Detection of antifungal drug-resistant and ERG11 gene mutations among clinical isolates of Candida species isolated from Khartoum, Sudan.

Alteration in the target enzyme

The azoles bind to and inhibit the activity of 14 α-demethylase, a key enzyme in the fungal ergosterol synthesis pathway, which belongs to the cytochrome P450 family¹⁰. One of the known potential resistance mechanism of azoles is alteration in the ergosterol syntheses pathway. Candida species can develop resistance by mutation/s in the gene (ERG11) which codes for the enzyme 14α-demethylase¹³. The point mutation in the ERG11 gene can result in an amino acid substitution which in turn produces a conformational change in the enzyme and decreases the affinity to azoles, however, susceptibilities are not affected equally by different mutations as the presence of some mutations such as Y132H, R467K, and I471T confirm resistance, on the other hand, mutations such as E266D does not affect the resistance¹⁴. In addition, drug resistance might develop through overexpression of the ERG11 gene through increased mRNA level which might increase the concentration of the enzyme in comparison to sensitive isolates¹⁵.

Efflux pump

Efflux pumps are the basic mechanism in most eukaryotic cells by which unwanted toxic materials are forced out of the cell. Two types of efflux pumps have been identified: ATP binding cassette (ABC) transporters and major facilitator superfamily (MFS)¹⁶. ABC transporters are pumps that are actively associated with the efflux of potentially toxic molecules to the cell, and they are primarily hydrophobic and lipophilic¹⁷. Many different pumps are known to belong to the different families, such as Candida resistance (CDR1&2) genes that are related to the PDR5 family, and known to be associated with resistance to antifungals¹⁵. The MFS pumps work by antiproton power i.e. proton pumped inside the cell and hydrophobic and/or lipophilic material pumped outside the cell. This is coded by multidrug resistance gene (MDR1) which found to be overexpressed in fluconazole resistant isolates, however, the gene was not overexpressed in other azoles, ketoconazole and itraconazole, resistant strains¹⁸. Some authors have tried to link overexpression of CDR1, 2 and MDR1, concluding that deletions of these genes will result in more susceptible isolate than each gene alone¹².

Among different antifungals, Fluconazole was the most commonly prescribed, amphotericin and itraconazole remains to be the cornerstone in the management of systemic fungal infections in Sudan since echinocandins are not yet registered (Sudan National Essential Medicine List- 2014)¹⁹. Due to the scarcity of reports about the rate of drug resistance and resistant genes among Candida species in the study area, this study was conducted to screen the susceptibility of different Candida species towards commonly used antifungals, and to identify the role of ERG11 gene mutation/s in the development of fluconazole resistance. To this end, we collected and identified isolates of Candida species, selected the most resistant isolates, amplified and sequenced the conserved domain of the ERG11 gene and detected the impact of the mutation(s) on the enzyme 14α-demethylase’s structure and function using in silico tools^20,21.

Ethical statement

This study was reviewed and approved by the Research ethical committee, Faculty of Medicine & Health Science, International University of Africa (IUA) (6-2017). Oral informed consent was obtained from the participating patients, when their hospital laboratory result was positive for Candida species. Oral consent was obtained over written consent (where it was recorded), since the majority of the patients included in this study were illiterate (in case of minors, consent was obtained from parents or guardians). The structure of the consent was approved by the Research Ethics Committee.

Study design, area and participants

This was a cross-sectional laboratory-based study using clinical isolates of Candida species. Clinical isolates were collected from different Khartoum state hospitals in a period between September 2017 to September 2018, all clinical isolates primarily identified as Candida species regardless of age, gender, and site of isolations, were included. Samples and demographic data were obtained directly from the patients within each hospital after consent was obtained by the principle investigators.

Sample size calculation

Sample size was calculated using the following formula on cross-sectional studies²²:

n = Z² * P (1-P) / d²

Where, n = desired sample size, Z = critical value and a standard value for the corresponding level of confidence (1.96), P = expected frequency of resistance obtained from previous studies (7%)^23,24, d = margin of error (0.05).

n = 1.96² * 0.07 (1-0.07) / (0.05)² = 100 samples.

Sample collection and storage

A total of 100 clinical isolates of Candida species were collected from Khartoum state hospitals, Sudan. The isolates were pre-identified at each hospital’s laboratory using conventional methods such as wet mount, gram stain, germ tube, and growth on Sabouraud Dextrose Agar media (SDA). Immediately after collection, the isolates were grown into SDA (M063, HIMEDIA, Mumbai, India) at 32°C for 24–48 hours. From the grown culture, colonies were picked and streaked over a slant of SDA in screw-cap tubes, each slant was filled with sterile liquid glycerol and tightly closed and stored in 4 C° until recovered.

Identification

Chromogenic media Hi-Chrome Candida differential agar media supplemented with chloramphenicol 0.5g/L (M1297A, HIMEDIA, Mumbai, India) was used to differentiate between Candida species based on colonies’ color and morphology. A subculture from the stock culture was allowed to grow on SDA for 24 hour, subsequently one well isolated colony from the grown culture was picked out and streaked over the prepared Hi-Chrome media and incubated at 32°C for 24–48 hours, the result was interpreted as per manufacture instructions (C. albicans—light green, C. glabrata—cream to white, Candida krusei—pale, fuzzy and C. tropicalis—blue to purple, C. dupliensis—pale green)²⁵.

Antimicrobial sensitivity testing

Sensitivity testing to all isolated Candida species was carried out as recommended by the Clinical Laboratories and Standard Institute (CLSI)²⁶. A modified Mueller Hinton Agar media (M173, HIMEDIA, Mumbai, India) supplemented with 2% glucose and methyl blue 5µg/mL was used. Using overnight culture on SDA, the inoculum was prepared by suspending 4 well-isolated colonies in 5mL sterile saline, inoculum size was adjusted by matching the turbidity with standard McFarland which was prepared by adding 0.5 mL BaCl2 (0.048 mol/L) to 99.5 mL H₂SO₄. Within 15 minutes after adjusting the turbidity and by using sterile cotton swab, the microorganisms were streaked from the center of the petri dish to the top, each time the plate was rotated 60° to ensure that the agar surface is at least double streaked. Within 15 minutes after streaking, three discs, namely fluconazole 25µg, itraconazole 10µg and amphotericin 10µg (SD232, SD221, SD111. HIMEDIA, Mumbai, India) were applied to each inoculated petri dish, gently pressed into the agar using sterile forceps, incubated at 32C° for 24–48 hours, zone dimeter around each disc were measured using calipers and result was interpreted as per CLSI²⁶.

Genomic DNA extraction, gene amplification, detection and sequencing

Genomic DNA was extracted using guanidine chloride method, briefly, DNA extraction was carried out using 48 hours grown culture on SDA media, three to five colonies were washed with 5 mL phosphate buffer saline (PBS) three times, then 2 mL white cell lysis buffer and 20 μL of proteinase K (10 mg/mL; iNtRON Inc, Korea) were added to the pellet in a Falcon tube, vortexed and incubated at 37°C overnight. Then 1 mL from guanidine chloride (7M; iNtRON Inc, Korea) and 350 μL of ammonium acetate (7M; Loba Chemie, India) were added. The tubes were vortexed and incubated at 65°C in a water bath for 2 hours. After that 2 mL pre chilled chloroform (sd Fine-Chem limited, India) was added, and centrifuged at 6000 RPM for 20 minutes and the supernatant was transferred into a new Falcon tube and completed to 10 mL volume with pre chilled absolute ethanol (Carlo Erba, France) and incubated overnight at -20°C for completion of DNA precipitation. After incubation the tubes were centrifuged at 6000 RPM for 20 minutes, then the ethanol was poured off and the same step was repeated with 70% ethanol. After that the tubes were left to air dry. Finally, DNA was suspended in 80 μL TE buffer (iNtRON Inc, Korea) and incubated at 4 °C until used²⁷, as a template for PCR. The ERG11 gene from C. albicans was amplified using previously published primers^14,28: forward: (5′-CAAGAAGATCATAACTCAAT-3′) and reverse (3′-AGAACACTGAATCGAAAG-5′) (Macrogen Inc. Korea). All PCRs were carried out in final volume of 20 µL containing Maxime PCR PreMix Kit i-Tag (2.5U i-TagTM DNA polymerase, 2.5mM each dNTPs, 1x reaction buffer and 1x Gel loading buffer), 1µL each forward and reverse primer (10pmol final concentration), 2.5µL genomic DNA, and the volume was completed with distilled water. The PCR was carried out in G-storm thermocycler with the following conditions: initial denaturation at 94°C for 4 min; 35 cycle of denaturation at 95°C for 30 s, primer annealing at 55°C for 30 s, and extension at 70°C for one min; followed by final extension step at 72°C for 10 min. The final product was visualized by loading 3µL in 0.8% agarose gel electrophoresis for 45 minutes under the voltage 100 V. Distinct bands were observed under UV light and photographed. The ERG11 gene from the most C. albicans resistant isolates (isolate 10, 13, and 14) and one sensitive isolate (24 randomly selected, double blinded by independent researcher) were selected for sequencing (Sanger sequencing in BGI, Shenzhen, China). All sequences were deposited in GenBank and accession numbers MT081007, MT081008, MT081009, MT081010 for isolate 10, 13, 14, and 24, respectively, were obtained. Sequences were aligned based on fluconazole susceptible strain SC5314 (GenBank accession number X13296) using BioEdit software version 7.2.5.

Collection and identifications

Out of 100 samples collected, 80 were from females and 20 from males with a mean age of 40. 66 isolates were from urine samples, 17 from sputum, 12 were high from vaginal swabs, and 5 from other sites. Overall, the most common species was C. albicans (n=51), while the most prevalent Non-albicans species was C. glabrata (n=31), followed by C. krusei (n=8). Table 1 provides a detailed description on the frequency of each species with respect to the site of isolation²⁹.

Antifungal sensitivity testing (AST)

Fluconazole was the least effective agent followed by itraconazole. Itraconazole resistance was observed among Non-C. albicans (NCA) species, high frequency of intermediate susceptibility dose-dependent (ISDD) was observed for itraconazole among all Candida species, while there was no amphotericin resistant isolate detected. Rate of resistance among female isolates 28.75% (23 out of 80) was higher than the male 15% (3 out of 20).

Fluconazole resistance was observed in 23% of C. albicans samples, only 2 isolates were ISDD, and the remaining isolates (72.5%) were sensitive, there were no itraconazole and amphotericin resistant C. albicans isolates. However, 31% were categorized as ISDD to itraconazole. Among NCA species, 19.4% of C. glabrata were fluconazole resistant, as well as all C. krusei isolates (8). Azole cross resistance was observed among 2 C. glabrata and 2 C. krusei isolates. One C. dupliensis was resistant to fluconazole while there were no C. tropicalis resistant isolates. Complete AST results are shown in Table 2²⁹.

C. albicans ERG11’s gene amplification, electrophoresis analysis and sequencing

The complete ERG11 gene coding region (1587 bp) from C. albicans resistant isolates (12) and one sensitive isolate (control) were amplified as shown in Figure 1³⁰. Sequence analysis revealed 15 different mutations, 12 of which were silent, and 3 were non-synonyms Table 3. T495A and G1609A were observed only in resistant isolates (isolate 10 and 14 respectively), while A945C was observed in sensitive and resistant isolates (isolate 13, 14, and 24). All mutations were previously reported (Table 3³¹).

Figure 1. Agarose gel Electrophoresis showing the bands of amplified ERG11 gene.

From left to right lanes: L, 100 bp DNA ladder, 1 to 12 lanes are the amplified Candida albicans ERG11’s gene (1587 bp).

Underlying data

All sequences were deposited in GenBank under accession numbers MT081007, MT081008, MT081009, and MT081010 for isolate 10, 13, 14, and 24, respectively.

Figshare: demographic, identification, sensitivity test data. https://doi.org/10.6084/m9.figshare.12449615.v1²⁹

This project contains the following underlying data:

Figshare: sequence data for ERG11 gene. https://doi.org/10.6084/m9.figshare.12600128.v1³¹

This project contains the following underlying data:

Figshare: candida identification using Hi-chrome media and gel electrophoresis for ERG11 gene. https://doi.org/10.6084/m9.figshare.12775769.v1³⁰

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).