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Probiotics Reduce the Prevalence of Oral Candida in the Elderly—a Randomized Controlled Trial

¹ Valio Ltd, R&D, PO Box 30, FIN-00039 Helsinki, Finland;
² Division of Infectious Diseases, Department of Medicine, Helsinki University Central Hospital;
³ Institute of Dentistry, University of Helsinki, Finland;
⁴ Department of Bacteriology and Immunology, University of Helsinki, Finland;
⁵ STAT-Consulting, Tampere, Finland;
⁶ Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland;
⁷ Department of Pharmacology, Institute of Biomedicine, University of Helsinki, Finland; and
⁸ Foundation for Nutrition Research, Helsinki, Finland

Correspondence: ^* corresponding author, katja.hatakka{at}valio.fi

	ABSTRACT

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Overgrowth of oral yeast is a common problem among the elderly. Probiotic bacteria are known to inhibit the growth of pathogenic microbes. We tested the hypothesis that cheese containing probiotic bacteria can reduce the prevalence of oral Candida. During this 16-week, randomized, double-blind, placebo-controlled study, 276 elderly people consumed daily 50 g of either probiotic (n = 136) or control cheese (n = 140). The primary outcome measure was the prevalence of a high salivary yeast count ( 10⁴ cfu/mL) analyzed by the Dentocult^® method. The prevalence decreased in the probiotic group from 30% to 21% (32% reduction), and increased in the control group from 28% to 34%. Probiotic intervention reduced the risk of high yeast counts by 75% (OR = 0.25, 95%CI 0.10–0.65, p = 0.004), and the risk of hyposalivation by 56% (OR = 0.44, 95%CI 0.19–1.01, p = 0.05). Thus, probiotic bacteria can be effective in controlling oral Candida and hyposalivation in the elderly.

Key Words: probiotics • Lactobacillus GG • Candida • the elderly • randomized trial • hyposalivation

	INTRODUCTION

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The elderly are vulnerable to Candida infection provoked by chronic diseases, medication, poor oral hygiene, reduced salivary flow, or the impairment of the immune system (Shay et al., 1997). As many as 75% of elderly people in Finland harbor oral yeast (Närhi et al., 1993). Even though the colonization by Candida may be asymptomatic, heavy growth usually leads to local candidosis, with various types of mucosal lesions and symptoms (Shay et al., 1997). It is important, therefore, that yeast proliferation be controlled.

Probiotic bacteria, such as Lactobacillus rhamnosus GG (Saxelin, 1997), may modify the microbial balance of the host by reducing the overgrowth of pathogens, such as Candida (Payne et al., 2003). Lactobacillus GG reduced the numbers of Candida cells in the alimentary tract of immunodeficient mice (Wagner et al., 1997) and, recently, the enteric colonization of Candida in pre-term neonates, as measured by colonies isolated from oropharyngeal, gastric aspirate, stool, and fecal specimens (Manzoni et al., 2006).

Certain strains of lactobacilli can adhere to the mucosal epithelium, and may thereby compete for adhesion sites with Candida (Strus et al., 2005). In addition, Lactobacillus species produce different metabolites, such as hydrogen peroxide (Strus et al., 2005), and antifungal cyclic peptides (Ström et al., 2002), which inhibit the in vitro growth of Candida. In mice inoculated orally with Candida, lactobacilli have shortened the duration of Candida colonization in the oral cavity, possibly by inducing the production of IL-4 and IFN- in lymph nodes and nitric oxide (NO) in the saliva (Elahi et al., 2005).

In the present study, we investigated whether treatment with cheese containing a mixture of probiotics (Lactobacillus rhamnosus GG, L. rhamnosus LC705, Propionibacterium freudenreichii ssp. shermanii JS) would reduce the growth of oral Candida in the elderly. Lactobacillus rhamnosus LC 705 and Propionibacterium JS have been previously used as preservatives against yeasts in the manufacture of sour milk products (Suomalainen and Mäyrä-Mäkinen, 1999).

	METHODS

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Participants
Independent elderly people aged 70–100 yrs were recruited by research assistants from old people’s homes and sheltered housing units in the Helsinki area of Finland. Exclusion criteria were the presence of dementia (mini-mental state (MMS) test of over three points) and the taking of current oral yeast medication. In total, 304 elderly participants from 12 old people’s homes and 37 sheltered housing units gave their written informed consent. The study protocol was approved by the Ethical Committee of Helsinki University Central Hospital.

Study Protocol and the Intervention
This randomized, double-blind, placebo-controlled study, with two parallel groups, was carried out between January, 2001, and March, 2002. Each participant was randomly allocated to the probiotic group or the control group, according to a computer-generated random permuted blocks method. A block size of four was used, and it was stratified according to the baseline yeast count.

During the 16-week intervention, after a three-week run-in period, the participants consumed daily either 50 grams of Emmental-type probiotic cheese (containing 15% fat) or 50 grams of edam-type control cheese, divided into two portions. In the probiotic cheese, Lactococcus lactis and Lactobacillus helveticus were used as starter cultures, and 10⁷ cfu (colony-forming units)/g of each of the probiotic strains, L. rhamnosus GG (ATCC 53103), L. rhamnosus LC705, and Propionibacterium freudenreichii ssp shermanii JS (Valio Ltd, Joensuu, Finland) were added. Control cheese contained only Lactococcus lactis as a starter culture, and no probiotic strains were added. For blinding purposes, the packaging of both cheeses was identical. The manufacturer did not reveal the identities of the treatment groups to the investigators until the intention-to-treat (ITT) analysis was performed. The use of other probiotic-containing products was forbidden during the study.

Clinical Examination
The oral and dental status of the participants was examined by an experienced dentist at Weeks 0 and 16, according to WHO criteria (WHO, 1997). The number of decayed, missing, and filled teeth and the number of prosthetic appliances were recorded. The Community Periodontal Index (CPI) was recorded for each sextant (0 = healthy gums; 1 = no periodontal pockets but bleeding occurs on probing; 2 = presence of pockets no deeper than 3 mm; 3 = pockets of 4–5 mm; 4 = pockets 6 mm or deeper) (WHO, 1997). Mucosal lesions were recorded as white oral lesions (leukoplakia), red oral lesions, hyperplasia, ulceration, and pigmented oral lesions. During a monthly visit, a research assistant collected information on each participant’s health, medication, oral pain, and sensations of dry mouth.

Saliva Samples
Sampling for oral yeasts was undertaken 4 times (at the beginning of the run-in period, and at Weeks 0, 8, and 16) between 8 and 11 a.m. The participants were told to refrain from eating, drinking (except water), and smoking for 1 hr prior to the investigation. The research assistant sampled for the yeasts by rotating a cotton wool swab in a standardized fashion on the oral mucosa of the cheeks, tongue, and gingival margin (dentate participants) or alveolar ridge (edentate participants), and immediately inoculating it onto a Dentocult^® CA slide (Orion Diagnostica, Espoo, Finland). After incubation for 48 hrs at 37°C, the growth was assessed semi-quantitatively according to the manufacturer’s visual scale and product instructions, with the following categories, where: 0 represented no visible colonies, 1 was equivalent to a colony density of 10³ cfu/mL, 2 was equivalent to 10⁴ cfu/mL, 3 was equivalent to 10⁵ cfu/mL, and 4 was equivalent to 10⁶ cfu/mL (the counts were extrapolated to salivary concentrations, cfu/mL). Category 1 (10³ cfu/mL) was used to distinguish between participants with low (<10⁴ cfu/mL) and high ( 10⁴ cfu/mL) prevalence. Further identification of the yeast species was made by subculturing them onto CHROMagar Candida (Becton Dickinson, Franklin Lakes, NJ, USA), which permitted the presumptive identification of C. albicans, C. glabrata, C. krusei, and C. tropicalis.

Stimulated and unstimulated salivary secretion rates and salivary buffering capacity were measured at Weeks 0 and 16. The participants were told to collect all their unstimulated secreted saliva in a measuring cylinder for 5 min. A paraffin-wax-stimulated salivary secretion was then collected for another 5 min. An unstimulated salivary flow rate of below 0.1 mL/min and a stimulated flow rate of below 0.8 mL/min was considered to indicate hyposalivation. The salivary buffering capacity was measured from stimulated saliva by the Dentobuff^® Strip method (Orion Diagnostica, Espoo, Finland), and was classified according to the manufacturer’s instructions (low, pH 4.0; intermediate, pH 4.5–5.5; high, pH 6.0).

Sample Size
Calculation of the sample size was based on the assumption that the consumption of cheese with probiotic bacteria results in a proportional reduction of 24% in the prevalence of yeast (from 75% to 57%). The results of a preventive oral hygiene program (Budtz-Jørgensen et al., 2000) were used as reference data. With a power of 80% and at a significance level of 0.05, 108 participants per group were needed for the difference between the groups (75% vs. 57%, OR 0.44) to be detected. After adjustment for drop-out (30%), 154 participants per group were needed.

Statistical Analysis
The primary outcome variable was the high yeast count ( 10⁴ cfu/mL = categories 2-4) after the intervention. The secondary outcome variables were hyposalivation, salivary buffering capacity, dry mouth, mucosal lesions, oral pain, and decayed teeth. We used logistic regression analysis to study the association between the intervention and the high yeast count, and the baseline yeast count (yes/no) was included as a categorical covariate. The possible confounding factors (baseline yeast category, age, diabetes, salivary flow rate, buffering capacity, denture wearing, number of drugs used daily, gender, and type of housing) were introduced to the stepwise multivariable model (criterion for entering, p < 0.15). The results are given as odds ratios (OR) with 95% confidence intervals (CI). The effect of the probiotic intervention on the secondary outcome variables was analyzed by separate logistic regression analysis, with the corresponding baseline included as a categorical covariate. The Student t test for independent samples and the chi-square test were used to compare the baseline characteristics between the groups.

All analyses were based on the intention-to-treat (ITT) population. In addition, the primary outcome variable was analyzed by the per protocol (PP) population, excluding participants who did not fulfill the inclusion criteria (18 participants were under 70 yrs old, and two participants used medication for Candida), who dropped out of the study, or who had cheese-eating compliance of under 80%. Thus, the number of participants included in the PP analysis was 151 (54.7%). Statistical analyses were performed with the use of SPSS software (Version 11.5, SPSS Inc., Chicago, IL, USA).

	RESULTS

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Participant Follow-up and Health Status at Baseline
A total of 304 persons volunteered to participate in the study, and 294 were included and randomized to the two study groups (Fig. 1). Of these, 276 participants began the study, but 84 (30%) subsequently dropped out. Those who dropped out carried more yeast (p = 0.08) and were older (p = 0.06) than those who completed the study. Pre-treatment characteristics and baseline oral health status are given in Table 1. Those who had regularly used lactic acid bacteria-containing products before the study had high yeast counts ( 10⁴ cfu/mL) less often than those who did not (25% vs. 38%; p = 0.03).

View larger version (19K): [in this window] [in a new window]   Figure 1. Trial profile during the 16-week study.

View larger version (9K): [in this window] [in a new window]   Figure 2. Percentage (95% confidence interval) of participants with high yeast counts (104 cfu/mL) in the probiotic (n = 92) and control (n = 100) groups at baseline, in the middle, and at the end of the intervention.

There were minor fluctuations, but C. albicans was the predominating species recovered during the entire study, found in 94% of the positive participants at the beginning of the study, and in 90% at the end. In the probiotic group, two participants who carried C. albicans at the beginning had been colonized by C. glabrata by the end. In the control group, C. glabrata was found in two participants at the beginning, and in seven participants at the end. C. tropicalis was found in only one person in the probiotic group, and Saccharomyces cerevisiae in two participants in the control group.

Effect of Probiotic Cheese on Secondary Outcome Variables
The median unstimulated salivary flow increased in the probiotic group, from 0.18 mL/min at the beginning to 0.22 mL/min at the end, and decreased in the control group, from 0.22 mL/min to 0.18 mL/min. Both the number of participants with hyposalivation and the risk of hyposalivation were reduced in the probiotic group (Table 2). Probiotic treatment tended to lower the risk of dry mouth, high salivary buffering capacity, mucosal lesions, and oral pain, although not significantly.

	DISCUSSION

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In an earlier study, probiotic cheese containing Lactobacillus GG and Lactobacillus rhamnosus LC705 tended to reduce the level of salivary yeasts in healthy adults (Ahola et al., 2002). This finding was confirmed in our study, and could possibly be explained by the addition of the strain Propionibacterium JS. The combination of Propionibacterium JS and Lactobacillus rhamnosus LC705 has been used as an active preservative against yeast in the manufacturing process (Suomalainen and Mäyrä-Mäkinen, 1999). In a recent study (Manzoni et al, 2006), oral supplementation with Lactobacillus GG also reduced the gastrointestinal colonization of Candida (23% vs. 49%) in pre-term neonates.

One interesting observation in our study was that probiotic treatment reduced the prevalence of hyposalivation and a subjective feeling of dry mouth. The gustatory stimulus caused by chewing cheese twice a day does not explain the observation, since hyposalivation increased in the control group. Based on the present study, the explanation for the reduced prevalence of hyposalivation in the probiotic group remains unclear. However, it could be hypothesized that probiotics might have somehow affected the composition of saliva, such as the concentrations of mucins and salivary immunoglobulins, as has been shown in animal (Negretti et al., 1997) and in in vitro (Mack et al., 2003) studies, thereby affecting the nature of saliva secreted.

Unlike in the probiotic group, yeast carriage was found to have increased in the control group. The major factors predisposing to oral candidosis, such as denture wearing (Närhi et al., 1999), or the use of steroid inhalers, systemic corticosteroids, and wide-spectrum antibiotics (Shay et al., 1997), did not differ between the groups, and cannot be the causative factors. The use of products containing lactic acid bacteria prior to the intervention was fairly common in both groups, and also a protective factor against Candida colonization. Therefore, the absence of the protective effects of probiotics, together with increased hyposalivation, might explain the increase in Candida growth in the control group.

It has been suggested that oral Candida incidence increases with age (Lockhart et al., 1999), possibly because of impaired immunity. Several elements in the immune system, such as T-lymphocytes, granulocytes, NK-cells, mast cells, and macrophages, account for the protection against Candida infections (Peterson, 1992). Lactobacillus GG and Propionibacterium JS cause enhanced T-cell and B-cell proliferation in mice (Kirjavainen et al., 1999). Probiotics have also stimulated the production of IFN-, enhanced phagocytic capacity (Arunachalam et al., 2000), and increased the proportions of helper T-lymphocytes and the activity of natural killer cells in elderly patients (Gill et al., 2001). In animal studies, lactobacilli have attenuated Candida infection by inducing the production of IL-4 and IFN- (Elahi et al., 2005), and by attenuating the production of pro-inflammatory IL-1β and TNF- (Brzozowski et al., 2005). Probiotics may also inhibit the Candida growth by producing antimicrobial compounds (Ström et al., 2002; Strus et al., 2005), and may inhibit its adhesion to epithelial cells (Reid et al., 1995). In an in vitro model mimicking gastrointestinal conditions, Lactobacillus suppressed the growth of Candida after antibiotic treatment (Payne et al., 2003), possibly by competing for the same receptor sites.

Our primary hypothesis, that probiotic bacteria can reduce the prevalence of oral Candida in the elderly, was confirmed in the present study. Oral candidosis is common in elderly people, and under certain circumstances can contribute to disseminated candidiasis (Shay et al., 1997). Probiotics could be regularly used as a prophylactic or therapeutic means, without side-effects, of reducing Candida. Probiotics also diminished the risk of hyposalivation and the feeling of dry mouth, and can therefore be considered beneficial to oral health in general.

	ACKNOWLEDGMENTS

 
We thank research assistants Mirkka Narva, PhD, Kajsa Kajander, MSc, and Mrs. Raita Koskenala-Tuominen, for participating in the sampling of saliva samples; Tiina Anttonen, MSc, for recording the data; Jonna Tallila, BSc, for technical help in the yeast culturing and identification of the yeast species; Mimi Ponsonby, MA, for language editing; the staff of the old people’s homes and all the participants for making this study possible. The study was supported by Valio Ltd and the Juho Vainio Foundation, Helsinki. The University of Helsinki participated in the funding by providing supervision and technical help. K. Hatakka and R.Korpela are currently working for Valio Ltd, and A. Ahola previously worked for the company. H. Yli-Knuuttila and T. Poussa were recompensed by Valio for their working time. The other authors have no conflicts of interest.

Received for publication December 5, 2005. Revision received October 22, 2006. Accepted for publication November 5, 2006.

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Journal of Dental Research, Vol. 86, No. 2, 125-130 (2007)
DOI: 10.1177/154405910708600204


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