Objectives: This research work intends to clarify the role of artificial saliva, in particularly the role of mucin, a salivary protein, on the surface properties and adhesion ability of Candida spp. oral clinical isolates to abiotic surfaces. Methods: Four oral clinical isolates of Candida spp. were used: two Candida albicans strains (AC; AM) and two Candida parapsilosis strains (AD; AM2). The strains were isolated from patients using oral prosthesis. The microorganisms were cultured in the absence or presence of mucin and artificial saliva, and their adhesion to an abiotic surface (coated with mucin and artificial saliva) was evaluated. Results: The presence of mucin per se onto the abiotic surface decreased the adhesion of all strains, although the combination of mucin with artificial saliva had reduced this effect. No direct correlation between adhesion and the surface free energies of adhesion of the microorganisms was found. Significance: Candida spp. were human commensal microorganisms that became pathogenic when the host immune defenses were compromised. Medical devices were colonized by Candida spp. particularly, oral prostheses, which might lead to the degradation of the prostheses and systemic infections. The salivary secretions that constantly cover the oral cavity influenced Candida spp. adhesion process. Therefore, it was important to understand the interactions between Candida spp., salivary proteins and the characteristic of oral prosthesis when developing materials for oral prostheses.
Candida spp. are opportunistic microorganisms present in the normal microbiota. On the right environment, these microorganisms are able to colonize, invade and multiply in tissues and organs, causing fungal infections that can go from superficial lesions to systemic infections [
Candida spp. are found in the oral cavity of more than 50% of the human population, and 80% of the clinical isolates are identified as Candida albicans. Candida parapsilosis is an emerging human pathogen that has dramatically increased in significance and prevalence over the past 2 decades, such that C. parapsilosis is now one of the leading causes of invasive candidal disease [
The human organism has many defence mechanisms in order to avoid colonization by microorganisms. Mucosal epithelial cells continuously secrete a mucosal fluid that acts as a barrier to maintain a healthy mucosa. In addition, saliva is formed by many defensive compounds, including mucins, antibodies, lysozyme or histatins that regulate the microorganism populations in the oral cavity [
Salivary secretions constantly cover the oral cavity, so, it is important that, during the investigation of oral colonization, the interactions between Candida spp. and salivary proteins are considered. This research work intends to clarify the role of the salivary protein mucin, as well as artificial saliva, on the surface properties and the adhesion ability of Candida spp. oral clinical isolates.
Four oral clinical isolates of Candida spp. were used in this study: two isolates of Candida albicans (strains AC and AM) and two isolates of Candida parapsilosis (strains AD and AM2). The strains were isolated from patients using an oral prosthesis. Candida albicans AC and Candida parapsilosis AD were obtained from different individuals and Candida albicans AM and Candida parapsilosis AM2 from the same individual. The oral isolates were obtained from a Dentistry Clinic and belong to the Biofilm Group of the Centre of Biological Engineering, Minho University, where they were identified by molecular methods.
Candida isolates were subcultured on Sabouraud dextrose agar for 24 h at 37˚C, after which each strain was inoculated in Sabouraud dextrose broth for 16 hrs at 37˚C in an orbital shaker at 120 rpm. Cells were then harvested by centrifugation at 8000 rpm and washed twice with phosphate saline buffer (PBS, pH 7, 0.1 M). The cell pellets were resuspended in the conditioning mediums, and the cell concentration adjusted to 1 × 107 cells ml−1.
Three different media were used in this study, namely artificial saliva without mucin (AS-Mu) and with mucin (AS + Mu) and mucin in PBS (Mu). The artificial saliva was prepared according to Lamfon et al. [
A 6-well polystyrene plate was used as an abiotic substrate to study the influence of the presence of mucin on the adhesion properties of Candida spp. The three different conditioning media were used for coating. The polystyrene surfaces were incubated with each conditioning medium for 4 hrs at 37˚C in an orbital shaker at 120 rpm, as described by Guggenheim et al. [
Contact angles were measured by the sessile drop technique, using a contact angle measurement apparatus model OCA 15 PLUS, DATAPHYSICS. Measurements were performed at room temperature using three different standard liquids (ultrapure water, formamide, and α-bromonaphthalene). Every assay was performed in triplicate and at least 10 measurements were performed for each sample.
Contact angles were measured on yeast lawns. Briefly, microorganisms were grown for 2 h in artificial saliva without mucin (AS − Mu) or in artificial saliva with mucin (AS + Mu). Subsequently, the cell suspensions were layered onto 0.22 mm pore sized filters and dried for 4 h at 37˚C, to standardize the humidity level [
The hydrophobicity and surface tension was determined using the Van Oss approach [
in which the AB component equals
where
Since a-bromonaphthalene is apolar (
Water and formamide are both polar liquids and their contact angles can be used to calculate
The free energy of adhesion ΔGadh can be separated into two components:
where
and
The adhesion assays were performed on non-treated polystyrene surfaces, and polystyrene surfaces treated with the three different conditioning mediums: AS − Mu, AS + Mu and Mu. Yeast suspensions grown in AS − Mu or AS + Mu were used. Briefly, 500 μl of the standardized cell suspensions (1 × 107 cells ml−1 in AS − Mu or AS + Mu) were placed on the coated or uncoated well-plates for 2 hrs at 37˚C in an orbital shaker at 120 rpm. Follow- ing this incubation period, the medium was removed and the wells washed with PBS to remove unattached cells. The wells were scraped to resuspend the adhered cells in PBS. The yeast cells were then sonicated for 45 sec, 30 W, in an Ultrasonic Processor. Viable counts for each Candida spp. were obtained by serial decimal dilutions in PBS, and plated on Sabouraud dextrose agar medium, followed by an incubation period of 24 hrs at 37˚C.
Statistical analysis was performed using the SPSS software. The one-way ANOVA test followed by a Bonferroni as a Post Hoc test (confidence level of 95%) was applied.
The treatments with AS − Mu, AS + Mu and Mu strongly affected the characteristics of the surface. The contact angles measured with the polar liquids (water and formamide) on the treated surfaces were lower than on the control surfaces (untreated), while the apolar liquid (α-bromonaftalene) formed higher contact angles (
The surface free energy (total surface tension, composed by the Lifshitz-van der Walls γLW and acid-base γAB components) and hydrophobicity of treated and non-treated polystyrene surfaces was determined (
θwater (˚) ± SE | θformanide (˚) ± SE | θα-bromonaftalene (˚) ± SE | |
---|---|---|---|
NT | 66.72 ± 2.08 | 49.84 ± 1.95 | <0 |
AS − Mu | <0 | 29.27 ± 1.46 | 28.17 ± 2.25 |
AS + Mu | <0 | 34.66 ±1.71 | 39.99 ± 1.34 |
Mu | 28.76 ± 1.52 | 35.65 ± 1.37 | 41.75 ± 3.32 |
Combining the contact angles and surface free energies (
The DGsws of the polystyrene surface was found to be −40.1 mJm−2 (
Interestingly, the four oral clinical isolates have different contact angle values, indicating different surface characteristics (
Moreover, the presence of mucin in the medium strongly influenced the water contact angles of all Candida spp. a decrease on the water contact angle value was observed for C. albicans AC and C. parapsilosis AM2, while the opposite effect occurred for C. albicans AM and C. parapsilosis AD. All the formamide contact angles increased, except for C. parapsilosis AD. The α-bromonaftalene contact angles also increased in the presence of mucin, except for C. parapsilosis AM2.
On the absence of mucin, C. albicans strains and C. parapsilosis AM2 had a γAB between 9 and 13 mJm−2, while C. parapsilosis AD presented a very small γAB (around 2 mJm−2) (
The electron-donating (γ−) and electron-accepting (γ+) parameters calculated for the Candida spp. are shown in
Cells | Strains | θw (˚) ± SE | θf (˚) ± SE | θb (˚) ± SE | |
---|---|---|---|---|---|
AS without mucin | C. albicans | AC | 48.03 ± 2.38 | 19.20 ± 1.98 | 17.24 ± 1.21 |
AM | 29.28 ± 1.97 | 27.75 ± 1.97 | 22.02 ± 2.02 | ||
C. parapsilosis | AD | 54.47 ± 215 | 47.16 ± 2.49 | 26.41 ± 2.02 | |
AM2 | 44.18 ± 2.82 | 28.04 ± 1.41 | 26.97 ± 3.24 | ||
AS with mucin | C. albicans | AC | 26.08 ± 1.80 | 39.33 ± 3.23 | 32.47 ± 1.54 |
AM | 38.14 ± 1.02 | 43.55 ± 2.48 | 23.34 ± 1.34 | ||
C. parapsilosis | AD | 57.62 ± 1.55 | 35.75 ± 1.44 | 33.59 ± 1.86 | |
AM2 | 28.97 ± 2.05 | 54.06 ± 3.54 | 23.34 ± 1.34 |
C. albicans AC | C. albicans AM | C. parapsilosis AD | C. parapsilosis AM2 | ||
---|---|---|---|---|---|
AS without mucin | γ− | 21.32 | 47.60 | 28.86 | 29.50 |
γ+ | 1.93 | 0.44 | 0.03 | 1.30 | |
AS with mucin | γ− | 60.79 | 49.78 | 17.00 | 76.20 |
γ+ | 0.04 | 0.00 | 1.69 | 0.00 |
C. albicans AC | C. albicans AM | C. parapsilosis AD | C. parapsilosis AM2 | ||
---|---|---|---|---|---|
AS without mucin | ΔGsws | ?19.9 | 20.2 | ?4.6 | ?4.7 |
AS with mucin | ΔGsws | 44.6 | 28.8 | ?22.2 | 62.5 |
On the absence of mucin, C. albicans AC and C. parapsilosis strains were found to be hydrophobic (DGsws < 0) (
When mucin is not present in the growth medium, the adhesion of all Candida spp. is reduced only on the polystyrene surface treated with mucin (Mu). The number of viable of C. parapsilosis AD cells increased when attached to the surface with AS-Mu treatment (
The two C. parapsilosis strains adhered at a higher extend to AS − Mu polystyrene than the two C. albicans strains. For the treatment of the polystyrene surface with Mu the adhesion followed the order: C. albicans AM > C. albicans AC > C. parapsilosis AD = C. parapsilosis AM2.
It is also important to observe that the number of C. albicans AC cells adhered to the polystyrene surfaces increased when mucin was added. The same adhesion trend was observed for C. albicans AM, although there were no significant differences between the treatments with AS − Mu and AS + Mu. For the two C. parapsilosis
strains only the treatment with Mu was able to significantly reduce the adhesion. Actually, the adhesion of C. parapsilosis AD and C. parapsilosis AM2 was increased for AS − Mu and AS − Mu/AS + Mu, respectively.
In the presence of mucin, the adhesion of C. albicans strains to the non-treated polystyrene surface was increased; while to the polystyrene treated with Mu was reduced. The adhesion to AS − Mu was not affected. The presence of mucin in the growth medium also reduced the adhesion of C. parapsilosis AD to all the surfaces, while the adhesion of C. parapsilosis AM2 was only reduced for the NT and Mu polystyrene surfaces.
In the absence and in the presence of mucin on the growth medium, the interaction energies between all the Candida spp. strains and the polystyrene surfaces treated with AS − Mu, AS + Mu and Mu were always unfavorable (ΔGadh > 0) (
The poor growth conditions on the oral environment oblige microbial cells to adhere in order to survive and colonize the oral cavity [
C. albicans AC | C. albicans AM | C. parapsilosis AD | C. parapsilosis AM2 | ||
---|---|---|---|---|---|
AS without mucin | NT | ?20.5 | 1.2 | ?15 | ?12 |
AS ? Mu | 13 | 38.8 | 28.2 | 22.7 | |
AS + Mu | 16.4 | 42.2 | 32 | 26 | |
Mu | 8.8 | 31.6 | 21.2 | 17.3 | |
AS with mucin | NT | 10.3 | 1.3 | ?24.1 | 18.2 |
AS ? Mu | 50.9 | 44.6 | 10.3 | 59.9 | |
AS + Mu | 54.2 | 48.2 | 13.6 | 63.4 | |
Mu | 41.9 | 36 | 6 | 50.1 |
facilitate adherence to surfaces [
This study gives a new insight on the effect of saliva on Candida spp. adhesion, and the precise role of a single component-mucin. The effect of mucin on the microorganism surface characteristics as well as its influence on the surface properties of polystyrene was assessed.
As expected, a high contact angle value (~67˚) and a low free energy (<10˚) were obtained for the non-treated polystyrene surface, demonstrating its hydrophobic nature. All the treatments performed (AS − Mu, AS + Mu, Mu) changed the nature of the polystyrene surface from hydrophobic to hydrophilic (
The contact angles measured on the yeast lawns showed important variations in the surface characteristics of the oral clinical isolates (
When Candida spp. were grown in artificial saliva with mucin, C. albicans AC and C. parapsilosis AM2 changed from hydrophobic to hydrophilic (
The energies of adhesion calculated based on the surface free energies of the Candida spp. and the polystyrene surfaces could not explain the adhesion profile on its own. Unfavorable energies of adhesion between the cells and the surfaces (
In the presence of artificial saliva with mucin, an increased number of C. albicans adhered to the non-treated polystyrene surface was observed, while for C. parapsilosis strains the adhesion was reduced (
As mentioned before, adhesion is a very complex process, mediated by several factors such as surface free energies and hydrophobic interactions, but the interaction between adhesins and surface binding sites play a determinant role on the adhesion process of yeast [
This study shows that the role of mucin on Candida spp. adhesion is complex and must be carefully examined. The four Candida strains used in this study behave differently in the presence of mucin, showing either increased or decreased adhesion depending on the presence of mucin on the growing medium or on the polystyrene surface. Actually, while the presence of adhered mucin onto the surface decreases the adhesion of all the strains, the combination of mucin with artificial saliva diminishes this effect.
Although there is not a direct correlation between adhesion and the surface free energies of adhesion of these particular Candida strains, the presence of artificial saliva affects the physicochemical characteristics of the adherent surface, as well the hydrophobicity behaviour of the strains.
This study clearly demonstrates that it is important to evaluate the surface characteristics, as they will enhance or decrease the microbial attachment.
The authors thank the Project “BioHealth-Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER. The would also like to thank the Fundação para a Ciência e Tecnologia for the Strategic Project Pest-OE/EQB/LA0023/2013 and Fundação para a Ciência e Tecnologia (FCT) for Ana Oliveira PhD Grant (SFRH/BD/68588/2010) and Catarina L. Seabra PhD Grant (SFRH/BD/89001/2012). The authors would also like to acknowledge Professora Rosário Oliveira, which is no longer with us, for her exceptional contribution and dedication to this work.
Catarina L.Seabra,11,11,11,Cláudia M.Botelho,MarianaHenriques,Ana C. N.Oliveira,11, (2015) Influence of Saliva and Mucin on the Adhesion of Candida Oral Clinical Isolates. Journal of Encapsulation and Adsorption Sciences,05,217-227. doi: 10.4236/jeas.2015.54018