Protective Activity of Probiotic Bacteria Against Candida albicans : An In Vitro Study

1Evidence-Based Phytotherapy and Complementary Medicine Research Center, Alborz University of Medical Sciences, Karaj, Iran 2Student Research Committee, Alborz University of Medical Sciences, Karaj, Iran 3Department of Microbiology and Immunology, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran 4Dietary Supplements and Probiotic Research Center, Alborz University of Medical Sciences, Karaj, Iran Int J Enteric Pathog. 2018 November;6(4):113-117


albicans.
Probiotics are live micro-organisms that produce health benefits when administered in certain amounts to the host. 8In recent years, there has been some evidence that probiotic bacteria reduce Candida infections in humans. 9It is important to note that the health benefits of probiotics are strain specific; therefore, appropriate probiotics against specific pathogens should be suggested for therapeutic purposes and their beneficial effects should not be generalized. 10Considering the strainspecific probiotic properties, a new strain was used in this study, which has not previously been tested for anticandidiasis.
In this context, the effects of probiotic Lactobacillus acidophilus on the adherence of C. albicans to the human epithelial cell line known as HEp-2 cells and the potential preventive effects of probiotic bacteria against the pathogenicity of C. albicans on the HEp-2 cells were studied.

Microbial Strains and Growth Condition
Candida albicans (PTCC 5027) and probiotic L. acidophilus strain La-5 were purchased from the Iranian Biological Resource Centre (IBRC).C. albicans was maintained on Yeast Mold Agar (YMA, Merck) and stored aerobically at 25°C; likewise, L. acidophilus was grown on the de Man Rogosa Sharp broth (MRS broth, Merck) and incubated anaerobically at 37°C. 11,12p-2 Cell Line Culture Condition A 3-week old human epidermoid laryngeal (HEp-2) cell line (ATCC ® CCL-23 ™ ) was used in this study.HEp-2 cells were cultured in Eagle's Minimum Essential Medium (EMEM) (ATCC ® 30-2003 ™ ) supplemented with 10% Fetal Calf Serum (Lablech 4-101-500), 10 000 units/mL of Penicillin, and 10 000 g/mL of Streptomycin (GIBCO 15140-122).Besides, the medium was replaced with a new one every 2 days.Next, the 12-well plates were incubated at 37°C in a humidified atmosphere containing 5% CO 2 and 95% air to reach a confluency of about 70%. 5 HEp-2 Cell Adhesion Assay Adhesion assay was carried out in the Department of Microbiology, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran.Adhesion of C. albicans strain to HEp-2 cells was tested as described by Negri et al. 13 To evaluate the effect of L. acidophilus on the adherence of C. albicans to HEp2 cells, 1.5 × 10 6 CFU/mL of yeast cells, 1.5 × 10 6 CFU/mL of probiotic bacteria, or both were added to each well of a 12-well plate with a coverslip at the bottom, covered with a dense layer of HEp-2 cells.After two hours of incubation at 37°C in 5% CO 2 , each well was rinsed to remove the non-attached microorganisms.The coverslips were removed from the wells, and cells were fixed, dehydrated, Gram stained and mounted on the slide.Next, 10 fields from each slide were observed under a light microscope (1000x) and the number of yeasts, probiotic bacteria, and HEp-2 cells was counted.Wells with no pathogens and probiotics were used as controls.

Cell Viability Assay
To determine the effect of C. albicans on the viability of HEp2 cells either in the presence or absence of L. acidophilus, the MTT (3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide) assay was conducted using the MTT-based Cell Titer 96 ® Non-Radioactive Cell Proliferation Assay kit (Promega) according to the manufacturer's protocol. 14First, 10 000 HEp2 cells were added to each well of a 96-well plate.After 24 hours, either 1.5 × 10 6 CFU/mL of C. albicans or 1.5 × 10 6 CFU/ mL of L. acidophilus or both were added to the wells and incubated for 3 hours.Uninfected cells were used as negative control (100% viability).The quantity of MTT is directly related to the number of metabolically active cells.Relative viability was calculated with regards to uninfected cells.

Statistical Analysis
Data were expressed as the mean ± standard error of the mean (SEM).Statistical analysis was assessed using SPSS software version 19.0 (SPSS Inc, Chicago, IL, USA).Unpaired sample Student's t test was used for comparison between two groups.The statistical significances were achieved when P < 0.05.

Results
The results showed that L. acidophilus La5 strain efficiently inhibited cell association of C. albicans strain with HEp-2 cells by 80% (Figure 1).The number of C. albicans adhered to 100 Hep-2 cells under light microscopy is presented in Table 1.The findings indicated that Lactobacillus acidophilus La-5 strains were able to significantly decrease the number of pathogen attached to the HEp-2 cells line (P < 0.001).

Cell Viability Assay
The present study showed that the viability of epithelial cells increased significantly in the presence of probiotic bacteria compared to Candida alone in the cell layer.The results indicated the protective effect of probiotic bacteria on Candida infection of epithelial cells (Figure 2).

Discussion
Among Candida species, C. albicans can be detected in about 50% of individuals in the general population and is the main cause of mucosal fungal infections. 10he use of probiotic bacteria is an alternative treatment for Candida infections.Probiotics are believed to improve the health status by decreasing pH, stimulating defense mechanism in epithelial cells, and preventing pathogen attachment. 1 A critical step in the pathogenesis and development of candidiasis is yeast adhesion to the epithelial cells. 1,15andida strains are able to adhere to different cell types, such as oral epithelial cells and cultured epithelial cell lines. 11,14,16In the present study, about 60% of C. albicans could adhere to the Hep2 epithelial cell lines.Similar results were reported by Holmes et al denoting the adhesion of C. albicans to the human epithelial monolayer. 17Slime, the extracellular polymeric substance (EPS) that is produced by Candida, is considered a virulence factor.When adhered to epithelial cells, Candida produces a polymeric substance that is considered a virulence factor and is related to their persistence and colonization of the host tissues when added to epithelial cells. 18ood adherence to the intestinal cells is related to many beneficial effects of probiotic bacteria, such as the exclusion of substance which glues the bacteria together and increases adherence. 19EPS may determine the cell surface properties and improve colonization.EPS is a long-chain polysaccharide that is suggested to have an impact on bacterial adhesion and colonization. 20,21In our experiment, we examined the effect of candida on epithelial cells via two methods of pathogen adhesion to the cell layer and checking with a light microscope.L. acidophilus La-5 showed a high adherence capability.Some probiotic strains produce and excrete a slime-like exopolysaccharide (EPS).Moreover, the obtained results indicated that L. acidophilus significantly improved the viability of contaminated Hep2 cells.Our findings showed that L. acidophilus had good anti-adherence activity against C. albicans.
Adherence inhibition is strain specific and totally depends on the probiotic strain type and the pathogen; thus, a probiotic mixture should be considered for oral candidiasis.The anti-adherence activity of L. acidophilus could be related to the competition of the bacteria and yeasts for eukaryotic cell receptors, and prevention of pathogen adherence by probiotic bacteria can protect the host cells from candida colonization. 8,22 recent study showed the positive effect of using oral probiotic containing Lactobacillus species on the effectiveness of an antifungal medicine as well its negative effects on Candida spp.4][25][26][27] Köhler et al reported that lactic acid at low pH adversely affected fungal growth and viability staining following cocultures with Lactobacilli indicated that metabolic activity of C. albicans was damaged. 28oreover, Rizzo et al showed that Lactobacillus   crispatus (ATCC 33820) boosts epithelial cell defense against C. albicans infection through the involvement of TLR2/4, IL-8 and human β-defensin 2 and 3; hence, they proposed a probiotic potential for Lactobacillus as an antiinfective agent against C. albicans. 29urthermore, the use of probiotics as a dietary supplement, local treatment, or therapeutic alternative to antibiotic treatment is a promising means to boost the host immune system and decrease pathogen adherence and its deleterious effects on the host cells.However, quality studies on the use of probiotics against candidiasis are still comparatively rare, particularly in Iran, and accurate knowledge of the most desirable characteristics of a probiotic species has not yet been obtained.These results should also encourage future studies aimed at improving the immune system, particularly in infectious diseases.

Figure 2 .
Figure 2. The Probiotic Strain La5 Increased the Viability of Infected Cells From 68.8% to 74%.

Table 1 .
Adherence of Microorganisms Individually and in Mixture to 100 Hep2 Cells a P < 0.001, b P < 0.005.