High Virulence and Antifungal Resistance in Clinical Strains of Candida albicans

Antifungal resistance and virulence properties of Candida albicans are a growing health problem worldwide. To study the expression of virulence and azole resistance genes in 39 clinical strains of C. albicans, we used a model of infection of human vaginal epithelial cells with C. albicans strains isolated from Mexican women with vulvovaginal candidiasis (VVC). The strains were identified by PCR amplification of the ITS1 and ITS2 regions of rRNA. The detection and expression of virulence genes and azole resistance genes MDR1 and CDR1 were performed using PCR and RT-PCR, respectively. All strains were sensitive to nystatin and 38 (97.4%) and 37 (94.9%) were resistant to ketoconazole and fluconazole, respectively. ALS1, SAP4–SAP6, LIP1, LIP2, LIP4, LIP6, LIP7, LIP9, LIP10, and PLB1-PLB2 were present in all strains; SAP1 was identified in 37 (94.8%) isolates, HWP1 in 35 (89.7%), ALS3 in 14 (35.8%), and CDR1 in 26 (66.6%). In nearly all of the strains, ALS1, HWP1, SAP4–SAP6, LIP1–LIP10, PLB1, and PLB2 were expressed, whereas CDR1 was expressed in 20 (51.3%) and ALS3 in 14 (35.8%). In our in vitro model of infection with C. albicans, the clinical strains showed different expression profiles of virulence genes in association with the azole resistance gene CDR1. The results indicate that the strains that infect Mexican patients suffering from VVC are highly virulent and virtually all are insensitive to azoles.

The increase of C. albicans resistant to antifungal agents, mainly to azoles, is a serious health problem and hampers the treatment of VVC. The mechanisms responsible for resistance to azoles include overexpression of CDR1 and CDR2 from the ABC transporter family and MDR1 which encodes a multidrug efflux pump. Mutations in ERG5 and ERG11, which cause alterations in C22-desaturase and sterol 14 -demethylase, respectively, are also associated with azole resistance [10].
The expression of ALS and SAP gene families in C. albicans of vaginal origin has recently been studied [11,12]. Likewise, the gene expression patterns of the LIP and PL family, which are associated with the formation of biofilm during infection, have been studied [13]. However, the expression patterns of ALS, HWP1, SAP, LIP, and PLB in association with azole resistance genetic determinants have been less studied in strains of vaginal origin. In this work, to analyse the expression patterns of ALS1-ALS3, HWP1, SAP1, SAP4-SAP6, LIP1-LIP10, PLB1-PLB2, CDR1, and MDR1, we implemented an in vitro model of infection of human vaginal cells with C. albicans strains isolated from women with VVC.

Patients Analysed and Sampling.
Our study included a group of 200 women (ages 18-57) who attended the gynaecology department of public hospitals in the State of Mexico, Mexico, and presented characteristic symptoms of VVC (itching, burning, dysuria, and curd-like discharge). In this study, adult women with/without active sexual life were included. The exclusion criteria were as follows: patients with cervical cancer, being pregnant, being under antibiotic treatment, or having ingested antifungal drugs in the last 30 days. In addition, the patients informed if they had been previously exposed to azoles at least once in the last year as treatment for VVC. All participating women signed a letter of informed consent. The ethics committee of each hospital approved the study. Two samples were taken from the cervicovaginal cavity of each patient, using sterile swabs. The first was used to observe the presence of yeast, hyphae, or pseudohyphae by light microscopy. The second was incubated for 24 h at 37 ∘ C in brain heart infusion (BHI) medium (BD Bioxon, Cuautitlan Izcalli, State of Mexico, Mexico). After 24 h, the samples were incubated at 37 ∘ C in Sabouraud agar plates (BD Bioxon) with 50 g/mL chloramphenicol.

2.2.
Identification of C. albicans. C. albicans strains were identified by the API 20 C Aux test (bioMerieux, Durham, NC, USA), germ tube test [11], and PCR amplification of ITS1 and ITS2 rRNA loci (the primers are shown in Table 1) [14]. The DNA of the strains was extracted by boiling, as described by Paniagua-Contreras et al. [15]. The C. albicans strain B311 (ATCC 32354, ATCC, Manassas, VA, USA) was used as a positive control.

Antifungal Susceptibility.
To test C. albicans antifungal susceptibility, we used diffusion disks impregnated with 25 g of fluconazole, 10 g of ketoconazole, and 100 U nystatin (HiMedia Laboratories, Mumbai, India). Antifungal susceptibility was established according to the interpretation criteria previously described (Table 2) [16].

In Vitro Infection Model.
To study the expression of virulence markers and azole resistance genes, we used the A431 cell line (ATCC CRL-1555), which is derived from a vulvar epidermoid carcinoma. The growth conditions of the strains were previously reported [22,23]. The A431 cell culture was inoculated with 50 L of phosphate-buffered saline (PBS) with 2 × 10 6 C. albicans cells. The mixed cultures were incubated for 72 h at 37 ∘ C in 5% CO 2 . The culture medium was changed every 24 h. C. albicans (ATCC 32354) and Staphylococcus epidermidis (ATCC 35984) were used as positive and negative controls, respectively.

RNA Purification and cDNA Synthesis.
After infection of A431 cells, the yeasts were harvested and suspended in 200 L of buffer Y1 (Qiagen, Hilden, Germany) with lyticase (50 U/10 7 cells). Samples were incubated for 20 min at 30 ∘ C with gentle agitation to facilitate the formation of spheroplasts. Total RNA was purified on a robotic QIAcube workstation (Qiagen) using the RNeasy Mini Kit (Qiagen), following the manufacturer's instructions, including DNase treatment. The concentration and purity of total RNA were analysed on a NanoDrop spectrophotometer 2000 (Thermo Scientific, Waltham, MA, USA). cDNA was synthesized with the QuantiTect Reverse Transcription kit (Qiagen) according to manufacturer's instructions.

Amplification and Detection of the Azole Resistance and
Virulence Genes. Amplification and detection of azole resistance and virulence genes were performed using real-time PCR with the Rotor-Gene SYBR Green PCR kit (Qiagen). The primer sequences used for the gene expression analysis are shown in Table 1. The final volume of each reaction was 25 L, consisting of 12.5 L of SYBR Green Master Mix, 1 L of forward primer (1 M), 1 L of reverse primer (1 M), 2 L of cDNA (∼20 ng), and 8.5 L RNase-free water. Amplification conditions were 95 ∘ C for 5 min, 40 cycles at 95 ∘ C for 5 s, and combination alignment/extension at 60 ∘ C for 10 s. The amplification conditions for the positive and negative controls were the same as described above.

Identification of C. albicans and Antifungal Resistance
Phenotypes. C. albicans was identified in 19.5% ( = 39) of the samples and, in the remaining ones (80.5%, = 161), bacteria such as Escherichia coli, Klebsiella spp., Staphylococcus aureus, or Staphylococcus epidermidis were found.

Discussion
In this study, we isolated and identified the virulence gene expression patterns of C. albicans strains in 19.5% of 200 vaginal samples. VVC is a condition that affects a quarter of young women [2,24]. The presence of E. coli, Klebsiella spp., S. aureus, or S. epidermidis in most vaginal samples ( = 161) agrees with previous reports showing bacterial vaginitis as the first cause of vaginal infections [24]. The chronicity of VVC or recurrent episodes by C. albicans is due to prolonged use of antifungals, which select for resistant strains, and due to the numerous virulence genotypes that promote adhesion to epithelial cells, production of hydrolytic enzymes, colonization, and invasion [9]. We found that each strain isolated that carried ALS1 (39/39) and ALS3 (14/14) was able to express these genes in the in vitro model of infection (Table 4). The frequency of expression of the ALS genes reported here is similar to those reported in vaginal candidiasis models [25]. The ALS proteins are extracellular components of the cell membrane which mediate adhesion and colonization of host cells [26,27]. Widespread expression of these proteins in the isolated strains supports their role in infection.
All strains positive for HWP1 (35/35) expressed this gene during infection of A431 cells ( Table 4). The frequency of expression is higher than results reported in C. albicans strains isolated from women with vaginal infection in Turkey [28]. The high frequency of expression of HWP1 and the ALS genes found in this study (Table 4) reveals the capacity of the isolated strains to cause chronic vaginal infections. This result is consistent with reports showing that the combined expression of the adhesion proteins HWP1 and ALS1-ALS3 significantly facilitates biofilm formation [29] and can participate in the resistance to antifungals by limiting their penetrance to the biofilm [30].
In this study, most strains expressed genes of the LIP family, except LIP3, LIP5, and LIP8 (Table 4). These data are consistent with those reported using an in vitro infection model of oral epithelial cells and in patient samples [34]. The specific role of LIP proteins in infection is unknown. However, it has been described that LIP family members are differentially expressed during infection in specific areas of affected mucosae [35].
All strains of C. albicans expressed PLB1 and PLB2 (39/39) (Table 4). Recently, phospholipase activity was reported in strains of C. albicans and in nonalbicans strains from patients with vulvovaginal infections [36], as well as in different Candida species isolated from patients with type 2 diabetes mellitus suffering from VVC [37]. The elevated expression of phospholipase genes in the C. albicans strains isolated in this study (Table 4) suggests that these genes are essential for vaginal infection by participating in the breakdown of the cell membrane, which allows the hyphae to penetrate the epithelial cell cytoplasm [7].
The use of azoles for the treatment of VVC over time has resulted in the selection of strains resistant to these Canadian Journal of Infectious Diseases and Medical Microbiology 5  [38]. In this study, the association of azole resistance phenotypes (fluconazole/ketoconazole) was identified in 94.9% ( = 37) of the strains (Table 3), whereas CDR1 was found in only 66.6% ( = 26) (Table 4). This gap in detection can be explained by the idea that azole resistance is not only conferred by CDR1, an ABC transporter, but may also be caused by the overproduction of an azole target, 14demethylase, which is encoded by ERG11. Other mechanisms of azole resistance are caused by mutations in ERG11 which decrease azole affinity to this enzyme [10,39]. Interestingly, all strains described in this study were sensitive to nystatin ( = 39, Table 3), as also reported in other studies [40,41]. This result suggests that nystatin can be an alternative for the treatment of vaginal infections caused by strains of C. albicans which are resistant to azoles [42]. The high frequencies of strains resistant to fluconazole and ketoconazole (Table 3) can be explained by the high use of these two drugs (and clotrimazole) in Mexico, given that this is the most commonly used therapy against VVC. Our results are consistent with the observation that Candida species isolated in different geographical regions differ in their sensitivity to fluconazole [43].
In this study, we identified 14 distinct expression patterns of ALS, HWP1, SAP, LIP, PLB, and CDR1 in A431 cells infected in vitro with strains of C. albicans (Table 5). Pattern 1, comprising 18 genes, was expressed by most strains (25.6%). These findings show that, during the pathogenesis of infection, most virulence markers are expressed (in 8 of the 14 patterns). The combined expression of the azole resistance gene CDR1 with the virulence markers indicates that these strains are highly virulent and should be treated with a nonazole therapy.

Conclusion
This is the first work done in Mexico on global expression of different markers of virulence and resistance to azoles in clinical strains of C. albicans. The results show that the strains that infect Mexican patients suffering from VVC are highly virulent and virtually all are insensitive to azoles.