Expression pattern of drug-resistance genes ERG11 and TAC1 in Candida albicans Clinical isolates

Candida albicans (C. albicans) is an opportunistic fungus and the most common cause of vulvovaginal candidiasis (VVC). In recent years, the use of antifungal drugs has led to the incidence of drug-resistant C. albicans strains. The purpose of this study is twofold: to determine the pattern of drug susceptibility and the relationship between demographic factors and the incidence of drug resistance among C. albicans isolates and to investigate the expression pattern of drug-resistance genes ERG11 and TAC1 in C. albicans isolates. This descriptive cross-sectional study was conducted on 50 C. albicans isolates from women with VVC. Antifungal susceptibility of the isolates was tested by M27-A3/S4 broth micro dilution method following the Clinical and Laboratory Standards Institute (CLSI) guidelines. High susceptibility rates were recorded for itraconazole and voriconazole (68%), followed by ketoconazole (46%). Fluconazole had the lowest susceptibility to C. albicans with susceptibility of 36%. The change in ERG11 and TAC1 genes expression was determined by qPCR. The mean ∆Ct values of ERG11 and TAC1genes were significantly different between fluconazole-resistant and susceptible groups (p < 0.001). Interestingly, we found that 77% of fluconazole-susceptible isolates had significantly upregulated ERG11 gene (2.9–99.0 fold). In addition, the expression of TAC1 was upregulated in 44% of fluconazole-susceptible isolates (3.86–89.8 fold). Our finding revealed that incidence of drug resistance in C. albicans is not simply controlled by genes but is a multi-factorial phenomenon, where several factors and mechanisms are involved in the process of drug resistance.


Introduction
Vulvovaginal candidiasis (VVC), is the most common mucosal infection caused by Candida albicans (C.albicans), affects women of reproducing age (17-55years) [1].Nearly 75 percent of women experience VVC at least once in their lifetime.Approximately, 40 to 50 percent of women have a history of VVC reinfection, and 5 percent of women develop recurrent vulvovaginal candidiasis (RVVC).The latter describes a situation where at least four discrete episodes occur in one year [2][3][4].Although the disease is rarely life-threatening, it can lead to high-cost treatment, and in cases, infertility [5].Even though the presence of itching and burning symptoms is regarded as a strong suspicion for the presence of VVC infection, these symptoms cannot differentiate between recurrent and acute types of the disease; therefore, it may be di cult to choose an appropriate treatment protocol [6].
Similar to other super cial fungal infections, VVC is typically treated with azole antifungal agents, which may include imidazoles (miconazole, clotrimazole, and ketoconazole) and/or triazoles ( uconazole and itraconazole) [7].While uconazole is a rst-line azole antifungal drug against VVC, susceptibility to this azole is decreased by various mechanisms of resistance [2,8].Indeed, the widespread use of azoles is assumed to be a factor that promotes drug resistance.Many factors are relevant to emerging azole resistance in C. albicans, including the expression of drug targets [9].For instance, upregulation in lanosterol 14-α-demethylase encoded by the ERG11gene is essential for the functioning of a microorganism cell [10,11].Therefore, overexpression of several genes is associated with uconazole resistance in Candida species.One of the mechanisms of resistance to azole drugs may result from the up-regulation of drug e ux pumps, which are encoded by the ATP-binding cassette transporter genes (CDR1, CDR2, and MDR1) [12,13].TAC1 gene is a classical zinc cluster transcription factor.This gene regulates the expression of e ux pump genes directly [6].
According to the literature, there is a keen interest in research on molecular mechanisms of antifungal resistance [14,15].A greater understanding of mechanism-speci c resistance and the biological factors relevant to resistance emergence is critical for developing better therapeutics and improving diagnostics and interventions that may overcome and prevent resistance [16].
This study attempted to test azole susceptibility among C. albicans isolates and to determine the relationship between risk factors and antifungal resistance of C. albicans clinical isolates.Moreover, in this study evaluated the relationship between ERG11 and TAC1 genes expressions and resistance to azoles to determine the pattern of molecular mechanism in C. albicans isolated from Iranian women with VVC.

Study design
This research was conducted on 50 C. albicans isolates obtained from women suspected of VVC, who were referred to the obstetrics and gynecological specialists in Birjand city from December 2018 to March 2019.In this regard, 250 patients suspected of VVC were identi ed and vaginal specimens were collected and were transferred to the laboratory for mycology diagnosis.The patient demographic characteristics including age, history of infection, symptoms, diabetes, method of contraception, and drug consumption were collected.Patients' identifying information was kept con dential.The study protocol was approved by the ethics committee of Birjand University of Medical Sciences, Iran (Ir.bums.REC.1398.350).All participants signed a written consent form.Besides, exclusion criteria comprised pregnant women, menstruating women, and women with a history of hysterectomy.

C. albicans strains
The study examined standard fungi strains, including C. albicans (ATCC10231) and 50 clinical isolates of C. albicans from women who suffered from VVC.The C. albicans isolates were con rmed using phenotyping method and con rmed by PCR.

Identi cation of Candida albicans isolates
The isolates for fungal culture was inoculated into Sabouraud Dextrose Agar (SDA Merck, Germany) medium and incubated at 30 • C. The identi cation of C. albicans was done by culture on CHROM agar (CAC, Becton Dickinson, Heidelberg, Germany) is ready to use (32 0 C for 24-48h) with which a presumptive identi cation of Candida species can be made on the basis of the morphology and colors of the colonies (C.albicans strain produced light green colonies and smooth colony) [17].

Pcr Assay
To con rm the detection of Candida albicans species using PCR technique which as carried out as repoted previously [18].In brief, PCR assay was optimized in a nal volume of 25µl consists target primers used to identify were as follows: CALBF: 5' CCATGTCGAACGTAGCGTATGC 3' , CALBR-5' AGATTATTGCCATGCCCTGAG 3' . of C. albicans isolates, DNA template and master mix (2X Blue Load master Mix, DNAbiotech, Iran).Genomic DNA from clinical isolates and standard species of candida were extracted by glassbead and lysis solution according to the method described previously by Nikoomanesh et al [19].The sequences of primer was BLAST in NCBI and approved.The primer was synthesized and shipped by Sinacolon Company (Tehran, Iran).Final volume of 25µl using Diethyl pyrocarbonate (DEPC) water.PCR ampli cation process was carried out with applied PeqSTAR 2X thermal cycler (PEQLAB, Germany) under the following condition: initial denaturation at 95 o C for 5min, followed by 35 cycle incubation at 95 o C for 45sec, 58 o C for 60sec and 72 o C for 60sec.and nally 72 o C for 5min as nal extension.C. albicans (ATCC10231) was used for positive control.PCR products were analyzed by electrophoresis through a 2% agarose gel.(Gel Doc XR+, BioRad, USA).Species identi cation was achieved by discrimination of amplicon sizes (606bp).

Antifungal Susceptibility Testing (Afst)
Assays for susceptibility of C. albicans isolates were performed using broth microdilution as per the guidelines of the Clinical and Laboratory Standards Institute (CLSI).Brie y, all isolates were tested against antifungal drugs, including uconazole, itraconazole, voriconazole, clotrimazole (Sigma-Aldrich, Canada), and amphotericin B (Sigma-Aldrich, Canada).The drugs were obtained from pure powder and prepared at concentrations speci ed in the M27-A3/S4 standard.The tests were performed in 96-well polystyrene microtitre plates.Antifungal drugs were diluted inspeci c dilutions.Afterward, 100µL of RPMI1640 medium with different concentrations of antifungals was added to a series of wells.In the end, 0.5×10 3 to 2.5× 10 3 CFU/ml were distributed in each well.The plates were closed and incubated at 35°C for 24 h.The nal concentration was in the range 64-0.125 µg/ml for uconazole and 16-0.03µg/ml for amphotericin B, itraconazole, voriconazole, and clotrimazole.Each C. albicans isolate was studied twice for each antifungal drug.The susceptibility tests were interpreted according to the CLSI M-27 (A3/S4) standard.Lastly,C.albicans (ATCC10231) was used for quality control purposes [20].
Total RNA was extracted from cultured colonies of C. albicans isolates with glass beads and lysis buffer as described earlier [22].The cDNA template was obtained from the total RNA usinga cDNA synthesis kit (Parstoos, Mashhad, Iran) as per the manufacturer's protocol.Quantitative real-time PCR was accomplished using AMPLIQON (Real Q plus 2 × master mixes Green High Rox, Sinnaclon, Tehran, Iran).To analyze PCR performance, the mixture containing 12.5 µl of master mix (Green High Rox), 0.25 µl of each speci c primer pmol), and 4 µl of cDNA template (10 ng) were adjusted to a nal volume of 25 µl using DEPC water.The PCR condition was started at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s.All steps were performed according to the ABI StepOne (Applied Biosystems, Rotkreus, Switzerland).All the experiments were carried out in triplicates.The expression of genes in RT-PCR was analyzed using REST 2009 software (Ver.2.0.13)(P < 0.001).

Statistical Analysis
The collected data were statistically analyzed by SPSS (V.22) software.Descriptive statistical tests, chisquare test, and t-test were used to analyze the data.The normality of numeric variables was checked by the Kolmogorov-Smirnov test.The association between antifungal resistance with potential risk factors and clinical symptoms was identi ed by the Chi-square test in univariate analysis.The signi cance level was set at P < 0.05.

Results
In this study, fty C. albicans isolates were con rm by molecular identi cation C. albicans-speci c primers which led to PCR product of 606bp for Candida albicans, and chromogenic medium were performed.According to the results of culturing on CHROM agar, 50 isolates which produced light green colonies.

Antifungal Susceptibility
The sensitivity of 50 C. albicans isolates to antifungals was tested for 1 polyene (amphotericin B) and 4 azoles ( uconazole, voriconazole, itraconazole, and ketoconazole) using the broth microdilution method.According to the Clinical and Laboratory Standards Institute (CLSI M27 (A3/S4) method, the C. albicans isolates were categorised as susceptible and resistance to uconazole; MIC ≥ 8 µg/mL was considered susceptible, MIC ≤ 2 µg/mL was considered resistant, and MIC = 4 µg/mL was dose-dependent susceptibility.Similarly, the isolates were categorized for other azoles; MIC ≤ 0.12 µg/mL was considered susceptible (),MIC ≥ 1 µg/mL was considered resistant.About of amphotericin B ; MIC ≤ 2 µg/mL was considered susceptible, and MIC ≥ 2 µg/mL was considered resistant.Amphotericin B exhibited an excellent e cacy against all isolates (100% sensitivity).Susceptibility to azoles varied among C. albicans isolates.The highest susceptibility was found for itraconazole and voriconazole (68%), followed by ketoconazole (46%) and uconazole (36%).It was observed that uconazole had the smallest impact on the C. albicans with 36% sensitivity.A detailed analysis of crossresistance among the four tested azoles revealed that seven C. albicans isolates were resistant to all azoles.Antifungal susceptibility is displayed in Table 1.  2. Statistical analyses showed a signi cant difference between the history of infection and the method of contraception with incidence of uconazole resistance (P < 0.05).
Individuals with a history of infection had a two-fold higher risk of developing uconazole resistance than those who did not have such a history (OR: 2.22, P = 0.006).Besides, the patients who used a method of contraception were at a higher risk for incidence of uconazole resistance (OR: 3.14, P = 0.002).However, no signi cant difference was seen among other characteristics and the incidence of resistance (P > 0.05).The expression levels of antifungal resistance genes (ERG11 and TAC1) in 50 C. albicans isolates susceptible and resistant to uconazole were quanti ed and normalized relative to the housekeeping gene B-actin (Act1).
C. albicans isolates showed the highest resistance to uconazole in this study; hence, they were selected for the evaluation of molecular mechanisms of resistance to uconazole.
The rates of overexpression and lowexpression of ERG11 gene among susceptible and resistant C. albicans isolates are shown in Fig. 1.The results show that overexpression of the ERG11 gene was observed in all isolates of the resistant group (100%).Overexpression of the TAC1 gene in the resistant group had the highest rate (96.8%) compared to the sensitive group (44.4%).The results of the t-test analysis showed a signi cant difference between the rate of overexpression and lowexpression of ERG11 and TAC1 genes (P < 0.001).
The mean relative gene expression levels of ERG11 in susceptible and resistant isolates were 135.75 ± 150.4 and 170 ± 152.64, respectively.The mean relative gene expression levels of TAC1 in susceptible and resistant isolates were 82 ± 128.21 and 173.85 ± 175.63, respectively.
Quantitative RT-PCR experiments revealed that the mean fold change in the expression levels of the ERG11gene was 1.1.The mean fold change in the expression levels of the TAC1 gene was a 3.1 fold change.There was a statistically signi cant difference in the levels of ERG11 and TAC1 expression in resistant isolates compared to susceptible isolates (P < 0.001) (Fig. 2).

Discussion
VVC is a widespread fungal infection that is induced primarily by C. albicans and affects reproductiveage women [2].Although VVC is primarily a challenging therapy worldwide, some socioeconomic factors could affect its incidence [4].Besides, service planning managers should utilize updated epidemiological data from across the world.
In the recent decade, the amount of fungal resistance to antifungal drugs has increased and has led to therapeutic problems [22].In the present study, the 50 C. albicans isolates that were tested for susceptibility to antifungals belonged to the polyenes group (amphotericin B) and azoles group ( uconazole, voriconazole, itraconazole, and clotrimazole) via the microdilution method.The results obtained from amphotericin B exhibited an excellent susceptibility to all C. albicans isolates.This nding agrees with the results reported in studies by Eski et al. and Mohammadi et al. concerning an absence of amphotericin B-resistant isolates [23,24].Besides, susceptibility to different azoles drugs varied among C. albicans isolates, with lower susceptibility of C. albicans isolates to uconazole (36%) followed by clotrimazole(52%).Meanwhile, itraconazole and voriconazole (68%) exerted similar effects.
The widespread use of uconazole against prophylaxis and Candida infections may be one of several factors contributing to antifungal resistance.C. albicans has developed multiple resistance mechanisms against azole antifungal drugs, leaving the way open to only a few antifungal agents available [31].
Therefore, it is of particular importance to investigate the mechanisms and patterns of antifungal resistance.In the current study, the analyses of the association between risk factors and incidence of uconazole resistance showed higher rates of anti fungal resistance in women with a history of vaginitis.Moreover, the patients who had used a contraception method were at a greater risk to develop drug resistance (Table 2).As a result, the history of infection indicates that a failure in the treatment of primary infection leads to the emergence of resistant species.Also, any factor that predisposes to infection can lead to the emergence of resistant species.
Findings that arise from such research can contribute to designing and developing alternative therapeutic strategies.Additionally, understanding the molecular mechanism of antifungal resistance and the role of genes relevant to drug resistance can be used to diagnose resistant isolates via molecular diagnostic tools.
Previous research has reported overexpression of ATP-binding cassette transporters, which are encoded by different genes, primarily CDR1 and CDR2, in C. albicans.TAC1 gene is the rst transcription factor that regulates e ux pump family genes [32,33].However, it is well documented that the most signi cant factor involved in C. albicans resistance to azole antifungal agents is the overexpression of the ERG11 gene [34,35].
According to our results, TAC1 gene expression was not merely increased in uconazole-resistant isolates; it was also abserved in sensitive isolates.The TAC1gene was found to have overexpression rates in resistant and sensitive isolates, respectively, with statistically signi cant differences between the two groups (P < 0.001).Moreover, uconazole-sensitive isolates had ERG11 gene overexpression.In fact, all resistant isolates had ERG11 gene overexpression at signi cantly higher levels than the ATCC 10231 strain.Based on the results, we witnessed an overexpression of ERG11 and TAC1 in susceptible isolates.Despite the increase in gene expression in susceptible isolates, these isolates are sensitive to uconazole.
In a previews study by Riberio et al. after uconazole treatment, they observed ERG11gene overexpression in uconazole-sensitive C. albicans isolates.They concluded that the overexpression of the ERG11 gene is accompanied by the overexpression of other e ux pump genes and that the ERG11 expression is not the only cause of increased resistance to uconazole [36].Teimouri et al. detected ERG11 gene overexpression in eight resistant isolates of C. albicans from infants [37].Another study conducted by Sasse et al. evaluated the expression of TAC1 and MRR1 transcription factors [38].They investigated mutations in the gene sequence after they observed an increase in the expression of TAC1 and MRR1 genes.The results of this study showed that the increase in TAC1 gene expression is sometimes up to 500-fold in the development of uconazole resistance, arguably due to mutations in this gene.Shi et al. investigated the expression rates of e ux genes (CDR1, CDR2, MDR1, Erg11, and TAC1) in the bio lm formation process on uconazole-resistant isolates of C. albicans [39].The results showed that the increase in TAC1 expression occurred in the early stages ( rst 8 hours) and that the expression increase was nearly two-fold.In a study by Chen 2010 and Xu in 2008, over 143 mutations,contributing to increased ERG11 gene expression, were reported in uconazole-resistant C. albicans isolates [40,41].
The results obtained from this study, indicate that the occurrence of drug resistance can be directly related to increased expression of TAC1 which as transcription gene.In addition to increasing the expression of drug resistance genes, the incidence of drug resistance can be directly related to patient risk factors such as the use of broad-spectrum antibiotics, immune system function, and underlying diseases such as diabetes [10].As observed, there is a signi cant relationship between the resistance to azole resistance and history of infection or the use of contraceptive methods.This study has some limitations.In this study, the mutations of the studied genes were not investigated due to limited facilities.Therefore, future research may aim to detect the frequency of de nite mutations in the C. albicans ERG11 and TAC1 genes and its targets in resistant isolates.

Conclusion
The results showed that C. albicans isolates were more resistant to uconazole than other antifungal agents.Also, the incidence of drug resistance is associated with some patient risk factors such as history of infection and the method of contraception.Molecular studies on ERG11 and TAC1 genes expression revealed that these genes had overexpression in some uconazole-sensitive isolates.The results also revealed that drug resistance in C. albicans is not simply controlled by several genes but is a multifactorial phenomenon, where several factors and mechanisms are involved in the process of drug resistance.It can be concluded that drug resistance is not merely controlled by genes but affectted mutations on genes expression.The fold change expression of ERG11 and TAC1 genes in resistant isolates compared to uconazole sensitive isolates.

Table 1
The mean age (± SD) of study subjects was 31.22 ± 8.45 years.The clinical characteristics of study subjects are summarized in Table

Table 2
Comparison of the relationship between patients' risk factors and uconazole resistance.