Action of disinfectant solutions on adaptive capacity and virulence factors of the Candida spp. biofilms formed on acrylic resin

Abstract Understanding the behavior of Candida spp. when exposed to denture disinfectants is essential to optimize their effectiveness. Changes in the virulence factors may cause increased resistance of Candida spp. to disinfectant agents. Objective To evaluate the microbial load, cellular metabolism, hydrolytic enzyme production, hyphae formation, live cell and biofilm quantification of Candida albicans, Candida tropicalis and Candida glabrata after exposure to disinfectant solutions. Methodology Simple biofilms were grown on heat-polymerized acrylic resin specimens, and divided into groups according to solutions/strains: distilled water (control); 0.25% sodium hypochlorite (NaOCl 0.25% ); 10% Ricinus communis (RC 10%); and 0.5% Chloramine T (CT 0.5%). The virulence factors were evaluated using the CFU count (microbial load), XTT method (cell metabolism), epifluorescence microscopy (biofilm removal and live or dead cells adhered), protease and phospholipase production and hyphae formation. Data were analyzed (α=0.05) by one-way ANOVA/ Tukey post hoc test, Kruskal-Wallis test and Wilcoxon test. Results NaOCl 0.25% was the most effective solution. CT 0.5% reduced the number of CFUs more than RC 10% and the control. RC 10% was effective only against C. glabrata. RC 10% and CT 0.5% decreased the cellular metabolism of C. albicans and C. glabrata. Enzyme production was not affected. Hyphal growth in the RC 10% and CT 0.5% groups was similar to that of the control. CT 0.5% was better than RC 10% against C. albicans and C. tropicalis when measuring the total amount of biofilm and number of living cells. For C. glabrata, CT 0.5% was equal to RC 10% in the maintenance of living cells; RC 10% was superior for biofilm removal. Conclusions The CT 0.5% achieved better results than those of Ricinus communis at 10%, favoring the creation of specific products for dentures. Adjustments in the formulations of RC 10% are necessary due to efficacy against C. glabrata. The NaOCl 0.25% is the most effective and could be suitable for use as a positive control.


Introduction
The complete denture is made with acrylic resin and widely used to replace all teeth, restoring function, aesthetics and comfort to the patient. However, acrylic resin in the oral environment favors the adherence of oral debris, bacteria, and fungi. Thus, there is a direct risk relationship between denture use, biofilm and the development of denture stomatitis ( parapsilosis or C. krusei) as infectious agents, which have different adaptive capacity, different susceptibility to antifungal agents 3,5 and potential for adhesion on acrylic surfaces. 6,7 Therefore, the relative value of cell surface hydrophobicity and the biofilm biomass of non-albicans Candida species is greater than C. albicans, considering that 92% of the non-albicans Candida species of oral isolates had the capacity to form biofilm against only 78% of C. albicans. 8 This fact indicates the need for a comprehensive approach to non-albicans species in individuals wearing dentures.
The adaptive capacity of Candida is influenced by virulence factors, such as the adhesion, biofilm formation, microbial load and cell viability, cellular metabolism, hydrolytic enzymes production, and phenotypic changes with formation of hyphae. 9,10 Change in the adaptive capacity is evident when infection is recurrent after therapy with antimicrobial agents, indicating resistance of the microorganism 1 and limiting treatment options. Infection control is even more difficult for denture users with inadequate hygiene and the presence of biofilm is clinically significant. Thus, disinfectant solutions have been proposed for the prevention of DS and for microbiota control 1,12,13 to reduce the need for systemic antifungal use.
Sodium hypochlorite (NaOCl) has proven antimicrobial activity 1,13-17 and has been recommended as a disinfecting agent for dentures by the American Dental Association (ADA) 18 . Satisfactory results have been reported with NaOCl at 0.25% concentration, which maintained the antimicrobial action, the ability J Appl Oral Sci. 2021;29:e20210024 3/11 significantly from each other; however, they would differ from the negative control.

Methodology
Specimen preparation and experimental design H e a t-p o l y m e r i z e d a c r y l i c r e s i n ( A r t i g o s Odontológicos Clássico Ltda., São Paulo, SP, Brazil) was mixed, packed in circular molds (13×3 mm) and conventionally polymerized. 17 The flask was placed in room temperature water, which reached 65°C in 1 hour. Thereafter, the temperature was raised to 100°C in half an hour and held for 1 hour, and then lowered to room temperature. The specimens were finished using rotary instruments and 180-grit abrasive paper in a polishing machine (Arotec, Aropol E, Cotia, SP, Brazil).
To simulate the inner surface of the denture, the surface roughness (Ra) was standardized between 2.7 and 3.7 µm 28 with a profilometer (Surftest SJ-201P, Mitutoyo Corporation, Kawasaki, Japan). Three readings were made, one in the central portion and two with 2 mm to the right and left (5 "cutoff" of 0.8 μm) of the center. The specimens were randomly divided into 12 groups (n=24)  In each group, nine specimens were used to assess microbial load (CFU), enzymatic activity and hyphae formation; nine to assess metabolic activity (XTT); and three for fluorescence microscopy. To confirm the sterility of the procedures and materials, three sterilized specimens were put in sterilized culture medium, and, to confirm biofilm formation, another three specimens contaminated by Candida spp. and not immersed in disinfection solution were used. Before biofilm formation, the specimens were sterilized by irradiation in a microwave oven (650W; 6 minutes). 30 The experiments were performed in triplicate on three different occasions. is the absolute value of dilution (0, 1, 2, or 3) and q is the quantity (mL) pipetted for each dilution at inoculation (0.05 mL). After 24 hours of incubation, the aliquots were used in hydrolytic enzyme and hyphae formation assays.

Evaluation of cell metabolism
The cell metabolism was evaluated by XTT assay [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)- Evaluation of enzyme production and hyphae formation After the biofilm had formed for 48 hours, the specimens were exposed to disinfectant solutions and transferred to the Letheen culture medium, and the tube was incubated for 24 hours. Next, the cellular concentration was counted in a Neubauer chamber.

Microbial load and cell metabolism
The NaOCl 0.25% reduced to zero the CFU count and the cellular metabolism of all Candida species.
CT0.5% was more effective in decreasing the CFU count when compared with RC 10% and the negative control. RC 10% differed from the distilled water (negative control) only against C. glabrata ( Figure   1). RC 10% and CT 0.5% decreased the cellular metabolism of C. albicans and C. glabrata in a similar manner, but, for C. tropicalis, CT 0.5% was more effective (Figure 2).

Enzyme production and hyphae formation
Only C. glabrata showed increased proteinase production after immersion in all solutions tested when compared with negative control (Figure 3). No difference was found in phospholipase production by Candida spp. after immersion of the specimens in the different disinfectant solutions (Figure 4). The NaOCl 0.25% reduced hyphal growth to zero. Hyphal growth in the other groups of solutions was similar to that in the control ( Figure 5). Even though they were statistically similar, the values of CT0.5% were lower than those of the RC10%.

Quantification of total biofilm and living cells adhered
NaOCl 0.25% showed the best results. CT 0.5% was better than RC 10% against C. albicans and C.
tropicalis for the total amount of biofilm and number of living cells. For C. glabrata, CT 0.5% was equal to RC 10% in the maintenance of living cells, where as RC 10% was better for biofilm removal (Table 1).

Discussion
In this study, ATCC strains were used to reduce the great diversification of factors influencing the analyzed variables, such as the expressive variability among organisms (hosts/ human physiology). As well as between clinical strains of the same species of Candida, which can show differences in the cell surface hydrophobicity and adhesion, 7,32 contributing in several states of disease in the human host, since superficial until and systemic infections. 6 Thus, this study cannot provide a representation of the microorganisms found in the oral cavity, due to its non-clinical origin.
However, it can be considered the first step towards an in-depth understanding of the mechanisms that permeate the relationship between Candida spp. and * Sodium hypochlorite (NaOCl 0.25%) reduced the number of metabolic activity to zero; ** Kruskal-Wallis, stepwise step-down post-test; AB Equal capital letters indicate statistical similarity between groups.   with a study that corroborates the results found in this study 12 and other that indicates promising results. 13,16,17 R. communis had a minimum inhibitory concentration of 0.0781% for C. albicans and C. glabrata and, when incorporated into a dentifrice, had a significant effect against bacteria (S. mutans; B. subtilis) that are important in biofilm formation. 22 The contrasting results *Kruskal-Wallis test; ** 0.25% sodium hypochlorite reduced the number of cells in the form of hyphae to zero.   may come from differences in the susceptibility of the microorganisms when in contact with the disinfectants or from methodological differences. The effects of R.
communis after long periods of immersion (overnight, 8 hours) could differ, and mixed biofilms could also lead to different results. Therefore, additional studies are necessary once R. communis is a natural products and it can be one therapeutic alternatives in the treatment of denture stomatitis 1,13 with low-cost and lower adverse effects as compared to antifungal drugs. CT 0.5% was better than RC 10%, and the negative control in terms of decreasing microbial load. Verardi, et al. 25 (2016) also reported a reduction of CFU/mL with titanium specimens after using the Chloramine T solution, which destroys the cellular material or disrupts important cellular processes of the microorganisms by oxidative reactions. 27 Microorganisms do not become resistant to Chloramine T as can happen with antibiotics and is effective even at low concentrations. 27 The data on cellular metabolism confirmed the findings on microbial load. The NaOCl 0.25% had 100% effectiveness for both variables, and these results were The solutions did not change the hydrolytic enzyme production (proteinase and phospholipase) from C. albicans or C. tropicalis. One of the enzymatic functions is to allow the fungus to invade and cause damage to tissues. 10 The maintenance of the production rates of these enzymes was similar to that of the negative control (distilled water) when C. albicans and C. tropicalis were exposed to hygiene solutions. This result can be considered good, since the solutions did not increase the pathogenicity of these species in the concentrations and immersion period: used in this study. However, C.
glabrata showed an increase in proteinase production when exposed to all solutions. This result suggests a greater specific capacity of this species to react to a given Action of disinfectant solutions on adaptive capacity and virulence factors of the Candida spp. biofilms formed on acrylic resin stimulus. 33 This characteristic can influence the survival or persistence of the microorganism between an initial exposure to an antimycotic agent and the acquisition of mutations that confer resistance, an adaptive response.
C. glabrata has a complex population structure, with genomic variants that may arise during the process of adaptation to environmental changes and persist over time, giving this species a greater pathogenicity. 11 Different results were reported by et al. 34 (2011), who stated that oral samples of Candida spp.
According to Gümrü, et al. 35 (2006)  Regarding biofilm removal, RC 10% reduced the amount of all species when compared with the negative control, but CT 0.5% was more effective against C. albicans and C. tropicalis, and less effective against C.
glabrata. This may be because of the lack of hyphae in this species, which is thus more easily eliminated by the detergent action of R. communis. Sánchez-Vargas, et al. 40 (2013) demonstrated that biofilm formation is dependent on Candida spp. and that, although C. albicans is more prevalent, biofilm production was higher in C. glabrata isolates, followed by C. tropicalis, C. albicans and C. krusei. These findings reinforce the importance of research such as the current study involving less prevalent species, but with a high potential for virulence. Conclusions NaOCl 0.25% was most effective than the other solutions in reduction of CFU count, cell metabolism, hyphae growth, living cells of all Candida species and biofilm removal. All solutions have not changed the enzymes productions by C. albicans and C. tropicalis, but NaOCl caused increased in proteinase production by C. glabrata. CT 0.5% was effective in decreasing of CFU count of C. tropicalis and cell metabolism of C. albicans and C. glabrata. RC 10% reduced only the CFU count of C. glabrata, but decrease the cellular metabolism of C. albicans and C. glabrata.
CT0.5% was better than RC10% in the biofilm removal and decrease of living cells of C. albicans and C. tropicalis, whereas RC 10% was more effective in the biofilm removal of C. glabrata. CT 0.5% and RC 10% were similar for the number of C. glabrata living cells and hyphal growth of C. albicans. In general, the Chloramine T at 0.5% achieved better results than Ricinus communis at 10%, favoring the creation of specific products for denture users. Adjustments in the formulations of RC 10% are necessary.