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Antimicrobial Agents and Chemotherapy, August 2007, p. 2733-2740, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.01249-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Synergistic Activity of Dispersin B and Cefamandole Nafate in Inhibition of Staphylococcal Biofilm Growth on Polyurethanes

G. Donelli,^1* I. Francolini,² D. Romoli,¹ E. Guaglianone,¹ A. Piozzi,² C. Ragunath,³ and J. B. Kaplan³

Department of Technologies and Health, Istituto Superiore di Sanità,¹ Department of Chemistry, University of Rome "La Sapienza," Rome, Italy,² Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey³

Received 5 October 2006/ Returned for modification 12 November 2006/ Accepted 25 May 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Antibiotic therapies to eradicate medical device-associated infections often fail because of the ability of sessile bacteria, encased in their exopolysaccharide matrix, to be more drug resistant than planktonic organisms. In the last two decades, several strategies to prevent microbial adhesion and biofilm formation on the surfaces of medical devices, based mainly on the use of antiadhesive, antiseptic, and antibiotic coatings on polymer surfaces, have been developed. More recent alternative approaches are based on molecules able to interfere with quorum-sensing phenomena or to dissolve biofilms. Interestingly, a newly purified ß-N-acetylglucosaminidase, dispersin B, produced by the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans, is able to dissolve mature biofilms produced by Staphylococcus epidermidis as well as some other bacterial species. Therefore, in this study, we developed new polymeric matrices able to bind dispersin B either alone or in combination with an antibiotic molecule, cefamandole nafate (CEF). We showed that our functionalized polyurethanes could adsorb a significant amount of dispersin B, which was able to exert its hydrolytic activity against the exopolysaccharide matrix produced by staphylococcal strains. When microbial biofilms were exposed to both dispersin B and CEF, a synergistic action became evident, thus characterizing these polymer-dispersin B-antibiotic systems as promising, highly effective tools for preventing bacterial colonization of medical devices.

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In the last decades, the development of novel polymeric biomaterials has allowed the manufacture of a large number of medical devices, including orthopedic prostheses, heart valves, intravascular catheters, and biliary stents, providing clinicians with useful means to improve health care. However, the continual increase in the use of these devices is associated with a significant risk of infectious complications, including bloodstream infections, septic thrombophlebitis, endocarditis, metastatic infections, and sepsis (7, 10, 13, 23). As is well known, microorganisms adhere to implanted medical devices by forming a sessile multicellular community encased in a hydrated matrix of polysaccharides and proteins, the so-called biofilm (6, 8, 14). As the biofilm thickness increases, there is a progressive dispersal of single cells and/or bacterial clusters of different sizes which can be considered to be the main promoters of the infectious process. In fact, recent experimental data on the growth and detachment of Staphylococcus aureus biofilms in in vitro catheter infection models (15, 17), as well as clinical data on device-bearing patients with septic pulmonary embolisms associated with active foci of extrapulmonary infection (5), support the hypothesis that bacterial clumps, detached from the mature biofilm, may become septic emboli that, dispersing throughout the bloodstream, can cause metastatic infections and sepsis (9).

These infections, usually not life threatening, are mostly resolved after device removal but involve a prolonged hospital stay and increased medical costs. Depending on the affected body site and the mode of implantation, different fungal and bacterial species may contribute to the establishment of a prevalently multispecies biofilm (28). The gram-positive organisms Staphylococcus epidermidis, S. aureus, and Enterococcus faecalis together constitute more than 50% of the species isolated from patients with medical device-associated infections; Candida spp., Pseudomonas aeruginosa, and a few other gram-negative organisms are the remaining causative agents.

Since the early 1980s, strategies to prevent device-related infections, based mostly on the use of antiadhesive, antiseptic, and antibiotic coatings on catheters, have been developed (11). However, results obtained so far have not reached enough clinical relevance to consider these medicated catheters to be definitive substitutes for the conventional ones. The recently proposed strategy of entrapping a combination of a pore former and an antifungal agent in properly functionalized polyurethanes to result in a controlled drug release over time appears to be a novel, promising approach to the development of medical devices refractory to fungal colonization (12). Other alternative approaches include the use of molecules interfering with quorum-sensing phenomena (26, 27, 30) or biofilm-dissolving substances (3, 22). In 2003, Ramasubbu and coworkers reported that during sessile growth, Actinobacillus actinomycetemcomitans produces a soluble ß-N-acetylglucosaminidase, named dispersin B (DspB), able to disperse and detach mature biofilms produced by S. epidermidis as well as some other bacterial species (25). In fact, numerous studies have shown that S. epidermidis biofilm-forming strains produce a linear poly-N-acetyl-1,6-beta-glucosamine (PNAG) which mediates bacterial intercellular adhesion. Originally defined as the polysaccharide intercellular adhesin on the basis of its main biological activity, PNAG plays a key role in biofilm formation and accumulation (19), protecting the pathogen from the innate host defense (29).

The study reported here focused on the development of polymeric matrices able to bind dispersin B alone or together with an antibiotic molecule in order to obtain medical devices refractory to bacterial colonization and biofilm formation. In particular, we compared the abilities of two properly functionalized polyurethanes and that of the commercially available PELLETHANE to adsorb cefamandole nafate (CEF) and dispersin B for synergistic action as antibiofilm agents.

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Polyurethanes. For our experiments, we employed three polymers possessing different chemical features: acidic, basic, and neutral.

Polyether urethane acid (PEUA) was synthesized by employing methylene-bis-4,4-phenyl-isocyanate (Polyscience Inc.), polypropylene oxide with a molecular weight of 1,118 (Fluka), and dihydroxymethyl-propionic acid (Aldrich) as previously reported (20).

Further, a primary amino group-containing polyurethane was obtained by the amidation of PEUA by activating its carboxyl groups with N-hydroxysuccinimide (Fluka) and dicyclohexylcarbodiimide (Fluka), added in equimolar amounts to a 5% (wt/wt) polymer solution in tetrahydrofuran (Fluka). Following activation for 3 h at 0°C, ethylenediamine (Fluka) was added in a 1:1 stoichiometric ratio with respect to PEUA carboxyl groups. The basic polymer PEUA-ethylenediamine (PEUAED), resulting after a 24-h reaction at room temperature with stirring, was recovered by precipitation in water and dried under a vacuum at 30°C for 3 days. The degree of amidation, determined by acidic-alkaline titration and ¹H nuclear magnetic resonance analysis, was 70%. PELLETHANE (Upjohn Co., New Haven, CT), a commercial polyurethane not possessing functional groups, was tested for comparison.

Cytotoxicity assay. To evaluate the possible cytotoxic effects of both dispersin B and the above-described polymers, human larynx carcinoma (HEp-2) ATCC CCL-23 cells were routinely grown in 25-cm² tissue culture flasks in minimum essential medium (GIBCO-Life Technologies, Milan, Italy) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 5 mM L-glutamine, and 1% penicillin-streptomycin and maintained in a humidified atmosphere of 5% CO₂ in air at 37°C.

Cells were seeded into a 24-well plate and treated with 40 to 400 µg of enzyme/ml or exposed to small (0.5-cm²) fragments of polymers. Cell viability and possible gross morphological changes (cell rounding, cell shrinkage, and cell detachment) were evaluated by light microscopy every hour for the first 6 h and then at 12-h intervals up until 48 h. To assess the possible effects of both dispersin B and the polymers on the organization of the actin cytoskeleton, cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS; pH 7.4) for 10 min at room temperature. After being washed in the same buffer, cells were permeabilized with 0.5% Triton X-100 (Sigma) in PBS (pH 7.4) for 10 min at room temperature and then stained with 5 µg/ml of fluorescein isothiocyanate-phalloidin (Sigma) in PBS. For the detection of microtubules, permeabilized cells were stained with the appropriate primary antibody (antitubulin from Sigma), washed in PBS, and incubated with the secondary fluorescein isothiocyanate-labeled antibody for an additional 30 min. Then, after being washed again in PBS, cells were mounted in glycerol-PBS (2:1) and observed with a Nikon Optiphot fluorescence microscope.

Dispersin B adsorption onto polyurethanes and quantitative determination. Dispersin B was adsorbed onto round disks (approximately 1 cm in diameter and 100 µm in thickness) made of the above-described polyurethanes. Disks were obtained by the casting of polymer solutions on Teflon plates and by subsequent solvent evaporation under a vacuum at 30°C. Following the sterilization of polymers by UV irradiation, disks were kept in contact with solutions of dispersin B at concentrations ranging from 20 to 600 µg/ml in PBS (pH 7.4) for 24 h at 4°C. Following the adsorption, polymers were washed for 2 h with PBS to rinse out the excess unbound enzyme and stored at 4°C. Experiments were performed in triplicate.

Dispersin B was quantitatively assessed by UV-visible (UV-VIS) spectroscopy measuring the absorbance at 420 nm of the p-nitrophenolate reaction product resulting from the enzymatic hydrolysis of the substrate 4-nitrophenyl-N-acetyl-ß-D-glucosaminide (Sigma). The enzymatic reaction was carried out for 30 min at 37°C in 2 ml of 50 nM PBS (plus 100 mM NaCl, pH 5.9) containing 4 mM substrate. The reaction was stopped by adding 10 µl of 10 N NaOH.

Next, the absorbance values for the solution before and after the reaction were compared with a standard curve (absorbance versus concentration) previously obtained in order to determine the amount of adsorbed dispersin B. The subtraction of the enzyme contents of the washing solutions from the concentration calculated as described above gave the effective amount of the drug adsorbed onto the polymer.

CEF adsorption onto polyurethanes. The binding of the antibiotic onto polyurethanes was performed by dipping polymer disks, 1 cm in diameter and approximately 100 µm in thickness, into an antibiotic-water solution (0.04 M) for 24 h at room temperature with mild stirring. Thereafter, to eliminate the unadsorbed antibiotic, polymers were washed twice for 1 h with saline solution. The amount of antibiotic bound was evaluated by UV-VIS spectroscopy by checking the absorbance of the solutions before and after the adsorption reaction as well as that of the washing solutions. To correlate the levels of absorbance of the solutions to the respective antibiotic concentrations, absorbance values were compared with a standard curve (absorbance versus drug concentration) constructed for a 270-nm wavelength and a range of concentrations from 10^–4 to 10^–5 M. The amount of adsorbed antibiotic was obtained by subtracting both the amount of the antibiotic still present in the solution after the contact with the polymer and that detected in the washing solutions from the initial drug concentration. Adsorbed amounts were expressed in micrograms per unit of polymer surface, including both the two exposed disk surfaces.

Bacterial strains and culture medium. Seven strains of S. aureus and S. epidermidis were routinely grown in tryptic soy agar and tryptic soy broth (TSB) and tested for methicillin resistance by means of both disk diffusion and standard molecular methods (Table 1). In particular, methicillin resistance was determined by the disk diffusion method according to the requirements of the Clinical and Laboratory Standards Institute by using 1-µg oxacillin disks (Oxoid, Basingstoke, United Kingdom). The presence of the mecA gene was detected by PCR. DNA was extracted using QIAamp DNA mini kit spin columns according to the instructions of the manufacturer (QIAGEN SpA, Milan, Italy), and PCR was performed as described by Murakami et al. (21) by using the Mastercycler Personal 5332 thermocycler (Eppendorf, Milan, Italy). Strains were also tested for their abilities to form biofilms, and exopolysaccharide production was assessed according to the protocol described by Baldassarri et al. (2). Briefly, bacteria were grown overnight at 37°C in TSB supplemented with 1% glucose. Polystyrene 96-well tissue culture plates (Corning Costar) were filled with 180 µl of fresh TSB supplemented with 1% glucose, and 20 µl of the culture grown overnight was added to each well. Plates were incubated overnight at 37°C to obtain cultures with a 0.5 McFarland standard. The culture medium was discarded, and the wells were carefully washed three times with 200 µl of PBS without disturbing the biofilms on the bottoms of the wells. Plates were dried for 1 h at 60°C and stained with 2% Hucker's crystal violet for 2 min. Excess stain was removed by rinsing the plates under tap water, and the plates were dried for 10 min at 60°C. The optical densities (ODs) of the biofilms at 570 nm were measured with a Novapath microplate reader (Bio-Rad, Italy). Each assay was performed in triplicate and repeated at least twice.

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TABLE 1. Results for staphylococcal strains tested for oxacillin susceptibility, the presence of the mecA gene, and slime production

Among the seven tested staphylococcal strains, four were used in the dispersin B and CEF experiments: the biofilm-forming S. epidermidis strain ATCC 35984 (16), the non-biofilm-forming S. epidermidis strain ATCC 12228 (1), and two clinical isolates, S. aureus strains 5683 and 10850, which are methicillin susceptible and methicillin resistant, respectively.

Assessment of antibiofilm activity of dispersin B-loaded polymers. To evaluate the activity of dispersin B-loaded polyurethanes against bacterial adhesion and biofilm formation, polymeric disks were immersed for 24 h in 2.5 ml of a bacterial suspension (0.5 McFarland standard) grown in TSB supplemented with 1% glucose to obtain massive exopolysaccharide production (2). Then polymer disks were collected and washed twice with PBS (pH 7.4) to remove loosely adherent cells, the remaining cells were sonicated for 5 min, and the disks were put into a vortex mixer for 10 s in test tubes with 10 ml of Ringer's solution to remove the biofilm cells. This procedure was repeated three times for each sample. Six 10-fold dilutions were prepared, and three 10-µl aliquots of each dilution were plated onto tryptic soy agar plates. Plates were then incubated at 37°C, and CFU were counted after 18 h.

Biofilm growth on the treated polymers was assessed by fluorescence microscopy and scanning electron microscopy (SEM). For fluorescence microscopy, polymers were layered onto glass coverslips and the coverslips were incubated in 24-well plates with a broth culture of the tested bacterial strain at a 0.5 McFarland standard. After 24 h of incubation, biofilms were washed with distilled water and stained with 3 µl/ml of LIVE/DEAD BacLight bacterial viability kit stain (L7007; Molecular Probes). Following incubation for 15 min at room temperature in the dark, samples were mounted with glycerol-PBS (1:2) and observed with a Nikon Optiphot fluorescence microscope. For SEM observations, samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature for 30 min, then postfixed in 1% OsO₄ for 20 min, dehydrated by using graded ethanols, critical-point dried in CO₂, and coated with gold by sputtering. Finally, samples were examined with a Cambridge 360 scanning electron microscope (Assing, Italy).

Image analysis was performed by using ImageJ software (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/). Biofilm surface area coverage was measured by image analysis of micrographs taken at a magnification of x1,500. The average area of coverage was obtained from five measurements taken at randomly sorted locations over the polymer surface. Means ± 1 standard deviation (SD) of results from three independent experiments are reported.

Assessment of synergistic effect of dispersin B and CEF. To evaluate the possible synergistic effect of dispersin B and CEF, unloaded or dispersin B-loaded PEUA disks were incubated for 24 h with a bacterial broth culture at a 0.5 McFarland standard. Then polymers were washed twice with PBS and incubated overnight with 1 ml of TSB containing CEF at a 0.25-µg/ml concentration (the minimal bactericidal concentration). Polymer disks were then washed twice with PBS (pH 7.4) to remove planktonic cells, the remaining cells were sonicated for 5 min, and the disks were put into a vortex mixer for 10 s in test tubes with 10 ml of Ringer's solution to remove the biofilm cells. In order to determine the number of adherent CFU per unit of surface area of the polymer, serial dilutions were prepared by the procedure described above.

Antibacterial activity of antibiotic-treated polymers. The antibacterial activity of the CEF-treated polyurethanes was assessed in vitro by a modified Kirby-Bauer test. Round polymeric disks were placed in petri plates containing Mueller-Hinton agar (Oxoid) seeded with 10⁸ S. epidermidis bacteria/ml (0.5 McFarland standard). After incubation at 37°C for 18 h, the zones of inhibition of bacterial growth around the antibiotic-loaded polymeric disks were measured.

Statistics. Analysis-of-variance comparisons were performed using MiniTab. Differences were considered significant for P values of <0.05. Data are reported as means ± 1 SD.

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Cytotoxicity assays of dispersin B and polyurethanes. Since the cytotoxicity of dispersin B and our newly synthesized polyurethanes was not previously assessed, the possible toxicity of dispersin B and the polyurethanes to eukaryotic cells was tested in vitro. Experiments showed that the exposure of HEp-2 cells to dispersin B (at doses ranging from 40 to 400 µg/ml) as well as to fragments of PEUA and PEUAED did not cause significant changes in the general morphology of the monolayer or in the major cytoskeletal components, i.e., F-actin and - and ß-tubulins (Fig. 1). As a positive control, the damaging effects on the cytoskeleton of a cytotoxic polyurethane are shown in Fig. 1.

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FIG. 1. Fluorescence micrographs of control and dispersin B-treated HEp-2 cells. F-actin (A) and tubulin (E) in untreated cells and F-actin (B and C) and tubulin (F and G) in dispersin B-treated cells after 24 h (B and F) and in PEUA-exposed cells after 24 h (C and G). Effects on F-actin (D) and tubulin (H) of a cytotoxic polyurethane matrix used as a positive control.

Dispersin B adsorption onto polyurethanes. PELLETHANE, PEUA, and PEUAED were loaded with enzyme concentrations ranging from 20 to 600 µg/ml. Despite their different chemical features, the three polymers showed similar abilities to adsorb dispersin B, reaching a saturation value of 40 µg/cm² when treated with an enzyme solution concentration of 200 µg/ml. As an example, the adsorption-concentration isotherm curve for dispersin B on PEUA is presented in Fig. 2.

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FIG. 2. Adsorption-concentration isotherm curve for dispersin B on PEUA. Values are reported as means ± SD.

Biofilm inhibition on the surfaces of dispersin B-treated polyurethanes. To evaluate the dispersing activity of dispersin B on microbial biofilms as a function of the amount of enzyme adsorbed onto the polymer, the experimental polymers, untreated or loaded with increasing amounts of dispersin B, were incubated for 24 h with a bacterial broth culture at a 0.5 McFarland standard. The numbers of CFU per square centimeter counted after sonication for the detachment of the biofilm grown on the untreated or loaded PEUA, as described in Materials and Methods, are reported in Fig. 3. It can be observed that dispersin B was not active at all against the non-biofilm-producing strain S. epidermidis ATCC 12228. In contrast, compared to the untreated PEUA (P < 0.01), the enzyme caused a significant reduction (around 1.5 logs) in the numbers of CFU of all the other bacterial strains per surface unit, even at small adsorbed amounts (12 µg/cm²). In fact, the enzyme activity seems not to be dependent on the amount of dispersin B adsorbed onto the polymer (P > 0.05). Similar results with PELLETHANE and PEUAED were obtained. The experimental data related to the activity of the polymers loaded with 25 µg of dispersin B/cm² against S. epidermidis ATCC 35984 are reported in Fig. 4.

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FIG. 3. Numbers of bacterial CFU adhered per surface unit of untreated PEUA and PEUA loaded with increasing amounts of dispersin B. Data are reported as means ± SD. Values on the y axis are on a logarithmic scale.

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FIG. 4. Numbers of S. epidermidis (ATCC 35984) CFU per square centimeter of untreated polyurethanes and polyurethanes loaded with 25 µg of dispersin B/cm2. Data are reported as means ± SD.

This decrease in the number of adherent bacteria was confirmed by fluorescence microscopy analysis of biofilms grown on polymers layered onto glass coverslips, incubated in 24-well plates, and stained with LIVE/DEAD BacLight bacterial viability kit stain (Fig. 5) (P < 0.05).

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FIG. 5. Fluorescence micrographs of S. epidermidis (ATCC 35984) cells adherent to untreated PEUA (A) and PEUA treated with 48 µg of dispersin B/cm2 (B). Cells were stained with the LIVE/DEAD BacLight bacterial viability kit stain. In the presence of dispersin B, a significant reduction in the number and size of bacterial clusters is evident.

SEM observations after 24 h of S. epidermidis (ATCC 35984) growth on the surfaces of dispersin B-treated PEUA and PEUAED revealed only single cells and few and very small bacterial clusters. In contrast, three-dimensional, growing biofilms on the surfaces of the untreated polymers were typically detected (Fig. 6). SEM image analysis demonstrated a significant reduction (P < 0.01) in the surface areas of dispersin B-treated polymers covered by bacteria compared with those of the untreated PEUA and PEUAED. As for PEUA, the covered surface area reached 13.8% ± 1.4% for the untreated polymer and 1.1% ± 0.2% for the dispersin B-treated PEUA.

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FIG. 6. SEM micrographs of S. epidermidis biofilms growing on PEUA (A) and single cells on dispersin B-loaded PEUA (B). The three-dimensional structure of an S. epidermidis biofilm growing on PEUAED, shown at a higher magnification than that for panels A and B (C), is also compared with a small cluster of bacterial cells appearing on the surface of PEUAED loaded with 48 µg of dispersin B/cm2 (D).

CEF susceptibility of bacterial cells grown on untreated or dispersin B-loaded polyurethanes. To evaluate the possible synergistic effect of dispersin B and CEF, unloaded or dispersin B-loaded PEUA disks were incubated for 24 h with a bacterial broth culture at a 0.5 McFarland standard and then treated with a 0.25-µg/ml (minimal-bactericidal-concentration) CEF solution.

Fig. 7 shows the numbers of adherent CFU of the two tested S. epidermidis strains (ATCC 12228 and 35984) per square centimeter of unloaded and dispersin B-loaded PEUA before and after treatment with the CEF solution. As expected, CEF showed a stronger killing effect against the oxacillin-sensitive S. epidermidis strain (ATCC 12228). However, in this case, the presence of dispersin B on the polymer did not contribute to a significant increase (P > 0.05) in the antibiotic activity, thus confirming the inefficacy of dispersin B against non-biofilm-forming strains. In contrast, the efficacy of dispersin B in enhancing CEF activity against the oxacillin-resistant and biofilm-forming S. epidermidis strain (ATCC 35984) was particularly evident. In fact, a reduction in CFU of 1.7 logs versus 1.0 log on the unloaded PEUA was observed (P < 0.05).

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FIG. 7. Numbers of S. epidermidis CFU per square centimeter of unloaded and dispersin B-loaded PEUA before and after treatment with a 0.25-µg/ml CEF solution. Data for the non-biofilm producer S. epidermidis strain ATCC 12228 and the strong biofilm producer S. epidermidis strain ATCC 35984 are reported.

To evaluate the relationship between the dispersin B effect in enhancing antibiotic activity and the degree of biofilm production, we compared the behavior patterns of three staphylococcal strains (Fig. 8) that produced different amounts of exopolysaccharides as evaluated by OD (Table 1). Compared with untreated PEUA, dispersin B significantly increased CEF activity by reducing the number of viable, biofilm-associated bacteria. In addition, a more pronounced effect with strains able to produce larger amounts of exopolysaccharides was observed.

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FIG. 8. Numbers of S. aureus CFU per square centimeter of unloaded and dispersin B-loaded PEUA before and after treatment with a 0.25-µg/ml CEF solution. Data for the methicillin-susceptible (strain 5683) and -resistant (strain 10850) S. aureus clinical isolates and the biofilm producer S. epidermidis strain ATCC 35984 are reported.

To further confirm the synergistic effect of CEF and dispersin B, we experimented with PEUA disks by loading them first with CEF (0.04 M) and then with dispersin B at a 400-µg/ml concentration. As measured by UV-VIS spectroscopy, PEUA adsorbed significant amounts of CEF (0.76 mg/cm²) even if no zone of inhibition was observed when the system was submitted to the Kirby-Bauer test. When PEUA-CEF was treated with dispersin B, the system was able to adsorb 44 µg of the enzyme/cm² and showed a zone of inhibition of 4 mm in diameter.

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A possible approach to the problem of medical device-associated infections involves finding molecules able to disaggregate the exopolysaccharide matrix that, as a biological glue, keeps the single bacterial cells within the biofilm together. The recently identified glycoside hydrolase dispersin B has been demonstrated to be able to cause the detachment of biofilms produced by several bacterial species. Here, we report data relative to the chemical adsorption of dispersin B onto properly synthesized and functionalized polyurethanes in order to develop medical devices refractory to bacterial colonization and biofilm formation. The results of proper in vitro testing of these polyurethanes for their possible cytotoxicity have demonstrated that they are safe and suitable for future application in clinics. A good chemical affinity of dispersin B for both PEUA and PEUAED, as well as for the commercially available PELLETHANE used as a control polyurethane, was evident. The amounts of enzyme adsorbed to the three polymers corresponded to the same order of magnitude, even if the strengths of the enzyme-polymer binding were substantially different. In fact, while PELLETHANE can establish hydrophilic interactions with dispersin B involving only its soft segment, the acidic PEUA and basic PEUAED can also bind dispersin B through the establishment of ionic interactions between the carboxyl and amino groups of both the polymers and the enzyme. The loading of polymers with different dispersin B concentrations (ranging from 20 to 600 µg/ml) allowed the achievement of surface saturation corresponding to 200 µg/ml, as shown in Fig. 2.

Repeated experiments focusing on the assessment of adhering bacteria revealed significantly lower numbers of CFU of biofilm-producing strains per unit of surface area on dispersin B-loaded polyurethanes than on the unloaded polymers (Fig. 3 and 4). In contrast, dispersin B was not efficient against S. epidermidis ATCC 12228, which does not produce biofilms.

On the basis of these results, since it is well known that most biofilm-producing staphylococcal strains produce a linear PNAG, a presumptive role of the dispersin B enzyme both in the detachment of already adhered bacteria and in the inhibition of their adhesion through its hydrolytic activity on the growing PNAG chains can be hypothesized. In fact, it has been demonstrated previously that dispersin B is able to degrade N-acetylglucosamine-containing polysaccharides (18), including those constituting the slime of S. epidermidis (2, 19).

SEM observations of growing biofilms on PEUA surfaces seem to support this hypothesis. In fact, as evident in Fig. 6, bacterial cells appeared to be glued by the exopolysaccharide matrix in the absence of dispersin B, soon giving rise to the typical three-dimensional biofilm structure. However, only single or few bacterial cells but not slime-embedded clusters were detectable on the surface of dispersin B-treated PEUA.

Given the well-known decreased antibiotic susceptibility of bacteria growing in a sessile mode, we carried out susceptibility experiments with S. epidermidis and S. aureus strains grown as single cells or small clusters and as biofilm colonies on untreated and dispersin B-loaded PEUA disks. To evaluate the possible effect of dispersin B in enhancing the killing activity of antibiotics, we chose CEF as a model molecule on the basis of its previously demonstrated short-term activity (lasting up to 4 h) against S. epidermidis ATCC 35984 in the PEUA-CEF system (24), which also allowed us to monitor small effects on microbial growth.

The 1.7-log reduction in CFU on the dispersin B-loaded PEUA disk versus the 1.0-log reduction on the control PEUA disk emphasized the significantly stronger activity of CEF against S. epidermidis ATCC 35984 in the presence of dispersin B. This finding can be attributed to the presumptive mechanism of action of dispersin B, which while exerting its antislime activity also improves the diffusion of CEF into bacterial clusters and promotes the reaching of antibiotic cell targets. The synergistic effect of dispersin B and CEF on staphylococcal strains with greater abilities for biofilm formation, and thus presumably producing large amounts of PNAG (4), was even more pronounced (Fig. 8).

These results suggested further experiments to investigate dispersin B adsorption onto polyurethanes along with CEF. As observed with the Kirby-Bauer test, CEF-treated polymers loaded with dispersin B caused a zone of inhibition of 4 mm in diameter after 24 h of incubation while the polymers treated only with CEF exhibited no activity. These findings seem to confirm our hypothesis on the possible dispersin B-CEF synergistic action, due presumably to the enzyme-dispersing effect during bacterial growth around the polymeric disk, which promotes antibiotic diffusion and killing activity. Further experiments involving polymers loaded with different antibiotic molecules will be planned to evaluate long-term synergistic effects also on either S. epidermidis or other biofilm-forming bacteria of medical relevance.

 
We thank the Ph.D. student Mariangela Bellusci for carrying out the preliminary tests on dispersin B adsorption onto the functionalized polyurethanes.

 
* Corresponding author. Mailing address: Department of Technologies and Health, Istituto Superiore di Sanità, Viale Regina Elena, 299-00161, Rome, Italy. Phone: 39 0649902228. Fax: 39 0649387141. E-mail: gianfranco.donelli{at}iss.it

Published ahead of print on 4 June 2007.

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Antimicrobial Agents and Chemotherapy, August 2007, p. 2733-2740, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.01249-06
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