Analysis of Biofilm-Related Genes and Antifungal Susceptibility Pattern of Vaginal Candida albicans and Non-Candida albicans Species

Background Vulvovaginal candidiasis caused by Candida species is a prevalent fungal infection among women. It is believed that the pathogenesis of Candida species is linked with the production of biofilm which is considered a virulence factor for this organism. The aim of this study was molecular identification, antifungal susceptibility, biomass quantification of biofilm, and detection of virulence markers of Candida species. Methods We investigated the molecular identification of 70 vaginal isolates of Candida species, antifungal resistance to amphotericin B, fluconazole, itraconazole, and voriconazole according to CLSI M27-A3 and M27-S4, biofilm formation, and frequency analysis of biofilm-related ALS1, ALS3, and HWP1 genes. Results Our findings showed that the most common yeast isolated from vaginal discharge was C. albicans (67%), followed by the non-Candida albicans species (33%). All C. albicans complex isolates were confirmed as C. albicans by HWP-PCR, and all isolates of the C. glabrata complex were revealed to be C. glabrata sensu stricto using the multiplex PCR method. FLC resistance was observed in 23.4% of C. albicans and 7.7% of C. glabrata. The resistance rate to ITC was found in 10.6% of C. albicans. The frequency of ALS1, ALS3, and HWP1 genes among Candida species was 67.1%, 80%, and 81.4%, respectively. Biofilm formation was observed in 54.3% of Candida species, and the highest frequency detected as a virulence factor was for the ALS3 gene (97.3%) in biofilm-forming species. Discussion. Our results showed the importance of molecular epidemiology studies, investigating antifungal susceptibility profiles, and understanding the role of biofilm-related virulence markers in the pathogenesis of Candida strains.


Introduction
Vulvovaginal candidiasis (VVC) is a common fungal infection in young women, and several factors such as age, diabetes, pregnancy, taking estrogen-containing birth control pills, antibacterial therapy, and the use of intrauterine devices have been identified as risk factors [1]. Vaginal candidiasis is associated with discharge, inflammation, burning, and redness. Candida albicans is the most important causative agent of VVC, followed by non-Candida albicans species [2]. The diagnosis of vulvovaginal candidiasis is often based on clini-cal signs and symptoms, and fluconazole is mostly used to treat Candida vaginitis. However, variable levels of resistance against antifungals may emerge in Candida species due to empirical administration or overuse of antimycotic agents [3,4]. In recent decades, drug resistance has been increasing due to the overuse of antifungals [5,6]. Therefore, understanding the antifungal susceptibility profiles of Candida species is key in guiding the selection of appropriate treatment for vulvovaginal candidiasis [6]. On the other hand, the pathogenicity of Candida species is increased with factors such as the ability to produce hyphae, binding and adhesion, extracellular enzyme production, and biofilm formation [7,8]. The agglutinin-like sequence (ALS) gene family is the largest gene family known in C. albicans and is considered one of the important factors in the adhesion of the organism and the formation of biofilm [9]. The ALS gene family consists of several genes including the ALS1-ALS7 and ALS9 genes which play a role in the production of cell surface glycoproteins, leading to increased adhesion to host cells [10]. ALS1 and ALS3 genes affect adhesion to host epithelial cells and endothelial cells [11,12]. Another protein affecting the adhesion and regulation of biofilm in C. albicans is the hyphal wall protein produced by the HWP1 gene [13].
HWP1 protein is a mannoprotein-linked glycosyl phosphatidylinositol that, similar to the protein encoded by the ALS family of genes, plays an important role in Candida adhesion [14].
Therefore, this protein is believed to have a significant link with the pathogenicity and virulence of C. albicans [14]. Several studies have shown that the HWP1-producing gene is expressed in the early stages of biofilm formation [15,16]. The objective of this study was molecular identification, antifungal susceptibility profiling, biomass quantification of biofilm, and analysis of the distribution pattern of ALS1, ALS3, and HWP1 virulence genes of Candida isolates from VVC.

Sample Collection.
A total of 135 genital clinical samples of the married women suspected of VVC were obtained from April to October 2020 who referred to the Kowsar gynecology clinic in Qazvin, Iran. The consent form was obtained from all participants. Pregnancy, douching history of less than past 48 hours, lack of patient contentment, and history of recent consumption of antimicrobials were considered exclusion criteria. The study was approved by the Ethics Committee of Qazvin University of Medical Sciences (IR.QUMS.REC.1397.085). A midwife was in charge of taking the vaginal specimens using sterile swabs, followed by rapid delivery of samples to the Qazvin Medical School mycology laboratory. The Sabouraud dextrose agar medium (SDA, Difco) was used to culture the vaginal sample which was later incubated at 30°C. The phenotypic test was performed using the chromogenic medium of CHROMagar Candida (CHROMagar Company, Paris, France).
2.3. PCR-RFLP Analysis. PCR products were digested with the MspI enzyme (Thermo Fisher Scientific, Lithuania). The 30 μL reaction mixture contained 1 μL of the MspI enzyme, 2 μL of the buffer, 10 μL of the PCR product, and sterile distilled water to the final volume, followed by incubation at 37°C for 3 h. The PCR-RFLP products were analyzed by agarose gel electrophoresis.
2.4. Hyphal Wall Protein 1 (HWP1) Gene and Multiplex Polymerase Chain Reaction. All C. albicans isolates were confirmed by PCR amplification of the HWP1 gene, using CR-f and CR-r primer pairs (Table 1). Conditions for HWP-PCR were as follows: initial denaturation at 95°C for 5 min, followed by 35 cycles of 45 s at 94°C, 40 s at 58°C, 55 s at 72°C, and a final extension of 10 min at 72°C. The identification of the C. glabrata complex including C. glabrata sensu stricto, C. bracarensis, and C. nivariensis was achieved using the universal reverse primer (UNI-5.8S), GLA-f, NIV-f, and BRA-f (Table 1) [16]. In brief, the reaction mixture contained 12.5 μL of Taq Mix Red 2X, 0.2 μM of each of the four primers, and 10 ng of genomic DNA. Multiplex amplification was performed as follows: initial denaturation at 95°C for 5 min, followed by 34 cycles of 30 s at 94°C, 40 s at 60°C, 50 s at 72°C, and a final extension of 10 min at 72°C.
2.5. Antifungal Susceptibility Test. Susceptibility assay was performed in 96-well plates by following the M27-A3 and M27-S4 guidelines of the Clinical and Laboratory Standards Institute [18] for in vitro testing [19,20].
2.6. Biofilm Formation. The biofilm production was evaluated using a 1% crystal violet (CV) solution in vitro. In brief, the yeast cell suspension and Sabouraud dextrose broth (SDB), supplemented with 60 g of glucose, were inoculated into wells of 96-well microplates [21]. After incubation at 35°C for 24 hours, the wells were washed three times with sterile PBS and stained with crystal violet for 15 min. Finally, fixation was performed using ethanol-acetone (80 : 20, v/v).
The absorbance values were determined using an ELISA reader at 590 nm. Biofilm formation assay was repeated three times for each strain.

Results
A total of 135 vaginal clinical samples of married women suspected of VVC were examined. The mean age of the patients was 31 ± 6:5 years (range: 19 to 46 years). Direct examination and culture results showed that out of 135 suspected women included in the present study, 70 (51.8%) were positive for VVC. Among women who suffered from VVC, the frequency of clinical symptoms included white discharge (83%), itching (77%), inflammation (60%), and irritation (54.3%), respectively.

Candida albicans and C. glabrata Complex Identification.
The multiplex PCR for the identification of 13 isolates of the Candida glabrata complex showed that the amplified products were solely a 397 bp sequence comparable with C. glabrata sensu stricto. None of the isolates belonged to C. nivariensis and C. bracarensis (Figure 1). Furthermore, the molecular identification using the HWP1 gene demonstrated that the PCR product of all isolates was a 1000 bp sequence belonging to C. albicans sensu stricto (Figure 1).   (Figure 1). The ALS1 gene was not detected in C. parapsilosis, whereas the HWP1 gene was observed in 50% of C. parapsilosis. The prevalence of the ALS3 gene was in C. albicans (n = 43/47; 91.5%), C. glabrata (n = 6/13; 46.2%), C. kefyr (n = 3/5; 60%), C. tropicalis (n = 3/3; 100%), and C. parapsilosis (n = 1/2; 50%) ( Figure 1). The simultaneous presence of ALS1, ALS3, and HWP1 genes was observed in 27% and 15.4% of fluconazoleand itraconazole-resistant biofilm-producing species, respectively ( Table 3). The result of SEM of the network of dense hyphal by C. albicans is shown in Figure 2. The simultaneous presence of ALS1, ALS3, and HWP1 genes was found in 95% of studied strains.

Discussion
It is reported that a quarter of women are exposed to vaginal candidiasis during their lifetime [23]. In recent years, there have been reports of non-Candida albicans agents isolated from women with VVC. Our findings showed that the most common yeast isolated from vaginal discharge was C. albicans (67%), followed by C. glabrata (18.5%), C. kefyr (7%), C. tropicalis (4.2%), and C. parapsilosis (2.8%) The distribu-tion of Candida isolates in our study was similar to other studies [24][25][26][27][28]. In our study, all the C. albicans complex isolates were confirmed as C. albicans based on the presence of a 1000 bp band, whereas no band was detected for either C.  [35]. In Greece, the antifungal susceptibility profile has shown that Candida isolates from patients with vulvovaginal candidiasis to AMB and VRC were 0.2% and 1.4%, respectively [36]. In addition, Ghaddar et al. revealed that 97.5% of C. albicans isolates from VVC patients were resistant to VRC [37]. Our findings demonstrated that 23.4% and 10.6% of C. albicans isolates were resistant to FLC and ITC, respectively. The fluconazole resistance rate was reported from Belgium, Ethiopia, and Taiwan at 21%, 53.7%, and 14.7%, respectively [25,33,38]. Similarly, Adjapong et al. reported that 10% of C. albicans isolates from VVC patients were resistant to ITC [39]. Edrees et al. showed that 29.6% of C. albicans isolates from VVC patients were resistant to ITC [40]. In addition, our findings showed that the resistance rate of C. glabrata to fluconazole was 7.7%. Miranda-Cadena et al. showed that the resistance rate to FLC and ITC in C. glabrata was 7.7% and 6.1%, respectively [41]. In Iowa, 15.2% of C. glabrata isolates from VVC were fluconazole resistant [42]. Biofilm formation, as a virulence factor, plays an important role in the pathogenesis and binding of Candida species to host cells [43]. In our study, the rate of biofilm production was detected in 54.3% of VVC-associated Candida species. Biofilm formation was also found in 26.3% and 13.2% of FLC-and ITCresistant isolates, respectively. Studies show that Candida species can help to reduce drug penetration and increase antifungal resistance by forming a dense network of pseudohyphae in biofilms [44,45]. The results of several studies showed that the ALS3 and ALS1 genes of the ALS family as well as the HWP1 gene play an important role in the Candida species adhesion and biofilm formation process [46]. Our results revealed that the highest frequency for the genes tested in the present study was obtained for the HWP1 gene (81.4%) of Candida species isolated from VVC. ALS3 and  Ardehali et al. reported that the highest virulence factor identified was the HWP1 gene, with a frequency of 95% in C. albicans isolated from blood and urinary infections. In their study, the presence of ALS3 and HWP1 genes in C. glabrata was higher than that of the ALS1 gene. In addition, the ALS1 gene was not detected in C. tropicalis [47]. Shrief et al. reported a frequency of 77% for the HWP1 gene and  BioMed Research International 65% for the ALS1 gene of C. albicans isolated from blood and urine cultures. They also described that 58% of C. albicans were capable of biofilm production [48]. The prevalence of the ALS1 and HWP1 genes was detected in 48% and 3.9% of the strains isolated from vaginal samples in Turkish women, respectively [49].
A study on Brazilian women showed that the frequency of ALS1 and HWP1 genes in the C. albicans collected from vaginal discharge was 73.6% and 21%, respectively [50]. Differences in the type of clinical samples and techniques used for diagnosis may cause variations in the frequency of genes in various studies. There are many reports that have been concerned with the expression of ALS and HWP genes. Cheng et al. indicated that the ALS1 and ALS3 genes were expressed in both the clinical vaginal discharge and the experimental model system for vaginal candidiasis [51]. The results of our study and several other studies have shown that the frequency of the presence of these genes and their expression in clinical isolates can indicate the role of these genetic markers in promoting the adhesion and biofilm formation in Candida species, especially C. albicans. Moreover, the simultaneous presence of ALS1, ALS3, and HWP1 genes plays a role in enhancing the synergistic effect on the performance of individual genes involved [13,52]. In our study, the frequency of the ALS3 gene was found in 94.7% of biofilm-positive species which was higher than the other genes examined. Furthermore, the simultaneous presence of ALS1, ALS3, and HWP1 genes in biofilm-producing Candida species was 52.8%. In Egypt, the biofilm capacity was observed in 58% of Candida albicans isolates from the chemotherapy patients. Also, the prevalence of ALS1 and HWP1 genes among the biofilmpositive C. albicans was 56.9% [48]. Inci et al. showed the presence of ALS1 and HWP1 genes in 83.1% and 11.3%, respectively, of biofilm-producing isolates [49].

Conclusion
In our study, C. albicans showed the highest frequency for ALS1, ALS3, and HWP1 genes. This high prevalence rate for virulence factors is significant in C. albicans vaginal specimens. It is of great concern that several studies have shown that Candida biofilm formation may be involved in resistance to antifungals. Since biofilm formation affects the process of vulvovaginal candidiasis infection, it is important to understand the role of virulence factors in the pathogenesis of Candida species.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors reported that they have no conflicts of interest in this paper.