Kinetics of antifungal activity of home-generated ozonated water on Candida albicans

Background and Purpose: Candida-associated denture stomatitis is one of the most common forms of oral candidiasis among denture wearers. Regarding this, the aim of the present study was to evaluate the antifungal effects of home-generated ozonated water on the adhesion of the C. albicans attached to the surface of the denture base acrylic resins. Materials and Methods: For the purpose of the study, different concentrations of C. albicans were added to the tubes containing acrylic resin blocks, and then incubated for 2 h at 35°C. The samples were assigned into three groups, each of which contained 42 samples, including normal saline (NS) solution as the negative control, nystatin (N) solution as the positive control, and ozonated water as the test group. The samples were washed and placed in an ultrasonic bath. Subsequently, the saline solution was cultured on Sabouraud dextrose agar. The concentrations of Candida were evaluated during the contact times. Results: The test group (i.e., ozonated water) with 114 colony-forming units (CFU) showed a significant reduction of Candida colonies, compared to the NS group with 2,172 CFU. The 120- and 1-minute incubation with ozonated water showed the highest and lowest effects on the viability of Candida adhered to the acrylic resin, respectively. Conclusion: Based on the findings, home-generated ozonated water can be applied to remove the Candida attached to the surface of the denture plates.


Introduction
se of dentures may alter the oral environment and provide a favorable condition for the proliferation and colonization of microorganisms, especially Candida-associated denture stomatitis [1]. This condition is characterized by the moderate to severe inflammation of the mucosa underneath the denture material. Some factors may influence the development and severity of denture stomatitis. These factors includes the extent of denturebase fit, patient's age and systemic conditions, poor diet and hygiene, and salivary flow [2].
Candida species, especially C. albicans, have a high affinity to adhere to and colonize on the acrylic resin denture base material, particularly rough surfaces [3,4]. Therefore, it is inevitable to control denture plaque to prevent the Candida-associated denture stomatitis. The microbial biofilms, especially those created by fungal agents, can be controlled by mechanical, chemical, or combined mechanochemical methods. Several chemicals are introduced for denture disinfection; however, they have their own drawbacks, such as high cost and exertion of deteriorative effects on the denture base material [5,6].
Ozone (O3), an allotrope of oxygen, is recognized as a strong oxidative antimicrobial agent. Ozonated water (OW) has been considered as an effective disinfectant agent against oral pathogens, including Candida [7,8]. Ozone is a versatile substance in dentistry [9] and can be used in caries control [10], root canal disinfection [11], avulsed teeth disinfection before re-implantation [12], surgery as a hemostatic agent, postsurgical wound healing [13], sterilization of implant and bone surface, and tissue regeneration stimulation at implantation site [14].
Ozone also has a high potential to be used as an antimicrobial agent in endodontics. Based on the evidence, ozonation can be beneficial if the ozone is utilized with adequate concentration, suitable time, and correct method for reaching root canals when the traditional cleansing, shaping, and washing of canals are completed. The potentiality of ozone, OW, and ozonated oil to be used in endodontic therapy has been frequently reported in the literature [15]. It has been shown that OW can be mutagenic if used for a long period and in high concentrations [7]. Home ozone generators are portable, inexpensive, and user-friendly devices for producing ozone gas for home use, such as disinfection of water, vegetables, or air. However, the capacity of home ozone generators is lower than that of the industrial and medical types. If the OW produced by home ozone generators shows antifungal activity against Candida, it can be introduced as an inexpensive and easy method for denture cleaning/disinfection to prevent the denture stomatitis. With this background in mind, the present study was conducted to evaluate the antifungal properties of OW produced by a commercialgrade ozone generator.

Fabrication of acrylic resin blocks
The bar-shape acrylic resin specimens (25×10×3 mm) were fabricated using a conventional flasking and pressure-pack technique [7]. Silicon-made patterns having the mentioned dimensions were embedded in type-III dental stone in a brass flask. The flasks were separated by the stone set, and the silicon molds were removed. Subsequently, the spaces were filled with a commercial heat-curing acrylic resin denture base material (powder/liquid: 3/1 v/v; Marlik, Marlik Medical Industries Co., Tehran, Iran).
The polymerization was accomplished in boiling water for 20 min based on the manufacturer's instruction and remained in water at ambient temperature for 24 h. The flask parts were separated, and the specimens were removed, cleaned from remaining stone, and then well-polished. Each specimen was washed vigorously under tap water, put in a test tube, and finally autoclaved at 121°C for 15 min.
In the next stage, each tube containing the specimen block was sealed and incubated for 2 h at 35°C. After the incubation, the blocks were washed three times with saline solution to remove the unattached Candida. Then, the samples were assigned into three groups, each of which containing 42 samples. Each group was further divided into seven sub-groups (n=6) to examine the effect of time on the antifungal activity. The study groups were as follows:  Group of normal saline: normal saline solution was used as a negative control. All tubes were filled with 5 ml saline solution.  Group of nystatin: nystatin solution (10.000 U/mL; Emad, Emad Darman Pars Co., Saveh, Iran) was used as a positive control (according to several reports) [18,19].  Group of OW: OW was used as the test group. The OW was prepared by a home air ozone generator (Sterile Air, ARDA, Tehran, Iran). The output tube was placed in a glass beaker containing 1.5 L deionized distilled water for 2 h. After various periods of time (i.e., 1, 5, 10, 20, 40, 60, and 120 min), the content of the tubes in each group was discharged, and the specimens were washed vigorously; subsequently, 5 ml fresh saline solution was added to each tube. All tubes were placed in an ultrasonic bath (DT 510, Bandelin Electronic, Germany) containing 30°C distilled water for 30 min to detach the Candida from the block specimens [20]. Then, from each tube, a volume of 10 µL saline was added to petri dishes containing SDA medium and incubated for 48 h at 35°C. Finally, the number of Candida colonies was counted in each group.

Statistical analysis
The relationship between the mean colony count in each group and time was estimated using a non-linear regression model. All statistical procedures were performed in SPSS software (version 16). P-value less than 0.05 was considered statistically significant. Table 1 shows the descriptive results for the mean colony count of Candida at different incubation times in each group. According to the results, Candida CFU had a positive relationship with the number of colonies attached to the surface of the specimens. Regardless of the primary Candida CFU, no fungal colony was grown in the presence of nystatin. Furthermore, Candida colonies in the normal saline group demonstrated no significant changes.

Results and Discussion
The OW group showed an inhibitory effect on the growth and colonization of C. albicans; therefore, the null hypothesis was rejected. In addition, the Candida CFU demonstrated the non-linear goodness of fit (r 2 >0.93, P<0.001) to the time ( Figure 1C) in these groups. Moreover, the changes in the mean Candida CFU showed a negative non-linear quadratic relationship with time from 40 to 120 min (r 2 =0.88, P<0.001) ( Figure 1D).
In Candida CFU of 2×10 5 , the mean Candida colony counts diminished between 40 and 60 min, while in Candida CFU of 2×10 6 , it took about 80 min to inhibit the colony growth. In lower suspensions of Candida CFU (2×10 3    growth was almost inhibited after 40 min (Table 1 and Figure 1). The aim of this study was to evaluate the antifungal activity of the ozonated water solution produced by a home ozone/water generator as a simple and inexpensive method for the disinfection of denture base acrylic resin. The results showed that the application of home-generated OW was effective in the removal of Candida attached to the surface of the denture plates.
Salerano et al. reviewed the Candida-associated denture stomatitis phenomena and recommended the correct oral hygiene for the control and prophylaxis of the Candida-associated denture stomatitis [5]. Among Candida species, C. albicans is reported as the most common causative agent of different Candida infections, especially for developing the denture stomatitis [5,[21][22][23][24][25].
According to the literature, the growth of Candida, especially C. albicans, on the denture surface is affected by several factors. These factors include denture roughness [4], salivary flow and proteins [1], oral cavity pH [1], permeability of denture base material [26], oral hygiene, and systemic conditions, such as diabetes mellitus and xerostomia [27]. Various disinfection methods, including chemical agents or microwave irradiation, have been evaluated to remove the yeast plaques on the surfaces of the denture disinfection. Nonetheless, these methods have their own drawbacks, such as costliness and exertion of deteriorative effects on the denture base material [22,28].
In the current study, the acrylic resin blocks were washed and moved to the ultrasonic device after certain periods of incubation in Candida suspension to detach the colonies attached to the material surface. Therefore, the number of isolated colonies at each time represented the Candida population survived on the surface of denture base material in the presence of OW. The results of our study showed that the homegenerated OW could inhibit the growth of C. albicans on the denture base material.
Previous studies have also shown the antimicrobial activity of OW against C. albicans in root canal system and acrylic denture base material [7]. Cardoso et al. [28] reported that OW decreased the number of C. albicans colony immediately after exposure. Arita et al. [7] showed that the number of C. albicans significantly decreased during the first 10 min and approached to zero after 30 min of the immersion of acrylic denture base material in OW. On the other hand, it is indicated that the poorer denture hygiene is associated with more oral colonization of Candida [29]. High levels of opportunistic microorganisms in the saliva have been recognized as an important predisposing factor for Candidaassociated stomatitis [30].
In this study, different concentrations of Candida suspension (denoted as the primary Candida CFU), representing the patient's oral hygiene, were used to evaluate the effect of infection load on the disinfection strength of OW. Based on the findings of the present study, higher concentrations of Candida can affect the colonies number growing on the denture base material. The relatively high inhibitory effect was achieved according to different primary Candida CFUs.
As a result, the use of OW method could rapidly decrease the Candida CFU during the first 20 min of storage. This value even reached to zero in lower Candida CFUs after 40 min. However, in higher Candida CFUs, it was able to reach to zero after 120 min. Therefore, the time recommended for the immersion of denture in OW depends on the patient's oral hygiene or other factors [5,31,32] affecting the load of infection.
One of the limitations of this study was the sole examination of C. albicans; however, this species is most commonly associated with denture stomatitis among the Candida species. Although home ozone generators have a lower capacity for ozone production for antifungal applications than industrial devices [7,28], they are accessible and effective.

Conclusion
As the findings of the present study indicated, the home-generated OW could inhibit Candida growth and colonization on the acrylic denture base material. Consequently, the home ozone/water generators can be used as inexpensive, portable, and effective storage medium for the disinfection of dentures, especially in high-risk patients.