The purpose of this study was to identify surface modifications that can prevent or inhibit Candida biofilm formation on medical devices. We compared the ability of C. albicans to form biofilms on surfaces modified by different SMEs or chemical modifications. We found that of all the surface modifications tested, the 6PEO SME was the only one that significantly inhibited Candida biofilm formation.
Surface modifications, including addition of SMEs and chemical modifications, have been previously tested for their ability to allow bacterial adhesion and biofilm formation on dental and catheter materials (10
). Olsson et al. (18
) investigated protein adsorption and saliva-mediated bacterial adherence on untreated, hydrophobic surfaces and on polyethylene oxide-coated glass and ceramic crown surfaces. These investigators showed that hydrophobic and PEO-treated surfaces exhibited much lower (or no) colonization and pellicle and plaque formation. Dijk et al. (8
) reported similar results, demonstrating that biofilm formation by C. albicans
and Candida tropicalis
on a silicone rubber voice prosthesis treated with a colloidal palladium/tin solution (resulting in a thin metal coat) was significantly less than that on untreated prostheses. Nikawa et al. (17
) showed that fluoric and heat-cured silicone denture lining materials promote the lowest colonization by C.albicans
. In this study, we identified surface modifications that can eliminate or reduce the ability of C. albicans
to form biofilm on biomaterial surfaces.
We demonstrated that the ability of C. albicans to form biofilm is influenced by the surface chemistries of the biomaterials used. Although all the surface modifications affected the ability of C. albicans to form biofilms, only one modification, the addition of the 6PEO SME, reduced the ability of C.albicans to form biofilm on the E80A surface. The metabolic activity and dry weight of C. albicans cells adhered to E80A-6PEO were reduced by 78% and 74%, respectively (compared to those of biofilm formed on nonmodified E80A). The low dry weight of C. albicans biofilm formed on 6PEO correlates well with the reduction in metabolic activity of biofilm formed on the same material. Moreover, when using CSLM, we were unable to detect any biofilm formation by C.albicans on the E80A-6PEO surface. Since very low, yet noticeable, metabolic activity and dry weight were observed for biofilm formed on E80A-6PEO, the low XTT values as well as inability of CSLM to detect biofilm on this surface may be because fungal cells did not adhere strongly to the modified biomaterial and were likely detached during manipulations performed for confocal analysis.
We observed statistically significant increases in the dry weights of biofilms formed on cationic and hydrophilic PET surfaces compared to those of biofilm formed on the nonmodified PET surface. In a different study, Brodbeck et al. (4
) showed that adhesion of monocytes/macrophages to hydrophilic and anionic PET substrates is greatly reduced compared to that with nonmodified PET surfaces. Since the cell surface hydrophobicity of Candida
cells is known to be an important factor in its adherence to acrylic surfaces (19
), this factor may also play a role in the formation of biofilm on PET surfaces.
We employed XTT assay and total biomass determination to show that PEO-modified polyetheruerthane significantly inhibited biofilm formation by C. albicans
. Using these established methods, it is possible to demonstrate a correlation between XTT and dry weight analyses. We found that metabolic activity and total biomass correlated well for Candida
biofilms formed on the polyurethanes and two (anionic and hydrophobic) PET surfaces but not for those formed on cationic and hydrophilic PET surfaces. In a previous publication, Kuhn et al. (16
) showed that the relationship between XTT colorimetric signal and organism number is not always linear. This observation can explain the apparent lack of correlation between XTT activity and dry weight of biofilms formed on some surfaces, since the number of cells contributes significantly to the total biomass of biofilms. Kuhn et al. (16
) also showed that while the XTT formazan product readily appears in solution, there can be a significant amount of retained intracellular product, which becomes soluble only after cell treatment with dimethyl sulfoxide. Moreover, the retention of residual formazan product varies with different species of Candida
; such variation may also be induced by the different substrates tested in this study. Since total biomass values are independent of formazan product formation, a lack of correlation between the metabolic activity and total biomass of biofilms formed on some surfaces is not entirely unexpected.
The mechanisms underlying the inability of C. albicans
to form biofilm on 6PEO-modified surfaces is unknown. The role of different surface chemistries in microbial adhesion and biofilm formation has been proposed to be a complex interplay between different microbial and host factors, including thermal cycling (17
), protein coating (saliva or serum) (17
), hydrophobicity (18
), and nonspecific physiochemical forces or specific ligand-receptor interactions (20
Our studies revealed that while more biofilm was formed on polyetherurethane surfaces with higher contact angles, no correlation was seen for PET biomaterials, indicating that the influence of contact angles on biofilm formation is surface dependent. Our results are in agreement with previous studies showing that the correlation between contact angle and biofilm formation is dependent on the surface tested (1
). Jansen and Kohnen (12
) investigated the influence of contact angles of modified polymers on adherence of Staphylococcus epidermidis
and showed a correlation between contact angle measurements and adherence. Similar correlations between contact angle and biofilm formation were noted for amalgam and resin composites (22
), poly(vinyl chloride) (PVC) catheters (25
), and glass (1
). However, other investigators have demonstrated no correlation between contact angle and biofilm formation. In this regard, Jones et al. (15
) showed that no correlation existed between contact angle and biofilm formation on PET and high-density polyethylene bottles. Balazs et al. (2
) reported complete inhibition of adhesion and colonization by Pseudomonas aeruginosa
of PVC with an ultrahydrophobic surface (contact angle o >120 degrees). In a separate study, Webb et al. (24
) demonstrated a positive correlation between adhesion of the fungus Aureobasidium pullulans
and the contact angle of unplasticized PVC. However, plasticized PVC, with a relative reduction of 13° in contact angle (compared to that of unplasticized PVC), showed enhanced adhesion and biofilm formation. Taken together, these studies clearly demonstrate that the correlation between contact angle and biofilm formation is dependent on the substrate surface.
Patel et al. (20
) showed that the adhesion of S. epidermidis
to hydrophobic surfaces is greater than the adhesion of this bacterial species to hydrophilic surfaces (e.g., 6PEO). These investigators used a rotating disk model to characterize adhesion of leukocytes and S. epidermidis
on polycarbonateurethanes and polyetherurethanes modified with SMEs (6PEO, 6FC, and silicone) under dynamic flow conditions. These studies showed that modification of materials with polydimethylsiloxane and PEO SMEs reduces bacterial adhesion, while fluorocarbon SMEs enhance adhesion. A different effect of 6PEO on bacterial adhesion to glass and ceramic surfaces was demonstrated by Olsson et al. (18
). These investigators determined the effect of immobilized PEO on protein adsorption and bacterial adherence in vitro to glass and in vivo to ceramic crown surfaces. In vitro, more protein and bacteria bound to untreated glass than to hydrophobic and PEO-treated glass. On the other hand, in vivo, pellicle and plaque formation was similar on the untreated ceramic and PEO surfaces, but less plaque formed on these surfaces than on adjacent normal tooth surfaces. Almost no plaque accumulated on the hydrophobic crown surface, and it was virtually devoid of stainable pellicle. The different effects of 6PEO SME on bacterial adherence to materials may be due to the different substrates used in these studies and suggested that the effect of the 6PEO SME is dependent on the substrate material to which this SME is applied.
In the only study focusing on fungal adhesion to substrates with modified surfaces, Nikawa et al. (17
) investigated the growth of C. albicans
on seven saliva-coated, serum-coated, or protein-free (uncoated) thermocycled commercial soft lining materials. For control resilient liners (not thermocycled and uncoated), fungal colonization was found to be dependent on the type of commercial resilient liner used; fluoric and heat-cured silicone materials promoted the lowest colonization, while cold-cured silicone materials and heat-cured acrylic resin exhibited the highest colonization capacity. Thermal cycling and protein coating (saliva or serum) significantly promoted fungal colonization on the materials. These investigators suggested that aging of the materials and the presence of host biological fluids promote yeast colonization on denture lining materials.
Based on the above discussion, it is likely that the mechanism(s) responsible for inhibition of the ability of C. albicans to form intact biofilm on 6PEO-modified surfaces is multifactorial and calls for further investigations which are beyond the scope of this study. It is possible that this inhibition is mediated by the prevention of Candida adhesion to the substrate surface.
In conclusion, we identified the 6PEO SME as a surface-modifying agent that inhibits C. albicans biofilm formation. Examination of the ability of 6PEO to inhibit biofilm formation in vivo is warranted. The results reported in this study may have important clinical implications in the design of novel biomaterials that have antibiofilm properties.