Analysis of CDR1 and MDR1 Gene Expression and ERG11 Substitutions in Clinical Candida tropicalis Isolates from Alexandria, Egypt

Introduction Candida tropicalis is a common non-albicans Candida (NAC) species that causes numerous fungal infections. Increasing antifungal resistance to azoles in NAC is becoming a major health problem worldwide; however, in Egypt, almost no data is available regarding fluconazole resistance mechanisms in C. tropicalis. The current study aims to investigate two possible important molecular mechanisms involved in fluconazole resistance in C. tropicalis isolates. Materials Fifty-four clinical C. tropicalis isolates were included. Identification and antifungal susceptibility profiles of the isolates were carried out using the VITEK 2 compact system. The molecular investigation of fluconazole resistance included the expression of the CDR1 and MDR1 genes by quantitative real-time RT-PCR as well as the sequence analysis of the ERG11 gene. Results Antifungal susceptibility testing identified 30 fluconazole-non-susceptible isolates. Statistically, CDR1 gene expression in fluconazole-non-susceptible isolates was significantly higher than that in fluconazole-susceptible isolates, with MDR1 gene expression levels that were similar in both non-susceptible and susceptible isolates. Sequence analysis of the ERG11 gene of 26 fluconazole-resistant isolates identified two missense mutations: A395T (Y132F) and G1390A (G464S). Conclusions This study has highlighted the role of overexpression of the CDR1 gene and ERG11 gene mutations in fluconazole non-susceptibility. Further studies in Egypt are required to investigate other possible molecular mechanisms involved in azole resistance. Supplementary Information The online version contains supplementary material available at 10.1007/s42770-023-01106-y.


Introduction
Candidiasis represents one of the most common causes of invasive fungal infections and is associated with high morbidity and mortality rates, especially in immunocompromised patients [1][2][3].In the past decades, changes in the epidemiology of Candida infections have been noted.Although Candida albicans remains the most common isolated species in patients with invasive candidemia, a shift towards non-Candida albicans Candida (NCAC) species has been noted [4,5].Candida tropicalis has become a predominant NCAC species, causing invasive candidiasis [3,6].Currently, C. tropicalis ranks as the first or second NCAC species causing candidemia and candiduria according to the geographical region, such as East Asia and Latin America [5,[7][8][9].After C. albicans, C. tropicalis is considered to be the second most virulent Candida species [10].Patients infected with C. tropicalis usually have longer hospitalizations and higher mortality rates in comparison to those infected with C. albicans [6].
Among the different classes of antifungal agents used for the management of fungal infections, azoles are the most commonly used drugs for both treatment and prophylaxis.Azoles act through inhibition of lanosterol C14αdemethylase (Erg11p), which is the main enzyme involved in the ergosterol biosynthesis pathway.Erg11p is encoded by the gene ERG11.Fluconazole represents one of the most widely used antifungal agents, as it has minimal side effects in addition to its affordable cost compared to other classes with higher costs, such as echinocandins [11,12].
Studies have reported a significant increase in azole resistance among C. tropicalis isolates, which could be attributed to the wide use of azoles as agents of antifungal prophylaxis and their widespread use in agriculture.This leaves a limited number of treatment options, endangering patients, especially in developing countries with limited resources [13,14].
There are numerous molecular mechanisms that could lead to azole resistance.Alterations and/or overexpression of the lanosterol C14α-demethylase (ERG11) gene, upregulation of efflux transporter genes, the major facilitator superfamily (MFS) family gene (MDR1), and the ATP-binding cassette (ABC) transporter family (CDR1) are some of the most common molecular resistance mechanisms reported.Also, mutations in either the sterol D5,6-desaturase (ERG3) gene or the acquisition of functional mutations and/or overexpression of different transcription factors are involved in azole resistance [14,15].
The current knowledge of azole resistance in C. tropicalis isolated from Egyptian hospitals is very scanty.Our study aimed to investigate some of the different molecular mechanisms involved in azole non-susceptibility in clinical C. tropicalis isolates.

Clinical isolates
A total of 54 C. tropicalis species clinical isolates were included from a previous study [16].The clinical specimens were collected from various ICU patients admitted to different medical facilities, including the Medical Research Institute (MRI), Alexandria Main University Hospital (AMUH), and Mabaret Al-Asafra Hospital.Identification of isolates was carried out by the CHROMagar Candida and VITEK 2 compact system.The antifungal susceptibility testing of the isolates was determined using the VITEK 2 system AST-YS07 card.Out of the included isolates, 30 samples were fluconazole-non-susceptible (4 isolates were susceptible-dose-dependent (SDD) and 26 isolates were fluconazole-resistant), and 24 samples were susceptible to fluconazole.Regarding voriconazole, 28 isolates were non-susceptible (23 isolates were SDD and 5 isolates were voriconazole-resistant), and 26 isolates were susceptible to voriconazole.The interpretation of MIC readings was according to the CLSI species-specific clinical breakpoints (SS-CBPs) (CLSI M27-S4) for fluconazole, voriconazole, caspofungin, and micafungin [17], and the epidemiological cut-off values (ECVs) for flucytosine and amphotericin B [18].(Supplementary Table 1) All isolates were stored as frozen stocks with 30% glycerol in a deep freezer at −20°C until used.

Expression analysis of CDR1 and MDR1 genes by quantitative real-time RT-PCR (RT-qPCR)
All C. tropicalis clinical isolates were cultured on SDA for 48 hours.RNA extraction and purification for all the isolates were performed from the grown fungal cells using the YeaStar™ RNA Kit (Zymo Research Corp., USA), according to the manufacturer's instructions.Samples were treated with RNAse-free DNAse (Qiagen, Hilden, Germany).Extracted RNA concentrations and purities were calculated using a NanoDrop 2000 spectrophotometer (Thermo Scientific) for the studied samples.
Samples were analyzed for relative expression of CDR1 and MDR1 genes using the SensiFAST SYBR® Hi-ROX One-Step kit (Bioline, UK), where cDNA synthesis and PCR amplification were carried out in the same tube using a fixed concentration of RNA template (0.1 μg/μL) for each sample.The protocol was followed according to the manufacturer's instructions.All the primers used for real-time RT-PCR were designed (Table 1) using the primer-BLAST (primer3) at www. ncbi.nlm.nih.gov, based on the CDR1, MDR1, and Actin (internal control) genes sequences of C. tropicalis MYA-3404 (GenBank accession no.GG692395) [19].Erg11-R TAC TTA GCA ACA ACT TCT AGTG Amplification of target genes was carried out using the StepOne ™ System (Applied Biosystems, USA) according to the following thermal profile: cDNA synthesis: one cycle at 45°C for 10 min, Polymerase activation: one cycle at 95°C for 2 min, followed by 40 cycles of amplification: Denaturation at 95°C for 5 sec, annealing at 55.5°C for 5 sec, and extension at 72°C for 5 sec.A melt curve analysis was performed at the end of the PCR cycles, confirming the specificity of the reaction and ensuring that only a single PCR product was amplified.(Supplementary figures 1-3).
Statistical analysis of gene expression was carried out using a one-sample t-test where appropriate by the Statistics Package for Social Sciences (SPSS) software version 21; a p value less than 0.05 (95% confidence interval of the difference) was considered significant.

Sequencing of the ERG11 gene and mutation analysis
Genomic DNA extraction and purification from the 26 fluconazole-resistant isolates were performed using the YeaStar Genomic DNA Kit™ (Zymo Research Corp.) according to the manufacturer's instructions.Extracted DNA concentrations and purities were checked using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA).
Primers were designed using the primer-BLAST (primer3) at www. ncbi.nlm.nih.gov, based on the Erg11 gene sequence of C. tropicalis ATCC 750 (GenBank accession no.M23673) (Table 1).Erg11 gene amplification was performed using the MyTaq™ HS Mix Kit (Bioline, UK).Amplification of target genes was carried out using the Veriti® Thermal Cycler (Applied Biosystem, CA, USA) according to the following thermal profile: Initial denaturation at 95°C for 1 min, followed by 35 cycles: Denaturation at 95°C for 15 sec, annealing at 55°C for 15 sec, and extension at 72°C for 10 sec.PCR products were purified using a PureLink® PCR purification kit (Invitrogen) according to the manufacturer's instructions.

Sequencing and Data Analysis:
Sequencing of both strands was performed, and the nucleotide sequences obtained were analyzed using the BioEdit® sequence alignment editor and analysis program.Raw DNA chromatograms were visually checked and scrutinized for heterozygosity, which was defined as the presence of overlapping peaks in the forward and reverse chromatograms, and compared with C. tropicalis ATCC 750 (GenBank accession no.M23673) Erg11 gene sequence.(Supplementary figures 4-9).

Expression analysis of CDR1 and MDR1 genes by quantitative real-time RT-PCR (RT-qPCR)
RT-qPCR showed that the relative CDR1 gene expression in fluconazole-non-susceptible isolates was significantly higher compared to fluconazole susceptible isolates (average fold expression level 2.19 vs. 1.07, p < 0.01).However, no statistical difference was observed regarding the relative MDR1 gene expression in fluconazole-non-susceptible isolates when compared to fluconazole-susceptible isolates (p = 0.589).(Figure 1).(Supplementary Tables 2-5)

Sequencing of the ERG11 gene and mutation analysis
The analysis of the chromatograms of the 26 fluconazoleresistant isolates revealed seven different mutations, two of which were missense mutations: A395T (Y132F), observed in only one clinical isolate, and G1390A (G464S), detected in 24 clinical isolates, while the other five mutations identified in all the tested isolates were silent mutations: T225C, G264A, G1362A, C1464T, and T1554C.(Table 2).

Discussion
Resistance to azoles, particularly fluconazole, is rising worldwide [20,21].In Egypt, limited data is available regarding the underlying mechanisms of azole resistance in C. tropicalis.In this study, we aimed to investigate some of the possible azole resistance mechanisms in C. tropicalis as mutations in the ERG11 gene as well as the upregulation of the major facilitator gene MDR1 and the ATP-binding cassette transporter gene CDR1.
Different studies reported various profiles of CDR1 and MDR1 gene expression.The current study investigated the role of efflux pumps in azole resistance by comparing quantitative relative gene expression for CDR1 and MDR1 genes in both fluconazole-susceptible and non-susceptible isolates.Our results showed that relative CDR1 gene expression in fluconazole-non-susceptible isolates was statistically and significantly higher than that of susceptible isolates.On the other hand, relative MDR1 gene expression in fluconazolenon-susceptible isolates was not statistically or significantly greater than susceptible isolates.
In agreement with our finding, Vandeputte et al. [22] stated that no difference was found in expression of the MDR1 gene between fluconazole-susceptible and resistant isolates.This finding could be attributed to the fact that MDR1 genes encode an MFS transporter, with a relative specificity for fluconazole.Thus, it is unlikely that MDR1 gene overexpression could alter fluconazole susceptibility pattern in comparison to the CDR1 gene, which encodes ABC transporters and is responsible for azole cross-resistance [23].On the contrary, You et al. and Jin et al. found overexpression of the MDR1 gene expression level in contrast to the CDR1 gene, which showed no difference in gene expression [24,25].
Although overexpression of both the CDR1 and MDR1 efflux pump genes was demonstrated in C. tropicalis isolates, highlighting that efflux pumps play a significant role in azole resistance in many studies [26][27][28][29][30], opposing results were reported in other studies demonstrating that efflux pumps do not play a vital role in azole resistance in C. tropicalis, as the CDR1 and MDR1 gene expression levels did not significantly differ between azole susceptible and resistant isolates [29,31].The inconsistencies and discrepancies in the literature's gene expression analysis may be attributed to different factors.There is an interplay of different resistance mechanisms, including ones that were not investigated.Overexpression of efflux pumps seems not to be the main azole resistance mechanism, as the EGR11 missense point mutation is the primary one, yet it contributes to the overall resistance.Some researchers observed that C. tropicalis isolates only had overexpression of a single gene target.Moreover, the difference may be caused by heterogeneity between the isolates.Other factors that may be considered are the number of included isolates, the MIC levels of fluconazole and other azole members of the tested isolates, reflecting the degree of cross-resistance between azole members, and the overall molecular mechanisms contributing to the azole resistance of each isolate [29][30][31].
Several studies have investigated ERG11 gene mutations as one of the possible mechanisms for azole resistance in C. tropicalis.In the present study, sequencing analysis of the ERG11 gene in 26 fluconazole-resistant isolates showed that seven different mutations took place.Two missense mutations were detected leading to amino acid substitutions: A395T (Y132F) and G1390A (G464S), which were located in ERG11 hot spots I and III, respectively, as previously described [32].The other five mutations were silent mutations: T225C, G264A, G1362A, C1464T, and T1554C.
The G1390A (G464S) mutation was the most frequent type of mutation identified in our study.This amino acid substitution was also reported in other studies [28,29].Another amino acid substitution at the same site was also reported [33].The A395T (Y132F) mutation may lead to decreased binding affinity for azoles [22].However, it was only demonstrated in one isolate in our research; the A395T (Y132F) mutation was frequently observed in other previous studies [22,30,31,33,34].
Additionally, silent mutations have been demonstrated in our study, including G1362A and T1554C in 25 isolates (96.15%).However, these mutations have been reported previously in other studies; being synonymous mutations, they are usually considered silent and cannot therefore be linked to azole resistance [22,33,35].It is worth mentioning that the percentage of silent mutations in relation to the total number of mutations detected in the current study was in agreement with a Chinese study conducted by Fan et al. (71.8%) [29].Our study revealed the first reported silent mutation, C1464T (I488I).Although the role of synonymous mutations in azole resistance is not established, different reports have begun to unravel the role of synonymous mutations in altering the nucleic acid sequence and consequently its structure.synonymous mutations may affect RNA secondary structure, stability, and regulatory elements such as ribosome binding sites or splicing signals, giving rise to changes at the gene expression levels as well as substrate specificity, rendering them not entirely silent.Furthermore, synonymous mutations can affect codon usage and the accuracy and speed of translation.Changes in codon usage may reflect on protein expression levels, stability, and folding, which can indirectly influence resistance mechanisms.
Studies have revealed that the presence of synonymous codons is non-random and may participate in antimicrobial resistance [36][37][38][39][40].According to our best knowledge, the various possible effects of synonymous mutations have not been described or investigated in clinically relevant fungi.Further studies are warranted to investigate any possible mechanisms.
The main limitation of the current study, due to financial restrictions, was the inability to investigate the role of other molecular mechanisms of azole resistance.For instance, the ERG11 gene expression in addition to the sequence analysis of efflux pumps genes CDR1 and MDR1 are examples of these limitations.Furthermore, the role of UPC2 (expression regulator) and ERG3 mutations in azole resistance, as well as gene expression, should be investigated.
Despite the molecular-level investigations' limitations, they provided valuable, clinically relevant information.Molecular analysis detects only known resistance-encoding genes or mutations.Novel molecular mechanisms may not be detected unless they share a high level of similarity with a known gene.Moreover, the results of molecular tests may not fully correlate with the phenotypic variations that contribute to resistance.In addition, molecular analysis alone may not provide a complete understanding of the clinical outcomes and responses to antifungal treatments.It is crucial to consider the interplay of genetic modifications, phenotypic characteristics, and patient-related factors, allowing for a more comprehensive assessment of azole resistance [41,42].
Beyond that, azole resistance could develop through mechanisms that molecular analysis may be unable to discover.Alterations in membrane permeability or changes in drug efflux pumps activities can contribute to azole resistance.Evaluating ergosterol levels, which are essential components of fungal cell membranes, can help assess the role of membrane-related modifications in resistance.Assessing efflux pump activity through efflux pump assays also provides important information on the functional aspects of resistance mechanisms.It helps determine whether resistant strains have increased efflux pump activity, leading to reduced intracellular drug accumulation [33,43].
In the meantime, this is the first Egyptian study to investigate molecular mechanisms involved in azole resistance in clinical C. tropicalis isolates.The study has highlighted the contribution of the upregulated CDR1 efflux pump and the ERG11 gene substitutions, Y132F and G464S, as possible molecular mechanisms in fluconazole non-susceptibility in C. tropicalis.
Eventually, further studies are required to evaluate other molecular mechanisms involved in azole resistance, such as mutation and/or overexpression of ERG3, which is a C5 sterol desaturase enzyme that is involved in ergosterol biosynthesis and linked with azole resistance.In addition, evaluation of ERG11 gene expression levels and the possible substitutions in UPC2, which is the transcription factor that regulates the majority of ergosterol biosynthetic genes, as well as gene sequence analysis of MDR1, CDR1, and its transcription factor TAC1, may give a comprehensive overview on azole resistance in C. tropicalis clinical isolates.
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Fig. 1
Fig. 1 Relative gene expression of (A) CDR1, (B) MDR1 efflux pumps genes, and comparison between the two groups (C) according to CDR1 and MDR1 relative gene expression levels in fluconazole-non-susceptible and susceptible C. tropicalis clinical isolates.* P ≤ 0.05

Table 1
Primers used in qRT-PCR and Erg11 gene amplification

Table 2
The ERG11 gene substitutions in fluconazole-resistant C.