Correspondence: Helena Bujdáková, Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University, Mlynska dolina B-2, 842 15 Bratislava, Slovakia. Tel.: +421 2 6029 6436; fax: 421 2 6029 6480; e-mail: firstname.lastname@example.org
The lack of work dealing with possible ways of reducing biofilm production via inhibiting Candida albicans adherence in the first stage of biofilm formation was a motivation for this study. The study was focused on two questions: (1) can a decrease in adherence affect the quantity of mature biofilm? and (2) can blocking the surface C. albicans complement receptor 3-related protein (CR3-RP) with polyclonal anti-C3-RP antibody or monoclonal antibody OKM1 significantly contribute to a reduction in adherence during biofilm formation? The presence and quantity the CR3-RP expressed in the biofilm was confirmed by immunofluorescence, immunocytometry and enzyme-linked immunosorbent assay. To determine the changes in adherence of C. albicans CCY 29-3-162 and C. albicans catheter isolate, 30-, 60-, 90- and 120-min time points were selected and viability was determined by XTT assay. The strains were preincubated with both antibodies to block CR3-RP, which proved to be effective at reducing adhesion and the formation of a mature biofilm (64.1–74.6%). The duration of adhesion, between 30 and 120 min, seems to have a significant effect on the mature biofilm. The blocking of CR3-PR by antibodies before adherence affected the fitness of biofilm, which was not able to revitalize in the later stages.
Recently, biofilm-associated infections have been generally classified as a new group of diseases directly connected with the use of medical devices (Kojic & Darouiche, 2004). At present, the high percentage of bloodstream and urinary infections has been related to catheter application (Kojic & Darouiche, 2004; Opilla & Grove, 2008). Candida albicans is the major fungal pathogen isolated from the human body, but it is also the most frequent catheter-isolated Candida sp. that is able to form a biofilm (Chandra et al., 2001; Ramage et al., 2006). The development of the biofilm structure is a process composed of four different phases: adhesion, formation of sessile colonies, maturation and the production of dispersal cells (Chandra et al., 2001; Blankenship & Mitchell, 2006). Generally, adhesion to an animate surface is a fundamental step in the interaction between the pathogen and host cells. In this process, several genes which code for proteins that enhance the adherence capacity of C. albicans as well as its physicochemical interactions are involved (Ibrahim et al., 2005; Nailis et al., 2006; Nobile et al., 2006; Henriques et al., 2007). Similarly, adherence to inanimate surfaces such as polystyrene or silicone has been proposed not only to be the first phase in biofilm formation but also may be critical for the whole of biofilm development from a qualitative and quantitative point of view (Seneviratne et al., 2009). Candida albicans expresses many virulence factors, including hyphal wall protein (Hwp1) and agglutinin-like proteins (ALS), which significantly contribute to the fitness of biofilm (Green et al., 2004; Nobile et al., 2006, 2008). Candida complement receptor 3-related protein (CR3-RP) has been described to be a ‘mimicry’ antigen functionally comparative with the human CR3 protein expressed in neutrophils, macrophages and monocytes, with the ability to bind human complement fragment iC3b (Gilmore et al., 1988; Hostetter et al., 1990; Hostetter, 1996). The human CR3 antigen can be detected via the monoclonal antibody (mAb) OKM1, which recognizes the α chain of CR3 and CD11b (Wright et al., 1983), but also cross-reacts with Candida CR3-RP (Heidenreich & Dierich, 1985; Bujdákováet al., 1997, 1999). The sequence of this antigen contains the DINGGG motif, which is characteristic of proteins belonging to the DING family (Bujdákováet al., 2008). This motif has already been mentioned in prokaryotic as well as in high eukaryotic organisms (Berna et al., 2009), but not in eukaryotic microorganisms. The CR3-RP has been recently reported to be a surface antigen participating in adherence to buccal epithelial cells as well as in in vitro biofilms. Moreover, the immunomodulation properties of CR3-RP and the novel CR3-RP glycoconjugate effectively triggered an enhancement of immune responsiveness in the rabbit model (Bujdákováet al., 2008; Paulovičováet al., 2008). While many reports have reviewed the antifungal susceptibility/resistance of C. albicans in a mature biofilm (Henriques et al., 2005; Seidler et al., 2006) only a few have mentioned inhibition during the adherence phase using antifungals or antibodies (Rodier et al., 2003; Cateau et al., 2007; Dorocka-Bobkowska et al., 2009; Maza et al., 2009). The lack of information about adherence and the possibility of decreasing biofilm production via a reduction in C. albicans adherence capability in the first stage of biofilm development was our motivation for searching the answer to two questions: (1) can a decrease in adherence (the first biofilm stage) affect the quantity of a mature biofilm? and (2) can blocking the C. albicans CR3-RP surface antigen by antibodies contribute significantly to a reduction in adherence during biofilm formation?
Materials and methods
Strains, antibodies and the preparation of mature biofilm in Petri dishes or in 96-well microtiter plates
In this study, the standard C. albicans strain was used (CCY 29-3-162 from the CCY Culture Collection of Yeasts, Chemical Institute, Slovak Academy of Sciences, Slovakia), originally recovered from a patient with mycotic colpitis. This strain was selected because of its high CR3-RP expression (Bujdákováet al., 1997). For comparison, the clinical isolate C. albicans with a high ability to form biofilm obtained from the urinary catheter of a patient with candidiasis was tested. Different antibodies were applied: polyclonal anti-CR3-RP antibody, prepared as described by Bujdákováet al. (2008) and OKM1 mAb (hybridoma cell culture ATCC, CRL-8026), purchased as previously described by Bujdákováet al. (1999). Titers of the antibodies were determined by enzyme-linked immunosorbent assay (ELISA) in 96-well plates (Sarstedt, Germany) (Voller, 1978). The mAb TIB111 (mAb IgG2a, kindly provided by the Division of Hygiene and Medical Microbiology, Department of Hygiene, Microbiology and Social Medicine, Medical University, Innsbruck, Austria) was used as a nonrelevant negative control.
The mature biofilm was prepared according to the protocol of Li et al. (2003) with minor modifications. Briefly, the polystyrene Petri dishes (3 cm diameter, Sarstedt) were inoculated with 1 × 107 cells mL−1 in 3 mL of yeast–nitrogen base (YNB) medium with amino acids (Sigma-Aldrich) supplemented with 0.9%d-glucose (AppliChem, Darmstadt, Germany) at 37 °C for 90 min (adhesion phase). Biofilm was also formed in polystyrene 96-well plates (flat bottom, Sarstedt) in the same medium with the cell concentration of 107 mL−1. A 100-μL aliquot of this suspension was then applied to each well. Nonadherent cells were then removed and adherent cells were washed three times with 1 × phosphate-buffered saline (PBS). Finally, 3 mL or 100 μL of YNB medium was added and cultivation continued at 37 °C for 48 h to obtain a mature biofilm.
Indirect immunofluorescence, immunocytometry and ELISA
The mature biofilm was washed with 3 mL of 1 × PBS three times and then blocked with 1% gelatin (w/v, Oxoid, Ogdensburg, NY) dissolved in 1 × PBS at 37 °C. After 1 h, the plates were washed once with PBS–0.05% v/v Tween 20 (Sigma-Aldrich), followed by incubation with 100 μL of polyclonal anti-CR3-RP antibody diluted 1 : 100 and OKM1 mAb or TIB111 mAb (used as the control), both diluted 1 : 10 in 1 × PBS for 1 h on ice. Then samples were washed three times in PBS–0.05% v/v Tween 20 followed by centrifugation to remove unbound antibody. Specific immunocomplexes were developed with goat anti-rabbit or goat anti-mouse immunoglobulin G (IgG)-(H+L) fluorescein isothiocyanate (FITC)-conjugated antibody (Bethyl Laboratories Inc., Montgomery, TX) for 1 h in the dark at room temperature. After three washing steps, the immunofluorescence signal was directly observed by microscopy (Axio Imager A.1, Carl Zeiss, Oberkochen, Germany). Parallel plates with the biofilm preincubated with all antibodies were scraped and submitted for immunocytometric assay, using an indirect staining (FITC-secondary anti-rabbit IgG and anti-mouse IgG antibodies) and evaluated by flow cytometry using a Beckman Coulter FC 500 flow cytometer (Beckman Coulter Inc., Fullerton, CA) equipped with a 488-nm argon laser and a 637-nm HeNe collinear laser, and controlled by cxp software. Candida biofilm cells were gated on the basis of forward light scatter (FSC) and side light scatter (SSC) using a logarithmic scale. Gates were set to exclude debris and intact cells on a forward scatter vs. side scatter dot plot. Additionally, gates were previously optimalized on properly prepared cultures of the yeasts, budding yeasts and hyphae from Candida strains CCY (29-3-163) according to the protocol of Bujdákováet al. (1999). For each sample, fluorescence histograms of 10 000 cells were generated and analyzed (green fluorescence, 525-nm band-pass filter, FL1 channel). All samples were tested twice. The data are expressed as mean±SD.
The expression of CR3-RP was evaluated by ELISA. The mature biofilm was developed in 96-well polystyrene plates (Sarstedt) according to the protocol of Li et al. (2003). The wells were then washed three times with 1 × PBS and unspecified epitopes were blocked with 100 μL of 1% gelatin as described previously. After a single-step washing with PBS–0.05% Tween 20, wells were coated with 100 μL (per well) of the anti-CR3-RP antibody (1 : 100 in 1 × PBS) or OKM1 mAb (1 : 10 in 1 × PBS) mAb or control antibody TIB111 (1 : 10 in 1 × PBS) and incubated for 1 h in ice. After three washing steps with PBS–0.05% v/v Tween 20, goat anti-rabbit (for the polyclonal anti-CR3-RP antibody) or goat anti-mouse IgG (for the OKM1 and TIB111 mAb) conjugated with alkaline phosphatase was added in a final dilution of 1 : 30 000 and the plates were incubated for 1 h at room temperature. After four additional washing steps, an alkaline phosphatase substrate containing p-nitrophenylphosphate (pNPP, Sigma-Aldrich) was used for development. The reaction was stopped with 3 M NaOH and evaluated at 405 nm using a microplate reader (MRX™, Dynex, Chantilly, VA). The experiment was repeated twice with five parallel wells for every antibody. Final results were calculated as mean±SD.
Kinetic of adhesion
A kinetic of adhesion was performed in polystyrene 24-well plates (Sarstedt) with five selected time points (0, 30, 60, 120, 240 min), according to the protocol of Sohn et al. (2006) with some modifications. Briefly, the loop of 48-culture of yeasts grown on Sabouraud dextrose agar (Biomark Laboratories, Pune, India) was inoculated in 20 mL of YNB medium with amino acids and incubated overnight at 28 °C with shaking. The inoculum was diluted to 0.2 (OD570 nm) in 20 mL of fresh YNB medium. After the subsequent 4-h cultivation at 30 °C with shaking, the density of cell was adjusted to OD570 nm 1 and then diluted 1 : 50 000. YNB medium (250 μL) and 50 μL of diluted strains was added per well and incubated at 37 °C. After every time point as well as at the starting point (time 0), planktonic cells in 300 μL of YNB medium were inoculated on Petri dishes (diameter 10 cm) with 20 mL yeast–peptone–dextrose (YPD) agar. Wells were then washed once with 1 × PBS, followed by scraping the adherent cells in 300 μL of PBS and inoculating on YPD agar medium. The cultivation of both adherent and nonadherent cells was performed at 28 °C for 48 h. The percentage of adherent cells was calculated in terms of CFU according to the formula: [(adherent cells)/(adherent cells+nonadherent cells)] × 100 for each time point. The experiment was performed in two independent biological replicas and in duplicates for each strain. The results were expressed as mean±SD.
Blocking of CR3-RP by antibodies and determination of viability in adherence phase and mature biofilm
This experiment was performed based on the protocol according to Li et al. (2003) described above. However, prior this experiment, both C. albicans strains were adjusted to 107 cells mL−1. The appropriate amount of this cell suspension was centrifuged again and the pellet blocked with 1% gelatin at 37 °C for 1 h with shaking. After a single washing step in 1 × PBS and centrifugation, pelleted cells were resuspended in 200 μL PBS with polyclonal anti-CR3-RP antibody (diluted 1 : 100), and mAb OKM1 (diluted 1 : 10). Control samples were resuspended in mAb TIB111 (diluted 1 : 10 in PBS). After 1-h incubation in ice, unbound antibodies were removed by centrifugation and cells were resuspended in a precise volume of YNB medium with amino acids containing 0.9%D-glucose (cell concentration, 107 mL−1). A 100-μL aliquot of this suspension was then applied to 96-well plates to undergo the adherence phase in biofilm formation for 30, 60, 90, and 120 min at 37 °C. At these time points, nonadherent cells were removed, adherent cells were washed with 1 × PBS in three washing steps and the viability of the adherent cells was evaluated by their ability to reduce 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) sodium salt to water-soluble formazan (Sigma-Aldrich). The parallel experiments were continued; after the adherence phase (90 min), nonadherent cells were removed and adherent cells washed three times with 1 × PBS. Adherent cells were then overlaid with 100 μL of the new YNB medium and incubation continued at 37 °C for 48 h. The viability of the mature biofilm was evaluated as described above. Every experiment was performed in five parallel wells and performed twice. The results were expressed as mean±SD.
Results were calculated as average±SD. Statistical significance in the difference between the samples was compared using Student's t-test. A P-value of <0.05 was considered significant, a P-value of <0.01 highly significant and a P-value of <0.001 extremely significant.
Results and discussion
Expression and quantification of the C. albicans CR3-RP antigen in biofilm
Although the formation of a biofilm in the environment is a natural process important for the survival of many microorganisms, medical microbiology regards this complex structure as a serious complication during patient treatment or convalescence. Current trends in biofilm studies are aimed at possible ways to eliminate them, mainly via the application of antifungal agents (Kuhn et al., 2002; Al-Fattani & Douglas, 2004; Seidler et al., 2006; Borecká-Melkusová & Bujdáková, 2008). However, some authors have published different thoughts on biofilm treatment, such as photodynamic effects (Müller et al., 2007; Dovigo et al., 2009) or using antibodies (Rodier et al., 2003; Fujibayashi et al., 2009; Maza et al., 2009). In this study, we were focused on two different aspects: whether decreasing the ability of C. albicans to adhere to a plastic surface can reduce the production of the mature biofilm, and whether blocking the C. albicans surface antigen (CR3-RP) participating in adherence can significantly affect adherence, the first stage of biofilm formation. For experiments, one standard strain was selected, together with a C. albicans clinical isolate obtained from the urinary catheter of patient with candidiasis. The catheter isolate was chosen from the collection of catheter isolates (Department of Microbiology and Virology) because of its high biofilm production (H. Bujdáková, unpublished data). The C. albicans CR3-RP has been already noted as being involved in adhesion to buccal epithelial cells. Additionally, preliminary experiments suggested that blocking this antigen resulted in a decrease in the biofilm (Bujdákováet al., 2008). To confirm the hypothesis about CR3-RP participation in the adhesion process, it was necessary to prove that this antigen is expressed in the biofilm. Three different experiments were carried out to confirm the expression of the CR3-RP antigen in the adhesion phase as well as in mature biofilm. The polyclonal anti-CR3-RP antibody was prepared according to the peptide sequence of CR3-RP (Bujdákováet al., 2008). The already characterized OKM1 mAb (former iC3b-like protein, Bujdákováet al., 1999) was also used in every experiment. Figure 1 (left) documents the strong immunofluorescence when using anti-CR3-RP antibody in C. albicans CCY 29-3-162 in a mature biofilm. The reaction with OKM1 mAb was lower (Fig. 1, left, despite lower dilution – 1 : 10), but it must be kept in mind that this antibody only cross-reacts with the C. albicans antigen. The results from immunocytometry (Fig. 2) were in agreement with those observed in fluorescence microscopy; the detection of the CR3-RP using polyclonal anti-CR3-RP antibody was higher than with OKM1 mAb. Moreover, the evaluated samples could be categorized according to the morphology of the yeasts, the budding yeasts or small hyphae, and the long hyphae (FSC and SSC distribution). The fluorescence signal was detected in all morphological forms with strong expression in the hyphae and a weaker expression in the yeasts or germ tubes. Additionally, the difference between the anti-CR3-RP antibody and OKM1 mAb signals showed the higher specificity and potency of the polyclonal antibody to interact with the CR3-RP antigen. A similar result was observed with the catheter isolate (data not shown). The quantification of the total CR3-RP expression was performed using ELISA in both C. albicans strains. In this experiment, the CR3-RP was detected in adherence phase (90 min) as well as in the mature (48 h) biofilm. Figure 3 documents that CR3-RP is manifested in both phases of the biofilm. Of course, the expression of this antigen is markedly higher in the mature biofilm because of the presence of the hyphal morphological form, which has, however, already been proved to be expressed in a higher quantity than the yeast form (Bujdákováet al., 1999).
Kinetic adhesion curve
It has been already proposed that the adhesion phase is the key step affecting the whole process of biofilm formation (Chandra et al., 2001; Nobile et al., 2008; Soll, 2008). However, the colonization of a particular surface is not only dependent on the primary adherence of planktonic cells, this process can also be time-dependent. With this in mind, the kinetic of adhesion were studied using six time points, 0, 30, 60, 90, 120 and 240 min (Fig. 4); a 90-min adhesion time is assumed to be sufficient for the occupation of a surface with irreversibly attached cells (Li et al., 2003; Seneviratne et al., 2009). This experiment was performed with the standard strain as well as the catheter isolate on polystyrene plates. No significant differences were observed between the strains (P<0.001). As indicated in Fig. 4, 30 min can be considered to be critical for both C. albicans strains to saturate a free surface, with about 60% of the yeasts attached and with a prolonged adhesion maximum until 120 min with approximately 69% adhesion. On the basis of these results, the changes in the adhesion phase during biofilm development after incubation of both C. albicans yeasts with polyclonal anti-CR3-RP antibody, OKM1 mAb as well as control antibody were selected at 30, 60, 90 and 120 min of adhesion.
Reduction in adhesion during biofilm formation and influence of adherence time on mature (48 h) biofilm
The main focus of this manuscript was on the hypothesis of whether a reduction in biofilm production can be achieved by decreasing cell attachment to the surface in the first stage of biofilm development – adhesion. For this experiment, the yeasts of both tested C. albicans strains were incubated with polyclonal anti-CR3-RP antibody or OKM1 mAb and compared with control samples incubated with TIB111 mAb. The results summarized in Fig. 5 clearly show that the adhesion of the yeasts was reduced after incubation with both antibodies, although this process appeared to be strain-dependent. In the standard C. albicans CCY 29-3-192 strain, the proportion of the reduction in adherence using polyclonal anti-CR3-RP antibody or OKM1 mAb compared with the control antibody (0% of reduction) proved to be very similar with regard to time points: 39.4%, 55.8%, 42.3% and 48.1% (P<0.001) and 6.3%, 33.9%, 24.6% and 28.1%, respectively, at 30, 60, 90 and 120 min (P<0.01), with the exception for 30 min, where P>0.05). The antibodies were observed to have different effects on the catheter isolate. Generally, both antibodies reduced its adherence to a greater extent than in the standard strain. While polyclonal anti-CR3-RP antibody showed an approximately similar reduction in adherence (71.6%, 73.8%, 67.0% and 62.6%, respectively, at 30, 60, 90 and 120 min, P<0.001), for the OKM1 mAb it increased continuously (63.9%, 66.9%, 77.0% and 83.9%, respectively, P<0.001). It is interesting to note that the proportional reduction of mature biofilm (Fig. 6) was very similar in both strains and antibodies used: 74.5/69.7% for polyclonal anti-CR3-RP antibody and 72.7/64.1% for OKM1 mAb for C. albicans CCY 29-3-162 and the clinical catheter isolate, respectively. For mature biofilm, the duration of adhesion between 30 and 120 min when the maximal number of cells is attached to the plastic surface, seems to have a significant effect on total biofilm production. However, the kinetics of adhesion are strain-dependent and therefore a longer time (90 or 120 min) is recommended. Before the formation of C. albicans biofilm layer on invasive medical devices, yeasts colonize the surface, for example a central venous or urinary catheter. In this step, C. albicans begins to express surface proteins promoting adhesion (Nobile et al., 2008; Soll, 2008). This step is a key to starting to build a biofilm. At this stage, the process of biofilm formation can be influenced very effectively. For example, echinocandins have been confirmed to be applied very successfully to inhibit adherence and reduce biofilm formation (Kuhn et al., 2002; Cateau et al., 2007). Other reports noted the ability of IgG purified from rabbit serum immunized with C. albicans cytoplasmatic extract to reduce the capacity of C. albicans to adhere to polystyrene (Rodier et al., 2003). This information supports our results, as specific IgG isotypic recognition was confirmed for the complex of the CR3-RP antigen and polyclonal anti-CR3-RP antibody by immunocytometry. Moreover, the higher specificity of our anti-CR3-RP can be predicted because the sequence of the CR3-RP fragment used to immunize the rabbit is known (Bujdákováet al., 2008). The higher specificity was also evidence of a lower dilution of OKM1 mAb (1 : 10; a higher dilution was not possible because of low activity) used in all experiments in comparison with polyclonal anti-CR3-RP antibody (1 : 100).
The reduction in the adherence capability of C. albicans due to blocking the CR3-RP surface antigen can effectively decrease biofilm formation. Additionally, despite the fact that adhesion takes a relatively short time, changes in the capability of C. albicans to interact with a surface affected the formation of the biofilm, which was not able to revitalize in the later biofilm stages, resulting in a decrease in final biofilm fitness.
This work was supported by financial contributions from EU project CanTrain MRTN-CT-2004-512481 as well as MVTS 6RP/MRTN-CT-2004-512481 and VEGA 1/0396/10 from the Slovak Ministry of Education.