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ISSN 0175-7598, Volume 88, Number 2

MINI-REVIEW

Prevention of bacterial adhesion

Per Klemm & Rebecca Munk Vejborg &

Viktoria Hancock

Received: 14 June 2010 /Revised: 26 July 2010 /Accepted: 27 July 2010 /Published online: 8 August 2010# Springer-Verlag 2010

Abstract Management of bacterial infections is becomingincreasingly difficult due to the emergence and increasingprevalence of bacterial pathogens that are resistant toavailable antibiotics. Conventional antibiotics generally killbacteria by interfering with vital cellular functions, anapproach that imposes selection pressure for resistantbacteria. New approaches are urgently needed. Targetingbacterial virulence functions directly is an attractivealternative. An obvious target is bacterial adhesion. Bacte-rial adhesion to surfaces is the first step in colonization,invasion, and biofilm formation. As such, adhesion repre-sents the Achilles heel of crucial pathogenic functions. Itfollows that interference with adhesion can reduce bacterialvirulence. Here, we illustrate this important topic withexamples of techniques being developed that can inhibitbacterial adhesion. Some of these will become valuableweapons for preventing pathogen contamination and fight-ing infectious diseases in the future.

Keywords Adhesins . Bacterial attachment . Biofilms .

Fimbriae

Introduction

Most bacteria live attached to or in close association withsurfaces. It has been estimated that at least 90% of allbacteria in the environment reside attached to a surface, andmany of these form sessile communities (Costerton et al.

1999). Essentially, all naturally occurring bacteria are ableto express adhesins. Adhesins enable bacteria to specificallyrecognize and bind to a diverse spectrum of molecular motifson target surfaces, ranging from surface components oftissues or cells, to substratum-coated abiotic materials suchas glass and plastic. By virtue of adhesins, bacteria can bindto any type of surface. Adhesion is of paramount importancein the life of bacteria and provides two vital roles; it allowstargeting of a given bacterium to a specific surface (e.g., aparticular epithelial surface in a mammalian host), and inflow environments, it enables bacteria to resist physicalremoval by hydrodynamic shear forces. The ability ofbacteria to resist removal by hydrodynamic shear forces isoften critical since many surfaces in nature are submitted tostrong flow forces. A plethora of bacterial adhesins havebeen characterized (Fig. 1, Table 1). Usually, bacterialadhesins exhibit exquisite selectivity for target moleculesand recognize molecular motifs in a lock-and-key fashionin line with enzymes and immunoglobulins. The targetspecificity is instrumental in guiding the bacterium to itsspecific ecological niche, for example, a specific tissuesurface in a specific host. This phenomenon is referred toas tissue and host tropism. Thus, the expression of agiven adhesin functions as an address indicator for themicrobe.

Attachment of pathogenic bacteria to host surfaces is acritical step in the pathogenesis of virtually all infections,for example, in infections of the respiratory, urinary, andgastrointestinal tracts. For many bacterial pathogens thatinfect such mucosal tissues, specific adhesins have beenimplicated as virulence factors. Several examples ofpathogens that have lost their adhesins are known andthese have become avirulent and even probiotic (Connellet al. 1997; Klemm et al. 2006, 2007; Roos et al. 2006).Sometimes, only one adhesin is expressed and is critical for

P. Klemm (*) :R. M. Vejborg :V. HancockMicrobial Adhesion Group, DTU Food, Bldg 301,Technical University of Denmark,2800 Lyngby, Denmarke-mail: pekl@food.dtu.dk

Appl Microbiol Biotechnol (2010) 88:451–459DOI 10.1007/s00253-010-2805-y

Author's personal copy

pathogenesis. However, many bacterial pathogens areendowed with the ability to express an array of differentadhesins, and it is the concerted action of these, oftenexpressed at different stages during infection, that isimportant for virulence.

The basic problem of positioning a receptor-specificprotein on the bacterial surface has been addressed innumerous ways by bacteria and accordingly, a plethora ofdifferent bacterial adhesins exists (Klemm and Schembri

2000). Many adhesins are single proteins located directlyon the bacterial surface, e.g., autotransporter adhesins likeTibA and AIDA of Escherichia coli (Benz and Schmidt1992; Sherlock et al. 2004, 2005); however, most bacterialadhesins are organized as thin thread-like organelles calledfimbriae or pili. Fimbriae are (hetero)polymers with lengthsof about 1 μm. Here, the bulk of the organelle is composedof a structural protein, which serves as a scaffold fordisplay of the actual adhesin—normally located at the tip of

Table 1 Examples of bacterial adherence structures of E. coli

Structure Adhesin Adherence to molecule/surface Role in biofilmformation

References

Afa/Dr fimbriae AfaE/DraE DAF receptor, integrin α1β5 Nd (Cota et al. 2006; Labigne-Roussel et al.1984; Nowicki et al. 1990)

AIDA AIDA Self-recognizing, binds to epithelial cells Yes (Laarmann and Schmidt 2003; Sherlocket al. 2004)

Antigen 43 Ag43 Self-recognizing Yes (Danese et al. 2000; Kjaergaard et al. 2000)

Auf fimbriae AufA Unknown Nd (Buckles et al. 2004)

CFA/I fimbriae CfaE Human intestinal cells Nd (Baker et al. 2009)

Curli fimbriae CsgA Fibronectin, plasminogen, humancontact phase proteins

Yes (Olsen et al. 1989)

ECP fimbriae Unknown Human epithelial cells Yes (Pouttu et al. 2001; Rendon et al. 2007)

EtpA EtpA Links flagella to intestinal cells Nd (Roy et al. 2009)

F1C fimbriae FocH Galatosylceramide, globotriaosylceramide Yes (Bäckhed et al. 2002; Khan et al. 2000)

F9 fimbriae Unknown Unknown Yes (Ulett et al. 2007)

P fimbriae PapG α-D-Gal-(1–4)–β-D-gal No (Hull et al. 1981; Kuehn et al. 1992)

Prs fimbriae PrsG α-D-Gal-(1–4)–β-D-gal No (Lund et al. 1988)

S fimbriae SfaS α-Sialyl-(2–3)–α-galactose Nd (Moch et al. 1987; Schmoll et al. 1989)

TibA Self-recognizing, binds to human intestinalepithelial cells

Yes (Lindenthal and Elsinghorst 2001; Sherlocket al. 2005)

Type 1 fimbriae FimH D-Mannose, fibronectin, collagen Yes (Klemm et al. 1985; Krogfelt et al. 1990;Pouttu et al. 1999; Sokurenko et al. 1992)

Type 3 fimbriae MrkD Type V collagen Yes (Gerlach et al. 1989; Ong et al. 2008)

Nd not determined

Fig. 1 Structures on the surfaceof E. coli involved in adherenceto surfaces and host cells.Bacterial adherence structuresare often very target specific,e.g., type 1 and P fimbriae, andmany autotransporters, e.g.,Ag43 and AIDA; however, anumber of surface structureslacking a specific targetmolecule have been implicatedin bacterial attachment includingcapsule, lipopolysaccharides(LPS), flagella, and cellulose

452 Appl Microbiol Biotechnol (2010) 88:451–459 Author's personal copy

the organelle (Hahn et al. 2002; Klemm et al. 2010; Klemmand Schembri 2000). Most wild-type strains are encapsulatedand enveloped in about 0.5-μm-thick capsule material.Arguably, one possible role for the fimbrial shaft would beto provide functional display of the tip-located adhesinbeyond the capsule. Most gram-negative and gram-positive bacterial pathogens produce fimbriae (Foster2004; Odenbreit 2005; Pizarro-Cerdá and Cossart 2006).

Bacterial adhesion is the first crucial step in biofilmformation. This usually takes place via an initial expansionstep resulting in microcolony formation and subsequentlyformation of a biofilm, i.e., a complex three-dimensionalstructure. In the medical field, the biofilm mode of growthhas attracted particular attention because many persistentand chronic bacterial infections are now believed to belinked to the formation of biofilms. Moreover, biofilmsoften serve as a source for recurrent infections. Bacterialbiofilm infections are particularly problematic becausesessile bacteria can withstand host immune responses andare highly resistant to antibiotics, biocides, and hydrody-namic shear forces compared with their planktonic counter-parts (Costerton et al. 1999). In industrial settings, bacterialbiofilms often cause serious problems due to contaminationof work surfaces and clogging of tubes and machinery(Kumar and Anand 1998; Mattila-Sandholm and Wirtanen1992).

The ability to prevent bacterial adhesion is an idealstrategy to interfere with bacterial pathogenesis andcolonization at an early stage. The subject has attractedgreat interest, and an impressive body of literature hasaddressed this topic over the years. Consequently, acomplete treatise of the subject is beyond the format ofthis review. Here, we shall use selected examples toillustrate some of the most important aspect of ways toinhibit bacterial adhesion.

Changing the surface

Bacterial adhesion to inert surfaces constitutes a significantproblem in a wide range of environmental, industrial, andmedical settings. The possibility of coating surfaces withsubstances that inhibit bacterial adhesion and colonizationtherefore seems like an attractive approach. One way ofachieving this is to coat the inert surfaces with macro-molecules. An example of this approach is the coating ofsurfaces with fish muscle α-tropomyosin.

It was serendipitously discovered that the coating ofstainless steel surfaces with a fish muscle extract reducedbacterial adhesion dramatically (Bernbom et al. 2006).Subsequently, it was shown that the responsible componentwas α-tropomyosin. The α-tropomyosin coating inhibitedbacterial adhesion to a range of materials including glass,

polystyrene, vinyl plastics, and stainless steel (Vejborg et al.2008). Tropomyosin belongs to a family of contractileproteins associated mainly with muscle tissue. Crystallo-graphic analysis has shown that it is composed of twoparallel α-helical strands interwoven in a coiled-coildimeric structure. α-Tropomyosin is also a highly nega-tively charged, hydrophilic protein. Given the fact that thesurfaces of most bacteria are negatively charged and hydro-philic, it was inferred that the ability of α-tropomyosin-coatedsurfaces to resist bacterial adhesion was primarily physi-ochemical and not adhesin specific. This tenet was bolsteredby the finding that the addition of a divalent cation like Ca(II)could counteract the inhibitory effect of α-tropomyosin tosome extent (Vejborg and Klemm 2008). α-Tropomyosin-coated surfaces were also shown to resist bacterial biofilmformation 100-fold better than non-coated surfaces (Fig. 2).The biofilm-reducing properties seem to be related primar-ily to the anti-adhesive effect of α-tropomyosin andpresumably to the initial adhesion phase of biofilmformation. Nevertheless, it is interesting to note that thisnon-toxic, cheap, readily available coating can significantlyblock both bacterial adhesion and biofilm formation onabiotic surfaces. Tropomyosin coatings might be beneficialon for example urinary catheters or contact lenses wherethey can delay bacterial attachment and colonization, or inindustrial settings on stainless steel surfaces. Due to theinherent instability of physically adsorbed protein coatings,short-term materials are appropriate; however, covalentattachment of the protein to the surface could extend thelifetime of the coating.

Prevention of adhesin biosynthesis

Fimbrial biosynthesis is a complex process that involvesassembly of large heteropolymeric organelles assisted byhelper proteins. One of the best characterized fimbriaegroups is assembled via the chaperone–usher-dependentpathway, which features a periplasmic chaperone thatescorts structural components to an outer-membrane-located usher where the actual organelle synthesis takesplace (Klemm et al. 2010; Waksman and Hultgren 2009).An attractive way to interfere with bacterial adhesion is toabolish biosynthesis of the fimbriae. Disruption of fimbrialorganelle formation of both P and type 1 fimbriae wasachieved by chemical interference of fimbriae biogenesis atthe assembly level in the chaperone–usher pathway(Pinkner et al. 2006). The activity of a family of bicyclic2-pyridones, termed pilicides, was successfully evaluated intype 1 and P fimbriae biogenesis. Adherence to bladdercells mediated by either type 1 or P fimbriae, and biofilmformation mediated by type 1 fimbriae, were all reduced byapproximately 90% in laboratory and clinical E. coli

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strains. The structure of the pilicide bound to the P fimbriaechaperone PapD revealed that the pilicide bound to thesurface segment of the chaperone is involved in theinteraction with the usher protein. Interestingly, pilicidescan be designed to target regions on chaperones and ushersthat are conserved. This makes it possible to interruptassembly of a range of fimbriae assembled by thechaperone–usher pathway (Åberg and Almqvist 2007;Larsson et al. 2005; Pinkner et al. 2006; Svensson et al.2001). Recently, bicyclic 2-pyridones were shown to inhibitassembly of curli (Cegelski et al. 2009). Curli are amyloidpolymers thinner than fimbriae and are involved inadhesion and biofilm formation (Chapman et al. 2002). Inline with the term pilicides, the bicyclic 2-pyridonesderivatives, which abolished curli biosynthesis, weretermed curlicides (Cegelski et al. 2009). Taken together,pilicides and curlicides target key virulence factors inpathogenic bacteria, i.e., fimbriae and curli, and representa promising new concept for development of novel anti-virulence drugs.

Inhibition with receptor analogs

Bacterial adhesins typically target sugar moieties ofglycoproteins or glycolipids. In several cases, the molecularstructure of the receptors targets of bacterial adhesins isknown (Table 1). Classic examples being the targets of twoadhesins that are involved in urinary tract infections, viz.type 1 and P fimbriae, are involved in bladder and kidneycolonization, respectively. The primary physiological recep-tors for the type 1 fimbrial adhesion, FimH, in the urinarytract are uroplakin Ia and Ib, two major high-mannose typeglycoproteins present on urothelial cells on the luminalsurface of the bladder (Min et al. 2002; Zhou et al. 2001).However, FimH also recognizes a wide range of glyco-

proteins carrying one or more N-linked mannose structures.The receptors for the P fimbrial adhesin PapG have beenidentified as the P1 blood group antigen (globo-A) and theForssman antigen (globopentosylceramide), both of whichpossess a Gal-α(1–4)Gal disaccharide core (Hultgren et al.1996). This type of information has been used to design anddevelop receptor-mimetics. Blocking of the FimH–receptorinteraction can be achieved by a variety of natural andsynthetic saccharides (Bouckaert et al. 2005; Firon et al.1987; Nagahori et al. 2002). In addition, such inhibitors canabolish or reduce type 1 fimbriae-based biofilm formation(Pratt and Kolter 1998; Schembri and Klemm 2001).Interestingly, alkyl-substituted mannosides have affinitiesfor FimH in the nanomolar range, e.g., butylmannosidebound so tight to FimH that it was impossible toremove by prolonged dialysis (Bouckaert et al. 2005).These findings may overcome the significant obstacle oflow affinity generally encountered in attempts to developcarbohydrate-based drugs. In the case of P fimbriae, α-D-galactopyranosyl-(1–4)-β-D-galactopyranosides inhibitPapG binding to receptor targets. New anti-adhesive drugscould be based on these leads to prevent bacterialattachment for example in the urinary tract to preventsome of the estimated 150 million yearly urinary tractinfections.

Anti-adhesive vaccines

An appealing approach to inhibit bacterial adhesion is toblock adhesin–receptor interaction by immunization withthe adhesion. If successful, this type of immunizationwould provoke production of adhesin-specific antibodiesin the host, which ideally would prevent bacterial adhesionand colonization. Many attempts have been made to makeanti-adhesive vaccines, or rather anti-fimbriae vaccines

Fig. 2 Bacterial adhesion andbiofilm formation on abioticsurfaces can be prevented bycoating with fishα-tropomyosin. Biofilmformation of E. coli ona non-treated or b α-tropomyosin-treated glass. Thebiofilms were grown for 30 h at37°C in minimalmedium

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(Levine et al. 1994). In most of these, the immune responsewas primarily directed against the major structural proteinsof the fimbriae, and often, this type of inroad has failed dueto sequence variation in these proteins. In some cases,fimbriae vaccines have been highly successful, for examplein the case of anti-diarrhea vaccine for piglets based on K88fimbriae from ETEC strains (Rutter and Jones 1973). Mostfimbriae are heteropolymers, and in these, the actualadhesion makes up a very small proportion of the organelle,about 0.1%. Consequently, very little of the immuneresponse is directed against the adhesin itself. Meanwhile,in such cases, one can target the adhesin itself. One suchexample is the attempt to make a vaccine against cystitisbased on the FimH adhesin from E. coli type 1 fimbriae.The foundation of this approach was based on observationsin animal models where adhesion-knock-out mutantsessentially were non-virulent and unable to colonize thehost (Connell et al. 1996). Vaccination with FimHcomponent vaccines significantly reduced UPEC infectionof the bladder in mice and monkeys (Langermann andBallou 2003; Langermann et al. 1997, 2000). From thesereports, it seemed that FimH would be an obvious candidatefor a vaccine against UPEC infections in humans. A FimH-based vaccine approach aimed at preventing UTI has shownpromising results when tested in animals and seems to beable to prevent bacterial adhesion to the bladder uroepithe-lium and thereby infection. Unfortunately, this strategy wasunsuccessful and further complicated by the ability ofFimH to trigger a severe auto-immune disease, pauci-immune focal necrotizing glomerulonephritis, due tomolecular mimicry. It turns out that FimH and the humanLAMP-2 protein, a heavily glycosylated membrane proteinexpressed by many human cell types, both contain the sameprominent immunological epitope (Kain et al. 2008).

Metal interference

Metallic cations are essential to bacterial growth, attach-ment, and biofilm formation (Banin et al. 2005; Brubaker1985; Hancock et al. 2008; Ratledge and Dover 2000).Metal chelators such as EDTA and citrate have been studiedas a means to disrupt bacterial surface adherence andbiofilm formation. One recent example is the use oftrisodium citrate, a high-affinity metal-binding chelator, inhemodialysis catheters, which proved efficient againstadherence of microbes in a randomized controlled humantrial (Bosma et al. 2010). Also, dispersal of Pseudomonasaeruginosa biofilms can be induced by the addition of ironchelators (Banin et al. 2006). The exact mechanisms behindinhibition of attachment and subsequently biofilm growthby metal chelators are not known. The major iron regulatorin E. coli is the ferric uptake regulator protein, Fur.

Activated Fur represses all known iron-transport systemsin E. coli. A typical high-affinity iron-uptake systemconsists of a low-molecular-mass Fe(III)-chelating com-pound, known as a siderophore, combined with its cognatemembrane-located receptor. In P. aeruginosa, siderophoreproduction and biofilm formation are linked traits (Harrisonand Buckling 2009). In E. coli, iron is also known to becrucial for efficient biofilm formation in vitro (Hancocket al. 2008), and the Fur-regulated siderophore receptor, Ihafound in pathogenic E. coli isolates, is known to promoteadherence to epithelial cells (Rashid et al. 2006; Tarr et al.2000). Another siderophore uptake receptor, the ferricyersiniabactin uptake receptor FyuA, which is also regulat-ed by Fur, has been shown to be involved in biofilmformation; the fyuA gene was required for efficient biofilmformation of urinary tract E. coli strains (Hancock et al.2008). However, the exact role of FyuA in biofilm formationis not known, and whether the effect is purely due to adecrease in intracellular iron concentration or a result of anyadditional mechanism remains to be elucidated. Furthermore,iron, through activation of Fur, has been reported to repressexpression of CFA/I fimbriae (Karjalainen et al. 1991) and toregulate type 1 fimbria expression (McHugh et al. 2003; Wuand Outten 2009) in E. coli.

Another intriguing way to inhibit iron-regulated attach-ment, apart from addition of chelators, is to fool thebacterial regulatory system for iron uptake, i.e., Fur. Furacts as a positive repressor, i.e., it represses transcription ofmore than 90 genes in E. coli upon interaction with its co-repressor, Fe(II), and causes derepression in the absence ofFe(II) (Hantke 2001). Except from regulating iron import,Fur also controls cell functions such as metabolism ofsuccinate and acetate, chemotaxis and flagella expression,as well as oxidative and acid stresses, and plays a role inpathogenesis (Carpenter et al. 2009; Hantke 1987; Masséand Gottesman 2002). Interestingly, the affinity of Fur forZn(II) and Co(II) is significantly greater than for Fe(II)(Mills and Marletta 2005). Activating Fur by addition ofhigh-affinity Fur-chelating metals ions will result inshutdown of Fur-controlled iron-uptake systems, includingsiderophores and adhesin factors such as the abovemen-tioned Iha and CFA/I fimbriae. It was recently discoveredthat addition of Zn(II) and Co(II) at non-growth-limitingconcentrations significantly inhibited biofilm formation ofE. coli and Klebsiella pneumoniae. Addition of 500 and50 μM of ZnCl2 to E. coli grown in microtitre plates andthe flow-cell system, respectively, reduced biofilm for-mation by 70% and 95%; also, addition of 500 μMZn(II) reduced attachment of urinary tract E. coli to Foleyurinary catheters by a factor 9.4 (Hancock et al. 2010). Inline with this, zinc oxide thin films have been seen toreduce E. coli biofilms, without inhibiting bacterialgrowth, likely due to diffusion of Zn(II) into surrounding

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environment (Gittard et al. 2009). Another reason forinterfering with the expression of siderophore uptakesystems regulated by Fur is the possible attachment ofsiderophores to metal surfaces—providing a link to whichthe bacteria can bind via siderophore receptors located attheir cell surface. Covalent bonds can form betweenbacterial siderophores and metal surfaces; the siderophorepyoverdine of P. aeruginosa has been shown to adsorb tometal oxide films of Fe2O3, CrOOH, and AlOOH(Upritchard et al. 2007).

Here, one can envision that matrix-coating with zinc-containing substances, perhaps in combinations with otheradherence-inhibiting compounds, could be used to reducebacterial attachment—for example on devises such asurinary catheters—and subsequently reduce the incidenceof urinary tract infections. Furthermore, the fact that themetal ions prove efficient at non-growth-inhibiting concen-trations makes them promising candidates in order to avoiddevelopment of resistant strains. Development of resistanceto heavy metals is widespread among bacteria and mightoccur in any system where bacteria are exposed to thesemetals (Nies 1999).

Conclusions and future developments

Conventional antibiotics have contributed extensively to themanagement of infectious diseases since the first large-scaleintroduction of for example penicillin during World War II.However, decades of antibiotic use in the medical andveterinary sectors has rendered a wide range of bacterialpathogens resistant to many conventional antibiotics,resulting in increased difficulties in the management ofinfectious diseases. Bacteria that are resistant to currentantibiotics have caused severe problems in our hospitalsand clinics and are now the primary cause of death inintensive care units (Livermore 2005). Methicillin resistantinfections have reached epidemic proportions in many partsof the world. The problem is further aggravated by theemergence of multiresistant strains of bacteria for examplemultiresistant Staphylococcus aureus, E. coli, and Klebsiellanot only in human infections but certainly also in theveterinary sector (Hancock et al. 2009; Skov et al. 2007).Indeed, the rapid spread of multiresistant strains could wellreturn us to the preantibiotic era. Furthermore, the pharma-ceutical companies have only introduced a handful of newantibiotics over the last decades, and we simply lack novelbroad-spectrum antibiotics.

Development of novel potent and safe drugs is thereforeurgently required for societal growth, development, andwelfare. Traditional antibiotics generally kill bacteria,thereby setting the scene for the emergence of resistantbacteria by virtue of the introduced selection pressure.

Typically conventional antibiotics target some cellularfunction, like cell-wall synthesis, ribosomal function, orDNA replication that is present in both saprophytic as wellas pathogenic bacteria. We believe that drugs, whichspecifically target virulence factors like bacterial adhesins,fit the bill for a new generation of anti-bacterial drugs. Theexamples given in this overview demonstrate that byunderstanding the detailed interactions between bacterialadhesins and their receptors, it is possible to develop newapproaches to prevent bacterial adhesion, thereby advertingdisease and reducing other problems related to bacterialattachment. In contrast to classical antibiotics that generallykill bacteria, such drugs modify the behavior of bacteria.Therefore, such approaches may also be an attractive meansof ecological management of bacterial diseases. Last butnot least, these might not be as prone to promote bacterialresistance as conventional antibiotics.

Acknowledgments This work was supported by the Danish MedicalResearch Council (Grant 271-07-0291) and the Lundbeck Foundation(Grant R19-A2191).

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