Virulence factors of Candida species from the oral mucosa and prostheses of elderly people from a riverside community in the Amazon state, Brazil

; mucosa oral; prótese dentária; virulência. Abstract Introduction: Candida albicans is the yeast most commonly affecting the oral cavity, sometimes causing infection. However, several factors may be associated with the onset of candidiasis, which may be related not only to the hygiene and health of individuals, but also to the pathogenicity of these microorganisms. Objective : To evaluate the virulence factors of Candida yeasts isolated from mucosa Amazonas state, Brazil. Material and method : Testes were performed to assess the production of urease, proteinase, phospholipase and hemolysin. Statistical analysis used the Fisher's test. Result : Prevalence of non-albicans species was observed. As for virulence factors, all isolates were negative ureases, and there was prevalence of very strong proteinase production, whereas most isolates did not produce this enzyme in the phospholipase test. All yeasts analyzed presented hemolysin production, with grade IV hemolysis as the most prevalent. There was no statistically significant difference the oral cavity and the prostheses of the elderly analyzed. Conclusion : Several virulence factors may present with high intensity in the presence of oral microbiota changes. In addition, non-albicans species present number of virulence factors similar to that of C . albicans , with high pathogenicity. This study allows a better analysis of candidiasis prevention strategies aiming to promote improvement in the health and quality of life for the elderly.


INTRODUCTION
Several physiological and metabolic changes of systemic order and in the oral cavity occur during aging. The use of dental prostheses that favor the colonization of C. albicans by altering the oral microbiota lead to the emergence of candidiasis [1][2][3][4] .
Candidiasis is an infectious process caused by fungi of genus Candida. This microorganism has a commensal relationship with the host, but when there is an imbalance in this flora, it can become pathogenic, affecting approximately 10% of elderly people with poor health. Candida albicans is the species most commonly associated with infection, accounting for over 80% of the fungi isolated from the oral cavity 5,6 .
Manifestations of oral candidiasis are classified as acute or chronic, with the latter being found more frequently in the elderly. The acute manifestation is pseudomembranous and erythematous, whereas the chronic manifestation presents lesions such as chronic hyperplasia, prosthetic stomatitis, medial rhomboid glossitis, angular cheilitis, and hairy tongue 5,6 .
Prosthetic stomatitis is an infectious process that affects the palatal mucosa in close contact with the prosthesis. It has a multifactorial etiology and may also be associated with poor hygiene of the prostheses with consequent biofilm accumulation 7 .
However, not only host-related factors can cause candidiasis, but also the virulence factors of these microorganisms, including secretion of hydrolytic enzymes (phospholipases and proteinases), toxins, hemolysins, and adhesion and biofilm formation capacity [8][9][10] .
Colonization by fungi of the genus Candida in the human oral microbiota may cause some diseases when the patient immune system is weakened, resulting in local or even systemic impairment.
These facts demonstrate the clinical relevance of the analysis of the main virulence factors of Candida species, highlighting the knowledge of these mechanisms involved in the pathophysiology of candidiasis, which may provide prevention and treatment strategies.

MATERIAL AND METHOD
Forty isolates of Candida yeast from the palate and prostheses of elderly people residing in the "Comunidade Lago do Limão", municipality of Iranduba, Amazonas state, Brazil, were used in this study. The samples were collected for a master's thesis entitled Oral health conditions of the elderly population in a rural community of Amazonas state (process no. CEP 61618016.0.0000.5020) and transferred to the Amazon Fungi Collection of "Instituto Leônidas e Maria Deane" (CFAM-ILMD-FIOCRUZ-Amazonia). The isolates were previously identified by CHROMagar Candida ® chromogenic medium (BioMerieux, France), a presumptive method for identifying Candida, and stored under refrigeration at 20 °C in Saboraud Agar (K25-610103-Kasvi, Italy) with glycerol as a wall protector.
The virulence factors of these yeasts were evaluated through production of urease, proteinase, phospholipase and hemolysin tests. For the urease test, isolates were reactivated and seeded in Christensen 11 agar medium (Urea Agar) and soon after incubated at 37 °C with daily observation for seven days. A change in medium color to fuchsine-red is indicative of a positive result, otherwise the culture is negative. After seven days of incubation, the result was read.
The study of proteinase production was performed according to the methodology described by Teixeira et al. 12 , in which the test medium consisted of sterile plates containing agar, gelatin and skimmed milk, diluted in Citrate-Phosphate Buffer solution (pH 5.0).
Phospholipase production was analyzed according to the egg yolk plate method described by Price et al. 13 with modifications, where the medium consisted of Sabouraud Dextrose Agar plus sodium chloride, calcium chloride, and yeast extract. After sterilization of the medium, 30% egg yolk emulsion was aseptically added.
For enzymatic analysis, yeast fragments with 24 h of growth were inoculated on the surface of the specific medium for each enzyme investigated, and incubated for seven days at 37 ºC. Enzyme production was observed by the formation of transparent and opaque halos around the yeast colony for proteinase and phospholipase, respectively.
The tests were performed in triplicate and the precipitation zone value (Pz) was given as the mean of the evaluated diameters (colony/halo+colony). Production was classified according to the Pz value as very strong ++++ (Pz≤0.69), strong +++ (Pz between 0.70 and 0.79), average ++ (Pz between 0.80 and 0.89), or weak + (Pz between 0.90 and 0.99).
The hemolysin production test was performed according to the method by Malpezzi et al. 14 ; Berlinck et al. 15 ; Costa-Lotufo et al. 16 with modifications as follows: sheep blood was diluted in saline (0.85% NaCl + 10mM CaCl2), washed three to four times in saline, and then centrifuged (1500 rpm/10 min). An erythrocyte pellet was obtained and resuspended in saline to prepare the 2% erythrocyte solution (ES). The test was performed using 96-well microplates containing 150 µL of yeast solution and an additional 150 µL of ES. The negative control was obtained with 0.2% DMSO-Dimethyl sulfoxide, whereas the positive control was obtained with 0.2% Triton X-100.
After this process, the samples were incubated for 1 hour under constant agitation at room temperature (26 °C) and then centrifuged at 1500 rpm/10 min. Reading of the results was classified according to the hemolysis intensity compared with the control as grade I (without hemolysis), II (weak), III (strong), and IV (very strong) hemolysis.
Data were processed using R 3.6.0 and Rstudio 1.1.4, with various packages (tidyverse, epiDisplay, gridExtra, and sjPlot) 17 . A significance level of 0.05 was adopted in all statistical analyses.
The Fisher's Exact and Chi-squared tests were used to analyze the relationship between the "Experiment Location" and the qualitative (categorical) variables.
As test hypotheses, we used H0: the variables are independent; H1: the variables are dependent. Decision Rule: Reject H0 if p-value is <0.05.

RESULT
The 40 yeasts analyzed (21 from samples collected from the mouth (palate) and 19 from the prostheses) in the elderly from "Comunidade Lago do Limão", municipality of Iranduba, showed prevalence of 80% Candida non-albicans species, and 21% of isolates originating from the palate of the elderly analyzed (Table 1). The results presented in Table 1 showed no evidence according to the p-values (none <0.05) found to reject the independence hypothesis.
In the virulence tests, the isolates did not present urease production. However, analysis of the production of hydrolytic enzymes showed prevalence of isolates collected from the mouth of the elderly with positive results for proteinase (57.1%), being classified as very strong, whereas prevalence of negative results (71.4%) was observed in the phospholipase test.
In this study, all isolates of Candida sp. found hemolysin production, with prevalence of grade IV hemolysis in samples from the prostheses of the elderly people (84.2%).
The species with the highest production of proteinases was C. glabrata, followed by C. krusei. In the phospholipase test, of the isolates that tested positive, C. parapsilosis showed the highest production. However, in the hemolysin test, all species analyzed had hemolytic potential, especially C. parapsilosis and C. krusei as major causes of grade IV hemolysis. Comparisons between the virulence tests and the Candida species found in the study are depicted in Figures 1, 2 and 3, respectively.

DISCUSSION
Despite the fact that Candida albicans is the yeast species that most affects the oral cavity, thus being the most pathogenic, the present study revealed a predominance of pathogenicity among non-albicans species, in line with the study conducted by Mohandas, Ballal 18 with hospitalized patients in southern India.
Some pathogenic fungi are able to produce urease to use urea as a source of nitrogen, but as a result of the breakdown reaction of urea, ammonia, a toxic substance to host cells, can cause the immune system to evade and, consequently, promote infection. A study by Seeliger 19 tested the 14 Candida species to identify urease production, but only one Candida humicola culture showed a positive result. However, studies have stated that most yeast species responsible for causing pathogens in humans are negative urease 20 .
Other virulence factors that Candida species may exhibit are the production of proteinases and phospholipases that facilitate the invasion of hyphae into the epithelium. In the case of proteinase, host protein degradation occurs by hydrolysis of peptide bonds, both those located on the mucosal surface and those related to the immune system (immunoglobulin, complement system proteins, and cytokines); and in the presence of phospholipases, phospholipid hydrolysis occurs resulting in the disintegration of cell membranes 8,9,21 .
Results of this study corroborate those by D'Eça et al. 21 , who found that 50% of the yeasts showed proteinase activity. However, in the study by Andreola et al. 9 , approximately 97% of Candida spp. were positive for proteinase. Regarding Candida species, studies 21 have shown prevalence of 64.2% of very strong proteinase activity by Candida albicans isolates, and even proteinase production by 100% of C. albicans isolates, whereas only 25% of isolates from non-albicans species obtained positive results 22 .
In the phospholipase test, there were difference between the results obtained in the study and those by D'Eça et al. 21 , who reported activity in 68.3% of Candida spp. and 76.6% prevalence of C. albicans, as well as those by Martins et al. 23 , who verified that all isolates were phospholipase positive; however, only 43% of the non-albicans species presented phospholipase production. In contrast, the results of this study were similar to those observed by Mohandas, Ballal 18 , who found higher phospholipase production in non-C. albicans species, differing only in their prevalence -44.1% of the isolates.
Nevertheless, although C. albicans is the most pathogenic species, studies have shown a considerable increase of non-albicans species involved in infectious processes, which may lead to systemic infection in debilitated patients 8 .
Candida fungi may also present hemolytic capacity, aiming to obtain iron from the host tissues for their metabolism through erythrocyte lysis, with hemoglobin being of great importance in this process. The oral cavity is rich in iron from both lactoferrin (a protein present in saliva) and ferritin (intracellular), facilitating the invasion of hyphae and possibly leading to systemic candidiasis 22 .
Regarding the hemolysin production test, the study by Rossoni et al. 22 indicated predominance of 100% Candida albicans in the production of this protein, thus being an important virulence factor responsible for causing systemic infections. However, the comparison between the hemolytic activity developed by non-albicans species and C. albicans showed no statistically significant difference, being characterized with the same hemolytic potential. Prevalence of hemolysin-producing non-albicans species was observed by Pakshir et al. 24 in a study with clinical analyses of Candida species.
In all associations tested in Table 1, no statistical dependence was found between the virulence factors and the fungi collected from the oral cavity and prostheses of elderly people from the "Comunidade Lago do Limão", municipality of Iranduba, Amazonas state, Brazil, thus accepting the H0 hypothesis, that is, the variables are not dependent.

CONCLUSION
Based on the methodology used, we conclude that Candida yeasts have several virulence factors that may present with high intensity in the presence of oral microbiota changes. Regarding the comparison between the Candida species analyzed, it was found that non-albicans species present number of virulence factors similar to that of C. albicans, with high pathogenicity, and may even resemble them with respect to infectious potential.
The study of host-related factors, as well as virulence factors in Candida species, allows a better analysis of candidiasis prevention strategies in order to promote improvement in the health and quality of life of these elderly people.