{'fraction_non_alphanumeric': 0.033733345932155344, 'fraction_numerical': 0.014268165926485873, 'mean_word_length': 4.929411764705883, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Probiotic Enterococcus durans Interference with Oral Candida albicans Adhesion: An in vitro Study

1. Student Research Committee, Kurdistan University of Medical Sciences, Sanandaj, Iran 2. Dept. of Microbiology, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran 3. Cellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran 4. Social Determinants of Health Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran 5. Dept. of Pediatric Dentistry, Faculty of Dentistry, Kurdistan University of Medical Sciences, Sanandaj, Iran


Introduction
Candida species are commensal microorganisms and members of normal oral cavity flora; however, these species are opportunistic pathogens which cause candidiasis under specific conditions (1). Candida albicans (C. albicans) is a microbiologic member of the oral cavity in children with dental caries (2). As reported by Odds, there is a relationship between adhesion rate of C. albicans to surfaces and its ability to form colonies and induce diseases (3).
As a new biologic technique, using probiotics has gained popularity in preventing and controlling diseases at present. It is considered as an alternative method to change the microbial ecology of the oral cavity. Competition among probiotics to replace pathogenic microorganisms makes it possible to simultaneously use systemic and local interventions (4, 5). The World Health Organization defines probiotic bacteria as viable microorganisms that are useful for the host's health when used appropriately (1). Different microorganisms have been used as probiotics. Lactic acid bacteria (LAB) is one of the most utilized and effective probiotics. Enterococci, as LAB family members, are ubiquitous due to their durability and resistance to growth inhibition factors, including their resistance to high acidity and biliary salts. They are naturally found in different environments including dairy products, meat, vegetables, and cereal (6-8). Also, they are the most predominant microorganisms found in traditional cheese produced from raw milk (7, 9).

Volume 28, September & October 2020
Journal of Advances in Medical and Biomedical Research Enterococcus Durans (E. durans) is a LAB group bacteria and is on the list of safe materials/agents (GRAS). This bacteria is one of the permanent residents of the gastrointestinal tract (10). Evidence has shown its useful antioxidative, antibacterial, and probiotic characteristics. It has a variety of characteristics, including potential adhesion and colonization of the mucosa, the ability to form biofilms, absence of aggressive potential, few virulence factors, specific antimicrobial properties against pathogens, and inducing immune responses (9, 11, 12). In addition, growth of this bacterial species in a selenium-rich environment results in selenium concentration in the bacterial cell mass. As a result, this bacterial species might be an alternative source for organic selenium in foodstuffs (12).
This is the first study on the effect of E. durans on C. albicans. One of the criteria for the selection of probiotic bacteria is adherence to dental tissue as part of biofilm and competition with the growth of cariogenic bacteria (4). Therefore, this study evaluated the effects of probiotic E. durans on the adhesion of C. albicans in vitro.

Isolation of Candida albicans
Reference bacteria strain of Candida albicans (PTCC-5027) (Pasteur Institute of Iran) and strains isolated from carious teeth of 31 children aged 6-12 years were used in this study. Children's parents received a complete explanation about the aims and methods of the study and then provided written consent for their children to be included in the study. The inclusion criteria consisted of no use of antimicrobial mouthwashes during the previous month, no use of antifungal agents, antihistamines, corticosteroids, and a caries index of >10 as previously reported (13).
Dental plaques were transferred from the carious teeth into Falcon tubes containing 5 mL of BHI (Brain heart infusion) culture medium (Ibresco Made in Italy) using a sterile microbrush. The tubes were incubated at 37ᵒC for 24 hours. The samples were cultured on Mycosel Agar (Quelab Made in UK) at room temperature for 24 hours. A colony of specimens grown in a Mycosel Agar was sub-cultured in chrome agar Candida (Ibresco) medium to separate C. albicans which is observed as green colonies on the culture medium (14).

Formation of Biofilms using the Microtiter Plate Technique
At this stage, the potential of biofilm formation of the clinical samples was determined using the microtiter plate technique. First, 0.5 McFarland concentrations of the clinical and standard samples were prepared in a BHI broth culture medium (based on standard CLSI) (15). Then, 200 µL of each suspension were transferred into 96 well plates in a microtiter plate (each sample in one raw with eight repetitions). Negative control well only contained BHI broth culture medium. After 24 hours, the contents of the wells were retrieved, and each well was washed with 200 µL of sterile physiologic serum three times. A total of 200 µL of ethanol was used in order to adhere bacteria to the wells for 15 minutes. The quantification of clinical isolates biofilm formation was performed using crystal violet (CV) (Merck, Germany) staining method, according to the protocol described by Silva et al (16). In a nutshell, following biofilm formation, the wells were washed with PBS, and CV (1% v/v) was added to the wells, followed by acetic acid (33% v/v) (17). An ELISA reader was used to determine the optical density of the CV stain in the stain solvent at a wavelength of 492 nm. Highly adhering strains with greater capacity to form biofilms were selected for the next stage (9, 18).

The Effects of E. durans on Inhibiting C. albicans Adhesion
The effects of E. durans (ATCC 6056) on inhibiting the adherence of clinical isolated C. albicans were evaluated using two techniques: by adding C. albicans and E. durans to the wells simultaneously (technique 1), and by adding E. durans 30 min before adding C. albicans (technique 2). For each technique, 100 µL of a C. albicans suspension and 100 µL of an E. durans suspension were added to each well (each sample in one raw with eight repetitions). Then, the differences between the optical densities of the control wells (standard C. albicans well in association with E. durans and E. durans well alone) and the wells containing clinical samples were evaluated to assess the effects of E. durans on adherence (9, 18).
The isolates were categorized as follows based on their optical density using Stepanovic et al. 's technique (9, 19).

Statistical Analysis
Data were analyzed with SPSS 20 (SPSS Inc., Chicago, IL., USA) using a repeated measure model. Statistical significance was set at P-value<0.01.

Isolation of the Samples and Determination of Adhesion Capacity
In this in vitro study, 10 of 31 clinical isolated samples were C. albicans and 21 samples were other microorganisms. All the isolates exhibited moderate adhesion capacity which was similar to the standard samples. The OD620 nm range was 0.45 to 0.49 and 0.33 for OD490 nm ( Table 1).

The effects of E. durans on C. albicans Adhesion using Microtiter Plate Technique
The results were indicative of a significant difference in the potential of adhesion, using both methods (Table).

Discussion
Biofilm formation is the first step in bacterial infections. They are able to attach to the dental lamina and produce destructive material. We aimed to introduce a methods to reduce the biofilm formation in dental caries. C. albicans adhesion to teeth surfaces has a major role in its pathogenicity (20). Therefore adhesion reduction can be an effective way to decrease its detrimental effects (21). present in food and dairy products, Enterococcus strains have been known for a long time as effective probiotics (9). Enterococci are a type of LAB species and are members of the normal flora of the gastrointestinal tract and fermented food (12). A study by Carasi et al. showed that E. durans isolated from kefir were able to inhibit different Grampositive and Gram-negative pathogens Hasslof et al. showed the inhibitory effects of commercial samples of probiotic Lactobacilli on oral S. mutans and Candida in vitro. In the mentioned study, all the Lactobacillus species inhibited the growth of Candida and S. mutans samples; however, the inhibitory effects on C. albicans were weaker than those of S. mutans, which might be attributed to the resistance of pathogenic bacteria (24).
In the present study, inhibitory effects of E. durans on C. albicans adhesion were evaluated. Adhesion was evaluated using the microtiter plate technique and the complete culturing of the probiotic agent. In the first technique, adhesion was evaluated by simultaneous addition of the bacteria and C. albicans to the culture medium; in the second technique, the probiotic bacteria culture was carried out 30 min before adding C. albicans. Based on the results of the present study, the presence of E. durans as a probiotic bacterial species resulted in a decrease in adhesion of C. albicans to the test surfaces for both techniques; However, in both techniques, there was a significant decrease in binding, which might be attributed to the inter-surface interactions (cell-to-cell and cell-to-surface) and the production of external metabolites that made the organization and structure of the biofilm unstable (1, 25).
Considering the useful systemic effects of E. durans on increasing the secretion of IgA, regulating the Journal of Advances in Medical and Biomedical Research immune system, and increasing the production of IL-10, further clinical studies are warranted (7, 11). The results of the present study indicate the positive effects of E. durans on decreasing C. albicans adhesion.

Conclusion
Using E. durans as a probiotic could reduce Candida albicans adhesion, which might be attributed to the inter-surface interactions and the release of bioactive substances which make the organization and structure of the biofilm unstable and, therefore, can be an effective way to decrease its pathogenicity.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.