INTRODUCTION

The incidence of fungal infections and also antifungal resistance in these types of infections have currently increased. Candida species are important fungal pathogens of humans that cause mucosal infections, especially in the vagina. Candida species are the most common pathogen seen in cervicovaginal smear. Ascomycola, Aspergillus, Sporidiobolaceae, Basidiomycota, Coprinellus, and Paracoccidioides are rarely found. Many pieces of evidence suggest that the majority of infections produced by Candida species are associated with biofilm growth. Biofilm formation is a major virulence factor in the pathogenicity of Candida species. Any biofilm or slime factor facilitates the colonization or invasive infections of Candida species in host cells, catheters, and prosthetic devices. Biofilm production is also associated with a high level of antifungal resistance in Candida species. When Candida species grow in vaginal samples, the detection of biofilm production as a criterion for antifungal resistance and virulence will guide the clinician for treatment. The method to be preferred for the detection of biofilm production should yield results in a short time, be easy to evaluate and be inexpensive. We, therefore, compared isolates from clinical vaginal discharge, representing different Candida species to each other for the capacity to form biofilms in glucose-containing medium such as 8% Sabouraud Dextrose Broth and Congo Red Agar (CRA).

MATERIALS AND METHODS

In our study, there were 192 routine patients who were examined in Sitonet Cyto-Pathology Center between September 2015 and August 2020. Two sets of examples were obtained using conventional smear cytology and wet smear (Figure 1). Conventional cervico-vaginal smears were scanned in Sitonet Cyto-Pathology and wet smears were studied in İstanbul Okan University Medical Faculty Hospital Microbiology Laboratory.

One hundred ninety-two Candida strain biofilm productions extracted from vaginal discharge samples were evaluated in the microbiology laboratory via two different methods. Vaginal samples were inoculated into Sabouraud Dextrose Agar (SDA) (Condalab, Spain) and then incubated for 24 hours at 37 ^oC. The suspected yeast colonies were subjected to Gram staining and then germ tube test. Subsequently, they were inoculated into Corn Meal Agar plate (Dalmau plate-Condalab, Spain) containing Tween-80 (Merck. Millipore, Germany). Isolates forming chlamydospore on germ tube and Corn Meal Agar plate were defined as C. albicans. Yeast samples from other non-albicans Candida (NAC) were identified with the VITEK 2 (BioMérieux, France) YST automated identification system.

Biofilm production in Candida strains was investigated using the tube adherence method (Sabouraud broth with 8% glucose/Sabouraud Dextrose Broth-SDB-) and CRA with glucose^1,2. SDB (Condalab, Spain) was prepared in accordance with the manufacturer’s recommended method by adding 60 g of glucose per liter (glucose density 80 g/L or 8%) and each sample was incubated at 35 ^oC for 24 hours at SDA. Then the grown yeast colonies were suspended in sterile saline, the concentration of 3x10⁷ CFU/mL was added to 9 mL of methylene blue and SDB suspension in 10 mL glass tubes and diluted to 3x10⁶ CFU/mL, incubated at 35 ^oC without shaking. Specimens were observed macroscopically at 24^th and 48^th hours. The specimens stained with methylene blue at the tube rim and the bottom were considered “positive” for biofilm production. In CRA, after 48 hours of incubation at 35 ^oC, the presence of black colonies and visually biofilm production was detected^1-5.

RESULTS

In the study, 70 of 192 Candida strains were identified as C. albicans (36.5%) and 122 as NAC (63.5%). Of the NAC species, 51 (41.8%) C. glabrata, 35 (28.7%) C. tropicalis, 18 (14.7%) C. pseudotropicalis, 9 (7%) 4) C. guillermondii and 9 (7.4%) were identified as C. krusei.

Fifty-eight (30.2%) biofilm positivity was detected at the 24^th hour in all Candida species in SDB and 109 (56.8%) at the 48^th hour (Figure 2). In CRA, 97 (50.05%) biofilm-positive Candida species were detected (Figure 3). Biofilm-positive Candida strain was found to be 88 (45.8%) and biofilm-negative Candida strain was found to be 74 (38.6%) with both methods (Table 1, Table 2, Table 3).

Of the 4 Candida strains that were positive at the 48^th hour in SDB and negative in CRA, 22 were found to be negative at the 24th hour and weakly positive (+1 positive) at the 48^th hour. No Candida strain that was positive in CRA but negative in SDB was found.

When all Candida species studied with CRA were evaluated, biofilm positivity was found to be 57.4% in NAC species and 38.6% in C. albicans. Biofilm positive NAC species were identified as 11 (15.7%) C. glabrata, 11 (15.7%) C. tropicalis, 6 (8.6%), C. guillermondii and 6 (8.6%) C. krusei (Figure 4).

DISCUSSION

The fact that some microorganisms, especially coagulase-negative staphylococci, produce biofilms under the name of slime factor was first reported by Christensen et al.¹. The determination of slime factor production in coagulase-negative staphylococci by the CRA method has been first described by Freeman et al.² in 1989. Today, to detect biofilm production in coagulase-negative staphylococci, microdilution plate and standard tube methods described by Christensen or CRA method are used. The effects of biofilm production on adhesion and antibiotic resistance in bacteria and therefore its clinical importance have been shown in various studies^3,4.

Similar studies were conducted on yeasts after coagulase-negative staphylococci. Dolapçi and Tekeli⁵ detected 15.14% slime factor positivity with the modified tube adherence method in a total of 350 Candida species (246 throat, 19 vaginal and 85 blood samples), and they also showed that the slime factor positivity was statistically significant. In the same study, they found that the slime factor positivity was significantly higher in C. famata, C. guillermondii, and C. krusei.

Li et al.⁶ demonstrated the quantitative variability of adhesion and biofilm formation that allowed adhesion to polystyrene plastic surfaces in 115 C. albicans strain, including 47 from the oral cavity of healthy volunteers, 31 from the environment and 37 from vaginal samples of patients with candidiasis, and its relationship with genotypes. Harriott et al.⁷ demonstrated in vivo biofilm formation and biofilm structure in the vaginal epithelium for the first-time. Calışkan et al.⁸ showed that Candida species in the vagina could cause vaginal infections by forming biofilms and that biofilm structures that might form in intrauterine devices might cause resistance to antifungal drugs.

In the study of Yücesoy and Karaman⁹, they investigated the biofilm production between C. albicans and NAC species with modified tube adherence and microplate methods. They showed a positivity rate of 65% with both methods. In our study, the biofilm positivity rate was found to be 45.8% with the standard tube adherence method and CRA. In the same study conducted by Yücesoy and Karaman⁹, the biofilm positivity rate in different Candida species was between 17% and 55% in the tube adherence test. In the microplate method, it varied between 0% and 48%. In our study, SDB was found between 41.4% and 65.6% in the tube adherence test and 38.6% and 57.4% in the CRA test among Candida species. In the study of Yücesoy and Karaman⁹_, it was reported that there was no significant difference between C. albicans and NAC species in the tube adherence test. However, in our study, biofilm positivity was 41.4% for Candida albicans and 65.6% for NAC species biofilm positivity. Biofilm positivity was found to be 38.6% for C. albicans and 57.4% for NAC species in the CRA test. In the studies of Yücesoy and Karaman⁹, it has been shown that biofilm production is a potential virulence factor for Candida species and is important in terms of antifungal sensitivity. In the study of Houdaii et al.¹⁰, a positive relationship was found between biofilm formation and amphotericin B resistance.

Kumari et al.¹¹ detected 30.6% Candida species as causative agents in vulvovaginitis samples obtained from a total of 232 patients. 32.4% of them were identified as C. albicans, 45.07% as C. parapsilosis, and 22.53% as C. glabrata. The NAC rate of 67.6% found by Kumari et al.¹¹ is consistent with the rate of 63.5% in our study. While biofilm formation among Candida species was 70.4% in the study in question, it was found as 56.8% in the SDB method and 50.5% in the CRA method in our study.

Araurio Paulo de Medeiros et al.¹² examined their adhesion capacity to epithelial cells in 62 C. albicans samples isolated from the vagina and anus, biofilm formation in polystyrene microtiter plates, and proteinase activities, and they showed that virulence factors played an important role in the transition from colonization to infection in vulvovaginal candidiasis.

Kalaiarasan et al.¹³, examined different virulence factors such as hemolysis, protease activity, and biofilm production, and they emphasized the importance of virulence factors, especially in NAC species. In our study, biofilm formation as a virulence factor in NAC species was found to be 63.5%.

In the study of Kıvanç and Er¹⁴ , although there were differences between CRA and SDB microtiter plate methods for detecting biofilm production, it was stated that biofilm production was higher in Candida species isolated from the vagina. In the same study, it was shown that lactic acid bacteria inhibited the production of biofilms formed by Candida species through competition in the adhesion area¹⁴.

CONCLUSION

In fungal infections, the biofilm produced by the agent is directly proportional to antifungal resistance and the invasion of the infection. Therefore, determining the biofilm production of the causative fungus is important in planning the treatment. According to the values obtained from our study, the probability of biofilm positive for those with positive CRA results is 100%, while it is 87% for those with negative CRA results. CRA method with glucose is a method that can be preferred in determining biofilm production as a virulence factor in NAC species, which is increasingly common among Candida species, because of its simpler and shorter application compared to other methods and its objective evaluation.

Ethics

Ethics Committee Approval: The study were approved by the Okan University of Local Ethics Committee (protocol number: 13, date: 23.06.2021).

Informed Consent: Consent form was filled out by all participants.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Surgical and Medical Practices: A.A., G.V., Concept: A.A., Design: G.V., Data Collection or Processing: A.A., G.V., Analysis or Interpretation: A.A., G.V., Literature Search: A.A., Writing: A.A., G.V.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.