The present study compares the retention of four species that are often isolated in association with biomedical device-related infections –Staphylococcus aureus, Streptococcus mutans, Pseudomonas aeruginosa, and Candida albicans– to three different surfaces. All four bacterial species were found to bind significantly less well to MPC-coated surfaces than to non-coated surfaces. We attribute this effect to the “superhydrophilicity” of MPC-coated surfaces, whereas hydrophobic surfaces are well known to reduce bacterial retention and thus to inhibit a crucial step in the formation of bacterial biofilms that lead to biomedical device-related infections and complications.
It is well known that microorganisms often survive within biofilms, which are implicated in environmental problems and numerous infectious diseases . Microbial infection associated with the formation of biofilms refractile to antibiotic therapy is a major reason for the failure of medical devices . On the other hand, considerable biofilm research is currently focused on those cellular functions that are modified during the cellular transition from the planktonic to the biofilm state . This initial interaction between microorganisms and artificial surfaces is a key determinant in biofilm formation. Since microbial adherence is an essential step in infection, inhibiting adherence is an effective strategy for preventing infectious diseases.
Many efforts have been made to remove biofilms from surfaces and thus to prevent further microbial adherence to such surfaces. Several methods of prevention have been developed, such as changing the wettability of a surface to increase its hydrophobicity, coating a surface with Teflon, or adding disinfectant to it [4,5]. The 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers are synthesized to match the structure of biomembranes . They effectively reduce protein adsorption and inhibit cell adhesion, even when they are in contact with whole blood in the absence of anticoagulants . These polymers are biocompatible materials and are completely harmless to humans .
Urinary tract infections are among the most common infections in women, with Escherichia coli being the predominant pathogen . Lewis et al.  reported that coating surfaces with MPC reduced the adherence of E. coli to such surfaces. The results of highly sensitive X-ray photoelectron spectroscopy indicated that coating polyethylene with MPC polymers greatly increased hydrophilicity . Lewis et al.  suggested that MPC polymer coating may be very useful for reducing the levels of bacteria that adhere to medical devices. It is therefore of interest to investigate the effects of coating surfaces with MPC on the adherence of opportunistic pathogens. For this investigation, we used one of the most common human pathogens, Staphylococcus aureus; a human cariogenic bacterium, Streptococcus mutans; and two of the most important opportunistic pathogens, Pseudomonas aeruginosa and Candida albicans. S. aureus is the etiologic agent of a wide range of diseases, from relatively benign skin infections to potentially fatal systemic disorders . S. mutans has been strongly implicated as an organism that can cause dental caries . P. aeruginosa is known as a typical pathogen in cases of opportunistic infection . C. albicans produces a broad range of serious illnesses, usually arising from indwelling devices in immunocompromised hosts . The goals here were to examine the usefulness of coating surfaces with MPC polymers by examining the levels of initial adherence of S. aureus, S. mutans, P. aeruginosa, and C. albicans to polyethylene telephthalate surfaces coated with MPC polymers.
2Materials and methods
2.1Preparation of microbial suspensions
The following microorganisms were used: S. mutans MT8148, S. aureus FHSAT1 (isolated from a patient treated at the Showa University Fujigaoka Hospital and kindly provided by that institute ), P. aeruginosa PAO1 [17,18], and C. albicans CAD1 (isolated from denture plaque). C. albicans CAD1 was identified using the CHROMagar Candida method (CHROMagar, Paris, France) , and the API-ID32C® (bioMérieux, Mercy l'Etoile, France) system , which is based on the assimilation of different carbohydrates. S. mutans MT8148, S. aureus FHSAT1, and C. albicans CAD1 were grown in brain heart infusion broth (BHI, Difco, Becton Dickinson, Sparks, USA) at 37°C for 18 h. P. aeruginosa PAO1 were incubated in Luria–Bertani broth [17,18]. After an 18-h incubation, the bacteria were harvested by centrifugation and washed twice in phosphate-buffered saline (PBS, 0.01 M, pH 7.5). For the adhesion experiments, the cells were suspended in PBS to a final concentration of 1 × 109 cells/ml.
2.2Preparation of cover slips
We used plastic coverslips (polyethylene telephthalate, 13.5 mm in diameter and 0.2 mm in thickness), with or without the MPC coating, as a substratum for the retention assays. The experimental coverslips were coated with MPC polymer in ethanol (0.5% wt/vol, NOF Corp., Tokyo, Japan) and dried in vacuo at 70°C for 4 h. Other coverslips, made of tetrafluoroethylene–ethylene pure polymer, were used as a hydrophobic substratum. The condition of a surface before bacterial retention occurs must first be defined in order to determine whether or not the MPC coating had an effect. In addition, contact angles have been shown to be useful in predicting the adhesion behaviors of specific pathogens to hydrophobic surfaces . As an index of hydrophobicity, surface contact angles were measured. The contact angles of distilled water on MPC-coated, non-coated, and hydrophobic coverslips – 0°, 72°, and 114°, respectively – were measured by the horizontal projection technique with the use of a contact angle meter (Model CA-A, Kyowa Co, Tokyo, Japan); each of these values was the mean of measurements at six points on a surface, as previously described .
2.3Microbial retention assays
After cultivation, the microbial cells were collected by centrifugation, resuspended in PBS, and used for the retention assays. To the coverslip in each well of a 24-well plate, 0.5 ml of microbial suspension was added, and the plate was then incubated at 37°C for 1 h. The cell concentration was 109 cfu/ml for S. mutans MT8148, S. aureus FHSAT1, P. aeruginosa PAO1, and C. albicans CAD1.
2.4Calculation of the number of cells adhering to a coverslip using fluorescence methods
The propensity of bacteria to adhere onto a surface has been estimated by counting the number of bacteria that remain attached to a surface following incubation for a specified length of time. In this experiment, we used fluorescence methods to quantify microbial retention. We selected this approach because eluted bacteria can be counted in colony-forming units by plating them on suitable selective agar plates, whereas non-eluted bacteria are difficult to count. On the other hand, fluorescence methods can be used to estimate the total number of bacteria that adhere to a surface. In this experiment, the coverslips were removed after incubation and washed three times in PBS to remove all non-adhering cells. The cells still adhering to the experimental surface were counted by laser scanning fluorescence microscopy (ACAS 570; Meridian Instruments, Okemos, MI, USA) as previously described , but with the following modification. S. mutans MT8148 was incubated with mAb H80 (diluted to 1:5) for 1 h. The mAb H80 (the epitope is N-acetyl-glucosamine) was obtained from hybridomas produced in our laboratory. Mouse mAb anti-S. aureus clone930 (Immunogen: S. aureus cells) was purchased from Chemicon International, Inc., Temecula, CA, USA). S. aureus was incubated with mAb930 (diluted to 1:1000) for 1 h and then with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgM (diluted to 1:1000; Cappel, Aurora, OH, USA) at room temperature for 1 h. Mouse mAb anti-P. aeruginosa clone B11 (Immunogen: outer membrane protein of P. aeruginosa) was purchased from Biogenesis (Poole, UK). P. aeruginosa PAO1 was incubated with mAb B11 (diluted to 1:1000) for 1 h, followed by FITC-labeled goat anti-mouse IgG (diluted to 1:1000; Cappel) at room temperature. For direct immunofluorescence of C. albicans, we used FITC-labeled anti-C. albicans antibody (diluted to 1:1000, Cosmobio, Tokyo, Japan) at room temperature for 1 h.
Each coverslip was washed in PBS three times, and the fluorescence was measured at 488 nm by laser scanning fluorescence microscopy. Data were processed by line analysis by using the complement data program of the ACAS software system (Meridian Instruments). In each of 10 fields selected at random on each plate of the restorative, the cells were counted and the number obtained was divided by the area of the view. From the values thus obtained, the average number of adherent cells and the standard deviation were calculated from five samples. (All of the numerical data obtained were analyzed by Student's t-test.)
A standard curve was constructed by plotting the fluorescence intensity against the number of colony-forming units inoculated on each agar plate. The units were counted using a Spiral System (Interscience, Waymouth, MA, USA). In order to count the colony-forming units (CFUs), adherent bacterial cells were recovered by 0.25% trypsin–EDTA. Aliquots of recovered bacteria were diluted in PBS and spirally plated in quadruplicate (using 50 μl volumes) onto each agar plate using a spiral plater (Model D, Spiral System Co., OH, USA) [24,25]. To 50 μl of the original sterilized fluid, microorganisms were added without dilution, and this suspension was inoculated onto a BHI agar plate in order to count the number of S. mutans MT8148. Likewise, a Mannitol salt agar (Eiken Co. Tokyo) plate was used to count the number of S. aureus FHSAT1, and a Candida GE agar (Nissui Co. Tokyo) plate was used to count the number of C. albicans CAD1. An NAC agar (Eiken, Co, Tokyo) plate was used for counting P. aeruginosa. After a 48-h incubation at 37°C, colonies appearing in the pattern of an Archimedes spiral were counted by using the spiral template. As an example, the case of S. aureus FHSAT1 is shown in Fig. 1. Other microorganisms were used for comparison with the standard curve, and the data were calculated according to the number of cells adhering to each coverslip using fluorescence methods.
2.5Scanning electron microscopy
Four examination of microorganisms that adhered to the MPC-coated, non-coated, and hydrophobic coverslips, samples were fixed in 2.5% glutaraldehyde solution for 1 h at room temperature, and were then rinsed with distilled water three times and dehydrated with aqueous ethanol solution. The ethanol concentration varied from 50% (vol/vol) to 100% in 5% steps. All samples were dried to the critical point with a critical point drier, coated with gold, and examined by scanning electron microscopy (SEM; JEOL JSM-5300; Tokyo, Japan) as previously described .
2.6Clinical observation of MPC-coated coverslips on a contiguous denture surface
Each MPC-coated, non-coated, and hydrophobic coverslip was attached to the centre of a contiguous maxillary denture surface. This denture was set in palatal tissue. The MPC-coated, non-coated, and hydrophobic coverslips were extracted from the denture after 1 or 24 h and were rinsed. To determine the number of colony-forming units, adherent bacterial cells were recovered by 0.25% trypsin–EDTA. Aliquots of recovered bacteria were diluted in PBS and spirally plated in quadruplicate (using 50 μl volumes) onto BHI blood agar plates using a spiral plater [24,25]. The BHI plates were incubated in a jar with CO2 gas pak (Unipath, Basingstoke, UK) for 4 days at 37°C, and colonies appearing along the Archimedes spiral were counted by using the spiral template.
3Results and discussion
The results of the S. aureus FITC intensity assay are shown in Fig. 1 and are a representative collection of spiral assays for each substrate, in which five samples (n) per assay were tested. These data indicated that the S. aureus FITC intensity assay can be used as a standard correlation curve between the rapid FITC intensity assay and conventional plating methods using a spiral plater. For each of the other microorganisms –S. mutans MT8148, P. aeruginosa PAO1, and C. albicans CAD1 – we created a standard correlation curve between the rapid FITC intensity assay and conventional plating methods (data not shown). For S. aureus FHSAT1, S. mutans MT8148, P. aeruginosa PAO1, and C. albicans CAD1, the number of bacteria adhering to the MPC-coated surface after 1 h differed significantly (p < 0.01) from the number of bacteria adhering to the non-coated and hydrophobic coverslips, as shown in Figs. 2(a) and (b), respectively. In this study, we demonstrated that the adherence of S. mutans MT8148, S. aureus FHSAT1, P. aeruginosa PAO1, and C. albicans CAD1 to MPC-coated polyethylene telephthalate was significantly lower than that to non-coated polyethylene telephthalate and to fluoroethylene–ethylene pure polymer. SEM was used to verify these results, and generally revealed the presence of a few isolated bacteria or none at all on the coated samples, whereas large numbers of bacteria adhered to the non-coated coverslips (Fig. 2). Here, we found significant reductions in the retention of S. aureus FHSAT4, S. mutans MT6R, P. aeruginosa FHPAN2048, and C. albicans Y2D (data not shown). In E. coli, the Cpx two-component signal transduction pathway has been shown to play a key role in regulating adhesion-induced genes . E. coli attachment would be expected to lead to a Cpx-dependent up-regulation of cell envelope components that alter the cell surface and mediate stable adhesion. Contact between E. coli and hydrophobic surfaces for 1 h induces the transcriptional activity of Cpx-regulated promoters. On the other hand, this result was not obtained when the bacteria adhered to a hydrophilic surface . The mechanisms underlying the highly significant difference (p < 0.01) in microbial retention between MPC-coated and non-coated substrates have yet to be elucidated. We attribute this effect to the “superhydrophilicity” of MPC-coated surfaces, whereas hydrophobic surfaces are well known to reduce microbial retention. To date, a number of adherence assays have been reported to generate models of microorganism transport [27–29]. The present simple model system was chosen for this experiment because dentures were used as a clinical sample material. In the near future, we wish to consider other types of medical devices in order to compare the levels of initial adherence of S. aureus, S. mutans, P. aeruginosa, and C. albicans to polyethylene telephthalate surfaces coated with MPC polymers using a flow chamber system and the present data.
In this investigation, non-adhering microorganisms were removed by gentle washing with PBS according to a simple model system . However, Gomez-Suarez et al.  reported that bacterial retention exerts more of an influence on the final development of a biofilm than does adhesion. When an attempt was made to shear off the adhering microorganisms by exposing them to a very high shear force by the deliberate passage of an air–liquid interface, many more microorganisms were found to detach from materials with low wettability, as compared to those with high wettability . In future experiments, the shearing of different microbial strains from MPC-coated surfaces by the deliberate passage of an air–liquid interface should be investigated in more detail.
MPC polymers are known to have excellent blood compatibility [32–34]. In previous studies, MPC polymers effectively reduced protein adsorption and denaturation, as well as inhibited cell adhesion, even when they came into contact with whole blood in the absence of anticoagulants. MPC has great potential for use in a number of new biomaterials applicable to biotechnology. The grafting of MPC onto the surfaces of medical devices has already been shown to suppress biological reactions, even when the devices are in contact with living organisms [6,33,35]. MPC grafting is now in clinical use on intravascular stents, intravascular guide wires, soft contact lenses, and oxygenators under the authorization of the Food and Drug Administration of the United States [11,36–38]. It would be of interest to extrapolate the results of this research on biomaterials in vivo in order to determine the additional potential of MPC grafting. In the present experiment, we used dentures as a sample material. Dentures, like teeth, accumulate plaque and calculus. The MPC-coated surface coverslips on the dentures were colonized by significantly fewer (p < 0.01) bacteria than the dentures that had non-coated and hydrophobic coverslips. SEM, which was used to verify these results, generally showed either only a few isolated bacteria or none at all on the coated samples, whereas large numbers of bacteria adhered to the uncoated controls (Fig. 3). The morbidity and mortality of the dependent elderly resulting from aspiration pneumonia are recognized as major geriatric health issues . A recent study revealed that the bacteria that commonly cause respiratory infection also colonized the dentures of dependent elderly subjects . Dentures should therefore be considered as an important reservoir of microorganisms likely to colonize the pharynx, and thus the importance of controlling denture plaque for the prevention of aspiration pneumonia cannot be overemphasized. Our data revealed that the use of MPC-coated coverslips significantly reduced the number of microorganisms on dentures. These results suggest that MPC-coated dentures may be used to prevent dentures from serving as a reservoir of potential respiratory pathogens that can be expected to colonize the pharynx.
Since microbial adherence is an essential step in infection, inhibiting such adherence would be is considered to be an effective strategy for preventing infectious diseases. Our present findings demonstrated that applying an MPC coating on surfaces could be a promising tool for the prevention of microbial retention to biomedical devices and to other surfaces that may harbor infectious diseases. Future work will focus on the potential usefulness of these materials as microbial inhibitors in a variety of applications.