Analysis of molecular resistance to azole and echinocandin in Candida species in patients with vulvovaginal candidiasis

Background and Purpose: Vulvovaginal candidiasis (VVC) is considered the most common mucosal infection caused by Candida species. Azoles were considered the first-line treatment for VVC or recurrent vulvovaginal candidiasis (RVVC) in both healthy and immunocompromised populations. Recently, azole-resistant isolates, especially among non-albicans Candida samples have been encountered. This study aimed to evaluate the antifungal susceptibility profile of Candida spp. isolated from VVC or RVVC patients and assess the molecular resistance mechanism of Candida spp. to azole and echinocandin. Materials and Methods: Point mutation analysis was performed on the ERG11 and FKS candidate genes of azole- and caspofungin-resistant Candida albicans and Candida glabrata isolates. Real-time polymerase chain reaction was performed to gain insight into the differential expression of ERG11 mRNA. Results: Variations in the amino acid D116E were observed in fluconazole- and itraconazole-resistant C. albicans strains, and changes in amino acid E517Q were observed only in fluconazole-resistant C. albicans strains. No polymorphisms were observed in the complete sequence alignment of the ERG11 gene in one azole-resistant C. glabrata isolate. The mutation triggered the changes in the amino acid serine in the reference gene FKS1 by the leucine at position 642 (S642L) of the isolates. Conclusion: In patients with persistent or recurrent infection, the choice of an antifungal agent is often challenging and requires monitoring of the antifungal susceptibility of the colonizing strain. C. albicans and C. glabrata isolates can be resistant to azole and caspofungin antifungal agents without mutations in the ERG 11 and HS1 regions of the FKS1 gene.


Introduction ulvovaginal candidiasis (VVC) is considered the most common mucosal infection caused by
Candida species [1]. It is believed that 75% of women experience VVC at least once in their lives [2]. As is the case with most infections caused by Candida spp., Candida albicans is isolated from 85-90% of cases [3]. However, non-albicans Candida spp. (NACs), which are less sensitive to the frequently administrated topical and oral azole agents, have also increased [1,[3][4][5]. In addition, NACs are more frequently involved in recurrent vulvovaginal candidiasis (RVVC), compared to C. albicans [4].
Azoles were considered the first-line therapy for VVC and RVVC in both healthy and immunocompromised populations. Recently, cases of azole-resistant isolates, especially among NACs, have been encountered [6,7]. Mutation in the gene that encodes the 14a-demethylase target, ERG11, can contribute to the resistance to azole in Candida spp. [8,9]. Alteration in the mRNA expression level of the genes encoding efflux pumps CDR1, CDR, and MDR created resistance to azole [10].
In most cases, loss of susceptibility to echinocandins was associated with the alternations in the hot spot region since echinocandins are insignificant substrates for drug efflux transporters [11]. Loss of susceptibility to echinocandins is facilitated through alternations in the FKS1 gene, which encodes the extraction of a key element in the cell wall [12]. The known mutations are associated mainly with hot spot 1 (HS1, at positions 641-649) and HS2 (at positions 1345-1365) regions [13]. The mutations in the FKS2 gene were found in C. glabrata and C. guilliermondii strains. Since the loss of Candida spp. susceptibility to antifungal treatment has become a major drawback in the management of recurrent infections, the use of other antifungals can be helpful in this regard.
Since various Candida spp., even different isolates of specific strains, have different sensitivity to an antifungal agent, it seems that identification of Candida at the species level is necessary for the selection of appropriate treatments and avoidance of antifungal failure. The present study aimed to evaluate the antifungal susceptibility profile of Candida spp. isolated from patients who were VVC and RVVCpositive. Furthermore, the molecular mechanism of resistance to azole and echinocandin was also assessed.

Study design
The current investigation was conducted on 75 clinical isolates of vaginal discharge from 147 patients suspected of VVC/RVVC who had referred to the Medical Mycology Laboratory of Razi Hospital, Tehran, Iran in 2019.

Sample collection and primary identification
The specimens were gathered using sterile cotton swabs. All specimens were subjected to direct microscopic examination to identify fungal elements. To access pure isolates, all specimens were cultured on CHROMagar Candida medium (CHROMagar, France) at 35 °C for 24 h. The specimens were stored in Tryptic Soy Broth (Liofilchem, Italy) at -70 ºC for further analysis.

DNA extraction
Genomic DNA of each positive isolate was extracted from fresh colony cultures on Sabouraud Dextrose Agar medium (Merck, Germany) at 35 ºC for 24 h using the isopropanol and proteinase K approach based on our previous setup [14]. The part of the yeast colony was transferred in a 1.5 ml microtube containing DNA extraction buffer and glass beads and vortexed. Subsequently, proteinase K and 20% Sodium dodecyl sulfate were added to it and it was kept warm at 55 ºC for 60 min. Afterward, 6 molar NaCl was added, and the total DNA was precipitated using isopropanol. The precipitate was rinsed in 300 μL ethanol 70%, air dehydrated, rehydrated in 50 μl TE buffer (Merck, Germany), and kept at -20 ºC.

Polymerase chain reaction amplification of internal transcribed spacers region and Restriction fragment length polymorphism assay
The amplification of the internal transcribed spacers (ITS)1-5.8S-ITS2 region was carried out using the ITS1 (5´-TCC GTA GGT GAA CCT GCG G -3´) and ITS4 (5´-TCC TCC GCT TAT TGA TAT GC-3´) [13] with the following program: 95 °C for 15 min; 35 cycles of 30 sec at 95 °C, annealing for 20 sec at 62 °C, 20 sec at 72 °C, and a final extension of 1 min at 72 °C.
The amplicons were seen using electrophoresis on 1.5 % agarose gel in Tris-borate-EDTA buffer (TBE) and DNA stain (Sim Bio, USA). Afterward, all appropriate products were subjected to the MspI (Fermentas, USA) restriction enzyme for Restriction Fragment Length Polymorphism (RFLP) assay. Restriction fragments were separated on 2% agarose gel electrophoresis in TBE buffer with DNA safe stain. To ensure precise identification of ambiguous fragments, polymerase chain reaction (PCR) sequencing with ITS primers was carried out by Noorgeen Company.

In-vitro antifungal susceptibility testing
Itraconazole, miconazole, fluconazole, butoconazole, and caspofungin were obtained from Sigma-Aldrich (St. Louis, MO, USA). The in-vitro activity of six antifungal drugs was assessed by means of the Clinical and Laboratory Standards Institute (CLSI) M-60 protocol [17]. The minimal inhibitory concentration (MIC) of antifungals was determined after 24 h. Candida parapsilosis ATCC 22019 was chosen as a control in every run. The results were analyzed using the CLSI M60 guideline.

RNA extraction
Total RNA was isolated from the yeast using the RNX-PLUS kit (Sinaclon, Iran). To remove any DNA pollution, RNA was cured with DNase1 (Fermentas, USA) based on the instructions of the manufacturer. The accuracy of cDNA was checked with Actin gene primers as housekeeping. The PCR condition was an initial denaturation step for 5 min at 94 °C, 30 cycles of denaturation for 30 sec at 94 °C, annealing for 30 sec at 60 °C, an extension for 7 min at 72 °C with a final extension of 72 °C.

Results of initial identification
Only 75 out of 147 specimens were positive for Candida spp. Through the use of the chromogenic media, 62 (82.66%) isolates with white to green colony color were identified as C. albicans, 11 (14.66%) isolates with pink color were identified as C. glabrata and C. krusei, and 2 (2.66%) isolates with blue colony known as C. tropicalis.

Results of polymerase chain reaction amplification of internal transcribed spacers region and Restriction fragment length polymorphism assay
The amplification of isolates using ITS1-F/ITS4-R was interpreted based on the production of a ~535 bp fragment of C. albicans, a ~871 bp C. glabrata, a ~510 bp C. krusei, and a ~524 bp C. tropicalis (Figure 1). The RFLP patterns indicated that C. albicans 62 (82.66%) was the predominant causative agent isolated from vaginal discharge followed by C. krusei 6 (8%), C. glabrata 5 (6.6%), and C. tropicalis 2 (2.1%) ( Figure 2

Polymerase chain reaction amplification HWP1 gene
The results of amplification of isolates with the HWP1 gene revealed that among C. albicans complex, 56 strains with ~900 bp were categorized as C. albicans, and two isolates with ~700 bp were identified as C. africana (Figure 3). It should be noted that no C. dubliniensis and C. stellatoidea isolates were identified. Table 1 presents the antifungal susceptibilities of the Candida spp. Three isolates of C. albicans were resistant to itraconazole (MIC≥8 µg/mL) and fluconazole (MIC>32µg/mL). All C. glabrata isolates were resistant to fluconazole (MIC≥16 µg/mL). Moreover, three strains of itraconazole (MIC≥4 µg/mL) and one isolate showed resistance (MIC 4 µg/mL) to caspofungin. No antifungal resistance was observed in C. tropicalis and C. krusei to the agents used in this study.

Analysis of amplification of ERG11 and FKS1 genes of C. albicans and C. glabrata
Augmentation of the ERG11 gene of C. albicans strains (two of which were resistant to fluconazole and itraconazole, one only showed loss of susceptibility to fluconazole, and 55 were sensitive to azole) was achieved using PCR. Although multiple silent single nucleotide polymorphisms were detected, missense mutations trigger amino acid alternations were observed in two strains. Changes in amino acid D116E were discovered in fluconazole-and itraconazoleresistant strains. Moreover, changes in amino acid E517Q were detected only in fluconazole-resistant strains.
No polymorphisms were found in the complete sequence arrangement of the ERG11 gene of one azoleresistant C. glabrata isolate. The piece of 450 and 725 bp of the FKS1 gene of C. albicans (3 caspofunginresistant and 2 caspofungin-sensitive) and C. glabrata (1 caspofungin-resistant and 1 caspofunginsusceptible) strains was subjected to PCR sequencing.
Nucleotide and protein alignments were performed to determine any amino acid changes. Single missense alternation was identified in all C. albicans and C. glabrata resistant strains, unlike the sensitive and reference strains. The mutation triggers the changes of serine in the reference FKS1 gene by the leucine in the isolates at position 642 (S642L).

Quantitative real-time PCR amplification
Quantitative real-time PCR was conducted to gain insight into the differential appearance of the ERG11 mRNA expression level. The expression degree of the target gene was standardized to the expression of Actin as a housekeeping gene, and finally, the data were analyzed by the REST© software (2009V2.0.13).
The expression of ERG11 mRNA in terms of resistance was relatively low, compared to the reference strains' expression of three C. albicans and one C. glabrata isolates (expression ratio: 0.129 up to 0.366-fold changes) (Figure 4).

Discussion
The obtained results revealed that three strains of C. albicans were resistant to itraconazole (MIC≥8µg/mL) and fluconazole (MIC>32µg/mL). Moreover, it was demonstrated that the isolates of C. glabrata were also resistant to fluconazole (MIC≥16 µg/mL) and itraconazole (MIC≥4 µg/mL). Therefore, these two drugs are not proper substitute medications for resistant pathogens.
The results of a study conducted by Carillo-Munoz et al. [22] indicated that terbinafine had excellent efficacy against resistant C. albicans, compared to fluconazole. However, greater MIC was found in C. glabrata (16 µg/ml), compared to other Candida spp. (0.125 µg/ml), and very weak MIC was observed in in vitro activity. In the present study, full sensitivity to miconazole and butoconazole was detected in Candida spp. (Table 1).
Caspofungin is an antifungal drug used for a wide spectrum of fungal infections, including Candida spp. infections [23]. Pfaller et al. collected Candida isolates (n=8197) from 91 therapeutic centers throughout the world and determined the sensitivity of caspofungin in the selected isolates [23]. In their study, which continued for over 4 years, the susceptibility to caspofungin remained unaffected and was prevented up to 99% (<1 µg/ml annually). In the present study, C. albicans (3/62) and C. glabrata (1/5) isolates did not show susceptibility to caspofungin (MIC 4 µg/mL), while all C. kruzei and C. tropicalis strains were sensitive to caspofungin.
Alternations in the ERG11 gene lead to azole resistance in Candida spp. [24]. More than 140 amino acid replacements have been verified until now, which are mainly located in three HS regions; specifically in 105-165, 266-287, and 405-488 amino acid regions [25]. Some of these mutations were associated with azole resistance [26,27].
In a study conducted by Morio et al., 23 amino acid alternations were reported in 60 out of 73 C. albicans strains [24]. Changes in K143R, R467K, V404I, V509M, and F449V amino acids were detected in fluconazole-resistant isolates, while substitutes for D116E, E226D, and V437I amino acids were identified in both susceptible and resistant isolates. Furthermore, three new amino acid replacements (i.e., F449V, V404I, and V509M) were recognized, two of which were sited outside the third HS region [24].
In addition, four substitutes were determined which included N136Y, Y221H, L276S, and Y555447H. In total, 11 out of the 23 amino acid changes were determined equally in azole-resistant and susceptible isolates. In another study, ERG11 gene mutations were reported in C. albicans isolates [28].
In the present study, the full-length ERG11 sequence of C. albicans strains was expanded to the image of any gene alternations occurring in the ERG11 gene. Point mutations were commonly observed in the ERG11 gene of azole-susceptible and azole-resistant strains; however, amino acid changes were observed only in a single azole-resistant strain.
The present data highlighted the role of other possible mechanisms in conferring resistance to azoles, including overexpression of genes encoding efflux transporters, overexpression of the ERG11 gene, and modification in the ergosterol biosynthetic pathway. According to the QRT results, the expression level of the ERG11 mRNA in three resistant C. albicans and two resistant C. glabrata isolates was upregulated (0.129 up to 0.366-fold changes), compared to the reference strains.
In another study, resistance to caspofungin in 85 isolates of C. albicans strains has been caused by mutations in the FKS1 gene [30]. However, in the present study, no mutations were observed in four C. albicans isolates that were resistant and susceptible to caspofungin, and only the substitution of polar amino acid serine with non-polar amino acid leucine (S642L) in the FKS1 gene was detected in the tested isolates. The substitution of serine with leucine in C. albicans isolates has been recorded as well Therefore, a mutation in the anticodon region led to the identification of the CUG codon with the seryl and leucyl-tRNA synthetases [31]. In the present study, the substitution of TCG with TTG led to a missense mutation.

Conclusion
The obtained results suggest that proper attention should be paid to the antifungal history of the individuals and future requirements for treatment when deciding about the best antifungal drugs for the treatment of VVC/ RVVC. In patients with continued or periodic illness, the prescription of the best antifungal drug is often difficult and requires monitoring of the antifungal sensitivity of the colonizing strain. Moreover, C. albicans and C. glabrata isolates showed resistance to azole and the caspofungin antifungals without any mutation in the ERG11 and FKS1 genes. The present data indicate the possible involvement of other mechanisms, including mutations in the HS2 region or chitin synthase genes.