In the intestine fluid flow is approximately 10mlmin (287) but flow rates at the topologically complex intesti-nal surface may be far lower especially in the crypts of Lieberkuumlhnand other crevices Flow rates of pressure-injectable catheters canrange from 4 to 6 mls or 240 to 360 mlmin and the pressure canbe 100 to 300 lbin2 (288) Indwelling urinary tract catheters mayhave relatively continuous high flow rates (289) whereas short-term indwelling intravenous catheters may have high flow ratesonly during therapeutic delivery or sampling (288 290) Fordrainage and therapy long-term catheters can be indwelling formore than 6 weeks The development of in vitro flow modelsespecially those with the rotating or rocking conditions used inmost in vitro models may therefore not mimic any in vivo condi-tions The effect of flow on bacterial biofilm formation was poi-gnantly demonstrated by Greenberg and colleagues in a compar-ison of biofilms formed by wild-type Staphylococcus aureus and amutant for the accessary gene regulator locus agr (279) Most agrisolates which are defective in quorum sensing were found to bedefective in biofilm formation (291) The number of cells in thebiofilm of the agrD mutant was less than one-third that in thewild-type biofilm in the static dish model but over two times thatin the wild-type biofilm in the spinning disc reactor (279) With aflow cell rather than spinning disc model the biofilms formed by theagrD mutant and the wild type were architecturally indistinguish-able after 1 and 5 days as revealed by CLSM (279) These bacterialresults suggest that flow rate must be considered an important vari-able in biofilm formation Hawser et al (179) clearly showed thatunder their conditions there was a significant increase in dry weightfor cultures rotated at 20 rpm on an orbital rotating platform com-pared to undisturbed cultures However rotation at speeds higherthan 30 rpm reduced dry weight Based on these results one mustwonder how rotation speeds of 200 rpm which have been employedaffect architecture One must also consider the possibility that rota-tion at 200 rpm rocking a culture 12 timesmin at a 6deg pitch andconstant flow across a biofilm in a flow apparatus result in differentshear forces with different effects on biofilm formation The point isthat the method used may lead to major differences in biofilm forma-tion and must therefore be taken into account in comparing datafrom different studies

VARIABILITY DUE TO DIFFERENCES IN DEVELOPMENTALTIMING

Although this is not always the case in the majority of studies of Calbicans biofilms the assessment of biofilm formation is per-formed at a single time point This has been true for comparisonsof biofilm formation between mutants and wild-type strains andfor analyses of antifungal drug susceptibility A single time pointmay not be sufficient The formation of C albicans biofilms in invitro models must be regulated by an underlying developmentalprogram involving cellular differentiation the divergence of cel-lular phenotypes complex cell-cell interactions and signaling thatchanges with time Time-dependent changes during biofilm for-mation have been demonstrated for gene expression (244ndash247292) cellular phenotype (168 179 183 184) architecture (183184) and the deposition of ECM (293ndash296) Hence by definition

there is an ordered sequence of dependent events and in somecases parallel pathways with a time table in complex develop-mental programs (297) For C albicans we know that there arechanges in adhesion yeast cell multiplication germination hyphaelongation and ECM deposition If there is a change resultingfrom a mutation in the length of time required for any of thedevelopmental stages in the underlying program of biofilm for-mation a difference in the genetic makeup between naturalstrains different model conditions or the absence or presence ofan antifungal drug a single measurement at a single time pointmay simply reflect a difference in when things happen not if theyhappen as outlined in the simple hypothetical example presentedin Fig 7 In this example strain ldquoardquo forms a basal yeast cell poly-layer during the first 5 h while strain ldquobrdquo does so during the first 20h Each then forms the upper hyphalECM layer over the subse-quent 43 h At 48 and 63 h respectively both have formed indis-tinguishable mature biofilms with similar pathogenic traits (Fig7) However if both were sampled at 15 h ldquoardquo would be extendinghyphae and depositing ECM while ldquobrdquo would be undergoing cellmultiplication in the yeast phase And if both were sampled at 33h ldquoardquo would have come close to completing a mature biofilmwhile ldquobrdquo would be in the middle stages (Fig 7) If ldquoardquo and ldquobrdquo werecompared at 4 and 15 h respectively or at 15 and 30 h respectivelyarchitecture and physiological measurements would be similarHigh-frequency epigenetic switching systems that selectively affectthe lengths of time between developmental stages but not the se-quence of stages that occur have been described for the model devel-opmental system Dictyostelium discoideum (298ndash300) Thereforeit seems reasonable to suggest that comparisons between dif-ferent natural strains wild-type strains and mutant derivativesuntreated and antifungal-treated cultures and cultures devel-oped under different model conditions should include multi-ple time points Just as importantly architectural landmarksshould be assessed in parallel at multiple points to verify phe-notypic comparability

EFFECTS OF MEDIA ON BIOFILM DEVELOPMENT

Hawser and Douglas (168) performed the first experiments on theimpacts of different media on biofilm development They firstcompared YNB medium containing either of two carbon sourcesie glucose and galactose Although dry weight [3H]leucine in-corporation and tetrazolium reduction increased and plateauedwith similar temporal dynamics the plateau values for galactosewere approximately twice those for glucose for all parameters Theextent of the difference was also strain dependent (179) In 2001Loacutepez-Ribot and colleagues introduced RPMI 1640 medium buff-ered with MOPS (181) RPMI 1640 medium was developed andoptimized specifically for the in vitro culture of human leukocytes(301) RPMI 1640 (MOPS) was also selected as a validated yeastsusceptibility testing medium after a direct comparison of Can-dida growth curves obtained using six candidate media (302) andwas recommended by the National Committee for Clinical Labo-ratory Standards (303ndash305) A number of media other than YNBand RPMI 1640 (MOPS) media have also been used to support Calbicans biofilm formation (Table 1) Compared to YNB mediumwith glucose Leersquos medium (185) supplemented with zinc andarginine (sLeersquos medium) (186) and YPD (yeast extract peptonedextrose) fail to support similarly robust 48-h biofilms (our per-sonal observations) It may be for this reason that the two mediaare rarely used in models to support biofilm formation in C albi-




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cans studies (Table 1) Spider medium which has been used in therat catheter model (117 193 202 306ndash308) and the Douglas wellmodel (309ndash311) was compared with RPMI 1640 (MOPS) me-dium for biofilm architecture (183) Spider medium supports for-mation of biofilms that lack a thick basal yeast cell polylayer Thebiofilms are composed of a carpet of yeast cells at the substratefrom which hyphae extend in relatively random directions withyeast cells formed throughout the upper layer The extracellularmatrix of these biofilms is less dense than that of biofilms formedin RPMI 1640 (MOPS) medium (183) Biofilms formed in RPMI1640 (MOPS) medium differ architecturally containing a thickbasal yeast-phase polylayer vertically oriented hyphae and adenser matrix (183) (Fig 3B) The lack of a thick basal yeast-phasepolylayer in Spider medium may be due to the rapid germination

of planktonic cells upon inoculation Agar containing Spider me-dium is a known facilitator of germ tube formation and hyphalgrowth (312ndash317) Interestingly even with the differences ob-served in architecture the biofilms formed by a cells in Spidermedium are relatively impenetrable by human white blood cellsand relatively resistant to fluconazole treatment although not tothe same degrees as the more organized biofilms formed in RPMI1640 medium (183) Spider medium becomes problematic how-ever when used to support MTL-homozygous biofilms in air asdescribed later in this review

VARIABILITY DUE TO THE MTL CONFIGURATION

Perhaps the most dramatic effect on the function and regulationof biofilms in C albicans relates to the configuration of the MTL

FIG 7 Example showing why a single time point might be invalid for comparing biofilms Strain differences mutations antifungal drugs and differentmodel conditions may affect when things happen not if they happen A hypothetical example is presented for a difference in timing in the first step of thebiofilm development program for two strains ldquoardquo and ldquobrdquo which results in an increase in the time it takes for the formation of the yeast-phase cellpolylayer for strain ldquobrdquo Germination hypha elongation and ECM deposition take the normal amounts of time but are shifted in terms of the total amountof time Data from a single time point at 15 or 33 h would reveal differences that could be interpreted as absolute when in fact they simply reflectdifferences in timing

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locus MTL-heterozygous yeast-phase cells and MTL-homozy-gous white-phase cells which are also in the yeast phase but differfunctionally form biofilms that are architecturally equivalent butdiffer in thickness and function (38 39 243 318 319) In thepreceding portions of this review we assumed that the majority ofstrains in nature were heterozygous (a) at the MTL locus eventhough the configuration of the MTL locus was rarely assessedThis assumption appears reasonable since in every major study ofthe MTL configuration the proportion of MTL-homozygousstrains fell between 5 and 10 of natural clinical strains (40 42255 256) In addition the C albicans strain which has been usedas the parent strain most often in mutational and molecular stud-ies is SC5314 which is a (320) However Rustad et al (256)found that 11 of 12 MTL-homozygous strains analyzed in a col-lection of 96 strains were resistant to azoles the major family ofanticandidal drugs Over half of the collection they studied had infact been selected for drug resistance The disproportionate rep-resentation of resistant strains among the MTL-homozygousmembers of the collection was reinforced by the discovery byOdds et al (41) that MTL-homozygous strains grown planktoni-cally are on average more resistant to the major azoles than MTL-heterozygous strains Hence the potential exists that collections ofisolates selected for a particular trait or collected from a particularhost niche may be enriched for MTL-homozygous strains More-over some a strains have a propensity to undergo homozygosisOn average a strains undergo homozygosis at the MTL locusspontaneously at a low frequency in vitro The predominantmechanism is the loss of one homolog of chromosome 5 whichharbors the MTL locus followed by duplication of the remaininghomolog (321) MTL-homozygous strains that persist in naturehowever originate primarily by mitotic recombination (322)Niche conditions that may stimulate homozygosis both in vitroand in vivo are not known Lockhart et al (40) found that one ofthree a strains injected into mice in the systemic mouse modelnamely strain P75063 underwent MTL homozygosis at an ex-tremely high rate in vivo Wu et al (321) showed that this strainalso underwent MTL homozygosis at an extremely high rate invitro However Wu et al (322) showed that MTL-homozygousstrains are less competitive than MTL-heterozygous strains in themouse systemic infection model and Ibrahim et al (323) foundthat tetraploids resulting from aa and mating were less viru-lent in that model as well Together these observations supportthe assumption that most natural and laboratory strains (90 to95) can be assumed to be a but they reveal that a signifi-cant minority of strains are MTL homozygous and that selecta strains can spontaneously undergo MTL homozygosis at ahigh frequency One environmental condition has been iden-tified that enriches for MTL-homozygous derivatives IndeedRustchenko and colleagues (324) found that on agar media inwhich the carbon source is sorbose a derivatives that loseone copy of chromosome 5 utilize sorbose and thus are en-riched in a population growing on medium containing sorboseas the carbon source

WHITE-OPAQUE SWITCHING MATING AND BIOFILMFORMATION

To understand the difference in C albicans biofilms based on theconfiguration of the MTL locus one must first understand thewhite-opaque transition and its role in mating (Fig 8) When Calbicans a cells undergo MTL homozygosis to aa or (320)

they still cannot mate (Fig 8A) To become truly mating compe-tent they must then switch from a traditional budding yeast-phase cell type referred to as the ldquowhiterdquo phase to an elongatedlarge pimpled cell type referred to as the ldquoopaquerdquo phase (Fig8A) (325 326) a phenotypic transition first described in 1987 forstrain WO-1 which was isolated from the bloodstream of a pa-tient at the University of Iowa Hospitals and Clinics in Iowa CityIA (327) It was subsequently found that the great majority ofMTL-homozygous strains but not heterozygous strains undergothis transition (40 326) In the mating process opaque aa cellsrelease a pheromone and opaque cells release pheromoneOpaque and aa cells then respond to these alternative pher-omones by evaginating to form shmoos that undergo chemotro-pism membrane fusion nuclear fusion evagination and daugh-ter cell formation (Fig 8B) (38 325 326 328 329) Interestinglywhite cells which themselves cannot normally mate play a role inmating White cells form robust biofilms that are architecturallysimilar to the biofilms formed by a cells but differ functionallyin that they support mating between minority opaque cells (24 38330) Opaque cells which themselves cannot form a biofilm playa stimulatory role in the formation of MTL-homozygous whitecell biofilms (39)

ARCHITECTURE OF MTL-HOMOZYGOUS BIOFILMS AND THEROLE OF PHEROMONE

When white MTL-homozygous cells are incubated in the modelemployed by Daniels et al (38 183 184) a biofilm is formed thatconsists of a basal yeast cell polylayer which accounts for the lower20 of the biofilm and an upper region of vertically orientedhyphae encapsulated in ECM which accounts for 80 of thebiofilm However MTL-homozygous biofilms formed by whitecells are on average 25 thinner than MTL-heterozygous bio-films (183 251) Most of the difference is erased if minority(10) MTL-homozygous opaque cells of the opposite matingtype are added to the white cell population (38) A mixture of11 aa and opaque cells is added because opposite matingtypes stimulate each other to release mating factor Howeveradding more than 40 opaque cells decreases biofilm thickness(38) probably because opaque cells which do not make bonafide biofilms physically interfere with white cell biofilm forma-tion Stimulation of white cell biofilm formation by minorityopaque cells led to the discovery that the white-to-opaque tran-sition regulates white cell biofilm formation (39) MTL-heterozygous biofilm formation is regulated by the RAS1cyclicAMP (cAMP) pathway while MTL-homozygous biofilm for-mation is regulated by the mitogen-activated protein (MAP)kinase pathway (39 243 318 331ndash333) It was hypothesizedthat minority opaque cells added to majority white cells stim-ulate the thickness of MTL-homozygous biofilms by releasingpheromone which activates the MAP kinase pathway (39) Itwas subsequently demonstrated that a small minority ofopaque cells (ie 1) which appear in a majority white cellpopulation by spontaneous switching (327 334 335) releasethe pheromone of the opposite mating type in an unorthodoxfashion (39) The secreted pheromone induces biofilm forma-tion in a paracrine system (39) In other words minority cells in a majority aa white cell population secrete phero-mone which stimulates biofilm formation by aa white cellsDeletion of the -pheromone gene results in the formation ofhighly defective aa white cell biofilms (39) The addition of




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10 opaque cells (11 mixture of aa and cells) to an aa or white cell population stimulates biofilm thickness pre-sumably by providing additional pheromone of the oppositemating type (39)

PERMEABILITY PENETRABILITY AND DRUG SUSCEPTIBILITYOF ALTERNATIVE BIOFILMS

Although the general architecture of biofilms formed by MTL-homozygous cells is similar to that of biofilms formed by MTL-heterozygous cells pathogenic traits and the capacity to facilitatemating differ When a 48-h MTL-heterozygous biofilm developedin RPMI 1640 (MOPS) medium in the Douglas model was over-laid with either Sypro Ruby (16 kDa) or ConA (104 to 112 kDa)and incubated for an additional 30 min there was minimal pen-etrance (15) by either the low- or high-molecular-mass mol-ecule (39) However when an MTL-homozygous biofilm was

treated similarly both the low- and high-molecular-mass mole-cules penetrated rapidly through approximately 80 of the bio-film (39) Similarly when 48-h biofilms were overlaid with DiI-labeled human polymorphonuclear leukocytes and incubated for3 h the leukocytes penetrated through only 5 of an MTL-heterozygous biofilm but through approximately 80 of an MTL-homozygous biofilm (39 183 264) Finally alternative biofilmsexhibit a dramatically different susceptibility to fluconazoleWhen a 48-h MTL-heterozygous biofilm was treated with 25 gper ml of fluconazole for 24 additional hours approximately 5of cells stained with Dead Red a dead cell nuclear stain Howeverwhen MTL-homozygous biofilms were treated similarly approx-imately 40 of cells in the biofilm stained with Dead Red (39) Allof the above differences were demonstrated for three unrelateda strains and their aa and derivatives the latter obtainedthrough spontaneous homozygosis at the MTL locus (39) MTL-

FIG 8 To understand the profound difference in biofilms formed by MTL-heterozygous and MTL-homozygous cells white-opaque switching opaquecell mating and the role of pheromone in the formation of MTL-homozygous biofilms must be taken into account (A) MTL homozygosis andwhite-opaque (Wh-Op) switching (B) Shmoo formation induced by pheromones of opposite mating types and pheromone-directed chemotropism inthe process of fusion (C) MTL-homozygous white cell biofilms are induced by the unorthodox release of pheromone of the opposite mating type by rareopaque cells which appear through switching In this example white aa cells produce minority opaque aa cells The latter release pheromone in anunorthodox fashion which induces white aa cells to form a biofilm Op opaque hy hyphae bp basal cell polylayer ECM extracellular matrix

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heterozygous and MTL-homozygous biofilms therefore representperhaps the most extreme example of biofilms that look alike butare functionally disparate

ldquoSEXUAL BIOFILMSrdquo LESSONS FROM BACTERIA

The relationship between mating and biofilm formation is not anovel aspect of C albicans It has long been recognized that mostbacteria live in biofilms in natural or host niches so it was likelythat gene transfer through conjugation occurred under these con-ditions Hausner and Wuertz (167) demonstrated that conjuga-tion occurs at higher frequencies in bacterial biofilms than inplanktonic cultures Conjugation in bacteria discovered approx-imately 70 years ago by Lederberg and Tatum (336) involves thetransfer of genetic material usually as a plasmid or transposonthrough cell-cell contact Licht et al (337) showed that donor cellsplaced on the upper surface of an E coli biofilm transfer geneticmaterial only to surface cells which may be due to the restrictedcontact Not only is conjugation facilitated by bacterial biofilmformation but mating in turn can enhance biofilm formationthrough conjugation pili as demonstrated for E coli (166) Fim-briae which are specialized pili function in adhesion (338 339) Ittherefore seemed logical to consider that if sex is facilitated bybiofilms for bacteria the same may hold true for C albicans Ob-viously the advantage of performing mating within a biofilm re-lates to proximity for both bacteria and C albicans Howeverthere is some question concerning the timing and location of bac-terial mating in bacterial biofilms since the encapsulation of cellsin a matrix (340) may prevent contact an issue dealt with byMolin and Tolker-Nielsen (341) Although the work on mating inbacterial biofilms began earlier than that on C albicans biofilmsthere have been no reports of the formation of functionally dis-tinct nonsexual and sexual biofilms of bacteria as is the case forMTL-heterozygous and MTL-homozygous C albicans biofilms

FACILITATION OF MATING BY SEXUAL BIOFILMS

Several observations led to the hypothesis that MTL-homozygouscells form ldquosexualrdquo biofilms First planktonic MTL-homozygouswhite cells in stationary phase are induced to adhere to plastic orcatheter material by pheromone (39 319) Second opaque cellssignal white cells through the release of pheromone to form awhite cell biofilm employing the same pheromone receptors tri-meric G protein complex and MAP kinase pathway as those thatregulate mating between opaque cells but with different targettranscription factors (243 319 332) Third and most impor-tantly when minority aa and opaque cells are seeded in ma-jority aa andor white cell biofilms they mate 50 to 100 timesmore frequently than minority aa and opaque cells seeded inmajority a biofilms (330) Furthermore Daniels et al (38)showed by vitally staining aa and cells with tags of differentcolors that an MTL-homozygous white cell biofilm supports che-motropism by conjugation tubes which leads to fusion (Fig 9)

The reasons for the differences in MTL-heterozygous andMTL-homozygous biofilms have been attributed to alternativefunctions and may be the result of differences in the ECM Thecharacteristics of MTL-heterozygous biofilms including imper-meability to high- and low-molecular-weight molecules includ-ing the azole fluconazole and impenetrability by white blood cells(39 183 184 251) are compatible with a pathogenic role in thelife history of this commensal but incompatible with the genera-tion of pheromone gradients that direct chemotropism in the

mating process and the directed extension of conjugation tubesduring chemotropism (33 38 52 82 330) In contrast the char-acteristics of MTL-homozygous biofilms including permeabilityto low- and high-molecular weight molecules including flucona-zole and penetrability by white blood cells are incompatible witha pathogenic role but compatible with chemotropism and hencea sexual role It seems reasonable to suggest that in an MTL-homozygous white cell biofilm spontaneous switching toopaque by a minority of cells results in mating which is facil-itated by the ECM of the white cell biofilm The observationthat opaque cells do not form biofilms is consistent with thisscenario (38) This specialization of white cell biofilm forma-tion may be the reason that white-opaque switching evolved inC albicans and related species including C dubliniensis and Can-dida tropicalis (44 342 343)

IMPACT OF ENVIRONMENTAL CONDITIONS ON MTL-HOMOZYGOUS BIOFILMS

As is the case for MTL-heterozygous biofilms the culture condi-tions used for the formation of an MTL-homozygous biofilm canaffect the biofilmrsquos architecture phenotypic transitions and func-tion Although the literature for MTL-homozygous biofilms ismore limited than that for MTL-heterozygous biofilms severalrecent studies focused on the effects of environmental conditionson MTL-homozygous biofilm formation using variations of theDouglas model In a comparison of two media used to supportbiofilm formation white aa biofilms of natural strain P37005were found to be three times thicker when formed in RPMI 1640(MOPS) medium than when formed in Spider medium (183) Inaddition whereas MTL-homozygous white cells formed a basalyeast cell polylayer and an upper region of vertically oriented hy-phae embedded in a dense ECM in RPMI 1640 medium at 37degC inair in Spider medium they formed a loose poorly adhesive and

FIG 9 Demonstration of chemotropism in an early MTL-homozygous whitecell biofilm The seeded aa opaque cell was stained green with fluorescein-conjugated ConA and the seeded opaque cell was stained red with rhod-amine-conjugated ConA The conjugation tubes that formed are stained blueNote how they extended toward each other by sensing gradients of alternativemating type pheromone released by the opaque cells in the process of chemot-ropism (Reproduced from reference 38 with permission)




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poorly cohesive polylayer of yeast cells with no significant upperhyphal region and with little ECM (183) The predominatelyyeast-phase cell biofilm formed in Spider medium was twice assusceptible to fluconazole as the already partially susceptible bio-film formed by MTL-homozygous cells in RPMI 1640 mediumunder otherwise identical conditions MTL-homozygous biofilmsformed in Spider medium were also far more penetrable by hu-man polymorphonuclear leukocytes than the partially penetrablebiofilms formed under the same conditions in RPMI 1640 me-dium (183) Furthermore the biofilms formed by white MTL-homozygous cells in Spider medium did not support matingbetween seeded minority aa and cells as did white MTL-homozygous biofilms formed in RPMI 1640 (MOPS) medium(183) However white aa cells did form biofilms in Spider me-dium if the air was replaced with 20 CO2 These biofilms werearchitecturally indistinguishable from a biofilms formed in Spi-der medium in air (183) For a cells in Spider medium similarbiofilms formed in either air or CO2 These results poignantlydemonstrate that a single parameter in this case the concentrationof CO2 dictates whether or not MTL-homozygous white cellsform bona fide biofilms in Spider medium CO2 however doesnot dictate whether a bona fide biofilm forms in RPMI 1640 me-dium Daniels et al (183) further demonstrated that a white aabiofilm formed in Leersquos medium for 24 h a condition used by Linet al (344) was not comparable to a biofilm formed in RPMI 1640medium for 48 h The biofilm formed in Leersquos medium was 110 asthick as the latter formed no cohesive basal yeast cell polylayerand had no upper layer dominated by hyphae encapsulated inECM Despite the dramatic decrease in thickness and lack of ar-chitecture and function the addition of pheromone was shownto stimulate thickness of MTL-homozygous biofilms formed inSpider medium (344) The stimulated biofilms however were stillless than half as thick as the one formed in RPMI 1640 (MOPS)medium and they lacked an organized architecture (183) Theseresults demonstrate that although white-phase aa cells are re-sponsive to pheromone in Leersquos medium in air they do notmake bona fide biofilms and in Spider medium will not form anupper hyphal region unless they are in an atmosphere containinghigh CO2 These results underscore the importance of knowingwhich environmental conditions are necessary for supporting for-mation of bona fide MTL-homozygous biofilms before experi-ments are performed on mutants or to determine drug suscepti-bility or gene expression

STRAIN VARIABILITY OF MTL-HOMOZYGOUS BIOFILMS

Using the Douglas model with RPMI 1640 (MOPS) medium Pu-jol et al (45) analyzed six aa and six strains for biofilm archi-tecture They found an extraordinarily high degree of architec-tural uniformity (Table 3) All 12 strains formed a basal yeast cellpolylayer an upper layer of vertically oriented hyphae and a denseextracellular matrix More surprisingly the means standarddeviations for 48-h biofilm thicknesses for the six aa and six strains were 56 3 m and 58 2 m respectively (45) Thestandard deviations were therefore 5 and 3 respectively of themeans (Table 3) reflecting an extremely high level of uniformityand therefore conservation among strains This level of variabilitywas far below that observed in other studies of variability amonga strains (Table 3) However the mean standard deviation forthe dry weights of biofilms formed by 10 MTL-homozygousstrains was 075 013 mg (Table 3) (45) The standard deviation

in this case was 17 of the mean reflecting a level of variabilityconsistent with that observed in the majority of other studies re-viewed in Table 3 This provides another example of the differ-ences in variability that can exist between different biofilm param-eters

CONCLUDING REMARKS

This review focused on the variability of C albicans biofilmsformed in in vitro models based on differences in strains methodsof assessment the conditions used to develop biofilms and theconfiguration of the MTL locus It was undertaken to engenderawareness that more attention must be paid to the strains em-ployed the methods used to assess biofilm development and themodels used to generate them in vitro Therefore it seems appro-priate to conclude with a summary of basic questions or issues thatC albicans researchers should consider in comparing the results ofdifferent studies and developing experimental protocols for newstudies First if one is analyzing a single strain of C albicans invitro does it form a representative biofilm One would not want toemploy a strain which forms a biofilm that exhibits a basic param-eter close to the lower or upper extreme for the species Extremescan vary 2-fold or more (Table 3) Second if one is assessing vari-ability among a collection of natural strains it would seem pru-dent to type them for the configuration of the MTL locus Itshould be recognized that between 5 and 10 of clinical isolatesare MTL homozygous Moreover some a strains undergo MTLhomozygosis at extremely high rates and environmental condi-tions such as the use of sorbose as the carbon source can select forMTL-homozygous derivatives The biofilms of MTL-homozygousstrains are functionally different from those of MTL-heterozygousstrains For some traits such as drug susceptibility a collection ofstrains may be distributed biphasically if the collection contains adisproportionate number of MTL-homozygous isolates For generegulation the differences between MTL-homozygous and MTL-heterozygous biofilms may be profound Third one time pointmay not suffice for assessing the effects of an antifungal drug amutation or an environmental condition on biofilm formation Abiofilm is developmentally programmed and we are still ignorantof the complexity of that program It appears that most studies donot take into account that biofilm traits architecture physiologyand function probably change with time during developmentTherefore multiple time points must be considered Fourth ar-chitectural phenotypic and ECM assessments should be per-formed in conjunction with studies on the effects of antifungaldrugs gene expression the physiology of cells the biochemistry ofthe matrix the effects of mutation and the effects of environmen-tal conditions If a mutation an antifungal agent or an environ-mental condition selectively inhibits a biofilm trait such as hyphaformation matrix deposition general growth or dispersal con-clusions must be formulated with that in mind In these cases ameasurement such as XTT reduction is not sufficient Fifth inter-pretations of data must consider the phenotypic complexity of thebiofilm and hence the possibility or probability that a measure-ment may represent an amalgam of phenotypes Sixth one mustconsider valid controls and comparisons in identifying biofilm-specific traits If a measurement is made for a particular charac-teristic such as gene expression on a 48-h biofilm preparationwhy is a planktonic culture of yeast-phase cells used as the solepreparation for comparison We know that hyphal growth is dis-tinct from yeast cell multiplication in terms of cellular architec-

Soll and Daniels



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nloaded from

ture gene expression molecular composition cell cycle andphysiology Since C albicans biofilms are composed of hyphae aswell as yeast-phase cells why not add hyphal growth in suspensionas a control Seventh researchers must be aware that biofilms havealmost completely stopped growing at 48 h in most in vitro mod-els Therefore 48-h time points provide information on biofilmdevelopment but also later on the final mature biofilm Eighthselecting a method of assessment solely for expediency may notprovide a valid answer XTT measurements are fraught with prob-lems and have been shown as early as 1998 not to correlate con-sistently with other parameters such as dry weight In addition itshould be realized that preparing cells for imaging such as forSEM can collapse a biofilm destroy the architecture or extractthe ECM CLSM preserves architecture but does not provide high-resolution images of cellular phenotype and can include stainingartifacts Gene expression profiles are not expedient and theirreproducibility especially for low-abundance transcripts has notbeen resolved Measurements of dry weight provide nothing morethan information on mass Obviously assessments of architec-ture mass cellular phenotypes gene expression and metabolismare synergistic in examinations of biofilm development The morethat is known about a preparation the more impact a study willhave Ninth perhaps the most important consideration is themodel employed Different conditions may result in different bio-films These include the conditions used for planktonic growththe concentration of the initial inoculum the period of initialadhesion the medium used to support biofilm formation thesubstratum temperature atmosphere (air versus increasing levelsof CO2) and fluid flow Hopefully concern for the above issueswill increase the relevance of future studies and facilitate morevalid comparisons of the results obtained in different in vitro stud-ies If nothing else this review should engender the notion that notall biofilms are created equal

ACKNOWLEDGMENTS

We are indebted to Claude Pujol for his suggestions and help in interpret-ing the data obtained in the genome-wide transcriptional profiling studiesand to Sandra Beck for help in assembling the manuscript

Work in the Soll laboratory is funded by the Developmental StudiesHybridoma Bank a National Resource created by the NIH

REFERENCES1 Costerton JW Lewandowski Z Caldwell DE Korber DR Lappin-

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30 Ramage G Tomsett K Wickes BL Loacutepez-Ribot JL Redding SW 2004Denture stomatitis a role for Candida biofilms Oral Surg Oral Med OralPathol Oral Radiol Endod 9853ndash59 httpdxdoiorg101016jtripleo200304002




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brasmorg

Dow

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31 Seneviratne C Jin L Samaranayake L 2008 Biofilm lifestyle of Can-dida a mini review Oral Dis 14582ndash590 httpdxdoiorg101111j1601-0825200701424x

32 Veerachamy S Yarlagadda T Manivasagam G Yarlagadda PK 2014Bacterial adherence and biofilm formation on medical implants a re-view Proc Inst Mech Eng H 2281083ndash1099 httpdxdoiorg1011770954411914556137

33 Soll DR 2009 Why does Candida albicans switch FEMS Yeast Res9973ndash989 httpdxdoiorg101111j1567-1364200900562x

34 Soll DR 2014 The evolution of alternative biofilms in an opportunisticfungal pathogen an explanation for how new signal transduction path-ways may evolve Infect Genet Evol 22235ndash243 httpdxdoiorg101016jmeegid201307013

35 Drenkard E Ausubel FM 2002 Pseudomonas biofilm formation andantibiotic resistance are linked to phenotypic variation Nature 416740 ndash743 httpdxdoiorg101038416740a

36 Seneviratne C Yip J Chang J Zhang C Samaranayake L 2013 Effectof culture media and nutrients on biofilm growth kinetics of laboratoryand clinical strains of Enterococcus faecalis Arch Oral Biol 581327ndash1334httpdxdoiorg101016jarchoralbio201306017

37 Webb JS Lau M Kjelleberg S 2004 Bacteriophage and phenotypicvariation in Pseudomonas aeruginosa biofilm development J Bacteriol1868066 ndash 8073 httpdxdoiorg101128JB186238066-80732004

38 Daniels KJ Srikantha T Lockhart SR Pujol C Soll DR 2006 Opaquecells signal white cells to form biofilms in Candida albicans EMBO J252240 ndash2252 httpdxdoiorg101038sjemboj7601099

39 Yi S Sahni N Daniels KJ Lu KL Srikantha T Huang G Garnaas AMSoll DR 2011 Alternative mating type configurations (aalpha versusaa or alphaalpha) of Candida albicans result in alternative biofilms reg-ulated by different pathways PLoS Biol 9e1001117 httpdxdoiorg101371journalpbio1001117

40 Lockhart SR Pujol C Daniels KJ Miller MG Johnson AD PfallerMA Soll DR 2002 In Candida albicans white-opaque switchers arehomozygous for mating type Genetics 162737ndash745

41 Odds FC Bougnoux M-E Shaw DJ Bain JM Davidson AD Diogo DJacobsen MD Lecomte M Li S-Y Tavanti A 2007 Molecular phylo-genetics of Candida albicans Eukaryot Cell 61041ndash1052 httpdxdoiorg101128EC00041-07

42 Tavanti A Davidson AD Fordyce MJ Gow NA Maiden MC OddsFC 2005 Population structure and properties of Candida albicans asdetermined by multilocus sequence typing J Clin Microbiol 435601ndash5613 httpdxdoiorg101128JCM43115601-56132005

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44 Pujol C Daniels KJ Lockhart SR Srikantha T Radke JB Geiger J SollDR 2004 The closely related species Candida albicans and Candidadubliniensis can mate Eukaryot Cell 31015ndash1027 httpdxdoiorg101128EC341015-10272004

45 Pujol C Daniels KJ Soll DR 2015 Comparison of switching andbiofilm formation between MTL-homozygous strains of Candida albi-cans and Candida dubliniensis Eukaryot Cell 141186 ndash1202 httpdxdoiorg101128EC00146-15

46 Sullivan D Coleman D 1998 Candida dubliniensis characteristics andidentification J Clin Microbiol 36329 ndash334

47 Desai JV Mitchell AP Andes DR 2014 Fungal biofilms drug resis-tance and recurrent infection Cold Spring Harb Perspect Med4a019729 httpdxdoiorg101101cshperspecta019729

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49 Fox EP Nobile CJ 2012 A sticky situation untangling the transcrip-tional network controlling biofilm development in Candida albicansTranscription 3315ndash322 httpdxdoiorg104161trns22281

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pathogenesis of Candida albicans J Oral Microbiol 622993 httpdxdoiorg103402jomv622993

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75 Garrett TR Bhakoo M Zhang Z 2008 Bacterial adhesion and biofilmson surfaces Prog Nat Sci 181049 ndash1056 httpdxdoiorg101016jpnsc200804001

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82 Soll DR 2011 Evolution of a new signal transduction pathway in Can-dida albicans Trends Microbiol 198 ndash13 httpdxdoiorg101016jtim201010001

83 Mahajan A Currie CG Mackie S Tree J McAteer S McKendrick IMcNeilly TN Roe A La Ragione RM Woodward MJ 2009 Aninvestigation of the expression and adhesin function of H7 flagella in theinteraction of Escherichia coli O157H7 with bovine intestinal epithe-lium Cell Microbiol 11121ndash137 httpdxdoiorg101111j1462-5822200801244x

84 Friedlander RS Vogel N Aizenberg J 2015 Role of flagella in adhesionof Escherichia coli to abiotic surfaces Langmuir 316137ndash 6144 httpdxdoiorg101021acslangmuir5b00815

85 Heydorn A Nielsen AT Hentzer M Sternberg C Givskov M ErsboslashllBK Molin S 2000 Quantification of biofilm structures by the novelcomputer program COMSTAT Microbiology 1462395ndash2407 httpdxdoiorg10109900221287-146-10-2395

86 Pratt LA Kolter R 1998 Genetic analysis of Escherichia coli biofilm for-mation roles of flagella motility chemotaxis and type I pili Mol Microbiol30285ndash293 httpdxdoiorg101046j1365-2958199801061x

87 Van Houdt R Michiels CW 2005 Role of bacterial cell surface struc-tures in Escherichia coli biofilm formation Res Microbiol 156626 ndash 633httpdxdoiorg101016jresmic200502005

88 Arciola CR Campoccia D Ravaioli S Montanaro L 2015 Polysac-charide intercellular adhesin in biofilm structural and regulatory as-pects Front Cell Infect Microbiol 57 httpdxdoiorg103389fcimb201500007

89 Chagnot C Zorgani MA Astruc T 2013 Proteinaceous determinantsof surface colonization in bacteria bacterial adhesion and biofilm forma-tion from a protein secretion perspective Front Microbiol 4303 httpdxdoiorg103389fmicb201300303

90 Heilmann C Schweitzer O Gerke C Vanittanakom N Mack D GoumltzF 1996 Molecular basis of intercellular adhesion in the biofilm-formingStaphylococcus epidermidis Mol Microbiol 201083ndash1091 httpdxdoiorg101111j1365-29581996tb02548x

91 Karatan E Watnick P 2009 Signals regulatory networks and materialsthat build and break bacterial biofilms Microbiol Mol Biol Rev 73310 ndash347 httpdxdoiorg101128MMBR00041-08

92 Costerton J Lewandowski Z DeBeer D Caldwell D Korber D JamesG 1994 Biofilms the customized microniche J Bacteriol 1762137

93 Davies D Geesey G 1995 Regulation of the alginate biosynthesis genealgC in Pseudomonas aeruginosa during biofilm development in contin-uous culture Appl Environ Microbiol 61860 ndash 867

94 Diggle SP Gardner A West SA Griffin AS 2007 Evolutionary theoryof bacterial quorum sensing when is a signal not a signal Philos Trans RSoc Lond B Biol Sci 3621241ndash1249 httpdxdoiorg101098rstb20072049

95 Okegbe C Price-Whelan A Dietrich LE 2014 Redox-driven regula-tion of microbial community morphogenesis Curr Opin Microbiol 1839 ndash 45 httpdxdoiorg101016jmib201401006

96 Seminara A Angelini TE Wilking JN Vlamakis H Ebrahim S KolterR Weitz DA Brenner MP 2012 Osmotic spreading of Bacillus subtilisbiofilms driven by an extracellular matrix Proc Natl Acad Sci U S A1091116 ndash1121 httpdxdoiorg101073pnas1109261108

97 Vroom JM De Grauw KJ Gerritsen HC Bradshaw DJ Marsh PDWatson GK Birmingham JJ Allison C 1999 Depth penetration and

detection of pH gradients in biofilms by two-photon excitation micros-copy Appl Environ Microbiol 653502ndash3511

98 Vlamakis H Chai Y Beauregard P Losick R Kolter R 2013 Stickingtogether building a biofilm the Bacillus subtilis way Nat Rev Microbiol11157ndash168 httpdxdoiorg101038nrmicro2960

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101 Karimi A Karig D Kumar A Ardekani AM 2015 Interplay of physicalmechanisms and biofilm processes review of microfluidic methods LabChip 1523ndash 42 httpdxdoiorg101039C4LC01095G

102 de Beer D Stoodley P Lewandowski Z 1994 Liquid flow in heteroge-neous biofilms Biotechnol Bioeng 44636 ndash 641 httpdxdoiorg101002bit260440510

103 Bowden GH Li YH 1997 Nutritional influences on biofilm devel-opment Adv Dent Res 1181ndash99 httpdxdoiorg10117708959374970110012101

104 Davey ME Caiazza NC OrsquoToole GA 2003 Rhamnolipid surfactantproduction affects biofilm architecture in Pseudomonas aeruginosaPAO1 J Bacteriol 1851027ndash1036 httpdxdoiorg101128JB18531027-10362003

105 McNally L Brown SP 2015 Building the microbiome in health anddisease niche construction and social conflict in bacteria Philos Trans RSoc Lond B Biol Sci 37020140298 httpdxdoiorg101098rstb20140298

106 Flemming H-C Wingender J 2010 The biofilm matrix Nat Rev Mi-crobiol 8623ndash 633 httpdxdoiorg101038nrmicro2415

107 Magee B Magee P 2000 Induction of mating in Candida albicans byconstruction of MTLa and MTL strains Science 289310 ndash313 httpdxdoiorg101126science2895477310

108 Renner LD Weibel DB 2011 Physicochemical regulation of biofilmformation MRS Bull 36347ndash355 httpdxdoiorg101557mrs201165

109 Grandclement C Tannieres M Morera S Dessaux Y Faure DD 2016Quorum quenching role in nature and applied developments FEMSMicrobiol Rev 4086 ndash116 httpdxdoiorg101093femsrefuv038

110 Lawrence J Korber D Hoyle B Costerton J Caldwell D 1991 Opticalsectioning of microbial biofilms J Bacteriol 1736558 ndash 6567

111 Mika F Hengge R 2014 Small RNAs in the control of RpoS CsgD andbiofilm architecture of Escherichia coli RNA Biol 11494 ndash507 httpdxdoiorg104161rna28867

112 Serra DO Richter AM Hengge R 2013 Cellulose as an architecturalelement in spatially structured Escherichia coli biofilms J Bacteriol 1955540 ndash5554 httpdxdoiorg101128JB00946-13

113 Yoon MY Lee K-M Park Y Yoon SS 2011 Contribution of cellelongation to the biofilm formation of Pseudomonas aeruginosa duringanaerobic respiration PLoS One 6e16105 httpdxdoiorg101371journalpone0016105

114 Abee T Kovaacutecs AT Kuipers OP Van der Veen S 2011 Biofilmformation and dispersal in Gram-positive bacteria Curr Opin Biotech-nol 22172ndash179 httpdxdoiorg101016jcopbio201010016

115 Mah T-F Pitts B Pellock B Walker GC Stewart PS OrsquoToole GA2003 A genetic basis for Pseudomonas aeruginosa biofilm antibiotic re-sistance Nature 426306 ndash310 httpdxdoiorg101038nature02122

116 Whiteley M Bangera MG Bumgarner RE Parsek MR Teitzel GMLory S Greenberg E 2001 Gene expression in Pseudomonas aeruginosabiofilms Nature 413860 ndash 864 httpdxdoiorg10103835101627

117 Caiazza NC OrsquoToole GA 2004 SadB is required for the transition fromreversible to irreversible attachment during biofilm formation by Pseu-domonas aeruginosa PA14 J Bacteriol 1864476 ndash 4485 httpdxdoiorg101128JB186144476-44852004

118 Purevdorj-Gage B Costerton WJ Stoodley P 2005 Phenotypic differ-entiation and seeding dispersal in non-mucoid and mucoid Pseudomo-nas aeruginosa biofilms Microbiology 1511569 ndash1576 httpdxdoiorg101099mic027536-0

119 Friedlander RS Vlamakis H Kim P Khan M Kolter R Aizenberg J2013 Bacterial flagella explore microscale hummocks and hollows toincrease adhesion Proc Natl Acad Sci U S A 1105624 ndash5629 httpdxdoiorg101073pnas1219662110

120 Guttenplan SB Kearns DB 2013 Regulation of flagellar motility during




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nloaded from

biofilm formation FEMS Microbiol Rev 37849 ndash 871 httpdxdoiorg1011111574-697612018

121 Pesavento C Becker G Sommerfeldt N Possling A Tschowri NMehlis A Hengge R 2008 Inverse regulatory coordination of motilityand curli-mediated adhesion in Escherichia coli Genes Dev 222434 ndash2446 httpdxdoiorg101101gad475808

122 Yamamoto K Arai H Ishii M Igarashi Y 2012 Involvement offlagella-driven motility and pili in Pseudomonas aeruginosa colonizationat the air-liquid interface Microbes Environ 27320 ndash323 httpdxdoiorg101264jsme2ME11322

123 Chua SL Liu Y Yam JKH Chen Y Vejborg RM Tan BGC KjellebergS Tolker-Nielsen T Givskov M Yang L 2014 Dispersed cells representa distinct stage in the transition from bacterial biofilm to planktoniclifestyles Nat Commun 54462 httpdxdoiorg101038ncomms5462

124 Kim SK Lee JH 2016 Biofilm dispersion in Pseudomonas aeruginosa JMicrobiol 5471ndash 85 httpdxdoiorg101007s12275-016-5528-7

125 Kirisits MJ Parsek MR 2006 Does Pseudomonas aeruginosa use inter-cellular signalling to build biofilm communities Cell Microbiol 81841ndash1849 httpdxdoiorg101111j1462-5822200600817x

126 von Bodman SB Willey JM Diggle SP 2008 Cell-cell communicationin bacteria united we stand J Bacteriol 1904377ndash 4391 httpdxdoiorg101128JB00486-08

127 Williams P Winzer K Chan WC Camara M 2007 Look whorsquos talkingcommunication and quorum sensing in the bacterial world Philos TransR Soc Lond B Biol Sci 3621119 ndash1134 httpdxdoiorg101098rstb20072039

128 Hurle JM Ros MA Climent V Garcia-Martinez V 1996 Morphologyand significance of programmed cell death in the developing limb bud ofthe vertebrate embryo Microsc Res Tech 34236 ndash246

129 Zuzarte-Luiacutes V Hurleacute JM 2002 Programmed cell death in the devel-oping limb Int J Dev Biol 46871ndash 876

130 Asally M Kittisopikul M Rueacute P Du Y Hu Z Ccedilagatay T RobinsonAB Lu H Garcia-Ojalvo J Suumlel GM 2012 Localized cell death focusesmechanical forces during 3D patterning in a biofilm Proc Natl Acad SciU S A 10918891ndash18896 httpdxdoiorg101073pnas1212429109

131 Kolodkin-Gal I Verdiger R Shlosberg-Fedida A Engelberg-Kulka HBereswill S 2009 A differential effect of E coli toxin-antitoxin systemson cell death in liquid media and biofilm formation PLoS One 4e6785httpdxdoiorg101371journalpone0006785

132 Sadykov MR Bayles KW 2012 The control of death and lysis in staph-ylococcal biofilms a coordination of physiological signals Curr OpinMicrobiol 15211ndash215 httpdxdoiorg101016jmib201112010

133 Chen X Stewart P 2002 Role of electrostatic interactions in cohesion ofbacterial biofilms Appl Microbiol Biotechnol 59718 ndash720 httpdxdoiorg101007s00253-002-1044-2

134 Cooksey K 1992 Extracellular polymers in biofilms p 137ndash147 In MeloLF Bott TR Fletcher M Capdeville N (ed) Biofilmsmdashscience and tech-nology Springer ScienceBusiness Media Dordrecht The Netherlands

135 Davies D 2003 Understanding biofilm resistance to antibacterialagents Nat Rev Drug Discov 2114 ndash122 httpdxdoiorg101038nrd1008

136 Sherrard LJ Tunney MM Elborn JS 2014 Antimicrobial resistance inthe respiratory microbiota of people with cystic fibrosis Lancet 384703ndash713 httpdxdoiorg101016S0140-6736(14)61137-5

137 Stewart PS 1996 Theoretical aspects of antibiotic diffusion into micro-bial biofilms Antimicrob Agents Chemother 402517ndash2522

138 Cabral DA Loh BA Speert DP 1987 Mucoid Pseudomonas aerugi-nosa resists nonopsonic phagocytosis by human neutrophils and macro-phages Pediatr Res 22429 ndash 431 httpdxdoiorg10120300006450-198710000-00013

139 Kelly N Kluftinger J Pasloske B Paranchych W Hancock R 1989Pseudomonas aeruginosa pili as ligands for nonopsonic phagocytosis byfibronectin-stimulated macrophages Infect Immun 573841ndash3845

140 Leid JG Willson CJ Shirtliff ME Hassett DJ Parsek MR Jeffers AK2005 The exopolysaccharide alginate protects Pseudomonas aeruginosabiofilm bacteria from IFN--mediated macrophage killing J Immunol1757512ndash7518 httpdxdoiorg104049jimmunol175117512

141 Mahenthiralingam E Campbell ME Speert DP 1994 Nonmotility andphagocytic resistance of Pseudomonas aeruginosa isolates from chroni-cally colonized patients with cystic fibrosis Infect Immun 62596 ndash 605

142 Wolcott RD Rhoads DD Dowd SE 2008 Biofilms and chronic woundinflammation J Wound Care 17333ndash341 httpdxdoiorg1012968jowc200817830796

143 Schmid T Burkhard J Yeo B-S Zhang W Zenobi R 2008 Towardschemical analysis of nanostructures in biofilms I Imaging of biologicalnanostructures Anal Bioanal Chem 3911899 ndash1905 httpdxdoiorg101007s00216-008-2100-2

144 Sutherland IW 2001 Biofilm exopolysaccharides a strong and stickyframework Microbiology 1473ndash9 httpdxdoiorg10109900221287-147-1-3

145 Aguilar C Vlamakis H Losick R Kolter R 2007 Thinking aboutBacillus subtilis as a multicellular organism Curr Opin Microbiol 10638 ndash 643 httpdxdoiorg101016jmib200709006

146 Flemming HC Neu TR Wozniak DJ 2007 The EPS matrix the ldquohouseof biofilm cellsrdquo J Bacteriol 1897945ndash7947 httpdxdoiorg101128JB00858-07

147 Hobley L Harkins C MacPhee CE Stanley-Wall NR 2015 Givingstructure to the biofilm matrix an overview of individual strategies andemerging common themes FEMS Microbiol Rev 39649 ndash 669 httpdxdoiorg101093femsrefuv015

148 Montanaro L Poggi A Visai L Ravaioli S Campoccia D Speziale PArciola CR 2011 Extracellular DNA in biofilms Int J Artif Organs34824 ndash 831 httpdxdoiorg105301ijao5000051

149 Okshevsky M Regina VR Meyer RL 2015 Extracellular DNA as atarget for biofilm control Curr Opin Biotechnol 3373ndash 80 httpdxdoiorg101016jcopbio201412002

150 Schlafer S Meyer RL 11 March 2016 Confocal microscopy imaging ofthe biofilm matrix J Microbiol Methods httpdxdoiorg101016jmimet201603002

151 Whitchurch CB Tolker-Nielsen T Ragas PC Mattick JS 2002 Extra-cellular DNA required for bacterial biofilm formation Science 2951487httpdxdoiorg101126science29555591487

152 McCrate OA Zhou X Reichhardt C Cegelski L 2013 Sum of the partscomposition and architecture of the bacterial extracellular matrix J MolBiol 4254286 ndash 4294 httpdxdoiorg101016jjmb201306022

153 Cegelski L Pinkner JS Hammer ND Cusumano CK Hung CSChorell E Aringberg V Walker JN Seed PC Almqvist F 2009 Small-molecule inhibitors target Escherichia coli amyloid biogenesis and bio-film formation Nat Chem Biol 5913ndash919 httpdxdoiorg101038nchembio242

154 Lin Y Sharma P van Loosdrecht M 2013 The chemical and mechan-ical differences between alginate-like exopolysaccharides isolated fromaerobic flocculent sludge and aerobic granular sludge Water Res 4757ndash65 httpdxdoiorg101016jwatres201209017

155 Wu C Lim JY Fuller GG Cegelski L 2012 Quantitative analysis ofamyloid-integrated biofilms formed by uropathogenic Escherichia coli atthe air-liquid interface Biophys J 103464 ndash 471 httpdxdoiorg101016jbpj201206049

156 Zogaj X Nimtz M Rohde M Bokranz W Roumlmling U 2001 Themulticellular morphotypes of Salmonella typhimurium and Escherichiacoli produce cellulose as the second component of the extracellular ma-trix Mol Microbiol 391452ndash1463 httpdxdoiorg101046j1365-2958200102337x

157 Neu TR 1996 Significance of bacterial surface-active compounds ininteraction of bacteria with interfaces Microbiol Rev 60151

158 Vu B Chen M Crawford RJ Ivanova EP 2009 Bacterial extracellularpolysaccharides involved in biofilm formation Molecules 142535ndash2554httpdxdoiorg103390molecules14072535

159 Costerton JW Cheng K Geesey GG Ladd TI Nickel JC Dasgupta MMarrie TJ 1987 Bacterial biofilms in nature and disease Annu RevMicrobiol 41435ndash464 httpdxdoiorg101146annurevmi41100187002251

160 Hooper LV Gordon JI 2001 Commensal host-bacterial relationshipsin the gut Science 2921115ndash1118 httpdxdoiorg101126science1058709

161 Izano EA Sadovskaya I Wang H Vinogradov E Ragunath C Rama-subbu N Jabbouri S Perry MB Kaplan JB 2008 Poly-N-acetyl-glucosamine mediates biofilm formation and detergent resistance in Ag-gregatibacter actinomycetemcomitans Microb Pathog 4452ndash 60 httpdxdoiorg101016jmicpath200708004

162 Davies DG Parsek MR Pearson JP Iglewski BH Costerton J Green-berg E 1998 The involvement of cell-to-cell signals in the developmentof a bacterial biofilm Science 280295ndash298 httpdxdoiorg101126science2805361295

163 Watnick P Kolter R 2000 Biofilm city of microbes J Bacteriol 1822675ndash2679 httpdxdoiorg101128JB182102675-26792000

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164 Landini P Antoniani D Burgess JG Nijland R 2010 Molecularmechanisms of compounds affecting bacterial biofilm formation anddispersal Appl Microbiol Biotechnol 86813ndash 823 httpdxdoiorg101007s00253-010-2468-8

165 de la Cruz F Davies J 2000 Horizontal gene transfer and the origin ofspecies lessons from bacteria Trends Microbiol 8128 ndash133 httpdxdoiorg101016S0966-842X(00)01703-0

166 Ghigo J-M 2001 Natural conjugative plasmids induce bacterial biofilmdevelopment Nature 412442ndash445 httpdxdoiorg10103835086581

167 Hausner M Wuertz S 1999 High rates of conjugation in bacterialbiofilms as determined by quantitative in situ analysis Appl EnvironMicrobiol 653710 ndash3713

168 Hawser SP Douglas LJ 1994 Biofilm formation by Candida species onthe surface of catheter materials in vitro Infect Immun 62915ndash921

169 Odds FC 1988 Candida and candidosis a review and bibliographyBailliere Tindall London United Kingdom

170 Laffery SF Butler G 2005 Phenotype switching affects biofilm forma-tion by Candida parapsilosis Microbiology 1511073ndash1081

171 Wilson LA Sawant AD Ahearn DG 1991 Comparative efficacies ofsoft contact lens disinfectant solutions against microbial films in lenscases Arch Ophthalmol 1091155ndash1157 httpdxdoiorg101001archopht199101080080115043

172 Neu TR Verkerke GJ Herrmann IF Schutte HK Van der Mei HCBusscher HJ 1994 Microflora on explanted silicone rubber voiceprostheses taxonomy hydrophobicity and electrophoretic mobilityJ Appl Bacteriol 76521ndash528 httpdxdoiorg101111j1365-26721994tb01111x

173 Keyf F Anil N Ercan M Etikan I Yener O 1995 Persistence of 99mTc-labelled microorganisms on surfaces of impression materials J NihonUniv Sch Dent 371ndash7 httpdxdoiorg102334josnusd1959371

174 Greenfield RA Troutt DL Rickard RC Altmiller DH 1988 Compar-ison of antibody antigen and metabolite assays in rat models of systemicand gastrointestinal candidiasis J Clin Microbiol 26409 ndash 417

175 McCourtie J Douglas LJ 1981 Relationship between cell surface com-position of Candida albicans and adherence to acrylic after growth ondifferent carbon sources Infect Immun 321234 ndash1241

176 Nikawa H Hayashi S Nikawa Y Hamada T Samaranayake L 1993Interactions between denture lining material protein pellicles and Can-dida albicans Arch Oral Biol 38631ndash 634 httpdxdoiorg1010160003-9969(93)90132-6

177 Samaranayake L MacFarlane T 1980 An in vitro study of the adherenceof Candida albicans to acrylic surfaces Arch Oral Biol 25603ndash 609 httpdxdoiorg1010160003-9969(80)90075-8

178 Prosser B Taylor D Dix BA Cleeland R 1987 Method of evaluatingeffects of antibiotics on bacterial biofilm Antimicrob Agents Chemother311502ndash1506 httpdxdoiorg101128AAC31101502

179 Hawser SP Baillie GS Douglas LJ 1998 Production of extracellularmatrix by Candida albicans biofilms J Med Microbiol 47253ndash256 httpdxdoiorg10109900222615-47-3-253

180 Pierce CG Uppuluri P Tristan AR Wormley FL Mowat E RamageG Loacutepez-Ribot JL 2008 A simple and reproducible 96-well plate-basedmethod for the formation of fungal biofilms and its application to anti-fungal susceptibility testing Nat Protoc 31494 ndash1500 httpdxdoiorg101038nprot2008141

181 Ramage G Vande Walle K Wickes BL Loacutepez-Ribot JL 2001 Stan-dardized method for in vitro antifungal susceptibility testing of Candidaalbicans biofilms Antimicrob Agents Chemother 452475ndash2479 httpdxdoiorg101128AAC4592475-24792001

182 Ramage G VandeWalle K Loacutepez-Ribot JL Wickes BL 2002 Thefilamentation pathway controlled by the Efg1 regulator protein is re-quired for normal biofilm formation and development in Candida albi-cans FEMS Microbiol Lett 21495ndash100 httpdxdoiorg101111j1574-69682002tb11330x

183 Daniels KJ Park YN Srikantha T Pujol C Soll DR 2013 Impact ofenvironmental conditions on the form and function of Candida albicansbiofilms Eukaryot Cell 121389 ndash1402 httpdxdoiorg101128EC00127-13

184 Daniels KJ Srikantha T Pujol C Park YN Soll DR 2015 Role of Tec1in the development architecture and integrity of sexual biofilms of Can-dida albicans Eukaryot Cell 14228 ndash240 httpdxdoiorg101128EC00224-14

185 Lee K Buckley HR Campbell CC 1975 An amino acid liquidsynthetic medium for the development of mycelial and yeast forms of

Candida albicans Med Mycol 13148 ndash153 httpdxdoiorg10108000362177585190271

186 Bedell GW Soll DR 1979 Effects of low concentrations of zinc onthe growth and dimorphism of Candida albicans evidence for zinc-resistant and -sensitive pathways for mycelium formation Infect Immun26348 ndash354

187 Merson-Davies L Odds F 1989 A morphology index for characteriza-tion of cell shape in Candida albicans J Gen Microbiol 1353143ndash3152

188 Uppuluri P Loacutepez-Ribot JL 2010 An easy and economical in vitromethod for the formation of Candida albicans biofilms under continuousconditions of flow Virulence 1483ndash 487 httpdxdoiorg104161viru1613186

189 Uppuluri P Loacutepez-Ribot JL 2016 Go forth and colonize dispersalfrom clinically important microbial biofilms PLoS Pathog 12e1005397httpdxdoiorg101371journalppat1005397

190 Nobile CJ Fox EP Hartooni N Mitchell KF Hnisz D Andes DRKuchler K Johnson AD 2014 A histone deacetylase complex mediatesbiofilm dispersal and drug resistance in Candida albicans mBio5e01201-14 httpdxdoiorg101128mBio01201-14

191 Schinabeck MK Long LA Hossain MA Chandra J Mukherjee PKMohamed S Ghannoum MA 2004 Rabbit model of Candida albicansbiofilm infection liposomal amphotericin B antifungal lock therapy An-timicrob Agents Chemother 481727ndash1732 httpdxdoiorg101128AAC4851727-17322004

192 Andes D Nett J Oschel P Albrecht R Marchillo K Pitula A 2004Development and characterization of an in vivo central venous catheterCandida albicans biofilm model Infect Immun 726023ndash 6031 httpdxdoiorg101128IAI72106023-60312004

193 Nobile CJ Nett JE Andes DR Mitchell AP 2006 Function of Candidaalbicans adhesin Hwp1 in biofilm formation Eukaryot Cell 51604 ndash1610 httpdxdoiorg101128EC00194-06

194 Nett JE Guite KM Ringeisen A Holoyda KA Andes DR 2008Reduced biocide susceptibility in Candida albicans biofilms Antimi-crob Agents Chemother 523411ndash3413 httpdxdoiorg101128AAC01656-07

195 Nett JE Lepak AJ Marchillo K Andes DR 2009 Time course globalgene expression analysis of an in vivo Candida biofilm J Infect Dis 200307ndash313 httpdxdoiorg101086599838

196 Robbins N Uppuluri P Nett J Rajendran R Ramage G Loacutepez-RibotJL Andes D Cowen LE 2011 Hsp90 governs dispersion and drugresistance of fungal biofilms PLoS Pathog 7e1002257 httpdxdoiorg101371journalppat1002257

197 Desai JV Bruno VM Ganguly S Stamper RJ Mitchell KF Solis NHill EM Xu W Filler SG Andes DR Fanning S Lanni F Mitchell AP2013 Regulatory role of glycerol in Candida albicans biofilm formationmBio 4e00637-12 httpdxdoiorg101128mBio00637-12

198 Ding C Vidanes GM Maguire SL Guida A Synnott JM Andes DRButler G 2011 Conserved and divergent roles of Bcr1 and CFEM pro-teins in Candida parapsilosis and Candida albicans PLoS One 6e28151httpdxdoiorg101371journalpone0028151

199 Green JV Orsborn KI Zhang M Tan QK Greis KD Porollo A AndesDR Long Lu J Hostetter MK 2013 Heparin-binding motifs andbiofilm formation by Candida albicans J Infect Dis 2081695ndash1704 httpdxdoiorg101093infdisjit391

200 Nobile CJ Fox EP Nett JE Sorrells TR Mitrovich QM Hernday ADTuch BB Andes DR Johnson AD 2012 A recently evolved transcrip-tional network controls biofilm development in Candida albicans Cell148126 ndash138 httpdxdoiorg101016jcell201110048

201 Taff HT Marchillo K Andes DR 2013 Preparation of Candida albicansbiofilms using an in vivo rat central venous catheter model Biol Protoc3e823

202 Uppuluri P Nett J Heitman J Andes D 2008 Synergistic effect ofcalcineurin inhibitors and fluconazole against Candida albicans biofilmsAntimicrob Agents Chemother 521127ndash1132 httpdxdoiorg101128AAC01397-07

203 Nett JE Brooks EG Cabezas-Olcoz J Sanchez H Zarnowski RMarchillo K Andes DR 2014 Rat indwelling urinary catheter model ofCandida albicans biofilm infection Infect Immun 824931ndash 4940 httpdxdoiorg101128IAI02284-14

204 Uppuluri P Chaturvedi AK Loacutepez-Ribot JL 2009 Design of a simplemodel of Candida albicans biofilms formed under conditions of flowdevelopment architecture and drug resistance Mycopathologia 168101ndash109 httpdxdoiorg101007s11046-009-9205-9




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205 Nett JE Marchillo K Andes DR 2012 Modeling of fungal biofilmsusing a rat central vein catheter Methods Mol Biol 845547ndash556 httpdxdoiorg101007978-1-61779-539-8_40

206 Nett JE Zarnowski R Cabezas-Olcoz J Brooks EG Berhardt JMarchillo K Mosher DF Andes DR 2015 Host contributions toconstruction of three device-associated Candida biofilms Infect Immun834630 ndash 4638 httpdxdoiorg101128IAI00931-15

207 Kucharikova S Van Dijck P Lisalova M Bujdakova H 2010 Effect ofantifungals on itraconazole resistant Candida glabrata Cent Eur J Biol5318 ndash323

208 Ricicova M Kucharikova S Tournu H Hendrix J Bujdakova H VanEldere J Lagrou K Van Dijck P 2010 Candida albicans biofilm for-mation in a new in vivo rat model Microbiology 156909 ndash919 httpdxdoiorg101099mic0033530-0

209 Olsen I Bondevik O 1978 Experimental Candida-induced denturestomatitis in the Wistar rat Eur J Oral Sci 86392ndash398 httpdxdoiorg101111j1600-07221978tb00642x

210 Nett JE Marchillo K Spiegel CA Andes DR 2010 Development andvalidation of an in vivo Candida albicans biofilm denture model InfectImmun 783650 ndash3659 httpdxdoiorg101128IAI00480-10

211 Johnson CC Yu A Lee H Fidel PL Noverr MC 2012 Developmentof a contemporary animal model of Candida albicans-associated denturestomatitis using a novel intraoral denture system Infect Immun 801736 ndash1743 httpdxdoiorg101128IAI00019-12

212 Lee H Yu A Johnson CC Lilly EA Noverr MC Fidel PL Jr 2011Fabrication of a multi-applicable removable intraoral denture system forrodent research J Oral Rehabil 38686 ndash 690 httpdxdoiorg101111j1365-2842201102206x

213 Chandra J Kuhn DM Mukherjee PK Hoyer LL McCormick TGhannoum MA 2001 Biofilm formation by the fungal pathogen Can-dida albicans development architecture and drug resistance J Bacteriol1835385ndash5394 httpdxdoiorg101128JB183185385-53942001

214 Cole G Lynn K Seshan K 1990 An animal model for oropharyngealesophageal and gastric candidosis Mycoses 337ndash19

215 Kamai Y Kubota M Kamai Y Hosokawa T Fukuoka T Filler SG2001 New model of oropharyngeal candidiasis in mice AntimicrobAgents Chemother 453195ndash3197 httpdxdoiorg101128AAC45113195-31972001

216 Mosci P Pericolini E Gabrielli E Kenno S Perito S Bistoni FdrsquoEnfert C Vecchiarelli A 2013 A novel bioluminescence mouse modelfor monitoring oropharyngeal candidiasis in mice Virulence 4250 ndash254httpdxdoiorg104161viru23529

217 Solis NV Filler SG 2012 Mouse model of oropharyngeal candidiasisNat Protoc 7637ndash 642 httpdxdoiorg101038nprot2012011

218 Srinivasan A Gupta CM Agrawal CM Leung KP Loacutepez-Ribot JLRamasubramanian AK 2014 Drug susceptibility of matrix-encapsulated Candida albicans nano-biofilms Biotechnol Bioeng 111418 ndash 424 httpdxdoiorg101002bit25120

219 Srinivasan A Leung KP Loacutepez-Ribot JL Ramasubramanian AK 2013High-throughput nano-biofilm microarray for antifungal drug discov-ery mBio 4e00331-13 httpdxdoiorg101128mBio00331-13

220 Srinivasan A Uppuluri P Loacutepez-Ribot J Ramasubramanian AK2011 Development of a high-throughput Candida albicans biofilm chipPLoS One 6e19036 httpdxdoiorg101371journalpone0019036

221 Berridge MV Herst PM Tan AS 2005 Tetrazolium dyes as tools in cellbiology new insights into their cellular reduction Biotechnol Annu Rev11127ndash152 httpdxdoiorg101016S1387-2656(05)11004-7

222 Scudiero DA Shoemaker RH Paull KD Monks A Tierney S NofzigerTH Currens MJ Seniff D Boyd MR 1988 Evaluation of a solubletetrazoliumformazan assay for cell growth and drug sensitivity in cul-ture using human and other tumor cell lines Cancer Res 484827ndash 4833

223 Massa EM Farias RN 1983 Effect of auto-oxidized phospholipids onoxidative enzyme assays based on tetrazolium salt reduction BiochimBiophys Acta 746209 ndash215 httpdxdoiorg1010160167-4838(83)90076-6

224 Roslev P King GM 1993 Application of a tetrazolium salt with awater-soluble formazan as an indicator of viability in respiring bacteriaAppl Environ Microbiol 592891ndash2896

225 Wogulis M Wright S Cunningham D Chilcote T Powell K RydelRE 2005 Nucleation-dependent polymerization is an essential compo-nent of amyloid-mediated neuronal cell death J Neurosci 251071ndash1080httpdxdoiorg101523JNEUROSCI2381-042005

226 Taff HT Nett JE Zarnowski R Ross KM Sanchez H Cain MT

Hamaker J Mitchell AP Andes DR 2012 A Candida biofilm-induced pathway for matrix glucan delivery implications for drugresistance PLoS Pathog 8e1002848 httpdxdoiorg101371journalppat1002848

227 Kuhn D George T Chandra J Mukherjee P Ghannoum M 2002Antifungal susceptibility of Candida biofilms unique efficacy of ampho-tericin B lipid formulations and echinocandins Antimicrob Agents Che-mother 461773ndash1780 httpdxdoiorg101128AAC4661773-17802002

228 Li X Yan Z Xu J 2003 Quantitative variation of biofilms among strainsin natural populations of Candida albicans Microbiology 149353ndash362httpdxdoiorg101099mic025932-0

229 Thompson SW Hunt RD 1966 Selected histochemical and histopatho-logical methods C C Thomas Springfield IL

230 Nett JE Sanchez H Cain MT Ross KM Andes DR 2011 Interface ofCandida albicans biofilm matrix-associated drug resistance and cell wallintegrity regulation Eukaryot Cell 101660 ndash1669 httpdxdoiorg101128EC05126-11

231 Liu Y Schubert D 1997 Cytotoxic amyloid peptides inhibit cellular3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT)reduction by enhancing MTT formazan exocytosis J Neurochem 692285ndash2293

232 Gorman SP Mawhinney WM Adair CG Issouckis M 1993 Confocallaser scanning microscopy of peritoneal catheter surfaces J Med Micro-biol 38411ndash 417 httpdxdoiorg10109900222615-38-6-411

233 Nickel J Ruseska I Wright J Costerton J 1985 Tobramycin resistanceof Pseudomonas aeruginosa cells growing as a biofilm on urinary cathetermaterial Antimicrob Agents Chemother 27619 ndash 624 httpdxdoiorg101128AAC274619

234 Zhao X Daniels KJ Oh SH Green CB Yeater KM Soll DR Hoyer LL2006 Candida albicans Als3p is required for wild-type biofilm formationon silicone elastomer surfaces Microbiology 1522287ndash2299 httpdxdoiorg101099mic028959-0

235 Talmon Y Steinbrecht R Zierold K 1987 Cryotechniques in biologicalelectron microscopy p 64 ndash 86 Springer-Verlag Berlin Germany

236 Little B Wagner P Ray R Pope R Scheetz R 1991 Biofilms an ESEMevaluation of artifacts introduced during SEM preparation J Ind Micro-biol 8213ndash221 httpdxdoiorg101007BF01576058

237 Bridier A Meylheuc T Briandet R 2013 Realistic representation ofBacillus subtilis biofilms architecture using combined microscopy(CLSM ESEM and FESEM) Micron 4865ndash 69 httpdxdoiorg101016jmicron201302013

238 Khajotia SS Smart KH Pilula M Thompson DM 2013 Concurrentquantification of cellular and extracellular components of biofilms J VisExp 2013e50639 httpdxdoiorg10379150639

239 Kuehn M Mehl M Hausner M Bungartz HJ Wuertz S 2001 Time-resolved study of biofilm architecture and transport processes using ex-perimental and simulation techniques the role of EPS Water Sci Tech-nol 43143ndash150

240 Hanrahan O Harris J Egan C 2011 Advanced microscopy laserscanning confocal microscopy Methods Mol Biol 784169 ndash180 httpdxdoiorg101007978-1-61779-289-2_12

241 Herth W Schnepf E 1980 The fluorochrome calcofluor white bindsoriented to structural polysaccharide fibrils Protoplasma 105129 ndash133httpdxdoiorg101007BF01279855

242 Hamer E Moore C Denning D 2006 Comparison of two fluorescentwhiteners calcofluor and blankophor for the detection of fungal ele-ments in clinical specimens in the diagnostic laboratory Clin MicrobiolInfect 12181ndash184 httpdxdoiorg101111j1469-0691200501321x

243 Sahni N Yi S Daniels KJ Huang G Srikantha T Soll DR 2010 Tec1mediates the pheromone response of the white phenotype of Candidaalbicans insights into the evolution of new signal transduction pathwaysPLoS Biol 8e1000363 httpdxdoiorg101371journalpbio1000363

244 Bonhomme J Chauvel M Goyard S Roux P Rossignol T drsquoEnfert C2011 Contribution of the glycolytic flux and hypoxia adaptation to effi-cient biofilm formation by Candida albicans Mol Microbiol 80995ndash1013 httpdxdoiorg101111j1365-2958201107626x

245 Fox EP Bui CK Nett JE Hartooni N Mui MC Andes DR Nobile CJJohnson AD 2015 An expanded regulatory network temporally con-trols Candida albicans biofilm formation Mol Microbiol 961226 ndash1239httpdxdoiorg101111mmi13002

246 Garciacutea-Saacutenchez S Aubert S Iraqui I Janbon G Ghigo J-M drsquoEnfert C2004 Candida albicans biofilms a developmental state associated with

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specific and stable gene expression patterns Eukaryot Cell 3536 ndash545httpdxdoiorg101128EC32536-5452004

247 Murillo LA Newport G Lan CY Habelitz S Dungan J Agabian NM2005 Genome-wide transcription profiling of the early phase of biofilmformation by Candida albicans Eukaryot Cell 41562ndash1573 httpdxdoiorg101128EC491562-15732005

248 Yeater KM Chandra J Cheng G Mukherjee PK Zhao X Rodriguez-Zas SL Kwast KE Ghannoum MA Hoyer LL 2007 Temporal analysisof Candida albicans gene expression during biofilm development Micro-biology 1532373ndash2385 httpdxdoiorg101099mic02007006163-0

249 Finkel JS Xu W Huang D Hill EM Desai JV Woolford CA Nett JETaff H Norice CT Andes DR Lanni F Mitchell AP 2012 Portrait ofCandida albicans adherence regulators PLoS Pathog 8e1002525 httpdxdoiorg101371journalppat1002525

250 Xu W Solis NV Ehrlich RL Woolford CA Filler SG Mitchell AP2015 Activation and alliance of regulatory pathways in C albicans duringmammalian infection PLoS Biol 13e1002076 httpdxdoiorg101371journalpbio1002076

251 Srikantha T Daniels KJ Pujol C Kim E Soll DR 2013 Identificationof genes upregulated by the transcription factor Bcr1 that are involved inimpermeability impenetrability and drug resistance of Candida albicansaalpha biofilms Eukaryot Cell 12875ndash 888 httpdxdoiorg101128EC00071-13

252 Canales RD Luo Y Willey JC Austermiller B Barbacioru CC BoysenC Hunkapiller K Jensen RV Knight CR Lee KY 2006 Evaluation ofDNA microarray results with quantitative gene expression platformsNat Biotechnol 241115ndash1122 httpdxdoiorg101038nbt1236

253 Draghici S Khatri P Eklund AC Szallasi Z 2006 Reliability andreproducibility issues in DNA microarray measurements Trends Genet22101ndash109 httpdxdoiorg101016jtig200512005

254 An D Parsek MR 2007 The promise and peril of transcriptional pro-filing in biofilm communities Curr Opin Microbiol 10292ndash296 httpdxdoiorg101016jmib200705011

255 Legrand M Lephart P Forche A Mueller FMC Walsh T Magee PMagee BB 2004 Homozygosity at the MTL locus in clinical strains ofCandida albicans karyotypic rearrangements and tetraploid formationMol Microbiol 521451ndash1462 httpdxdoiorg101111j1365-2958200404068x

256 Rustad TR Stevens DA Pfaller MA White TC 2002 Homozygosity atthe Candida albicans MTL locus associated with azole resistance Micro-biology 1481061ndash1072 httpdxdoiorg10109900221287-148-4-1061

257 Soll DR Lockhart SR Zhao R 2003 Relationship between switchingand mating in Candida albicans Eukaryot Cell 2390 ndash397 httpdxdoiorg101128EC23390-3972003

258 Jain N Kohli R Cook E Gialanella P Chang T Fries B 2007 Biofilmformation by and antifungal susceptibility of Candida isolates fromurine Appl Environ Microbiol 731697ndash1703 httpdxdoiorg101128AEM02439-06

259 Villar-Vidal M Marcos-Arias C Eraso E Quindoacutes G 2011 Variationin biofilm formation among blood and oral isolates of Candida albicansand Candida dubliniensis Enferm Infecc Microbiol Clin 29660 ndash 665httpdxdoiorg101016jeimc201106009

260 Hartwell LH 1971 Genetic control of the cell division cycle in yeast IIGenes controlling DNA replication and its initiation J Mol Biol 59183ndash194

261 Hartwell LH 1974 Saccharomyces cerevisiae cell cycle Bacteriol Rev38164

262 Hartwell LH Culotti J Reid B 1970 Genetic control of the cell-divisioncycle in yeast I Detection of mutants Proc Natl Acad Sci U S A 66352ndash359 httpdxdoiorg101073pnas662352

263 Cottrell SF Avers CJ 1970 Evidence of mitochondrial synchrony insynchronous cell cultures of yeast Biochem Biophys Res Commun 38973ndash980 httpdxdoiorg1010160006-291X(70)90817-X

264 Bertoli C Skotheim JM de Bruin RA 2013 Control of cell cycletranscription during G1 and S phases Nat Rev Mol Cell Biol 14518 ndash528httpdxdoiorg101038nrm3629

265 Hartwell L 1973 Synchronization of haploid yeast cell cycles a preludeto conjugation Exp Cell Res 76111ndash117 httpdxdoiorg1010160014-4827(73)90425-4

266 Manukyan A Abraham L Dungrawala H Schneider BL 2011 Syn-chronization of yeast Methods Mol Biol 761173ndash200 httpdxdoiorg101007978-1-61779-182-6_12

267 Soll DR 1986 The regulation of cellular differentiation in the dimorphic

yeast Candida albicans Bioessays 55ndash11 httpdxdoiorg101002bies950050103

268 Hulst HC Van De Hulst H 1957 Light scattering by small particlesWiley amp Sons Inc New York NY

269 Spellman PT Sherlock G Zhang MQ Iyer VR Anders K Eisen MBBrown PO Botstein D Futcher B 1998 Comprehensive identificationof cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by mi-croarray hybridization Mol Biol Cell 93273ndash3297 httpdxdoiorg101091mbc9123273

270 Hereford LM Osley MA Ludwig JR McLaughlin CS 1981 Cell-cycleregulation of yeast histone mRNA Cell 24367ndash375 httpdxdoiorg1010160092-8674(81)90326-3

271 Kusch H Engelmann S Bode R Albrecht D Morschhaumluser J HeckerM 2008 A proteomic view of Candida albicans yeast cell metabolism inexponential and stationary growth phases Int J Med Microbiol 298291ndash318 httpdxdoiorg101016jijmm200703020

272 Farrell MJ Finkel SE 2003 The growth advantage in stationary-phasephenotype conferred by rpoS mutations is dependent on the pH andnutrient environment J Bacteriol 1857044 ndash7052 httpdxdoiorg101128JB185247044-70522003

273 Finkel SE 2006 Long-term survival during stationary phase evolutionand the GASP phenotype Nat Rev Microbiol 4113ndash120 httpdxdoiorg101038nrmicro1340

274 Rechsteiner M 1988 Regulation of enzyme levels by proteolysis the roleof pest regions Adv Enzyme Regul 27135ndash151 httpdxdoiorg1010160065-2571(88)90015-5

275 Bojsen R Regenberg B Folkesson A 2014 Saccharomyces cerevisiaebiofilm tolerance towards systemic antifungals depends on growthphase BMC Microbiol 14305 httpdxdoiorg101186s12866-014-0305-4

276 Watts H Veacute A-A Perera T Davies J Gow N 1998 Thigmot-ropism and stretch-activated channels in the pathogenic fungus Can-dida albicans Microbiology 144689 ndash 695 httpdxdoiorg10109900221287-144-3-689

277 Crusz SA Popat R Rybtke MT Caacutemara M Givskov M Tolker-Nielsen T Diggle SP Williams P 2012 Bursting the bubble on bacterialbiofilms a flow cell methodology Biofouling 28835ndash 842 httpdxdoiorg101080089270142012716044

278 Hentzer M Teitzel GM Balzer GJ Heydorn A Molin S Givskov MParsek MR 2001 Alginate overproduction affects Pseudomonas aerugi-nosa biofilm structure and function J Bacteriol 1835395ndash5401 httpdxdoiorg101128JB183185395-54012001

279 Yarwood JM Bartels DJ Volper EM Greenberg EP 2004 Quorumsensing in Staphylococcus aureus biofilms J Bacteriol 1861838 ndash1850httpdxdoiorg101128JB18661838-18502004

280 Uppuluri P Chaturvedi AK Srinivasan A Banerjee M Ramasubra-maniam AK Koumlhler JR Kadosh D Loacutepez-Ribot JL 2010 Dispersionas an important step in the Candida albicans biofilm developmentalcycle PLoS Pathog 6e1000828 httpdxdoiorg101371journalppat1000828

281 Ramage G Wickes BL Loacutepez-Ribot JL 2008 A seed and feed model forthe formation of Candida albicans biofilms under flow conditions usingan improved modified Robbins device Rev Iberoam Micol 2537 httpdxdoiorg101016S1130-1406(08)70009-3

282 Belardinelli PA Morelatto RA Benavidez TE Baruzzi AM Lopezde Blanc SA 2014 Effect of two mouthwashes on salivary pHActa Odontol Latinoam 2766 ndash71 httpdxdoiorg101590S1852-48342014000200004

283 Duckworth RM Jones S 2015 On the relationship between the rate ofsalivary flow and salivary fluoride clearance Caries Res 49141ndash146 httpdxdoiorg101159000365949

284 Rad M Kakoie S Brojeni FN Pourdamghan N 2010 Effect of long-term smoking on whole-mouth salivary flow rate and oral health DentRes Dent Clin Dent Prospects 4110 ndash114 httpdxdoiorg105681joddd2010028

285 Dawes C 1972 Circadian rhythms in human salivary flow rate andcomposition J Physiol 220529 ndash545 httpdxdoiorg101113jphysiol1972sp009721

286 Massey BT 2001 Potential control of gastroesophageal reflux by localmodulation of transient lower esophageal sphincter relaxations Am JMed 111186 ndash189 httpdxdoiorg101016S0002-9343(01)00829-4

287 Brener W Hendrix TR McHugh PR 1983 Regulation of the gastricemptying of glucose Gastroenterology 8576 ndash 82




httpmm

brasmorg

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nloaded from

288 Sanelli PC Deshmukh M Ougorets I Caiati R Heier LA 2004 Safetyand feasibility of using a central venous catheter for rapid contrast injec-tion rates Am J Roentgenol 1831829 ndash1834 httpdxdoiorg102214ajr183601831829

289 Lose G Thunedborg P Joslashrgensen L Colstrup H 1986 A comparisonof spontaneous and intubated urinary flow in female patients NeurourolUrodyn 51ndash 4 httpdxdoiorg101002nau1930050102

290 Reuss M Rudolph A 1979 Distribution and recirculation of umbilicaland systemic venous blood flow in fetal lambs during hypoxia J DevPhysiol 271ndash 84

291 Vuong C Saenz HL Gotz F Otto M 2000 Impact of the agr quorum-sensing system on adherence to polystyrene in Staphylococcus aureus JInfect Dis 1821688 ndash1693 httpdxdoiorg101086317606

292 Sellam A Al-Niemi T McInnerney K Brumfield S Nantel A SuciPA 2009 A Candida albicans early stage biofilm detachment event inrich medium BMC Microbiol 925 httpdxdoiorg1011861471-2180-9-25

293 Jin Y Zhang T Samaranayake YH Fang HH Yip HK Samaranayake LP2005 The use of new probes and stains for improved assessment of cellviability and extracellular polymeric substances in Candida albicans bio-films Mycopathology 159353ndash360 httpdxdoiorg101007s11046-004-6987-7

294 Martins M Uppuluri P Thomas DP Cleary IA Henriques M Loacutepez-Ribot JL Oliveira R 2010 Presence of extracellular DNA in the Candidaalbicans biofilm matrix and its contribution to biofilms Mycopathology169323ndash331 httpdxdoiorg101007s11046-009-9264-y

295 Thomas DP Bachmann SP Loacutepez-Ribot JL 2006 Proteomics for theanalysis of the Candida albicans biofilm lifestyle Proteomics 65795ndash5804 httpdxdoiorg101002pmic200600332

296 Zarnowski R Westler WM Lacmbouh GA Marita JM Bothe JRBernhardt J Lounes-Hadj Sahraoui A Fontaine J Sanchez H HatfieldRD Ntambi JM Nett JE Mitchell AP Andes DR 2014 Novel entriesin a fungal biofilm matrix encyclopedia mBio 5e01333-14 httpdxdoiorg101128mBio01333-14

297 Soll DR 1979 Timers in developing systems Science 203841ndash 849 httpdxdoiorg101126science419408

298 Kraft B Steinbrech D Yang M Soll DR 1988 High-frequency switch-ing in Dictyostelium Dev Biol 130198 ndash208 httpdxdoiorg1010160012-1606(88)90426-5

299 Soll DR 1990 The regulation of developmental timing p 1-82 In RothS (ed) Topics in developmental biology University of PennsylvaniaPress Philadelphia PA

300 Soll DR Kraft B 1988 A comparison of high frequency switching in theyeast Candida albicans and the slime mold Dictyostelium discoideum DevGenet 9615ndash 628 httpdxdoiorg101002dvg1020090438

301 Moore GE Gerner RE Franklin HA 1967 Culture of normal humanleukocytes JAMA 199519 ndash524

302 Radetsky M Wheeler R Roe M Todd J 1986 Microtiter broth dilu-tion method for yeast susceptibility testing with validation by clinicaloutcome J Clin Microbiol 24600 ndash 606

303 Espinel-Ingroff A Kish C Kerkering T Fromtling R Bartizal KGalgiani J Villareal K Pfaller M Gerarden T Rinaldi M 1992Collaborative comparison of broth macrodilution and microdilution an-tifungal susceptibility tests J Clin Microbiol 303138 ndash3145

304 National Committee for Clinical Laboratory Standards 1992 Perfor-mance standards for antimicrobial susceptibility testing National Com-mittee for Clinical Laboratory Standards Villanova PA

305 Wanger A Mills K Nelson PW Rex JH 1995 Comparison of Etest andNational Committee for Clinical Laboratory Standards broth macrodi-lution method for antifungal susceptibility testing enhanced ability todetect amphotericin B-resistant Candida isolates Antimicrob AgentsChemother 392520 ndash2522 httpdxdoiorg101128AAC39112520

306 Nobile CJ Andes DR Nett JE Smith FJ Jr Yue F Phan Q-T EdwardsJE Jr Filler SG Mitchell AP 2006 Critical role of Bcr1-dependentadhesins in C albicans biofilm formation in vitro and in vivo PLoS Pathol2e63

307 Nobile CJ Nett JE Hernday AD Homann OR Deneault J-S NantelA Andes DR Johnson AD Mitchell AP 2009 Biofilm matrix regula-tion by Candida albicans Zap1 PLoS Biol 7e1000133 httpdxdoiorg101371journalpbio1000133

308 Nobile CJ Schneider HA Nett JE Sheppard DC Filler SG Andes DRMitchell AP 2008 Complementary adhesin function in C albicans bio-

film formation Curr Biol 181017ndash1024 httpdxdoiorg101016jcub200806034

309 Nobile CJ Mitchell AP 2005 Regulation of cell-surface genes andbiofilm formation by the C albicans transcription factor Bcr1p Curr Biol151150 ndash1155 httpdxdoiorg101016jcub200505047

310 Norice CT Smith FJ Solis N Filler SG Mitchell AP 2007 Require-ment for Candida albicans Sun41 in biofilm formation and virulenceEukaryot Cell 62046 ndash2055 httpdxdoiorg101128EC00314-07

311 Richard ML Nobile CJ Bruno VM Mitchell AP 2005 Candidaalbicans biofilm-defective mutants Eukaryot Cell 41493ndash1502 httpdxdoiorg101128EC481493-15022005

312 Braun BR Johnson AD 2000 TUP1 CPH1 and EFG1 make indepen-dent contributions to filamentation in Candida albicans Genetics 15557ndash 67

313 Brown DH Jr Giusani AD Chen X Kumamoto CA 1999 Filamen-tous growth of Candida albicans in response to physical environmentalcues and its regulation by the unique CZF1 gene Mol Microbiol 34651ndash662 httpdxdoiorg101046j1365-2958199901619x

314 Calera JA Zhao X-J Calderone R 2000 Defective hyphal developmentand avirulence caused by a deletion of the SSK1 response regulator genein Candida albicans Infect Immun 68518 ndash525 httpdxdoiorg101128IAI682518-5252000

315 Gale CA Bendel CM McClellan M Hauser M Becker JM Berman JHostetter MK 1998 Linkage of adhesion filamentous growth and vir-ulence in Candida albicans to a single gene INT1 Science 2791355ndash1358 httpdxdoiorg101126science27953551355

316 Liu H Kohler J Fink GR 1994 Suppression of hyphal formation inCandida albicans by mutation of a STE12 homolog Science 2661723ndash1726 httpdxdoiorg101126science7992058

317 Lo H-J Koumlhler JR DiDomenico B Loebenberg D Cacciapuoti A FinkGR 1997 Nonfilamentous C albicans mutants are avirulent Cell 90939 ndash949 httpdxdoiorg101016S0092-8674(00)80358-X

318 Sahni N Yi S Daniels KJ Srikantha T Pujol C Soll DR 2009 Genesselectively up-regulated by pheromone in white cells are involved in bio-film formation in Candida albicans PLoS Pathog 5e1000601 httpdxdoiorg101371journalppat1000601

319 Yi S Sahni N Daniels KJ Lu KL Huang G Garnaas AM Pujol CSrikantha T Soll DR 2011 Utilization of the mating scaffold protein inthe evolution of a new signal transduction pathway for biofilm develop-ment mBio 2e00237-10 httpdxdoiorg101128mBio00237-10

320 Hull CM Johnson AD 1999 Identification of a mating type-like locusin the asexual pathogenic yeast Candida albicans Science 2851271ndash1275 httpdxdoiorg101126science28554311271

321 Wu W Pujol C Lockhart SR Soll DR 2005 Chromosome loss fol-lowed by duplication is the major mechanism of spontaneous mating-type locus homozygosis in Candida albicans Genetics 1691311ndash1327

322 Wu W Lockhart SR Pujol C Srikantha T Soll DR 2007 Heterozy-gosity of genes on the sex chromosome regulates Candida albicans viru-lence Mol Microbiol 641587ndash1604 httpdxdoiorg101111j1365-2958200705759x

323 Ibrahim AS Magee B Sheppard D Yang M Kauffman S Becker JEdwards JE Magee P 2005 Effects of ploidy and mating type on viru-lence of Candida albicans Infect Immun 737366 ndash7374 httpdxdoiorg101128IAI73117366-73742005

324 Janbon G Sherman F Rustchenko E 1999 Appearance and propertiesof L-sorbose-utilizing mutants of Candida albicans obtained on a selec-tive plate Genetics 153653ndash 664

325 Lockhart SR Daniels KJ Zhao R Wessels D Soll DR 2003 Cellbiology of mating in Candida albicans Eukaryot Cell 249 ndash 61 httpdxdoiorg101128EC2149-612003

326 Miller MG Johnson AD 2002 White-opaque switching in Candidaalbicans is controlled by mating-type locus homeodomain proteins andallows efficient mating Cell 110293ndash302 httpdxdoiorg101016S0092-8674(02)00837-1

327 Slutsky B Staebell M Anderson J Risen L Pfaller M Soll D 1987ldquoWhite-opaque transitionrdquo a second high-frequency switching system inCandida albicans J Bacteriol 169189 ndash197

328 Bennett RJ Miller MG Chua PR Maxon ME Johnson AD 2005Nuclear fusion occurs during mating in Candida albicans and is depen-dent on the KAR3 gene Mol Microbiol 551046 ndash1059 httpdxdoiorg101111j1365-2958200504466x

329 Daniels KJ Lockhart SR Staab JF Sundstrom P Soll DR 2003 Theadhesin Hwp1 and the first daughter cell localize to the aa portion of the

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conjugation bridge during Candida albicans mating Mol Biol Cell 144920 ndash 4930 httpdxdoiorg101091mbcE03-04-0264

330 Park YN Daniels KJ Pujol C Srikantha T Soll DR 2013 Candidaalbicans forms a specialized ldquosexualrdquo as well as ldquopathogenicrdquo biofilmEukaryot Cell 121120 ndash1131 httpdxdoiorg101128EC00112-13

331 Sahni N Yi S Pujol C Soll DR 2009 The white cell response topheromone is a general characteristic of Candida albicans strains Eu-karyot Cell 8251ndash256 httpdxdoiorg101128EC00320-08

332 Yi S Sahni N Daniels KJ Pujol C Srikantha T Soll DR 2008 Thesame receptor G protein and mitogen-activated protein kinase path-way activate different downstream regulators in the alternative whiteand opaque pheromone responses of Candida albicans Mol Biol Cell19957ndash970

333 Yi S Sahni N Pujol C Daniels KJ Srikantha T Ma N Soll DR 2009A Candida albicans-specific region of the alpha-pheromone receptorplays a selective role in the white cell pheromone response Mol Micro-biol 71925ndash947 httpdxdoiorg101111j1365-2958200806575x

334 Anderson JM Soll DR 1987 Unique phenotype of opaque cells in thewhite-opaque transition of Candida albicans J Bacteriol 1695579 ndash5588

335 Rikkerink E Magee B Magee P 1988 Opaque-white phenotype tran-sition a programmed morphological transition in Candida albicans JBacteriol 170895ndash 899

336 Lederberg J Tatum EL 1946 Gene recombination in Escherichia coliNature 158558

337 Licht TR Christensen BB Krogfelt KA Molin S 1999 Plasmid transferin the animal intestine and other dynamic bacterial populations the roleof community structure and environment Microbiology 1452615ndash2622 httpdxdoiorg10109900221287-145-9-2615

338 Doig P Todd T Sastry PA Lee K Hodges RS Paranchych W Irvin R1988 Role of pili in adhesion of Pseudomonas aeruginosa to human re-spiratory epithelial cells Infect Immun 561641-1646

339 Rothbard JB Fernandez R Wang L Teng N Schoolnik GK 1985Antibodies to peptides corresponding to a conserved sequence of gono-coccal pilins block bacterial adhesion Proc Natl Acad Sci U S A 82915ndash919 httpdxdoiorg101073pnas823915

340 Hung C Zhou Y Pinkner JS Dodson KW Crowley JR Heuser JChapman MR Hadjifrangiskou M Henderson JP Hultgren SJ 2013Escherichia coli biofilms have an organized and complex extracellularmatrix structure mBio 4e00645-13 httpdxdoiorg101128mBio00645-13

341 Molin S Tolker-Nielsen T 2003 Gene transfer occurs with enhancedefficiency in biofilms and induces enhanced stabilisation of the biofilmstructure Curr Opin Biotechnol 14255ndash261 httpdxdoiorg101016S0958-1669(03)00036-3

342 Porman AM Alby K Hirakawa MP Bennett RJ 2011 Discovery of aphenotypic switch regulating sexual mating in the opportunistic fungalpathogen Candida tropicalis Proc Natl Acad Sci U S A 10821158 ndash21163httpdxdoiorg101073pnas1112076109

343 Xie Z Thompson A Sobue T Kashleva H Xu H Vasilakos J Don-gari-Bagtzoglou A 2012 Candida albicans biofilms do not trigger reac-tive oxygen species and evade neutrophil killing J Infect Dis 2061936 ndash1945 httpdxdoiorg101093infdisjis607

344 Lin CH Kabrawala S Fox EP Nobile CJ Johnson AD Bennett RJ2013 Genetic control of conventional and pheromone-stimulated bio-film formation in Candida albicans PLoS Pathog 9e1003305 httpdxdoiorg101371journalppat1003305




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Can the term as applied to prokaryotic communities that rimbodies of water clog pipes form on the surface of your petrsquos waterbowl and obstruct the respiratory tracts of cystic fibrosis patientshave the same meaning in regard to the C albicans communitiesthat coat dentures and form plaque on teeth spread along thewalls of intestines and line the walls of catheters Is the meaning ofthe term so general that it can apply to any population of micro-organisms on a substratum including a colony of cells on nutrientagar Or does the term ldquobiofilmrdquo have specific functional and ar-chitectural connotations that apply to all species both prokaryoticand eukaryotic In delving into the literature on C albicans bio-films formed in vitro and relating it to that on the more intensivelystudied bacterial biofilms it became apparent that the definitionof the term had indeed evolved into something more than simplya film or colony of microorganisms supported by a substratum inan aqueous environment It connoted a community of interactingcells anchored tightly to a substratum made up of multiple cellphenotypes embedded in a self-generated extracellular matrix(ECM) It connoted a population in a self-established microenvi-ronment with specialized functional and architectural character-istics and a mechanism for dispersal And as is the case for allcommunities there appeared to be the potential for a significantdegree of plasticity depending upon the genotype and differentenvironmental pressures It became clear that there is a lack ofappreciation of the variability of C albicans biofilms due to straindifferences and the plasticity of these biofilms resulting from dif-ferent conditions employed in vitro Attempting to integrate datafrom different studies that employed different strains differentculture conditions and different quantitative methods of assess-ment can indeed lead to problems of interpretation without con-sidering the possibility that the biofilm preparations might differphysiologically and developmentally There can also be a majorproblem in interpreting the body of knowledge that has emergedfor bacterial biofilms formed in vitro (35ndash37) The problems for Calbicans are particularly poignant when attempts are made to gen-erate general models of biofilm development signal transductionpathways regulating biofilm formation and drug resistance Theproblem is further exacerbated for C albicans by the discoverythat depending upon the configuration of the mating type locus(MTL) (a versus aa or ) cells form biofilms that have dis-tinctly different functional characteristics (38 39) In reviewingthe literature it became clear that researchers rarely test whetherthe strain they employ is representative and rarely assess the con-figuration of the MTL locus For C albicans the latter omissionhas not had a profound effect on most studies since 90 to95 of strains are MTL heterozygous (a) (40ndash42) Howeverfor the closely related species C dubliniensis which shares mostof the developmental processes exhibited by C albicans (43ndash46)the omission is indeed worrisome given that one-third of all nat-ural strains are MTL homozygous (44)

The goal of this review is to consider the variability and plas-ticity of C albicans biofilms formed in in vitro models with theintent of engendering awareness that they are developmentallycomplex and depending on the strain and culture conditions notcreated equal This review does not cover in detail the literature onthe signal transduction pathways that regulate gene expressionduring biofilm formation the genes that are differentially regu-lated or the molecular mechanisms regulating matrix forma-tion or drug resistance unless that information pertains to thespecific aim set forth The reader is directed to a number of

excellent reviews on these subjects published in the past sev-eral years (47ndash55)

DEFINING A C ALBICANS BIOFILM LESSONS FROMBACTERIA

Considering what a C albicans biofilm represents may best beaccomplished by first reviewing bacterial biofilms given that de-tailed studies of their formation in vitro appeared in the literatureapproximately 2 decades prior to studies of C albicans biofilmsThe first report of the cellular nature of a microbial biofilm wasthat of Antonie van Leeuwenhoek in 1684 when he described tothe Royal Society of England the variety of microorganisms indental plaques which he examined with his newly developed mi-croscope (56 57) However not until the 1940s did researchersrealize that the bacterial slime covering surfaces in aquatic ecosys-tems represented more than disorganized detritus Rather theywere complex and organized communities of cells (58 59) In1978 J W Costerton and his coworkers (60 61) published thefirst formal description of a bacterial biofilm in researching howbacteria adhered to surfaces in aquatic systems They defined abiofilm as a matrix-encapsulated population of microorganismsadhering to a surface in a nutrient-sufficient environment Subse-quently they and others described the fundamental differencesbetween bacteria in biofilms and bacteria that are free-living(planktonic) (62ndash64) They noted (60) that over 99 of the bac-terial biomass in the natural aquatic ecosystems they first studiedwere in the form of biofilm communities on wet or submergedsurfaces rather than free and independent in suspension Becausethe bacteria include so many species one general biofilm descrip-tion cannot encompass the variety of their forms HoweverCosterton and others extracted a number of generally commonsteps in the formation of bacterial biofilms in vitro as well asseveral shared attributes (21 65ndash67) which are outlined briefly inFig 1 As will become evident in this review many of the steps ofbacterial biofilm formation and the attributes of these biofilmshave proven relevant to the emerging descriptions of C albicansbiofilm formation in vitro The first step in generating a bacterialbiofilm in vitro is to grow cells planktonically for inoculation intothe biofilm model The concentration of cells in the inoculum canbe adjusted to generate an initial monolayer on the substratum ofthe model employed The second step is to add the inoculum ofplanktonic cells to an adherent substratum to facilitate adhesionStrong adherence is necessary for anchoring especially when thereare fluid flow and mechanical shear forces that could release singlecells not firmly attached to the substratum during initial biofilmdevelopment It should be noted that although most in vitro mod-els include a solid substratum for adherence cells in natural set-tings may also converge at air-liquid or liquid-liquid interfaceswhere differences in density hydrophobicity and water structurecan result in a discrete physical interface that can be assessed bycell surface receptors (68 69) Cells may also use each other assurfaces through cohesion to form suspended aggregates or floc-cules exhibiting properties of a biofilm on a substratum (70ndash73)In vitro adherence to a substrate is basic to the establishment of ageographically confined population Bacterial adhesion has beenseparated into two phases primary reversible adhesion some-times referred to as ldquodockingrdquo and secondary irreversible adhe-sion sometimes referred to as anchoring or ldquolockingrdquo (74ndash77)(Fig 1) For biofilm formation in vivo a question arises as towhether one cell a number of independent cells or a fragment of

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BIOFILM DISPERSAL



OTHER MODELS










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STRAIN VARIABILITY







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Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











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CONCLUDING REMARKS


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ACKNOWLEDGMENTS





































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BIOFILM DISPERSAL



OTHER MODELS










mediume






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STRAIN VARIABILITY







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Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











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CONCLUDING REMARKS


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ACKNOWLEDGMENTS





































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Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


BIOFILM DISPERSAL



OTHER MODELS










mediume






httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from





httpmm

brasmorg

Dow

nloaded from





Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from














Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


STRAIN VARIABILITY







httpmm

brasmorg

Dow

nloaded from








Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from








httpmm

brasmorg

Dow

nloaded from


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


BIOFILM DISPERSAL



OTHER MODELS










mediume






httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from





httpmm

brasmorg

Dow

nloaded from





Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from














Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


STRAIN VARIABILITY







httpmm

brasmorg

Dow

nloaded from








Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


BIOFILM DISPERSAL



OTHER MODELS










mediume






httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from





httpmm

brasmorg

Dow

nloaded from





Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from














Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


STRAIN VARIABILITY







httpmm

brasmorg

Dow

nloaded from








Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from


BIOFILM DISPERSAL



OTHER MODELS










mediume






httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from





httpmm

brasmorg

Dow

nloaded from





Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from














Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


STRAIN VARIABILITY







httpmm

brasmorg

Dow

nloaded from








Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from





httpmm

brasmorg

Dow

nloaded from





Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from














Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


STRAIN VARIABILITY







httpmm

brasmorg

Dow

nloaded from








Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from





httpmm

brasmorg

Dow

nloaded from





Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from














Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


STRAIN VARIABILITY







httpmm

brasmorg

Dow

nloaded from








Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from





Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from














Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


STRAIN VARIABILITY







httpmm

brasmorg

Dow

nloaded from








Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from














Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


STRAIN VARIABILITY







httpmm

brasmorg

Dow

nloaded from








Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from














Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


STRAIN VARIABILITY







httpmm

brasmorg

Dow

nloaded from








Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from


STRAIN VARIABILITY







httpmm

brasmorg

Dow

nloaded from








Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from








Mediumd




YNB medium(glucose)






8503 2684 32 2672ndash10225 38

MTT A540 098 027 28 049ndash124 32

Kuhn et al2002 (26)











Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from




Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from















































httpmm

brasmorg

Dow

nloaded from












































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from




















httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from










httpmm

brasmorg

Dow

nloaded from






Soll and Daniels



httpmm

brasmorg

Dow

nloaded from














httpmm

brasmorg

Dow

nloaded from





CONCLUDING REMARKS


Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


ACKNOWLEDGMENTS





































httpmm

brasmorg

Dow

nloaded from
















































Soll and Daniels



httpmm

brasmorg

Dow

nloaded from


















































httpmm

brasmorg

Dow

nloaded from













































Soll and Daniels



httpmm

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Plasticity of Candida albicans Biofilms · protocols for new ones. ... culture conditions, and...

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Transcript of Plasticity of Candida albicans Biofilms · protocols for new ones. ... culture conditions, and...