Drug Resistance and Biofilm Formation in Candida Species of Vaginal Origin.

Background: Candida species are normal vaginal flora in healthy women, which can cause vulvovaginal candid-iasis (VVC). The formation of biofilm is a cause of drug resistance in Candida species of vaginal origin. We aimed to specify Candida species cause VVC, detect their biofilm-forming ability, and antifungal susceptibility pattern.
Methods: Overall 150 vaginal samples were collected from suspected cases of referring to Bahar Hospital of Shahroud, Iran between Jan 2018 and Jan 2019. Samples were cultured on Sabouraud dextrose agar (SDA), Chrome gar Candida and Corn meal agar (CMA). PCR-RFLP was performed to confirm the identification. Bio-film formation of the identified species was measured by the Crystal Violet method. The susceptibility to fluconazole, clotrimazole, and miconazole was determined based on the CLSI document M27-A3.
Results: Of 50 women (33.3%) were suffering from VVC. C.albicans was the predominant species isolated in this study (n=39, 78%) followed by C. glabratia (n=11, 22%). In addition, in 25 (50%) of positive samples, bio-film formation was determined. The mean MIC of fluconazole and clotrimazole for C. albicans was 5.02 μg/mL and 3.92 μg/mL, respectively. Furthermore, the mean MIC related to these drugs for C. glabrata was 12.45 μg / mL and 4.1μg / mL, respectively. The mean diameter of miconazole inhibition zone for C. albicans and C. glabra isolates was 25.13 mm and 24.5mm, respectively and all of them were susceptible to this drug.
Conclusion: C.albicans was the predominant Candida species isolated from patients with VVC and also was the predominant biofilm producer species.

Introduction

Vulvovaginal candidiasis (VVC) often referred to a mucosal infection and a common gynecologic problem caused by Candida species (1, 2). About 75% of all women will experience an episode of VVC in their lifetime (3). This infection affects most women of reproductive age and is one of the main reasons for referral to a doctor.

C. albicans, a dimorphic normal vaginal flora, is responsible for 85–90% of clinical significance in patients with positive vaginal fungal infections (4). However, it seems that there is an increase in the frequency of other Candida species (C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, and others) in certain populations (5). In fact, where the balance between the normal vaginal flora and host cells is disturbed, they can cause vulvovaginal infections. Socio-demographic characteristics, use of antibiotics and oral contraceptives, diabetes mellitus, dietary practices, personal hygiene, sexual activity, and specific immunological defects have been identified as potential risk factors (6).

Imidazoles and polyene macrolides are used commonly as effective drugs for eradicating fungal infections (1). The use of these drugs is topically or systemically and they are safe and efficacious (7). The Chemical structures of azole anti-fungal agents contain either two or three nitrogens. Therefore, classified as imidazoles (ketoconazole, miconazole, clotrimazole, econazole, and butoconazole) or triazoles (itraconazole, fluconazole, terconazole). The azoles inhibit ergosterol synthesis in the fungal cell membrane through their action on the cytochrome P450- dependent enzyme lanosterol 14α-demethylase. Differences among various azoles are related to their pharmacokinetics as well as their affinity for the target enzymes (8). Therefore, there is no equal effect for all imidazoles against all Candida species. For example, C. tropicalis and C. glabrata are 10 times less sensitive to the action of miconazole than C. albicans (9). According to the report, up to 7.5% of Candida species isolated from the genital area are resistant to one or more of the commonly used azoles (7).

The excessive use of antifungal drugs as well as a lack of proper drug therapy has led to drug resistance in recent years. One of the resistance factors to antifungal drugs is the formation of biofilms. Biofilms, by preventing the binding of antifungals to microorganisms, produce high resistance to antifungal drugs. Therefore, for the correct diagnosis and timely treatments, it is necessary to identify the factors causing and investigate the pattern of drug sensitivity (10).

In this study, we isolated and identified Candida species in the vaginal area of patients referred to Shahroud Bahar Hospital and tested in vitro activities of fluconazole, clotrimazole, and miconazole against isolated Candida species. Therefore, this study can provide a deeper understanding and a better perspective of biofilm production and antifungal susceptibility in Candida spp. isolated from patients with vaginal candidiasis and assist in its control.

Materials and Methods

Ethics approval

This study was approved by the Ethical Committee of Tehran University of Medical Science (the number of Ethics Committee protocol: IR.TUMS.SPH.REC.1397.154). Written informed consent was obtained from all subjects before sample collection.

Sampling

During a cross-sectional study, from Feb 2018 to Jul 2018, 150 patients with vvulvovaginitis symptoms such as itching, burning sensation, pain, inflammation, and excessive and stinging discharge were enrolled in this study. Laboratory test for determining the presence of infection and evaluation the in vitro susceptibility to three anti-fungal agents was done in Shahroud University of Medical Sciences, Iran.

In the first step, demographic data of the patients were obtained through questionnaires, and then the collection of posterior vaginal fornix or vaginal wall specimens by the senior clinicians was done for laboratory testing. Patients who were previously treated with anti-fungal drugs within two months ago were excluded from the study. Samples were prepared by KOH 10%, cultured on Sabouraud’s dextrose agar (Merck, Germany), and kept at 37 °C for one week. Isolates were identified based on production chlamydoconidia in cornmeal agar (Becton, France) and colony color on chromogenic CHROMagar Candida medium (CHROMagar, Paris, France). In addition, for confirmation of species identification isolates were subjected to the polymerase chain reaction– restriction fragment length polymorphism (PCRRFLP) technique (11).

Crystal violet method for assessment biofilm formation

Growth from Sabouraud dextrose agar (SDA) was taken in 5 mL of sterile brain heart infusion (BHI) broth and incubated overnight. It was followed by 1:100 dilutions in BHI. Then, 100 μL of diluted broth was incubated at 37 °C overnight in commercially available pre sterilized, polystyrene, round-bottomed, 96-well microtiter plate (Cat no. 2101; Thermo Fischer Scientific, Waltham, MA, USA) for biofilm production. The microtiter plates were washed with distilled water. After washing with distilled water, the microtiter plate was stained with 120 μL of 0.1% aqueous crystal violet solution (crystal violet, Hi-Cert/ACSGRM114-10G) and incubated at room temperature for 15 min. Each well was washed four times with sterile distilled water, blot dried, immediately de-stained with 125 μL of 95% methanol, and incubated for 15 min at room temperature. After de-staining, 100 μL of the de-staining solution was transferred to a new well, and the de-staining solution was measured spectrophotometrically using the enzyme-linked immunosorbent assay (ELISA) reader at 570 nm. Each sample was run in duplicates (12). The following were the values for the biofilm formation:

OD≤ODC=non-adherent

ODC

2ODC

4ODC

Assessment of antifungal drug susceptibility

Three clinically important antifungal agents were used in this study. All drug solutions were prepared immediately before use. Antifungal susceptibility testing for fluconazole and clotrimazole was determined by the broth microdilution method using the guidelines outlined in the Clinical and Laboratory Standards Institute (CLSI) document M27-A3. Both of them were dissolved in dimethyl sulfoxide (DMSO) and filtered into the growth medium. Freshly prepared stock solutions of the drugs were diluted in the growth medium (RPMI with MOPS buffer, pH 7). Testing was performed in sterile flat-bottomed 96-well microplates, containing RPMI-1640 medium with L-glutamine, without sodium bicarbonate supplemented with glucose (18 mg/mL) and buffered with M3-(Nmorpholine)-propane sulfonic acid (MOPS) at pH 7.0. A final yeast concentrations after inoculation 1 × 103 cells/ml. 50 μl of this suspension was transferred to an ampule of microplates and 100 μl of the inoculated medium was transferred into each cupule. Microplates were incubated at 35 °C for 48 h. Readings were made both visually and spectrophotometrically at 490 nm with a microplate reader.

Disk diffusion (DD) testing of miconazole was performed as described previously in the CLSIM27-A3 protocol (13). We prepared plates with Muller Hinton Agar + 2% Glucose + 0.5 mcg/mL Methylene Blue Dye (M1825) for carrying out the susceptibility of antifungal discs. The medium in the plates was sterile and had a depth of about 4 mm. The agar surface was inoculated by using a swab dipped in a cell suspension adjusted to the turbidity of a 0.5 McFarland standard. Miconazole disks (Becton Dickinson, Sparks, MD) were placed onto the surfaces of the inoculated plates, and the plates were incubated in the air at 35 °C to 37 °C and read at 18 to 24 h. Zone diameter endpoints were read at 80% growth inhibition by using a Biomic image analysis plate reader system (Giles Scientific) (13–15).

Statistics analysis

Data analysis was performed by t-test method with SPSS (Chicago, IL, USA) Statistics version 21. The level of statistical significance was set at P<0.05. The Chi-square test was used to compare significantly percentages.

Results

From 150-suspected cases of vulvovaginal disease, 50 women (33.3%) were suffering from VVC. C. albicans was the predominant species isolated in this study (n=39, 78%) followed by C. glabratia (n=11, 22%).

All 50 clinical isolates were subjected to biofilm production. Out of 39 C. albicans isolates, 19 (48.7%) strains were positive for biofilm production.

Among the total 11 C. glabrata isolates, 6 (54.5%) isolates were biofilm producers. We calculated the OD Cut off = 0.13.

The highest inhibitory concentration of fluconazole for C. albicans was 16 ug/mL, while the lowest inhibitory concentration was 0.5 ug/ml. Furthermore, the highest inhibitory concentration of this drug for C. glabrata was 32 ug/mL, while the lowest inhibitory concentration was 1 ug/mL.Furthermore, the highest inhibitory concentration of clotrimazole for both isolated species was 8 ug/mL, while the lowest inhibitory concentration was 1 1 ug/mL for both of them.

The diameter range of miconazole inhibition zone against C. albicans strains was 20–40 mm and the diameter range of miconazole inhibition zone against C. glabrata was 19–40 mm. Moreover mean diameter of miconazole inhibition zone for C. albicans and C. glabra isolates was 25.13 mm and 24.5 mm, respectively and all of them were susceptible to this drug.

There was a statically significant relationship between the MIC of clotrimazol and the ability of biofilm production in C. albicans and C. glabrata isolates (P<0.05). In addition, there was not a statically significant relationship between the MIC of fluconasole and the ability of biofilm production in C. albicans and C. glabrata isolates. Concerning miconazole disk the results of C. albicans and C. glabrata isolates were not statistically significant.

Discussion

Candidiasis is an infection caused by a yeast (a type of fungus) called Candida. Candida normally lives in anatomical sites of the body such as the mouth, gut, vagina, and skin without causing any problems. However, sometimes it can multiply and cause a vaginal yeast infection (16–18). VVC due to different Candida species is a very common type of fungal infection. It remains a significant problem worldwide estimated to be the most common fungal infection in a number of countries (19–21).

Recent evidence suggested that the majority of infections produced by these fungal elements are associated with biofilm growth. Biofilms are biological communities with a high degree of organization, in which microorganisms form structured, coordinated, and functional communities. These biological communities are embedded in a self-created extracellular matrix. Biofilm production is also associated with a high level of antimicrobial resistance of the associated organisms. The ability of Candida species to form drug-resistant biofilms is an important factor in their contribution to human disease.

In this study, according to other studies, C. albicans was the predominant Candida species isolated from vaginal specimens (22–27). However, one study provided evidence of the increasing prevalence of non-C. albicans species contribute to VVC (28).

Moreover, C. albicans and C. glabrata are capable of producing biofilms. The majority of disease produced by C. albicans is associated with biofilm growth (29). C. glabrata isolates were also capable of producing biofilms (30).

Antifungal susceptibility testing revealed that clotrimazole had the highest antimicrobial activity against isolated Candida species. In addition, miconazole demonstrated potent inhibitory activity against all of the Candida strains tested, indicating that no miconazole resistant strains were detected among any of the species.

On the contrary, 2 C. glabrata species (18.18%) were resistant to fluconazole. Totally, with the exception of fluconazole, all of the comparator drugs demonstrated activity against the tested isolates.

Conclusion

For the correct diagnosis and timely treatment, it is necessary to identify the factors causing drug resistance and investigating the pattern of drug sensitivity. In the present study, C. albicans was the predominant Candida species isolated from patients with VVC, which also was the predominant biofilm producer species. Resistance of vaginal Candida species isolates to antifungal agents was infrequent.

Journalism Ethics 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.