Blocking of Bacterial Biofilm Formation by a Fish Protein Coating

Bacterial biofilm formation on inert surfaces is a significanthealth and economic problem in a wide range of environmental,industrial, and medical areas. Bacterial adhesion is generallya prerequisite for this colonization process and, thus, representsan attractive target for the development of biofilm-preventivemeasures. We have previously found that the preconditioningof several different inert materials with an aqueous fish muscleextract, composed primarily of fish muscle ${alpha}$ -tropomyosin, significantlydiscourages bacterial attachment and adhesion to these surfaces.Here, this proteinaceous coating is characterized with regardsto its biofilm-reducing properties by using a range of urinarytract infectious isolates with various pathogenic and adhesiveproperties. The antiadhesive coating significantly reduced ordelayed biofilm formation by all these isolates under everycondition examined. The biofilm-reducing activity did, however,vary depending on the substratum physicochemical characteristicsand the environmental conditions studied. These data illustratethe importance of protein conditioning layers with respect tobacterial biofilm formation and suggest that antiadhesive proteinsmay offer an attractive measure for reducing or delaying biofilm-associatedinfections.

INTRODUCTION

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INTRODUCTION

Many bacteria live in highly complex sessile communities, referredto as biofilms, as well as or even rather than existing as solitaryplanktonic cells (10, 18). These microbial consortia are ubiquitousin nature; almost all surfaces that are exposed to naturallyoccurring fluids will inevitably become subject to microbialcolonization (10, 45). As a result, microbial biofilms causea variety of problems in different medical, environmental, andindustrial settings, ranging from the fouling of ship hullsand the blocking of industrial piping to the colonization ofartificial medical implants (11, 26). Many chronic or persistentbacterial infections have also been linked to the formationof microbial biofilms (11). Biofilm-associated bacteria arenotoriously resistant to removal and often tolerate conditionsthat would readily eliminate their planktonic counterparts (15,45). They frequently display increased resistance to hydrodynamicshear forces, phages, and different biocides. Moreover, in themedical field, biofilm-associated bacteria regularly displaygreat tolerance toward antibiotics and host immune defense mechanisms.Consequently, they are often intractable and cause multiplerecurrent infections (11, 15).

In the human body, a number of highly efficient defense mechanismsnormally counter the buildup of microbial biofilms. Simple anatomicalbarriers, such as the sloughing off of epithelial cells, e.g.,from the bladder epithelium, and the secretion of antiadhesiveproteins and mucin, are generally very efficient at preventingthe establishment of biofilms on mucosal surfaces (28, 41).Notably, one of the only natural surfaces in the human bodythat is regularly exposed to bacteria but is not protected bysuch mechanisms is tooth enamel. The buildup of dental plaque,essentially comprising multispecies biofilms, readily testifiesto what can happen in the absence of these defense mechanisms(14, 32).

Artificial implants are also very prone to microbial colonization.Urinary catheters, stents, and contact lenses, for example,are readily colonized by innocuous or pathogenic microorganisms.Implant-associated infections often compromise the functionand safety of the devices and regularly do so with devastatingconsequences (11, 34, 36). Moreover, implant-associated biofilmsoften serve as a source of recurrent infections. Today, theleading type of hospital-acquired infection is the catheter-associatedurinary tract infection (CAUTI) (34, 35, 45). Up to 50% of allpatients undergoing short-term catheterization and essentiallyall patients carrying an indwelling urinary catheter for morethan 30 days will develop a urinary tract infection (40, 45).Catheter-associated biofilms are usually extremely difficultto eradicate by conventional antimicrobial treatment. Followinga course of treatment, bacteria originating from any residualbiofilms will often reinfect the urinary tract (29, 39, 40).Most short-term CAUTIs are caused by a single microbial species,such as Escherichia coli, Proteus mirabilis, or Klebsiella pneumonia,while long-term CAUTIs are often polymicrobial (30, 45, 46).Given that E. coli is one of the most frequently occurring speciesin implant-associated urinary tract infections, much efforthas been focused on understanding and controlling this particularmicroorganism (30).

Bacterial biofilm formation is a remarkably complex processentailing a range of molecular and physiological events. Itis generally considered to proceed through several steps, includingadhesion, microcolony formation, and structural maturation (31,47). While strain- and niche-specific variations largely determinethe complex interactions that occur within monoclonal and polyclonalmicrobial biofilms, the initial requirement for bacterial attachmentand adhesion appears to be universal. Without adhesion, thefollowing events leading to biofilm formation will generallynot take place (25). Bacterial adhesion itself is governed bya wide range of parameters pertaining to the environmental conditionsand the bacterial cell surface properties (1, 23). The capacityof a microorganism to adhere, however, also depends on the natureof the surface and any conditioning (23). When a clean, abioticsurface is exposed to a complex physiological fluid, a conditioningfilm will form almost instantaneously (33, 37, 38). This conditioninglayer may in turn influence the extent of bacterial adhesion(1, 23). Recently, we have found that the preconditioning ofinert materials with an aqueous extract of fish muscle proteins(FMPs) can significantly reduce bacterial attachment to thesesurfaces (5, 43). Based on this finding, we have tested thebiofilm-reducing activity of this nontoxic coating against arange of urinary tract infectious (UTI) E. coli and Klebsiellastrains.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Bacterial strains and growth conditions.
All bacterial strains used in this study are listed in TableS1 in the supplemental material. Cultures were established (at30 or 37°C) in either supplemented FAB minimal medium (24),modified Luria-Bertani (LB) medium (43), or sterile human urine.The human urine samples were collected from three to four healthywomen who had no prior history of urinary tract infections orantibiotic use within the previous 2 months. The urine sampleswere pooled, sterile filtered, and stored at 4°C.

Preparation of the aqueous extract.
The aqueous fish muscle extracts were prepared as describedpreviously (5, 12). Briefly, pieces of cod fillet were cookedfor 5 min in 0.5 liter of water per kg of fillet, and the aqueoussolution was passed through a strainer and recooked. The resultingsuspension was subsequently filtered through standard coffeefilters, and H₂KPO₄ (0.056 M) and HK₂PO₄ (0.044 M) were added.The pHs of the final solutions were then adjusted to 6.5, andthe solutions were sterilized for 30 min at 100°C. All aqueousextracts were stored at 4°C and used within 1 to 2 months.

Isoelectric fractionation.
Isoelectric fractionation was achieved with a pH adjustmentbuffer (pH 5) prepared from a 50 mM acetic acid buffer (pH 3;5 mM NaCl) and a 50 mM MES (morpholineethanesulfonic acid) buffer(pH 6; 5 mM NaCl). The aqueous fish muscle extract was dilutedfive times in the adjustment buffer, and the pH of the mixturewas readjusted to 5. The protein precipitate was then retrievedby centrifugation and resuspended in Tris buffer (10 mM; pH6.6) according to volume. All fractions were sterile filteredand stored at 4°C prior to use.

SDS-PAGE.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)was performed under denaturing conditions, and the gel was runand stained with Coomassie brilliant blue as described previously(27). All extract samples were diluted five times prior to theloading of the gel.

Microtiter plate biofilm assay.
Biofilm formation was assayed during growth in FAB minimal medium(24) containing 0.2% glucose, 0.2% Casamino Acids, and 1 µgof thiamine ml^–1; in LB medium; or in human urine. Briefly,48-well non-cell-culture-treated polystyrene microtiter plates(Nunc) were preconditioned for 1 h at room temperature and washedtwice with 1x phosphate-buffered saline. Aliquots of the cultures(diluted 100-fold in fresh medium) were added to the wells,and the plates were incubated for 16 h at 37°C. Biofilmformation was subsequently quantified by staining with crystalviolet (0.1%). The stain was dissolved with ethanol (96%), andthe absorbance at 595 nm was measured. When necessary, hydrodynamicconditions were simulated by incubating the plates with agitation(100 rpm).

Inspection of microplate biofilms.
For the microscopic inspection of biofilm formation under staticconditions, the biofilms were grown in coated or uncoated sonicseal slide dishes (Nunc), which have removable chambers. Followingbiofilm growth, each well was washed with phosphate-bufferedsaline and inspected by microscopy. Pictures were edited usingthe IMARIS software package (Bitplane).

Cultivation of flow cell biofilms.
All flow chamber biofilms were cultivated (at 30 or 37°C)either in FAB minimal medium (24) supplemented with 0.02% glucose,thiamine (1 µg ml^–1), and proline (10 µg ml^–1)or in human urine. The flow system was assembled and preparedmuch as described previously (8), using either glass (KnittelGläser) or vinyl plastic (Rinzl; Electron Microscope Sciences)coverslips as the substrata. Prior to inoculation, selectedchannels were preconditioned in situ for 1 h with the extractsand the entire system was washed with medium for 1 to 2 h. Theflow chambers were subsequently inoculated with diluted overnightcultures (optical density at 600 nm, 0.05), and adhesion wasallowed to take place for 1 h under static conditions. Laminarflow conditions were subsequently established using a WatsonMarlow 205S peristaltic pump. Biofilm development was inspectedusing a Zeiss LSM510 confocal scanning laser microscope, andthe pictures were edited using the IMARIS software package (Bitplane).

Biofilm formation on catheters.
Biofilm formation was studied much as described previously byusing commercial all-silicone Foley catheters (Coloplast Ltd.)(16). Briefly, small pieces of the catheters (4.5 cm) were connectedto the flow system and preconditioned in situ for 1 h at roomtemperature. The entire system was subsequently washed withprewarmed (37°C) human urine for 1 to 2 h at 37°C. Eachsegment was inoculated with an overnight culture of E. coliCFT073cfp (diluted 100-fold in fresh urine), and the cells wereallowed to adhere for 1 h under static conditions. The biofilmswere then grown for 24 h at 37°C. Following biofilm formation,the catheter pieces were disconnected, stained with 0.1% crystalviolet, and photographed. Biofilm formation was quantified bydissolving the stain with ethanol (96%) and measuring the absorbance(at 595 nm).

Adhesion assay.
Bacterial adhesion on preconditioned non-cell-culture-treatedpolystyrene microtiter plates was examined as described previously(43). The bacterial cells (E. coli ZK2686 grown overnight inLB medium) were harvested, washed twice, and resuspended in1/4-diluted Ringer's solution (3) containing various concentrationsof CaCl₂ prior to the adhesion assay. The adhesion assay wasrepeated three times. The relative adhesion values were calculated,based on the uncoated, unsupplemented control, prior to anyinternal comparisons.

Data analysis.
For the quantification of surface coverage, selected confocalscanning laser microscope pictures were analyzed using the computersoftware program COMSTAT (21). Surface coverage calculationsare based on pictures obtained from a total of four separatechannels for each coating and the control (two channels fromeach of two independent experiments). Five pictures were analyzedfor each channel at each time point.

RESULTS

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RESULTS

The FMP coatings significantly reduce biofilm formation.
During the preconditioning of various materials with the aqueousfish muscle extract, a significant antiadhesive proteinaceousconditioning film is formed (5, 43). This coating layer waspreviously found to consist primarily of ${alpha}$ -like tropomyosins(43). These fibrous muscle proteins can be crudely isolatedfrom the extract by isoelectric fractionation (at pH 5) (Fig.1A) and display significant antiadhesive properties (43). Tocharacterize the biofilm-reducing potentials of the aqueousextract and the isoelectric fraction, an E. coli K-12 strain,SAR19 (CSH26cfp), was studied during biofilm growth in a simplecrystal violet microtiter plate assay. The polystyrene microtiterplates were preconditioned with either of the two protein extractsand subsequently inoculated. The biofilms were then allowedto develop in minimal medium under static conditions (Fig. 1B).As seen in Fig. 1B, the preconditioning of the polystyrene surfaceswith the extracts significantly reduced biofilm formation bySAR19 compared to that on the uncoated controls (paired two-tailedt test; P < 0.001). The inspection of SAR19 during biofilmformation in the coated and uncoated sonic seal slide dishesconfirmed this result (Fig. 1C). The biofilm-reducing capacitiesof the two fractions were of quite similar magnitudes, indicatingthat the fish tropomyosin proteins contribute significantlyto the biofilm-reducing activity. Other proteins may, however,also have contributed to the activity. The repelling effectsof the two extracts were not linked to any growth inhibitionin itself, and the coatings in fact even appeared to increasethe overall bacterial density in the surrounding suspensions.Presumably, this relates to the general complexity and nutritionalvalues of the extracts. In order to account for such variations,all absorbance values were correlated with regards to the overallgrowth (2).

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FIG. 1. (A) SDS-PAGE analysis of the total aqueous fish extract (FJ) and the isoelectric fraction obtained at pH 5 (IEF). (B) Reduction of biofilm formation by SAR19 by preconditioning with the total aqueous extract or the isoelectric fraction. The biofilms were grown statically in minimal medium at 37°C. The absorbance values relative to that for the uncoated control are shown. Standard deviations of the results obtained from three independent experiments are indicated. Abbreviations: NC, not coated; FC, coated with the aqueous fish extract; IEF, coated with the isoelectric fraction. Asterisk, statistical significance indicated by a P value of <0.001 (paired two-tailed t test). (C) Biofilm formation by SAR19 on uncoated or coated sonic seal slides.

Analysis of FMP-mediated biofilm reduction in flow chambers.
To study the biofilm-reducing activities of the coatings inmore detail and to characterize the biofilm-reducing effectsunder hydrodynamic conditions, SAR19 was grown in flow chamberbiofilms. To also examine the significance of the material physicochemicalproperties in relation to the biofilm-reducing activity, twodifferent substrata were included in the study, specifically,glass and vinyl plastic. When studied under these conditions,the crude extract and the isoelectric fraction significantlyreduced or delayed biofilm formation (Fig. 2). The isoelectricfraction, in particular, proved to be very efficient. Even following76 h of incubation, few if any microcolonies were seen, comparedto the near-mature biofilms observed at 20 to 30 h on the uncoatedcontrols. This result most likely reflects the integrity andstability of the coating.

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FIG. 2. Confocal scanning micrographs of biofilm formation by E. coli CSH26cfp (SAR19) on uncoated and coated glass (A) and vinyl plastic (B) surfaces. The biofilms were grown in flow cells in minimal medium at 30°C. The experiment was repeated at least three times, using different batches of the aqueous extract. Abbreviations: NC, not coated; FC, coated with the aqueous fish extract; IEF, coated with the isoelectric fraction.

The biofilm-repelling effect also showed some substratum-dependentdifferences. To quantify these differences, the levels of bacterialcoverage of the different surfaces were calculated using theCOMSTAT software (Fig. 3) (21). These data clearly showed thedelay in the initiation or onset of biofilm formation and theefficiency of the isoelectric fraction coating. The substratum-dependentdifferences were also apparent. Overall, the biofilm-reducingactivity appeared to be more stable on glass than on vinyl plastic.While this finding may relate to differences in protein adsorptionpatterns, previous studies had not revealed any major compositionalor quantitative differences in the conditioning layers formedon these materials (43). Other differences, therefore, suchas substratum-dependent conformational changes, seem to accountfor the effect. The significance of protein conformational stateswith regards to the biofilm-reducing activity, however, remainselusive. Moreover, glass is in itself often less favorable tobacterial adhesion (data not shown) and may thus contributeindirectly to the activity.

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FIG. 3. Levels of substratum coverage by E. coli SAR19 biofilms formed on glass (open symbols) or vinyl plastic (filled symbols) substrata. The surfaces were either preconditioned with the total extract (triangles) or the isoelectric fraction (squares) or left uncoated (circles). The biofilms were grown in minimal medium in flow cells at 30°C. Standard deviations for four distinct channels are indicated.

The inspection of the biofilms formed on the coated and uncoatedsurfaces revealed very few, if any, morphological and behavioraldifferences, indicating that once the bacteria did establishon the coated surfaces, biofilm formation proceeded as normal.This observation correlates well with previous data (from atomicforce microscopy and surface topography analyses) which indicatedthat the coating layer is typically incomplete (5). Arguably,bare sites may become loci for bacterial adhesion and subsequentmicrocolony formation.

FMPs significantly reduce biofilm formation by several urinary tract isolates.
To characterize the biofilm-reducing properties of the proteincoatings against various wild-type bacteria, a set of UTI isolateswas selected. These bacterial isolates display a wide rangeof biofilm-forming and virulence properties; some are highlyvirulent, while others are generally considered avirulent (seeTable S1 in the supplemental material). To study the biofilm-reducingactivities against these isolates, the efficiencies of the coatingswere initially assayed in minimal medium. As seen in Fig. 4,the proteinaceous coatings significantly reduced biofilm formationby all the UTI isolates (paired two-tailed t test; P < 0.001).Under these conditions, the activities of the two coatings appearedto be of quite similar magnitudes.

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FIG. 4. Reduction of biofilm formation by a range of pathogenic and asymptomatic-infection-causing UTI isolates by preconditioning of the polystyrene microtiter plates with the total extract (white bars) or the isoelectric extract (black bars). The biofilms were grown statically in minimal medium for 16 h at 37°C. The absorbance values indicated were calculated relative to that for the uncoated control (NC). Standard deviations of the results obtained from at least three independent experiments are indicated. ABU, asymptomatic-bacteriuria strains.

Having shown that the tropomyosin coating significantly reducesbiofilm formation in a simple lab minimal medium, we subsequentlyexamined the activity in a more realistic context. The UTI isolatesstudied here normally grow in a complex and highly hydrodynamicenvironment; the urinary tract is usually subjected to verystrong hydrodynamic shear forces (an average adult human produces2 liters of urine per day). To mimic these in vivo conditions,the activity was studied during the growth of bacteria in sterilefiltered human urine. This setup takes into account the preconditioningwhich occurs in complex physiological fluids. To simulate thehydrodynamic flow conditions normally encountered in the urinarytract, the polystyrene microtiter plates were also incubatedunder shaking conditions. The same set of strains (except strainSAR19, which grows quite poorly in urine) and another uropathogenicE. coli (UPEC) strain, NU14, were studied. As seen in Fig. 5,both of the two protein coatings displayed considerable biofilm-reducingcapacities (paired two-tailed t test; P < 0.01). Under theseconditions, however, the isoelectric fraction proved to be farsuperior.

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FIG. 5. Reduction of biofilm formation by the UTI isolates during growth in human urine on polystyrene microtiter plates preconditioned with the total extract (white bars) or the isoelectric fraction (black bars). The biofilms were grown with agitation for 16 h at 37°C. The absorbance values indicated were calculated relative to that for the uncoated control (NC). Standard deviations of the results obtained from at least three independent experiments are indicated. ABU, asymptomatic-bacteriuria strains.

Analysis of FMP-mediated biofilm reduction in flow chambers during growth in urine.
To characterize the biofilm-reducing effect further, two ofthe UTI E. coli strains, 83972yfp and CFT073yfp, were studiedin flow cell chambers during growth in urine. These two prototypicstrains have previously been found to show very different biofilmcharacteristics (16, 20). Preconditioning with either of thetwo fractions significantly delayed the initiation of biofilmformation by these two isolates (Fig. 6). Interestingly, underthese conditions, the activity of the total aqueous extractcoating appeared to be somewhat superior to that of the isoelectricfraction.

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FIG. 6. Confocal scanning micrographs of E. coli CFT073yfp (A) and ABU83972yfp (B) biofilms formed on vinyl plastic coverslips in flow cells during growth (at 37°C) in human urine. Abbreviations: NC, not coated; FC, coated with the aqueous fish extract; IEF, coated with the isoelectric fraction.

FMPs significantly reduce biofilm formation by CFT073 on urinary catheters.
Some of the most commonly used urinary catheters are all-siliconeFoley catheters. Silicone materials display a range of desirableproperties, including great flexibility and reduced immunologicalreactivity (4). However, silicone catheters are not imperviousto bacterial colonization and may in some cases even selectfor UPEC strains (16). To probe the biofilm-reducing activitiesof the extracts on this type of material, small urinary cathetersegments were coupled to the flow system and preconditionedwith the two extracts. The biofilm-reducing activities againstCFT073yfp during growth in sterile filtered human urine werethen assayed. This highly pathogenic strain was previously foundto be an excellent biofilm former on several types of commercialsilicone catheters (16). When studied under these conditions,biofilm formation by CFT073 was significantly reduced by eitherof the two coatings compared to the formation on the uncoatedcontrol (paired two-tailed t test; P < 0.001) (Fig. 7).

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FIG. 7. Reduction of biofilm formation by the prototypic UPEC strain CFT073 on coated urinary catheters during growth in human urine. The biofilms were grown under laminar conditions for 24 h at 37°C. (A) Representative catheter segments after crystal violet staining. Abbreviations: B, buffer; FC, coated with the aqueous fish extract; IEF, coated with the isoelectric fraction. (B) Absorbance values (A₅₉₅) standardized according to the uncoated control (NC). Standard deviations of the results obtained from at least three independent experiments using different batches of urine are indicated. Asterisk, statistical significance as indicated by a P value of <0.001 (paired two-tailed t test).

Taken together, these data suggest that the ability of UTI E.coli and Klebsiella strains to form biofilms on inert surfacescan be efficiently impeded by fish muscle proteinaceous coatings.

Probing the biofilm-reducing activity.
In order to probe the biofilm-reducing activity further, a rangeof strains and mutants were tested for their abilities to establishon the coated surfaces. These strains either overexpressed orcarried deletions in various well-defined surface structuresknown to influence adhesion and biofilm formation (e.g., typeI fimbriae, conjugative pili, curli, PGA [poly-β-1,6-N-acetyl-D-glucosamine],lipopolysaccharide, and capsule). While some of these mutants,notably, E. coli strains overexpressing type I fimbriae (MS18),conjugative pili (SAR19R1drd19), curli (MG1655ompR234), andPGA adhesin (MG1655csrA::kan) and the E. coli or Klebsiellastrains carrying deletions relating to lipopolysaccharide (UTI536rfaHwaaG manB) or capsule production (UTI536 ${Delta}$ kps_K15 and Klebsiellapneumoniae C105NCV), did display increased buildup on coatedsurfaces compared to the wild types (in LB), they also establishedbetter on the uncoated control surfaces (data not shown). Thisresult, therefore, presumably relates to a general increasein biofilm formation rather than to any specific binding. Inan effort to identify other potential adhesins, two transposonmutant libraries were also enriched and screened for mutantsdisplaying increased adhesion to the preconditioned surfaces.Two bacterial strains were tested: the UPEC strain CFT073, whichis predicted to encode several uncharacterized adhesins, andan environmental isolate, Pseudomonas fluorescens AH2, previouslystudied in relation to fish probiotics (19). This enrichmentscheme (in which 300 mutants from each enrichment were screened)did not, however, identify any mutants showing increased oreven equivalent biofilm formation on the coated surfaces comparedto the formation on the uncoated controls (data not shown).Rather, this screening also identified mainly mutants with overallincreased adhesion and biofilm formation in general.

Preliminary experiments using the total extract also indicatedthat the extract did not contain any antibiofilm compounds perse (data not shown). The biofilm-reducing activity of the coating,therefore, appears to be linked primarily to the bacterium-repellingproperties of the FMPs, which presumably in turn relate to thephysicochemical properties of ${alpha}$ -tropomyosin. ${alpha}$ -Tropomyosin isa highly charged protein (predominantly negative), which mayincrease the electrostatic repulsion of negatively charged bacteria(17). In line with this idea, we did find that adhesion to thecoated surfaces increased slightly with increasing ionic strength(40 mM CaCl₂ increased the relative adhesion of ZK2686 to theaqueous fish extract- and isoelectric fraction-coated surfacesby 8.8% ± 1.9% and 5.8% ± 2.6%, respectively,compared to the relative adhesion in the presence of the unsupplementedbuffer, while slightly reducing adhesion [by 7.8% ± 0.6%]to the uncoated control). The influence of protein conformationalchanges cannot, however, be excluded. The coating has also previouslybeen shown to increase the wettability of the surfaces, suggestinga reduction in surface hydrophobicity (5). These characteristicssuggest that the biofilm-reducing activity of the coating isprobably linked to changes in electrostatic repulsion and surfacehydrophobicity, as also suggested previously for other proteins(42).

DISCUSSION

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DISCUSSION

Bacterial biofilms are a growing concern in the medical fielddue to the increasing use of artificial medical devices fortherapeutic and restorative purposes. CAUTIs are a notable exampleof this and illustrate perfectly the significant economic andmedical problems associated with the use of such devices (34).These problems have also spurred a growing interest in the fieldof microbial biofilms and in the search for biofilm-preventivemeasures. Various strategies to reduce the accumulation of microbialbiofilms have been studied or suggested over the years, withvarious degrees of success. In different industrial settings,a range of biocides and toxic metals (e.g., tin and copper)have been used for antifouling coatings and sanitizing purposes(7, 9). These substances are, however, not appropriate in relationto the food industry or medical implants, and other strategieshave been sought. In relation to urology, a range of urinarycatheters have been developed which incorporate and/or releaseantimicrobial compounds, e.g., metals (such as silver salts)and antibiotics (13, 22). Nevertheless, the general risk ofantibiotic resistance development suggests that these compoundsmay be of only limited use. Here, we studied the biofilm-reducingproperties of a nontoxic proteinaceous coating obtained froman aqueous extract of fish muscle tissue. This cheap, readilyavailable coating, which is composed primarily of tropomyosins,was found to exhibit significant biofilm-reducing or -delayingactivity against both UTI E. coli and Klebsiella strains onseveral different materials. In effect, a 100-fold reductionin the biofilm accumulation was frequently observed. The biofilm-reducingeffect was, however, also found to vary depending on the extract,the substratum, the growth medium, and the particular bacterialstrain studied. Overall, the biofilm-reducing effect was mostsignificant when assayed in minimal medium. This result is notentirely unexpected, considering that complex physiologicalfluids such as urine contain a wide range of compounds and macromolecules,some of which are known to affect bacterial adhesion and mayalso affect protein adsorption and competition as well (6).The biofilm-reducing capacity of the isoelectric fraction wasalso found to be superior to that of the crude aqueous extractin most cases. However, during growth in urine under continuous-flowconditions, CFT073 and 83972 formed less biofilm on the surfacescoated with the crude aqueous extract than on those coated withthe isoelectric fraction. The reason for this discrepancy remainselusive but may relate to differences in the bacterial adhesivecapacities or to differences in the general stability of theprotein layers with regards to flow and/or competitive adsorptionunder the different experimental conditions studied. High-molecular-weightproteins are often believed to bind more favorably to surfacesthan lower-molecular-weight proteins and may in some cases evendisplace them (44). Whether the content of high-molecular-weightproteins in the total extract, however, affects the stabilityof the coating differentially under these conditions remainselusive. Regardless, the results clearly demonstrate the importanceof material physicochemical properties and suspension medium,in relation to protein adsorption, and the impact of these parameterson bacterial adhesion and biofilm formation.

Several different experiments were also performed to characterizethe biofilm-reducing activity further. These results indicatedthat the effect was linked primarily to the bacterium-repellingproperties of the FMPs and not to any antibiofilm compoundsper se. This finding correlates well with the absence of anyobvious morphological or behavioral differences in the flowcell biofilms and implies that the biofilm-reducing activityis mainly physicochemical and/or steric. The biofilm-reducingproperties of the extract, therefore, seem to be related mainlyto the initial stages of biofilm formation and presumably tochanges in hydrophobicity and/or electrostatic repulsion. Whenbiofilm formation on the coated surfaces finally does take place,uncoated sites may well act as loci for bacterial establishment.To what extent these physicochemical characteristics correlatewith the surface properties of the bacterial strains and mutantsstudied, in terms of hydrophobicity and zeta potential, remainsunknown. The aggregative phenotype of several of the mutantsand the incomplete coverage of the protein coating complicatesuch a correlation.

Competitive protein adsorption may also contribute to the activitiesof the coatings, at least in the more complex urine medium.While the idea remains purely speculative, we did find thatwhen the flow system was filled with urine prior to preconditioningwith the aqueous extract, the subsequent biofilm-reducing activityof the FMP coating was reduced (data not shown). While thisresult may obviously relate to differences in the general proteinadsorption patterns (in the different coating suspensions),it may also relate to the adsorption of urine macromolecules,some of which may directly or indirectly influence bacterialadhesion or biofilm formation. Whether preconditioning withthe fish extracts in turn could also reduce urine conditioningfilm formation, however, remains unknown. Studies are in progressto investigate the influence of the proteinaceous coating withregards to the competitive adsorption of other proteins. Meanwhile,our results indicate that surface-conditioning films are importantfor the development of bacterial biofilms and that they arelargely defined by both environmental conditions and materialphysicochemical properties. Nevertheless, it is interestingthat an efficient nontoxic antibiofilm coating can be obtainedby this cheap, low-technology inroad.

ACKNOWLEDGMENTS

This work was financed by a grant from the Danish Food Agency(DFFE 3304-05-66).

FOOTNOTES

* Corresponding author. Mailing address: Microbial Adhesion Group, Center for Biomedical Microbiology, DTU, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark. Phone: 45 4525 2506. Fax: 45 4593 2809. E-mail: pkl{at}biocentrum.dtu.dk

Published ahead of print on 18 April 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

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