Candida albicans is an important opportunistic yeast that can cause mucosal infections, such as thrush and oropharyngeal candidiasis (OPC) in AIDS patients, as well as systemic life-threatening infections in immunocompromised patients . The use of potent antifungal agents is crucial for the management of these potentially fatal infections. Azole antifungal agents, such as fluconazole, are the treatment of choice for these infections .
However, the increased use of these antifungal agents, especially in HIV/AIDS patients receiving long-term therapy, has resulted in the emergence of azole-resistant isolates of C. albicans. Azole resistance mechanisms are different and may contribute to mutations in encoding gene ERG11. It is based on the spatial configuration alterations of the target enzyme 14a-demethylase (ERG11p) [3, 4]. Erg11p is a significant protein of the cytochrome P450 superfamily enzyme and has an important role in the ergosterol synthesis pathway of C. albicans. Ergosterol (ergosta-5,7,22-trien-3β-ol) is a kind of sterol found in the fungal cell membranes and play a major role in maintaining the integrity and function of C. albicans membrane .
Mutations in ERG11 result in changes in the ERG11p spatial construction and reduce the affinity between the protein and azole . The aim of the present study was to determine the susceptibility pattern of C. albicans clinical isolates to fluconazole using microdilution method. This study was also targeted toward the detection of mutations that might be responsible for fluconazole resistance by the amplification and sequencing of ERG11 gene.
Materials and Methods
This study was conducted on a total of 216 clinical isolates obtained from Tehran (n=67), Isfahan (n=37), and Mashhad (n=112) cities in Iran, from July 2016 to February 2018. All strains were sub-cultured on Sabouraud glucose agar (SGA; Difco, Detroit, MI, USA) and incubated at 37°C for 24-48 h.
Genomic DNA was extracted using the boiling method. Briefly, a bit of fresh colonies were suspended in 80 µL of distilled water and boiled for 20 min. They were then centrifuged for 5 min at 6,000 rpm. The resultant supernatant was used for polymerase chain reaction (PCR). Clinical isolates of Tehran and Isfahan were identified using the PCR-restriction fragment length polymorphism (PCR-RFLP) of the ITS1-5.8S-ITS2 regions as described previously [7, 8]. Furthermore, the identification of the clinical samples of Mashhad was performed through matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF or MT-MS) mass spectrometry (Bruker Daltonics, Bremen, Germany), which is a new method.
In vitro antifungal susceptibility testing
Minimum inhibitory concentration (MICs) was determined according to the clinical and laboratory standards institute M27-A3 and M27-S4 documents [9, 10]. Antifungal effect of fluconazole (Pfizer Central Research, Sandwich, United Kingdom) was evaluated on the clinical isolates of C. albicans. To this end, fluconazole was diluted in the RPMI-1640 medium (Sigma Chemical Co.), buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS; Sigma) with L-glutamine without bicarbonate.
A final concentration of 0.063-64 μg/ml was considered for fluconazole. The MIC results were read after 24 h of incubation at 35°C. These values were determined visually as the lowest concentration of drug that caused a significant (>50%) reduction in the microorganism growth. According to the M27-S4 document, a fluconazole concentration of > 8 was considered for the resistant isolates .
Amplification of ERG11 gene and sequencing
Three pairs of primers were used for the amplification of ERG11 gene . ERGSec1A (50-TTAGTGTTTTATTGGATTCCTTGGTT-30) and ERGSec1B (50-TCTCATTTCATCACCAAATAAAGATC-30) yielded an amplicon expanding from 295 to 777 bp of the ERG11 gene. Furthermore, ERGSec2A (50-ACCAGAAATTACTATTTTCACTGCTTCA-30) and ERGSec2B (50-AAGTCAAATCATTCAAATCACCA CCT-30) yielded a product extending from 723-1204 bp of the ERG11 gene. Finally, ERGSec3A (50-AGGTGGTGATTTGAATGATTTGACTT-30) and ERGSec3B (50-GAACTATAATCAGGGTCAGGCACTTT-30) provided an expected PCR product extending from 1179-1667 bp of the ERG11 gene.
One susceptible isolate (No. 27) was used as the control strain. The PCR mixture included 2.5 μl of 10X reaction buffer, 0.5 ml of 30 pmol/ml of each primer, 0.5 ml of 10 mM dNTP, 0.25 ml of Taq polymerase (5U/ml), and 2 μl of extracted DNA in a final volume of 25 μl. The PCR products were purified by means of the ethanol purification method. The cycle sequencing reactions were performed in forward and reverse directions (Bioneer, Korea).
The sequencing products were aligned and analyzed in comparison with the nucleotide sequence of cytochrome P450 L1A1 (lanosterol 14 alpha-demethylase) from C. albican  and published ERG11 sequence in GenBank by Lai and Kirsch (accession number: X13296) using MEGA (version 7) and Gene Runner software.
Out of 216 strains, 100 (46.3%) species were identified as C. albicans. The majority of the patients (23%) were in the age group of 31-40 years. Most of the specimens were collected from vaginitis (30%), onychomycosis (26%), and bronchoalveolar lavage (BAL; 21%) cases. The study population had a male to female ratio of 31:69. The most frequent predisposing factors among the patients included pregnancy (29%), cancer (9%), and diabetes mellitus (9%). An MIC range of 0.125-16 μg/ml was obtained for fluconazole having the MIC50 and MIC90 values of 0.5 and 1 μg/ml, respectively.
Table 1 summarizes the demographic and clinical data of the patients enrolled in the present study. Isolates number 3, 11, 15, 23, and 45 were resistant to fluconazole (MIC=16); therefore, they were applied for sequencing. However, isolate number 3 was contaminated with Aspergillus species and did not recover on SGA. Totally, 41 nucleotides changes were detected among the isolates. Out of 41 mutations, 4 mutations in codons caused changes in ERG11p (Table 1) [6, 12-17].
|No||City||Age||Gender||Source of specimen||Risk factor||MIC (µg/ml)||Site of nucleotide (bp) mutation||Nucleic acid mutation||Amino acid substitution||Change in ERG11p|
|Table 1. Continued|
|Table 1. Continued|
|*BAL: Bronchoalveolar lavage, MIC: minimum inhibitory concentration, M: male, F: female, N/C: no change, aa: amino acid, Phe: phenylalanine, Ser: serine, His: histidine, Leu: leucine, Val: valine, Lys: lysine, Glu: glutamine, Asp: aspartic acid, Tyr: tyrosine, Ala: alanine, Ile: isoleucine, Asn: asparagine|
Candidiasis is referred to both superficial and deep-tissue fungal infections often caused by C. albicans. Based on the epidemiological surveys performed in the Europe , United States , and the Middle East , the superficial types can affect the oral, oropharynx, esophagus, and vaginal mucosa. Candida albicans is the main cause of invasive Candida infections in the majority of obtained clinical specimens, accounting for 50-70% of the cases . The treatments of Candida infections differ considerably and are based on the patients' underlying disease and immune status, anatomic location of the infection, Candida species responsible for infection, predisposing factors, and, in some cases, the susceptibility of Candida species to antifungal agents [22, 23].
Nystatin, itraconazole, miconazole, voriconazole, echinocandins, flucytosine, and amphotericin B are the anti-Candida agents applied for the management of this infection. However, the most prevalently recommended antifungal used for the majority of C. albicans isolates is fluconazole, which is a member of the azole class . Azoles inhibit 14-α-sterol demethylase encoded by ERG11 gene, which is a significant enzyme involved in the biosynthesis of ergosterol (i.e., fungal-specific membrane sterol).
Some investigations have shown the ability of Candida species to expand high resistance to azole agents [25, 26]. Prior ﬂuconazole exposure has been shown to increase the risk of ﬂuconazole resistance in Candida species . Increased resistance to fluconazole is caused by different factors, such as the change of the target enzyme to diminish drug binding, reduction of the toxic effects of the antifungal, enhancement of the amount of the target enzyme, prevention from the intracellular accumulation of the antifungal agent , raised expression of ERG11 as a result of activating mutations in the gene encoding the zinc-cluster transcriptional regulator Upc2p , overexpression of drug efflux pumps , and inactivation of ERG3 gene .
Candida albicans strains isolated from patients with candidemia have the lowest incidence of azole resistance (0-5%) [31, 32]. However, the incidence of fluconazole resistance among the C. albicans clinical isolates of OPC is higher and is associated with prior OPC infections and previous fluconazole treatment . Out of the 100 C. albicans clinical isolates, 5 isolates were resistant to fluconazole (MIC=16 μg/ml). Interestingly, all resistant strains belonged to Mashhad.
Resistant isolates were obtained from BAL (n=2), vaginitis (n=2), and joint (n=1). One isolate (from BAL) did not grow after re-passaging on SGA; therefore, it was excluded from the study. Amino acid substitutions have been delineated in 61 parts of the ERG11p due to mutations in ERG11 gene . Erg11p mutations, such as D153E, E266D, D116E, K128T, K147R, E266Q, K287R, G129A, and G303D, do not cause fluconazole resistance [12-14, 34, 35]. However, ERG11p mutations, such as G464S, Y132H, S405F, R467K, T315A, and I471T, have been shown to cause resistance to azoles [34, 36-40].
The C-terminal and N-terminal of ERG11p are on the protein surface rather than on the active site; therefore, the 295-1667 bp part of ERG11 gene was amplified to look for possible mutations. Biofilms, multidrug transporters in Candida species, multiple drug resistance gene MDR1, drug segregation within intracellular vacuoles, and also overexpression of CDR1 and CDR2 as Candida drug-resistant genes can significantly reduce the intracellular drug concentration [4, 41, 42].
Susceptibility to fluconazole in C. albicans isolates may reach to 95.8% [43, 44]. However, as indicated in a number of investigations, the fluconazole resistance of such clinical isolates might be as high as 35% [45, 46]. In the present study, the incidence rate of fluconazole resistance in C. albicans was 5%. This low rate may be related to the administration of an appropriate dosage of antifungal agents or completion of the therapeutic course.
In this study, two types of mutations, including E266D and V488I, were detected among the resistant isolates. E266D mutation was also reported previously; nonetheless, this mutation does not result in fluconazole resistance . V488I (G1609A) mutation may not also lead to fluconazole resistance because V488 is located far from the active center of ERG11p . Khademi et al. found four resistant C. albicans strains among patients in Guilan, Iran. Missense mutations, such as C470G, K291N, and Q474R, were detected in all resistant isolates.
Similar to our results, Teymori et al.  detected 5 (5.1%) resistant C. albicans isolates (MIC≥64 μg/ml) out of 97 clinical samples. They revealed that ERG11 gene in the five fluconazole-resistant C. albicans isolates was up-regulated 4.15-5.84 folds, compared to the control strain. Eftekhari et al.  isolated 4 resistant C. albicans out of 40 strains and screened ERG11 gene mutations by PCR sequencing. Their isolated strains had D116E and V456G polymorphisms.
In another study carried out during April 2015-April 2016, Alizadeh et al.  isolated 28 strains of C. albicans from muco-cutaneous candidiasis among immunocompromised patients in Omidiyeh, Khuzestan, Iran. In the mentioned study, all isolates were resistant to fluconazole. They evaluated ERG gene expression by semi-quantitative reverse transcriptase-(RT) PCR. In line with the results of the present study, they showed no significant changes in fluconazole-resistant isolates in comparison with untreated controls and ERG11 reference sequence.
In 2017, Balabandi et al.  detected two missense mutations (i.e., D116E and E266D) in ERG11 gene among 20 resistant C. albicans in Rasht, Iran. Furthermore, Peron et al.  found two fluconazole-resistant C. albicans isolates from oral cavity (MIC=8 μg/ml) and esophageal cavity (MIC=64 μg/ml). They found six mutations encoding distinct amino acid substitutions (i.e., E116D, T128K, E266D, A298V, G448V, and G464S) that were previously reported to be associated with fluconazole resistance.
Moron and Cabrera  isolated 26 clinical strains from two tertiary hospitals in Metro Manila, Philippines, during November 2016 to January 2017. Out of 26 C. albicans, a high percentage of the isolates (73.08%) showed resistance to fluconazole. They detected the presence of point mutations T462C, A369C, and C558T. Mutations A369C and T462C have been also identified as possible factors associated with resistance to azole agents .
In accordance with many investigations in this field, our results demonstrated that the majority of C. albicans isolates were susceptible to fluconazole. Since the main mechanism of azole resistance is the mutation occurring through the ERG11 gene in C. albicans, this gene was selected for analysis in the present study. Our analysis resulted in the detection of 41 mutations in codons, only 4 of which caused changes in ERG11p. Nonetheless, these mutations (i.e., E266D and V488I) cannot lead to fluconazole resistance. Resistance among these isolates could be due to other mechanisms, such as the prevention of the intracellular accumulation of the antifungal agent and alteration of the target enzyme to diminish drug binding.
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