Effects of Probiotic Bacteria on Candida Presence and IgA Anti-Candida in the Oral Cavity of Elderly

Yeasts of Candida genus are normal constituents of the human flora, found as commensal on skin, genitourinary and gastrointestinal tracts (1). These microorganisms can be isolated in the oral cavity of 20% to 80% of healthy individuals (2,3). However, some factors, such as immunosuppression and use of broadspectrum antimicrobial drugs, can favor their overgrowth and the development of candidosis (4). Oral candidosis is a common problem among the elderly, denture wearers are more susceptible to the disease (5,6). Products known as probiotics have been consumed for many years for therapeutic and prophylactic reasons. The presence of probiotic microorganisms in mucosa can prevent the growth of other bacterial species by Effects of Probiotic Bacteria on Candida Presence and IgA Anti-Candida in the Oral Cavity of Elderly


INTRODUCTION
Yeasts of Candida genus are normal constituents of the human flora, found as commensal on skin, genitourinary and gastrointestinal tracts (1). These microorganisms can be isolated in the oral cavity of 20% to 80% of healthy individuals (2,3). However, some factors, such as immunosuppression and use of broadspectrum antimicrobial drugs, can favor their overgrowth and the development of candidosis (4). Oral candidosis is a common problem among the elderly, denture wearers are more susceptible to the disease (5,6).
Products known as probiotics have been consumed for many years for therapeutic and prophylactic reasons. The presence of probiotic microorganisms in mucosa can prevent the growth of other bacterial species by Imbalance in the resident microbiota may promote the growth of opportunistic microorganisms, such as yeasts of Candida genus and the development of diseases, especially in aged people. This study evaluated whether the consumption of the probiotic Yakult LB ® (Lactobacillus casei and Bifidobacterium breve) was able to influence on the specific immunological response against Candida and on the presence of these yeasts in the oral cavity of 42 healthy aged individuals. Saliva samples were collected before and after the probiotic use for 30 days, 3 times a week. The samples were plated in Dextrose Saboraud Agar with chloramphenicol, the colonyforming units (CFU/mL) were counted and the Candida species were identified. Anti-Candida IgA analysis was conducted using the ELISA technique. ANOVA and Student's t-test were used for normally distributed data and the Wilcoxon test was used for data with non-normal distribution (α=0.05). The results showed a statistically significant reduction (p<0.05) in Candida prevalence (from 92.9% to 85.7%), in CFU/mL counts of Candida and in the number of non-albicans species after consumption of the probiotic. Immunological analysis demonstrated a significant increase (p<0.05) in anti-Candida IgA levels. In conclusion, probiotic bacteria reduced Candida numbers in the oral cavity of the elderly and increased specific secretory immune response against these yeasts, suggesting its possible use in controlling oral candidosis.

Effects of Probiotic Bacteria on Candida Presence and IgA Anti-Candida in the Oral Cavity of Elderly
Key Words: Candida, probiotics, geriatrics, elderly. production of acid and antimicrobial products (7) or by interference on the mucosa immunity by modulating IgA synthesis and mucus production (8).
Experiments performed with probiotic bacteria, Lactobacillus acidophilus and Lactobacillus fermentum in rats infected with Candida albicans showed a significant reduction in amounts of yeasts in the oral cavity, suggesting a therapeutic capacity of these bacteria in candidosis (9).
Hatakka et al. (10) also observed a reduction of 32% on Candida prevalence in the oral cavity of elderly who consumed a cheese enriched with probiotic bacteria.
With the increase in life expectancy, geriatric problems became more frequent. Being candidosis a common disease among the elderly, the search for products that could help in its treatment is important Correspondence: Profa. Dra. Mariella Vieira Pereira Leão, Avenida Tiradentes, 500, Laboratório de Microbiologia, 12030-180 Taubaté, SP, Brasil. Tel:+55-12-3635-3466. e-mail: mariellaleao@yahoo.com.br / mariella.leao@unitau.com.br Therefore, the aim of this study was investigate the effect of Yakult LB ® consumption in Candida spp. presence and in anti-Candida IgA levels in the oral cavity of elderly.

Studied Population
After approval of the research project by the institutional Ethics Committee (Protocol #057/08), 42 institutionalized women aged 65 or older who lived in the city of Taubaté, SP, Brazil, were clinically healthy and had not used antibiotics or immunossupressors for the previous 3 months were selected for the study. The sample size was determined considering: the total number of clinically healthy aged women of the institutions (n=50), 5% of error, 95% of confidence level and 80% of expected prevalence of Candida based on previous pilot study. Preliminary analyses regarding the oral and dental conditions were performed, including presence of periodontal disease, periodontal pockets and mucosal lesions. When these lesions were detected, the patients were included in the study and treated only after enrolment to avoid interference in the results. Patients with emergency conditions were not included and were treated immediately. If any new fact that could influence the results (e.g.: antibiotics use) emerged during the course of the study, the samples of that patient were also excluded. All participants and their caregivers were fully informed about the objectives and methodology of the study and signed an informed consent form.

Saliva Samples and Probiotic consumption
Samples of volunteers' saliva were collected without stimulation, about 2 h after oral hygiene, in the morning or in the afternoon, depending on the rules designated by the institution. The samples were stored in sterile disposable collectors and kept on ice up to 3 h. Then, a fraction of the samples was plated in culture media for isolation and identification of Candida yeasts. Another fraction was kept in a vial added of phenylmethylsulfonyl fluoride 0.1 mM (Sigma Co., St Louis, MO, USA) and sodium azide 0.1% (Difco Laboratories Inc., Detroit, MI, USA) at -20ºC, for assessment of IgA levels.
The caregivers were instructed to mix 1 g (content of 1 envelope) of the probiotic Yakult LB ® (Yakult S/A Industria e Comércio, Caçapava, Brazil) (Lactobacillus casei and Bifidobacterium breve, 2x10 7 to 10 9 and 5x10 7 to 10 9 CFU/mL, respectively) with some kind of juice and give to the elderly 3 times a week, at the same hour, for 30 days. After this period, another saliva sample was collected from each participant.

Isolation, Counting and Identification of Candida Genus
The original samples or samples diluted 10-fold serially were plated in duplicate in Saboraud Dextrose Agar (Oxoid, Lawrence, KS, USA) with chloramphenicol (1 mg/mL) (Inlab, São Paulo, SP, Brazil) and incubated at 37ºC for 48 h at room temperature for 5 days. The number of colony forming units per mL of saliva (CFU/mL) was counted. Suggestive colonies from each plate were confirmed by smear tests stained by the Gram technique. The Candida species were identified by phenotypic (germinative tube formation and chlamydoconidia production) and biochemistry tests (fermentation and assimilation of carbohydrates and urase test).

IgA Analyses
The levels of anti-Candida IgA were also analyzed using the ELISA technique. Sensitization of ELISA plates was performed using surface antigens from Candida cells, prepared as described by Koga-Ito et al. (11). The plates were then incubated with diluted saliva (1:10), followed by an anti-human IgA labeled with horseradish peroxidase. The reaction was developed with orthophenylenediamine and H 2 O 2 as substrate. Absorbance was measured at 450 nm.

Statistical Analysis
First, the normality of the data (CFU/mL counts and IgA anti-Candida levels, before and after the use of probiotics) was evaluated using the D'Agostino test. ANOVA and Student's t-test were used for normally distributed data while the Wilcoxon test was used for data with non-normal distribution. A significance level of 5% was established for all analyses.

RESULTS
The results showed a reduction in the prevalence of Candida in oral cavity of elderly, since 39 (92.9%) individuals carried the microorganism before probiotic consumption and after that just 36 (85.7%). A statistically significant reduction in CFU/mL counts (p=0.02, Wilcoxon test) and in the log of CFU/mL counts (Fig. 1) (p=0.0, ANOVA test; p=0.03, Student's t-test) was also observed. There was a reduction in Candida numbers in 57.1% of the elderly and total elimination of the yeasts in 11.9% of them.
Among the identified species, C. albicans was the most frequently isolated one before and after the probiotics use. The species profile was slightly modified, since a reduction in the numbers of non-albicans species was observed (Table 1).
Immunological analysis showed a significant increase in the levels of anti-Candida IgA (p=0.00, ANOVA test and p=0.01, Student's t-test) (Fig. 2). The results showed that 26 (61.9%) individuals had their IgA levels increased, and from them 17 (65.4%) had a reduction in amounts of Candida in the oral cavity.

DISCUSSION
With the increase in life expectation, works involving geriatrics are becoming more and more important. Being the aged population more susceptible to Candida colonization, it was expected to find a high prevalence of the fungus in the oral cavity of the studied individuals. Nevertheless, the results showed a reduction in the prevalence and counts of Candida after probiotic use. Santos et al. (12) had already observed a similar effect with the same product (Yakult LB ® ), but in a younger population. Hatakka et al. (10), also studying elderly, observed a decrease from 30% to 21% in the prevalence of Candida in oral cavity after the consumption of a cheese enriched with probiotic bacteria. Although Candida presence could be influenced by many factors, it is important to highlight that the older people of the present study maintained the same habits and were accompanied throughout the experiment by the caregivers of the institution, who administered the probiotic in a blind manner, i.e., without knowledge of the elderly. With this, the placebo effect was minimized, a study with a placebo group would be appropriate. The   quality of the samples could also contribute to variations. However, in the whole experiment, it was maintained the same time for the collection of the samples and the maximum of 3 h for processing them, reducing the risk of interferences. The identification of analyses showed that C. albicans was the most prevalent species before and after the probiotic use. Its predominance in the oral cavity has been demonstrated (6,12,13). Several non-albicans species were also isolated, a phenomenon identified in long-term antibiotic users, immunossupressed and chronic pathologies patients (14)(15)(16). For example, Alberth et al. (15) observed the substitution of C. albicans for non-albicans species (C. kefyr, C. lusitaniae, C. sake, C. tropicalis) in children with cancer, especially in the stages of severe neutropenia. Costa et al. (16) observed an increase in non-albicans species in HIVinfected patients, although C. albicans was still the most frequent. Thus, the elderly of this work had a profile of Candida species more similar to patients with a predisposing factor. However, with the probiotic use there was a reduction in non-albicans species frequency, approximating the elderly to a healthy profile.
Controversial results regarding the role of secretory antibodies in the prevention of Candida infections and the correlation of Candida and anti-Candida IgA levels have been reported. Silva et al. (17) observed that patients with vaginal candidiasis showed lower levels of anti-Candida IgA in saliva when compared with the control group (absence of candidosis). In contrast, Kantardjiev and Popova (18) reported that the saliva samples of patients with denture stomatitis, mainly associated with Candida, showed higher titers of anti-Candida antibodies.
The present results revealed that after the period of probiotics use, a significant increase in the levels of specific IgA was observed. According to Boirivant and Strober (19), probiotics can improve the defense function of epithelial cells by the induction of cytokine secretion and the production of immunoglobulins and antimicrobial substances. Since some individuals in this study did not present the yeast or showed diminished amounts in the oral cavity after probiotics use, it is likely that Candida elimination could have occurred due to secretory immunological stimulation.
The maintenance and re-establishment of the internal balance, frequently compromised in older people, are the best alternative to avoid the development of candidosis and other opportunistic diseases.
The findings of this study suggest that the periodic consumption of probiotics could help recovering this equilibrium and eliminating less adherent Candida species, but with pathogenic potential.