In vitro activity of zinc oxide-eugenol and glass ionomer cements on Candida albicans Atividade in vitro dos cimentos de óxido de zinco e eugenol e ionômero de vidro sobre Candida albicans

The aim of this study was to evaluate in vitro the antimicrobial activity of glass ionomer (GIC) and zinc oxide-eugenol (ZOE) cements against Candida albicans. Standardized GIC and ZOE specimens were maintained in contact with C. albicans suspension (1  106 cells/ml) at 37C for 24 h, 48 h or 7 days. A control group without any testing cement was included. After the incubation period, aliquots of 0.1 ml were plated on Sabouraud’s agar, and then the number of colonies was counted. The results were expressed as values of logarithms of colonyforming units per milliliter (log CFU/mL) and were analyzed statistically by Kruskal-Wallis ANOVA. After 48 h of incubation, the ZOE group presented no growth of C. albicans. GIC and control groups presented similar mean values at all tested periods. According to the results obtained, it could be concluded that, under the experimental conditions, ZOE cement was more effective in vitro against C. albicans than GIC. DESCRIPTORS: Candida; Candidiasis, oral; Glass ionomer cements; Zinc oxide-eugenol cement; Antimicrobial activity. RESUMO: O objetivo deste estudo foi avaliar in vitro a atividade antimicrobiana dos cimentos de ionômero de vidro (CIV) e óxido de zinco e eugenol (OZE) sobre Candida albicans. Corpos-de-prova padronizados de CIV e OZE foram mantidos em contato com suspensão (1  106 células/ml) de C. albicans a 37°C por 24 horas, 48 horas ou 7 dias. Um grupo controle sem nenhum cimento teste foi incluído. Após o período de incubação, alíquotas de 0,1 ml foram semeadas em ágar Sabouraud e o número de colônias foi contado. Os resultados foram expressos em logaritmos de valores de unidades formadoras de colônias por ml (log UFC/mL) e analisados estatisticamente pelo teste ANOVA Kruskal-Wallis. Após 48 horas de incubação, o grupo OZE não apresentou crescimento de C. albicans. Os grupos CIV e controle apresentaram médias similares em todos os períodos testados. De acordo com os resultados obtidos, pode ser concluído que, sob as condições experimentais testadas, o cimento OZE apresentou-se mais efetivo in vitro sobre C. albicans em relação ao CIV. DESCRITORES: Candida; Candidíase bucal; Cimentos de ionômeros de vidro; Cimento de óxido de zinco e eugenol; Atividade antimicrobiana.


INTRODUCTION
Candida genus yeasts are usually isolated from the human oral cavity 21 and their prevalence among healthy individuals varies from 35-38% to 40-60% 3,12 .C. albicans may be isolated from the mouth of 5-7% of newborns few hours after birth, and from 14.2% of them one week later 17 .C. albicans is the most prevalent species in the mouth (60 to 70% of the isolates), followed by C. tropicalis and C. glabrata 21 .
Under specific situations, Candida spp.may cause oral and systemic pathologies 6 .The transformation from the saprophytic to the parasitic form is related to microbial, environmental and individual factors 18 .The pathogenicity of Candida species is related to tissue invasion, enzyme production and to the adherence to the oral cavity's epithelium 2 .
Systemic and local predisposing factors to candidosis, such as immunodepression, xerostomia, hormonal alterations, use of orthodontic devices or total dentures have been reported in the related literature 10,21,22 .Among immunocompromised patients, especially in AIDS cases, candidosis is related to increased rates of mortality and morbidity 22 .
Oral environmental stabilization procedures are commonly employed in Dentistry.The aim of these procedures is the elimination of pathogenic microorganisms, preventing the progression of oral diseases and creating conditions for the improvement of oral health.Glass ionomer and zinc oxideeugenol cements are the most frequently employed cements for this purpose 7,16 .
The glass ionomer cement has a fluoride-releasing property that is desirable in the carious process control.Also, it presents good chemical adherence to the dental structure 19 .The zinc oxide-eugenol cement is also frequently used for provisional restorations due to its low cost and easy manipulation.
The studies of the antimicrobial activity of these materials have been generally performed against cariogenic microorganisms, in particular Streptococcus mutans and lactobacilli, and dental biofilm formation 1,7,8 .Chandler, Heling 4 (1995) reported that a zinc oxide-eugenol cement showed effective antimicrobial activity against Enterococcus faecalis after 24 hours.
Previous studies concerning the effect of endodontic sealers on Candida spp.growth showed contradictory results.Kaplan et al. 11 (1999) reported that fluoride-releasing materials did not show any activity against these yeasts.On the other hand, Palenik et al. 14 (1992) showed the antimicrobial effect of fluoride-releasing materials.A previous study 20 reported the relative effectiveness of an eugenol-containing endodontic sealer against Candida.
The information about the effect of dental materials on Candida spp.yeasts seems to be important for candidosis prevention purposes.Jacob et al. 9 (1998), studying candidosis among HIV-infected patients, observed that the elimination of carious dentine promotes the reduction of Candida spp.colonization sites, reducing the risk of infections caused by these microorganisms.
In our previous study 16 , oral environmental stabilization procedures employing a zinc oxideeugenol cement were more effective to reduce the levels of yeasts in saliva than those employing a glass ionomer cement.Based on these results and on the lack of conclusive studies, the aim of the present research was to evaluate in vitro the antimicrobial activity of glass ionomer and zinc oxideeugenol cements on C. albicans.
Immediately after the curing process that was standardized in 1 hour for ZOE and 5 minutes for GIC, according to the manufacturers' instructions, specimens were transferred to tubes (Corning, New York, USA) containing 3 mL of Sabouraud's dextrose broth (Difco, Detroit, USA).Then, 0.1 ml of FIGURE 1 -Representative illustration of the aluminum matrix employed for obtaining standardized specimens (diameter = 0.5 mm and height = 0.3 mm). the standardized C. albicans suspension was inoculated in each tube.Tubes were incubated at 37°C for 24 h, 48 h or 7 days according to the experimental group.Nine experimental groups were included in this study, as shown in Table 1.Three groups were composed of zinc oxide-eugenol (ZOE) specimens incubated for different periods of time.GIC groups included glass ionomer specimens incubated for 24 h, 48 h and 7 days.Control groups were incubated for different periods of time without any specimen of the tested materials.
After the incubation period, serial decimal dilutions (10 -1 to 10 -5 ) were obtained from each initial suspension in sterile saline solution (0.85% NaCl).Then, aliquots of 0.1 ml were plated in duplicate on Sabouraud's dextrose agar (Difco, Detroit, USA) and incubated for 37°C for 48 h.After this period, the number of colony-forming units (expressed in values of logarithms of colony-forming units) per milliliter (log CFU/mL) was obtained.Values of mean, standard deviation and median of log CFU/ mL were calculated for each experimental group.

Statistical analysis
The results obtained after 24 h of incubation were analyzed statistically by Kruskal-Wallis ANOVA and Dunn's test (5%).Considering that the counts obtained for zinc oxide-eugenol after 48 h and 72 h of incubation were zero, the results of the glass ionomer cement and control groups at these periods were analyzed by two-way ANOVA and Tukey's test (5%).

RESULTS
The values of mean, standard deviation and median obtained for the groups at each period are shown in Table 2.
Counts of Candida albicans after 24 h were lower and statistically different (p = 0.000) in the ZOE group in relation to GIC and control groups.No differences were observed between GIC and control groups at any experimental period.
After 48 h of incubation, the ZOE group presented no growth of C. albicans.GIC and control groups presented similar mean values (p > 0.05).The same results were observed after 7 days of incubation.

DISCUSSION
Preventive antifungal therapy has been adopted to avoid the occurrence of systemic candidosis.However, this practice may contribute to the appearance of resistant isolates.In fact, some authors 13,23 correlate the widespread use of azolic drugs in the prevention of systemic mycoses in patients with low immunity (i.e., transplant recipients or HIV-infected patients) with the selection of resistant isolates.Therefore, other concomitant preventive measures could be of great importance for patients under risk of systemic candidosis development.Oral Candida spp.level control might be an important preventive measure since the occurrence of oral candidosis may be considered a potential risk for the occurrence of systemic diseases among immunocompromised patients.However, few studies of this subject have been reported in the related literature.
Oral stabilization procedures may contribute to Candida spp.level control.A previous study 9 showed that the elimination of dentine affected by carious process reduced the colonization sites of Candida spp., reducing the risk of infection by these yeasts.Considering that zinc oxide-eugenol and glass ionomer cements are the most frequently employed materials in oral stabilization procedures, they were included in this study.
The findings of the present study showed that the zinc oxide-eugenol cement was more effective in reducing Candida spp.colony counts than the glass ionomer cement.These results may be related to the high antimicrobial activity demonstrated by eugenol-containing materials.Previous literature reports on eugenol-containing materials showed their significant antimicrobial effect on Candida spp.Siqueira Júnior et al. 20 (2000), testing the antimicrobial activity of root canal sealers, showed that an eugenol-containing sealer presented relative effectiveness against Candida spp.However, this activity was observed only after a long interval of time (40 days).Also, Kaplan et al. 11 (1999), evaluating the antimicrobial effect of endodontic sealers, showed that eugenol was effective against Candida spp.
The lack of activity of the glass ionomer cement against Candida spp.may be related to the fluoride concentration released by this material.These results are similar to those showed by Ka-plan et al. 11 (1999).These authors, analysing fluoride-releasing endodontic sealers did not observe any inhibitory effect on Candida growth.These authors attributed this lack of activity to the low fluoride concentration in the studied materials.Some methods have been suggested for testing the antimicrobial effect of dental materials.The most frequently employed methods are those based on direct contact test (DCT) 1,6,15 .However, no standardization among methods is found in the literature.The agar diffusion test was also performed in previous studies 5,19 , although the differences between the diffusion velocities that are characteristic for each material seem to be a limitation for comparative purposes.
The methodology applied in this research was based on DCT 1,6,15 and focused on the standardization of the experimental conditions, in particular in relation to the specimens' dimensions and C. albicans suspension.
Our previous clinical study 16 showed that oral environmental stabilization procedures using zinc oxide-eugenol cement were effective to reduce the levels of Candida spp. in saliva.The present in vitro results corroborate these findings suggesting that the procedures employing zinc oxide-eugenol may be indicated for oral candidosis prevention purposes.

CONCLUSIONS
At all periods of time evaluated (24 h, 48 h and 7 days), the zinc oxide-eugenol cement showed effective antimicrobial activity on Candida albicans.The glass ionomer cement did not show any effect on the growth of Candida albicans.

TABLE 1 -
Experimental conditions included in the study.