# High Virulence and Antifungal Resistance in Clinical Strains ofCandida albicans

Authors: Eric Monroy-Pérez; Gloria Luz Paniagua-Contreras; Pamela Rodríguez-Purata; Felipe Vaca-Paniagua; Marco Vázquez-Villaseñor; Clara Díaz-Velásquez; Alina Uribe-García; Sergio Vaca
Journal: Canadian Journal of Infectious Diseases and Medical Microbiology
 (2016)

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## Abstract

Antifungal resistance and virulence properties ofCandida albicansare a growing health problem worldwide. To study the expression of virulence and azole resistance genes in 39 clinical strains ofC. albicans, we used a model of infection of human vaginal epithelial cells withC. albicansstrains 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 genesMDR1 andCDR1 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, andPLB1-PLB2 were present in all strains;SAP1 was identified in 37 (94.8%) isolates,HWP1 in 35 (89.7%),ALS3 in 14 (35.8%), andCDR1 in 26 (66.6%). In nearly all of the strains,ALS1,HWP1,SAP4–SAP6,LIP1–LIP10,PLB1, andPLB2 were expressed, whereasCDR1 was expressed in 20 (51.3%) andALS3 in 14 (35.8%). In our in vitro model of infection withC. albicans, the clinical strains showed different expression profiles of virulence genes in association with the azole resistance geneCDR1. The results indicate that the strains that infect Mexican patients suffering from VVC are highly virulent and virtually all are insensitive to azoles.


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## Body

## 1. Introduction

Candida albicans is an opportunistic fungus that infects the mucosae of oral and vaginal cavities [1]. Vulvovaginal candidiasis (VVC) is among the most common infections that affect women of childbearing age [2]. Clinical strains ofC. albicans possess an array of virulence genes that directly influence the pathogenesis of VVC, including genes responsible for phenotypic switching [3],HWP1 (hyphal wall protein 1) [4],ALS (agglutinin-like sequence) [5],SAP (secreted aspartyl proteases) [6],PL (phospholipases) [7], andLIP (lipases) [8]. The aspartyl proteases, phospholipases, and lipases are involved in the breakdown of cell membranes in host epithelia, which promotes colonization and invasion [9].The increase ofC. 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 ofCDR1 andCDR2 from the ABC transporter family andMDR1 which encodes a multidrug efflux pump. Mutations inERG5 andERG11, which cause alterations in C22-desaturase and sterol 14α-demethylase, respectively, are also associated with azole resistance [10].The expression ofALS andSAP gene families inC. albicans of vaginal origin has recently been studied [11, 12]. Likewise, the gene expression patterns of theLIP andPL family, which are associated with the formation of biofilm during infection, have been studied [13]. However, the expression patterns ofALS,HWP1,SAP,LIP, andPLB in association with azole resistance genetic determinants have been less studied in strains of vaginal origin. In this work, to analyse the expression patterns ofALS1–ALS3,HWP1,SAP1,SAP4–SAP6,LIP1–LIP10,PLB1-PLB2,CDR1, andMDR1, we implemented an in vitro model of infection of human vaginal cells withC. albicans strains isolated from women with VVC.


## 2. Materials and Methods

### 2.1. 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 50μg/mL chloramphenicol.


### 2.2. Identification ofC. 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]. TheC. albicans strain B311 (ATCC 32354, ATCC, Manassas, VA, USA) was used as a positive control.Table 1
Primers used in PCR and real-time PCR assays.
Gene Orientation Sequence 5′ to 3′ rRNA FW TTTATCAACTTGTCACACCAGA RV ATCCCGCCTTACCACTACCG  ALS1 FW CCATCACTGAAGATATCACCACA RV TGGAGCTTCTGTAGGACTGGTT  ALS3 FW CCAAGTGTTCCAACAACTGAAA RV GAACCGGTTGTTGCTATGGT  HWP1 FW CCATGTGATGATTACCCACA RV GCTGGAACAGAAGATTCAGG  SAP1 FW TCAATCAATTTACTCTTCCATTTCTAACA RV CCAGTAGCATTAACAGGAGTTTTAATGACA  SAP4 FW TTATTTTTAGATATTGAGCCCACAGAAA RV GCCAGTGTCAACAATAACGCTAAGTT  SAP5 FW AGAATTTCCCGTCGATGAGACTGG RV CAAATTTTGGGAAGTGCGGGAAGA  SAP6 FW CCCGTTTTGAAATTAAATATGCTGATGG RV GTCGTAAGGAGTTCTGGTAGCTTCG  LIP1 FW AGCCCAACCAGAAGCTAATGAA RV TGATGCAAAAGTCGCCATGT  LIP2 FW GGCCTGGATTGATGCAAGAT RV TTGTGTGCAGACATCCTTGGA  LIP3 FW TCTCACCGAGATTGTTGTTGGA RV GTTGGCCATCAAATCTTGCA  LIP4 FW GCGTCCTGTTGCTTTCACT RV ACACGGTTTGTTTTCCATTGAA  LIP5 FW TGGTTCCAAAAATACCGTGTT RV CGACAATAGGGACGATTTGATCA  LIP6 FW AAGAATCTTCCGACCTGACCAA RV ATATGCACCTGTTGACGTTCAAA  LIP7 FW AACTGATATTTGCCATGCATTAGAAA RV CCATTCCCGGTAACTAGCATGT  LIP8 FW CAACAATTGCTAAAATCGTTGAAGA RV AGGGATTTTTGGCACTAATTGTTT  LIP9 FW CGCAAGTTTGAAGTCAGGAAAA RV CCCACATTACAATTTGGCATCT  LIP10 FW CACCTGGCTTAGCAGTTGCA RV CCCAGCAAAGACTCATTTTATTCA  PLB1 FW GGTGGAGAAAGATGGCCAAAA RV AGCACTTACGTTACGATGCAACA  PLB2 FW TGAAACCTTTGGGCGACAACT RV GCCGCGCTCGTTGTTAA  CDR1 FW AAGAGAACCATTACCAGG RV AGGAATCGACGGATCAC  MDR1 FW GGAGTTTAGGTGCTGT RV CGGTGATGGCTCTCAA


### 2.3. Antifungal Susceptibility

To testC. albicans antifungal susceptibility, we used diffusion disks impregnated with 25 μg of fluconazole, 10 μg of ketoconazole, and 100 U nystatin (HiMedia Laboratories, Mumbai, India). Antifungal susceptibility was established according to the interpretation criteria previously described (Table 2) [16].Table 2
Interpretive criteria of susceptibility and resistance to antifungals used in this study.
Antifungal drugs Zone diameter (mm) Sensitive Dose-dependent Resistant Ketoconazole ≥30 23–29 ≤22 Fluconazole ≥19 15–18 ≤14 Nystatin ≥25 17–24 ≤14


### 2.4. Detection of Genetic Virulence Markers and Azole Resistance Genes by PCR

The primers and PCR conditions used were previously described: ALS1 and ALS3 [17], HWP1 [18], SAP1 [19], SAP4–SAP6 [20], LIP1–LIP10 and PLB1-PLB2 [13], and CDR1 and MDR1 [21] (Table 1).


### 2.5. 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 μL of phosphate-buffered saline (PBS) with 2 × 106C. albicanscells. The mixed cultures were incubated for 72 h at 37°C in 5% CO2. The culture medium was changed every 24 h.C. albicans (ATCC 32354) andStaphylococcus epidermidis (ATCC 35984) were used as positive and negative controls, respectively.


### 2.6. RNA Purification and cDNA Synthesis

After infection of A431 cells, the yeasts were harvested and suspended in 200μL of buffer Y1 (Qiagen, Hilden, Germany) with lyticase (50 U/107 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.


### 2.7. 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 Table1. The final volume of each reaction was 25 μL, consisting of 12.5 μL of SYBR Green Master Mix, 1 μL of forward primer (1 μM), 1 μL of reverse primer (1 μM), 2 μL of cDNA (~20 ng), and 8.5 μL 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.


## 2.1. 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 50μg/mL chloramphenicol.


## 2.2. Identification ofC. 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]. TheC. albicans strain B311 (ATCC 32354, ATCC, Manassas, VA, USA) was used as a positive control.Table 1
Primers used in PCR and real-time PCR assays.
Gene Orientation Sequence 5′ to 3′ rRNA FW TTTATCAACTTGTCACACCAGA RV ATCCCGCCTTACCACTACCG  ALS1 FW CCATCACTGAAGATATCACCACA RV TGGAGCTTCTGTAGGACTGGTT  ALS3 FW CCAAGTGTTCCAACAACTGAAA RV GAACCGGTTGTTGCTATGGT  HWP1 FW CCATGTGATGATTACCCACA RV GCTGGAACAGAAGATTCAGG  SAP1 FW TCAATCAATTTACTCTTCCATTTCTAACA RV CCAGTAGCATTAACAGGAGTTTTAATGACA  SAP4 FW TTATTTTTAGATATTGAGCCCACAGAAA RV GCCAGTGTCAACAATAACGCTAAGTT  SAP5 FW AGAATTTCCCGTCGATGAGACTGG RV CAAATTTTGGGAAGTGCGGGAAGA  SAP6 FW CCCGTTTTGAAATTAAATATGCTGATGG RV GTCGTAAGGAGTTCTGGTAGCTTCG  LIP1 FW AGCCCAACCAGAAGCTAATGAA RV TGATGCAAAAGTCGCCATGT  LIP2 FW GGCCTGGATTGATGCAAGAT RV TTGTGTGCAGACATCCTTGGA  LIP3 FW TCTCACCGAGATTGTTGTTGGA RV GTTGGCCATCAAATCTTGCA  LIP4 FW GCGTCCTGTTGCTTTCACT RV ACACGGTTTGTTTTCCATTGAA  LIP5 FW TGGTTCCAAAAATACCGTGTT RV CGACAATAGGGACGATTTGATCA  LIP6 FW AAGAATCTTCCGACCTGACCAA RV ATATGCACCTGTTGACGTTCAAA  LIP7 FW AACTGATATTTGCCATGCATTAGAAA RV CCATTCCCGGTAACTAGCATGT  LIP8 FW CAACAATTGCTAAAATCGTTGAAGA RV AGGGATTTTTGGCACTAATTGTTT  LIP9 FW CGCAAGTTTGAAGTCAGGAAAA RV CCCACATTACAATTTGGCATCT  LIP10 FW CACCTGGCTTAGCAGTTGCA RV CCCAGCAAAGACTCATTTTATTCA  PLB1 FW GGTGGAGAAAGATGGCCAAAA RV AGCACTTACGTTACGATGCAACA  PLB2 FW TGAAACCTTTGGGCGACAACT RV GCCGCGCTCGTTGTTAA  CDR1 FW AAGAGAACCATTACCAGG RV AGGAATCGACGGATCAC  MDR1 FW GGAGTTTAGGTGCTGT RV CGGTGATGGCTCTCAA


## 2.3. Antifungal Susceptibility

To testC. albicans antifungal susceptibility, we used diffusion disks impregnated with 25 μg of fluconazole, 10 μg of ketoconazole, and 100 U nystatin (HiMedia Laboratories, Mumbai, India). Antifungal susceptibility was established according to the interpretation criteria previously described (Table 2) [16].Table 2
Interpretive criteria of susceptibility and resistance to antifungals used in this study.
Antifungal drugs Zone diameter (mm) Sensitive Dose-dependent Resistant Ketoconazole ≥30 23–29 ≤22 Fluconazole ≥19 15–18 ≤14 Nystatin ≥25 17–24 ≤14


## 2.4. Detection of Genetic Virulence Markers and Azole Resistance Genes by PCR

The primers and PCR conditions used were previously described: ALS1 and ALS3 [17], HWP1 [18], SAP1 [19], SAP4–SAP6 [20], LIP1–LIP10 and PLB1-PLB2 [13], and CDR1 and MDR1 [21] (Table 1).


## 2.5. 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 μL of phosphate-buffered saline (PBS) with 2 × 106C. albicanscells. The mixed cultures were incubated for 72 h at 37°C in 5% CO2. The culture medium was changed every 24 h.C. albicans (ATCC 32354) andStaphylococcus epidermidis (ATCC 35984) were used as positive and negative controls, respectively.


## 2.6. RNA Purification and cDNA Synthesis

After infection of A431 cells, the yeasts were harvested and suspended in 200μL of buffer Y1 (Qiagen, Hilden, Germany) with lyticase (50 U/107 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.


## 2.7. 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 Table1. The final volume of each reaction was 25 μL, consisting of 12.5 μL of SYBR Green Master Mix, 1 μL of forward primer (1 μM), 1 μL of reverse primer (1 μM), 2 μL of cDNA (~20 ng), and 8.5 μL 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.


## 3. Results

### 3.1. Identification ofC. albicans and Antifungal Resistance Phenotypes

C. albicans was identified in 19.5% (n=39) of the samples and, in the remaining ones (80.5%, n=161), bacteria such asEscherichia coli,Klebsiellaspp.,Staphylococcus aureus, orStaphylococcus epidermidis were found.All 39 isolates were sensitive to nystatin, whereas 97.4% (n=38) and 94.9% (n=37) were resistant to fluconazole and ketoconazole, respectively (Table 3). Resistance to both azoles was found in 94.9% (n=37) of the strains.Table 3
Susceptibility and resistance ofCandida albicans strains to antifungal drugs.
Antifungal drugs ResistantNumber (%) SensitiveNumber (%) Nystatin 0 (0) 39 (100) Fluconazole 37 (94.9) 2 (5.1) Ketoconazole 38 (97.4) 1 (2.6)


### 3.2. Detection of Virulence Markers and Azole Resistance Genes

All of the isolated strains (n=39) were carriers of theALS1, SAP4-SAP6,LIP1,LIP2,LIP4,LIP6,LIP7,LIP9,LIP10, andPLB1–PLB2genes. Other genes detected wereSAP1 in 94.8% (n=37),HWP1 (89.7%, n=35),ALS3 (35.8%, n=14), andCDR1 (66.6%, n=26) of the strains (Table 4).Table 4
Percentages of detection and expression of virulence markers and azole resistance genes inCandida albicans strains.
Gene DetectionNumber (%) ExpressionNumber (%) ALS1 39 (100) 39 (100) ALS3 14 (35.8) 14 (35.8) HWP1 35 (89.7) 35 (89.7) SAP1 37 (94.8) 36 (92.3) SAP4 39 (100) 39 (100) SAP5 39 (100) 39 (100) SAP6 39 (100) 39 (100) LIP1 39 (100) 39 (100) LIP2 39 (100) 39 (100) LIP3 38 (97.4) 38 (97.4) LIP4 39 (100) 39 (100) LIP5 35 (89.7) 35 (89.7) LIP6 39 (100) 39 (100) LIP7 39 (100) 39 (100) LIP8 38 (97.4) 38 (97.4) LIP9 39 (100) 39 (100) LIP10 39 (100) 39 (100) PLB1 39 (100) 39 (100) PLB2 39 (100) 39 (100) CDR1 26 (66.6) 20 (51.3) MDR1 0 (0) 0 (0)


### 3.3. Expression Patterns of the Virulence Genes andCDR1

In nearly all of the isolates,ALS1,HWP1,SAP4–SAP6,LIP1–LIP10,PLB1, andPLB2 were expressed after infection of the cell line A431, whereasCDR1 was expressed in 20 (51.3%) andALS3 in 14 (35.8%) (Table 4).Fourteen distinct expression patterns ofALS,HWP1,SAP,PLB,LIP, andCDR1 were identified (Table 5). The most prevalent expression profile, Pattern 1, composed of 18 genes, was found in 25.6% (n=10) of the strains. Pattern 2 included 20 expressed genes and was found in 18% (n=7); Patterns 3 and 4, both expressing 19 genes, were detected in 12.8% (5 strains); Patterns 5 (18 genes) and 6 (19 genes) were detected in 5.1% (n=2) (Table 5). The following eight expression patterns (Patterns 7–14) were represented by a single strain.CDR1 was found in eight different patterns (n=20).Table 5
Expression patterns of virulence genes andCDR1 in Candida albicans strains.
Patternnumber Pattern Number of strains % 1 ALS1/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2 10 25.6 2 ALS1/ALS3/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 7 18.0 3 ALS1/ALS3/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2 5 12.8 4 ALS1/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 5 12.8 5 ALS1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 2 5.1 6 ALS1/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 2 5.1 7 ALS1/HWP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2 1 2.5 8 ALS1/ALS3/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2 1 2.5 9 ALS1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2 1 2.5 10 ALS1/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 1 2.5 11 ALS1/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP6/LIP7/LIP9/LIP10/PLB1/PLB2 1 2.5 12 ALS1/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 1 2.5 13 ALS1/ALS3/HWP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 1 2.5 14 ALS1/HWP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 1 2.5


## 3.1. Identification ofC. albicans and Antifungal Resistance Phenotypes

C. albicans was identified in 19.5% (n=39) of the samples and, in the remaining ones (80.5%, n=161), bacteria such asEscherichia coli,Klebsiellaspp.,Staphylococcus aureus, orStaphylococcus epidermidis were found.All 39 isolates were sensitive to nystatin, whereas 97.4% (n=38) and 94.9% (n=37) were resistant to fluconazole and ketoconazole, respectively (Table 3). Resistance to both azoles was found in 94.9% (n=37) of the strains.Table 3
Susceptibility and resistance ofCandida albicans strains to antifungal drugs.
Antifungal drugs ResistantNumber (%) SensitiveNumber (%) Nystatin 0 (0) 39 (100) Fluconazole 37 (94.9) 2 (5.1) Ketoconazole 38 (97.4) 1 (2.6)


## 3.2. Detection of Virulence Markers and Azole Resistance Genes

All of the isolated strains (n=39) were carriers of theALS1, SAP4-SAP6,LIP1,LIP2,LIP4,LIP6,LIP7,LIP9,LIP10, andPLB1–PLB2genes. Other genes detected wereSAP1 in 94.8% (n=37),HWP1 (89.7%, n=35),ALS3 (35.8%, n=14), andCDR1 (66.6%, n=26) of the strains (Table 4).Table 4
Percentages of detection and expression of virulence markers and azole resistance genes inCandida albicans strains.
Gene DetectionNumber (%) ExpressionNumber (%) ALS1 39 (100) 39 (100) ALS3 14 (35.8) 14 (35.8) HWP1 35 (89.7) 35 (89.7) SAP1 37 (94.8) 36 (92.3) SAP4 39 (100) 39 (100) SAP5 39 (100) 39 (100) SAP6 39 (100) 39 (100) LIP1 39 (100) 39 (100) LIP2 39 (100) 39 (100) LIP3 38 (97.4) 38 (97.4) LIP4 39 (100) 39 (100) LIP5 35 (89.7) 35 (89.7) LIP6 39 (100) 39 (100) LIP7 39 (100) 39 (100) LIP8 38 (97.4) 38 (97.4) LIP9 39 (100) 39 (100) LIP10 39 (100) 39 (100) PLB1 39 (100) 39 (100) PLB2 39 (100) 39 (100) CDR1 26 (66.6) 20 (51.3) MDR1 0 (0) 0 (0)


## 3.3. Expression Patterns of the Virulence Genes andCDR1

In nearly all of the isolates,ALS1,HWP1,SAP4–SAP6,LIP1–LIP10,PLB1, andPLB2 were expressed after infection of the cell line A431, whereasCDR1 was expressed in 20 (51.3%) andALS3 in 14 (35.8%) (Table 4).Fourteen distinct expression patterns ofALS,HWP1,SAP,PLB,LIP, andCDR1 were identified (Table 5). The most prevalent expression profile, Pattern 1, composed of 18 genes, was found in 25.6% (n=10) of the strains. Pattern 2 included 20 expressed genes and was found in 18% (n=7); Patterns 3 and 4, both expressing 19 genes, were detected in 12.8% (5 strains); Patterns 5 (18 genes) and 6 (19 genes) were detected in 5.1% (n=2) (Table 5). The following eight expression patterns (Patterns 7–14) were represented by a single strain.CDR1 was found in eight different patterns (n=20).Table 5
Expression patterns of virulence genes andCDR1 in Candida albicans strains.
Patternnumber Pattern Number of strains % 1 ALS1/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2 10 25.6 2 ALS1/ALS3/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 7 18.0 3 ALS1/ALS3/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2 5 12.8 4 ALS1/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 5 12.8 5 ALS1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 2 5.1 6 ALS1/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 2 5.1 7 ALS1/HWP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2 1 2.5 8 ALS1/ALS3/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2 1 2.5 9 ALS1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2 1 2.5 10 ALS1/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 1 2.5 11 ALS1/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP6/LIP7/LIP9/LIP10/PLB1/PLB2 1 2.5 12 ALS1/HWP1/SAP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 1 2.5 13 ALS1/ALS3/HWP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 1 2.5 14 ALS1/HWP1/SAP4/SAP5/SAP6/LIP1/LIP2/LIP3/LIP4/LIP5/LIP6/LIP7/LIP8/LIP9/LIP10/PLB1/PLB2/CDR1 1 2.5


## 4. Discussion

In this study, we isolated and identified the virulence gene expression patterns ofC. albicans strains in 19.5% of 200 vaginal samples. VVC is a condition that affects a quarter of young women [2, 24]. The presence ofE. coli,Klebsiellaspp.,S. aureus, orS. epidermidisin most vaginal samples (n=161) agrees with previous reports showing bacterial vaginitis as the first cause of vaginal infections [24].The chronicity of VVC or recurrent episodes byC. 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 carriedALS1 (39/39) andALS3 (14/14) was able to express these genes in the in vitro model of infection (Table 4). The frequency of expression of theALS 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 forHWP1 (35/35) expressed this gene during infection of A431 cells (Table 4). The frequency of expression is higher than results reported inC. albicans strains isolated from women with vaginal infection in Turkey [28]. The high frequency of expression ofHWP1and theALS 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].Likewise, all isolated strains expressedSAP4–SAP6 (39/39) and almost all expressedSAP1 (92.3%, (36/37)) (Table 4). These results are similar to those previously described forSAP expression in VVC in humans [31]. The expression ofSAP4–SAP6 has a relevant role in pathogenesis by promoting hyphae formation [32] and by the inhibition of phagocytosis [33].In this study, most strains expressed genes of theLIP family, exceptLIP3,LIP5, andLIP8 (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 thatLIP family members are differentially expressed during infection in specific areas of affected mucosae [35].All strains ofC. albicans expressedPLB1 andPLB2 (39/39) (Table 4). Recently, phospholipase activity was reported in strains ofC. albicans and in nonalbicans strains from patients with vulvovaginal infections [36], as well as in differentCandida species isolated from patients with type 2 diabetes mellitus suffering from VVC [37]. The elevated expression of phospholipase genes in theC. 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 compounds [38]. In this study, the association of azole resistance phenotypes (fluconazole/ketoconazole) was identified in 94.9% (n=37) of the strains (Table 3), whereasCDR1 was found in only 66.6% (n=26) (Table 4). This gap in detection can be explained by the idea that azole resistance is not only conferred byCDR1, an ABC transporter, but may also be caused by the overproduction of an azole target, 14α-demethylase, which is encoded byERG11. Other mechanisms of azole resistance are caused by mutations inERG11 which decrease azole affinity to this enzyme [10, 39].Interestingly, all strains described in this study were sensitive to nystatin (n=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 ofC. 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 thatCandida species isolated in different geographical regions differ in their sensitivity to fluconazole [43].In this study, we identified 14 distinct expression patterns ofALS,HWP1,SAP,LIP,PLB, andCDR1 in A431 cells infected in vitro with strains ofC. 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 geneCDR1 with the virulence markers indicates that these strains are highly virulent and should be treated with a nonazole therapy.


## 5. Conclusion

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



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