Knowing that antifungal susceptibility testing is not routine testing in many centers around the world, the azole resistance in
Several studies have shown that azoles inhibit the ergosterol biosynthetic pathway by interacting with the two CYP51 proteins (CYP51A and CYP51B) and alter the metabolism of sterols in cells (Alcazar-Fuoli et al. 2008). However, a risk of decisive infection may occur if resistance is acquired (Howard and Arendrup 2011; Hadrich et al. 2012a). Various studies focus on azole-resistant
This study evaluated the mRNA expression levels of
The 34 molecularly confirmed
The ETEST® strips (AES™, bioMérieux, France) were used as previously described to determine antifungal susceptibility profiles after 48 h of incubation (Hadrich et al. 2012b). Epidemiological cutoff values (ECVs) for POS (0.25 μg/ml), IT, and VOR (1 μg/ml) were previously described by five laboratories, according to the CLSI M38-A2 microdilution method (Espinel-Ingroff and Turnidge 2016).
DNA was extracted using a QIAamp® DNA Mini Kit (QIAGEN, Germany), as indicated by the manufacturer’s instructions.
The RNeasy® Mini Kit (QIAGEN, Germany) was used according to the manufacturer’s instructions to extract total RNA from the cell lysates. To avoid DNA contamination, RNA extracts were treated with RNase-free DNase (Promega, UK). The concentration and purity of the RNA were determined using a UV spectrophotometer by measuring the absorbance at 230 (A230), 260 (A260), and 280 nm (A280). The A260 nm/A280 nm ratio of the samples, reflecting the purity ranged from 2.06 to 2.21. The A260 nm/A230 nm ratio ranged from 1.90 to 2.50.
The transformation of the RNA extract from each isolate into cDNA was done by reverse transcription method (RT), according to the manufacturer’s instructions of the PrimeScript™ RT Reagent Kit (Perfect Real Time) from Takara Bio Inc. (Japan).
Specific primers and probes for gene expression analysis were designed using Primer3 software (
The sequences of primers and probes used in RT-qPCR.
Gene | Primers and probes | |
---|---|---|
F | 5’-GCGCGCATGAGGGAGAT-3’ | |
R | 5’-CAATGCATGAGGTTCCAGATCA-3’ | |
Probe | HEX-TCATTAACGAGCGCCGCAAGAACC-MGB | |
F | 5’-ATTCGACTCGACATTTGCTGAA-3’ | |
R | 5’-GCATCACGCTTGCGGTTAT-3’ | |
Probe | FAM-CATGATCTCGACATGGGTTTTGCCC-MGB | |
ANXC4 | F | 5’-CCAACCCATAAACGCTCTGT-3’ |
R | 5’-TGGTGGGAATCTTGGAGAAC-3’ | |
Probe | CY5-ATCGAAGCAGCCTGTCTCAT-MGB |
The measurement of the expression level and the number of copies of each of the two genes
Gene expression was analyzed using 10 μl TaqMan™ Universal PCR Master Mix (Applied Biosystems™; Thermo Fisher Scientific, Inc., USA) and 1 μl template (DNA or cDNA) with 20 pmol forward and reverse primers, 7 pmol hydrolysis probe. The PCR amplification conditions were as follows. The first step is 50°C for 2 minutes, 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds, and the final step is 54°C for 1 minute. All reactions were performed in triplicate using the StepOne™ Real-Time PCR instrument (Applied Biosystems™; Thermo Fisher Scientific, Inc., USA). StepOne™ software version 2.1 was used to collect Cq data and calculate relative quantification (RQ). We then used the published comparative 2
A 2.5-fold change was considered gene overexpression or an increase in gene copy number (Livak and Schmittgen 2001; VanGuilder et al. 2008).
The
The sequences of primers used in PCR sequencing.
Gene Primers | ||
---|---|---|
AP1F | 5’-ATGGCATCCTTCACTCTCGT-3’ | |
AP1R | 5’-CG ATCAACTTCATGCTTCCG-3’ | |
AP2F | 5’-TCTGGAACCTCATGCATTGT-3’ | |
AP2R | 5’-TCCCTCGAAACCAGCAATTA-3’ | |
AP3F | 5’-GGCAGGGTCGAAATCACGGA-3’ | |
AP3R | 5’-GCCCGGATGGAAGAACCCTT-3’ | |
BP1F | 5’-CTTTTATCGGAAGTACCATC-3’ | |
BP1R | 5’-CTTTATCAGGAACAACTTCG-3’ | |
BP2F | 5’-ACTCTTCGCATACATGCACC-3’ | |
BP2R | 5’-CCAACGTGCATGATATTGCC-3’ | |
BP3F | 5’-CGACTCGACATTTGCTGAAC-3’ | |
BP3R | 5’-CTCTTCGCATACATGCACCA-3’ |
Amplification reactions were performed with a final volume of 50 μl containing 10 μl 5 × reaction buffer (pH 8.5), 25 mM MgCl2, 0.2 mM (each) dATP, dCTP, dGTP and dTTP (Promega, UK), 20 pmol of each primer, 2.5 U of GOTaq® DNA Polymerase (Promega, UK) and 400 ng of genomic DNA. PCR was performed in a thermocycler (Eppendorf, Germany), with the following amplification conditions: 94°C for 5 min, followed by 30 cycles of 30 s at 94°C, 30 s at 58°C and 1 min at 72°C and a final extension at 72°C for 10 min. PCR products were analyzed by agarose gel electrophoresis and purified using the Purification PCR Wizard® Kit (Promega, UK). Both sequences of the triplicate PCR product were directly sequenced using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems™; Thermo Fisher Scientific, Inc., USA) with primers and automated sequencer (ABI 3730; Applied Biosystems™; Thermo Fisher Scientific, Inc., USA), according to the manufacturer’s instructions. Product sequences were compared with those of wild-type
The DNA sequences of
Antifungal susceptibility to the azoles, relative quantification of gene expression, gene copy number of
Strain | GenBank accession | Isolation site | Isolation date | Itraconazole | Posaconazole | RNA relative quantification | DNA relative quantification | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
MIC (μg/ml) | R/S | MIC (μg/ml) | R/S | ±SD | ± SD | ±SD | ± SD | GenBank accession | Punctual mutation | GenBank accession | Punctual mutation | ||||||||||
Patient 1 | Invasive aspergillosis | TN-1 | MN964040 | nasal | 15/10/2018 | 0.125 | 0.125 | S | 0.90 | ±0,02 | 1.16 | ±0,04 | 0.69 | ±0,05 | 0.57 | ±0,02 | ND | ND | ND | ND | |
TN-2 | MN964041 | sputum | 15/10/2018 | 0.125 | S | 0.125 | S | 2.15 | ±0,03 | 0.73 | ±0,02 | 0.89 | ±0,02 | 1.98 | ±0,04 | ND | ND | ND | ND | ||
TN-3 | MN964042 | sputum | 22/10/2018 | 0.125 | S | 0.125 | S | 0.58 | ±0,01 | 0.90 | ±0,02 | 1.37 | ±0,02 | 0.97 | ±0,02 | ND | ND | ND | ND | ||
Patient 2 | Invasive aspergillosis | TN-4 | MN964043 | nasal | 15/10/2018 | 0.125 | S | 0.19 | S | 1.53 | ±0,02 | 1.05 | ±0,04 | 1.27 | ±0,03 | 1.93 | ±0,02 | ND | ND | ND | ND |
TN-5 | MN964044 | sputum | 15/10/2018 | 0.125 | S | 0.064 | S | 0.51 | ±0,04 | 0.05 | ±0,02 | 1.05 | ±0,02 | 1.92 | ±0,06 | ND | ND | ND | ND | ||
TN-6 | MN964045 | nasal | 23/10/2018 | 0.032 | S | 0.094 | S | 0.95 | ±0,01 | 0.89 | ±0,02 | 1.69 | ±0,03 | 0.93 | ±0,02 | ND | ND | ND | ND | ||
Patient 3 | Invasive aspergillosis | TN-7 | MN964046 | nasal | 30/10/2018 | 1.5 | R | 0.125 | S | 1.65 | ±0,02 | 0.73 | ±0,03 | 2.51 | ±0,02 | 1.98 | ±0,02 | MN964023 | C-T*(183) | MN964034 | No mutation |
TN-8 | MN964047 | sputum | 30/10/2018 | 0.5 | S | 0.094 | S | 1.12 | ±0,02 | 0.73 | ±0,02 | 1.13 | ±0,07 | 5.97 | ±0,04 | ND | ND | ND | ND | ||
Patient 4 | Invasive aspergillosis | TN-9 | MN964048 | sputum | 30/10/2018 | 0.75 | S | 0.125 | S | 4.48 | ±0,02 | 3.89 | ±0,02 | 2.71 | ±0,02 | 2.67 | ±0,02 | ND | ND | ND | ND |
TN-10 | MN964049 | nasal | 30/10/2018 | 0.38 | S | 0.064 | S | 1.42 | ±0,02 | 1.97 | ±0,04 | 2.49 | ±0,03 | 2.01 | ±0,03 | ND | ND | ND | ND | ||
Patient 5 | Invasive aspergillosis | TN-11 | MN964050 | sputum | 10/11/2018 | 0.5 | S | 0.125 | S | 5.65 | ±0,01 | 2.31 | ±0,02 | 1.87 | ±0,02 | 1.02 | ±0,02 | MN964018 | No mutation | MN964029 | No mutation |
TN-12 | MN964051 | BAL | 17/11/2018 | 0.125 | S | 0.125 | S | 2.35 | ±0,02 | 2.31 | ±0,02 | 1.87 | ±0,02 | 1.02 | ±0,02 | MN964019 | C-T*(183) | MN964030 | No mutation | ||
Patient 6 | Invasive aspergillosis | TN-13 | MN964052 | BAL | 03/12/2018 | 0.75 | S | 0.19 | S | 5.68 | ±0,02 | 2.35 | ±0,05 | 2.83 | ±0,05 | 3.95 | ±0,02 | ND | ND | ND | ND |
TN-14 | MN964053 | sputum | 26/11/2018 | 0.5 | S | 0.125 | S | 0.63 | ±0,03 | 0.88 | ±0,06 | 6.95 | ±0,07 | 1.06 | ±0,02 | ND | ND | ND | ND | ||
TN-15 | MN964054 | nasal | 26/11/2018 | 1 | R | 0.19 | S | 0.90 | ±0,02 | 0.76 | ±0,02 | 0.53 | ±0,02 | 1.65 | ±0,05 | MN964024 | C-T*(183) | MN964035 | No mutation | ||
TN-16 | MN964055 | sputum | 10/11/2018 | 1 | R | 0.19 | S | 1.02 | ±0,02 | 1.22 | ±0,05 | 2.34 | ±0,04 | 2.84 | ±0,02 | MN964025 | C-T*(183) | MN964036 | No mutation | ||
Patient 7 | Invasive aspergillosis | TN-17 | MN964056 | sputum | 01/03/2017 | 0.75 | S | 0.125 | S | 0.48 | ±0,02 | 1.10 | ±0,02 | 0.77 | ±0,02 | 2.07 | ±0,06 | ND | ND | ND | ND |
TN-18 | MN964057 | BAL | 15/03/2017 | 0.5 | S | 0.125 | S | 2.18 | ±0,03 | 1.81 | ±0,02 | 2.11 | ±0,02 | 2.17 | ±0,02 | ND | ND | ND | ND | ||
TN-19 | MN964058 | sputum | 08/03/2017 | 0.75 | S | 0.125 | S | 0.92 | ±0,02 | 0.76 | ±0,02 | 0.57 | ±0,02 | 1.11 | ±0,02 | ND | ND | ND | ND | ||
TN-20 | MN964059 | sputum | 15/03/2017 | 0.38 | S | 0.064 | S | 2.27 | ±0,02 | 2.88 | ±0,05 | 0.64 | ±0,04 | 1.09 | ±0,02 | ND | ND | ND | ND | ||
Patient 8 | Invasive aspergillosis | TN-21 | MN964060 | sputum | 08/03/2017 | 0.38 | S | 0.125 | S | 0.45 | ±0,02 | 0.88 | ±0,02 | 6.87 | ±0,02 | 1.82 | ±0,02 | MN964020 | C-T*(183) | MN964031 | No mutation |
Patient 9 | Invasive aspergillosis | TN-22 | MN964061 | sputum | 01/03/2017 | 0.25 | S | 0.125 | S | 0.49 | ±0,02 | 0.55 | ±0,02 | 1.84 | ±0,02 | 1.68 | ±0,04 | ND | ND | ND | ND |
Patient 10 | Invasive aspergillosis | TN-23 | MN964062 | sputum | 15/03/2017 | 0.38 | S | 0.125 | S | 0.69 | ±0,02 | 0.95 | ±0,03 | 1.73 | ±0,03 | 1.59 | ±0,02 | MN964021 | C-T*(183) | MN964032 | No mutation |
Patient 11 | Invasive aspergillosis | TN-24 | MN964063 | sputum | 01/03/2017 | 0.38 | S | 0.125 | S | 0.51 | ±0,02 | 0.87 | ±0,02 | 1.62 | ±0,02 | 1.66 | ±0,09 | ND | ND | ND | ND |
TN-25 | MN964064 | sputum | 08/03/2017 | 0.75 | S | 0.125 | S | 0.81 | ±0,02 | 0.75 | ±0,01 | 1.62 | ±0,01 | 1.48 | ±0,02 | ND | ND | ND | ND | ||
TN-26 | MN964065 | BAL | 15/03/2017 | 0.75 | S | 0.125 | S | 2.21 | ±0,02 | 2.51 | ±0,02 | 0.67 | ±0,02 | 1.42 | ±0,02 | ND | ND | ND | ND | ||
Patient 12 | Invasive aspergillosis | TN-27 | MN964066 | BAL | 03/12/2018 | 0.25 | S | 0.125 | S | 0.65 | ±0,02 | 0.78 | ±0,04 | 5.95 | ±0,03 | 1.03 | ±0,08 | ND | ND | ND | ND |
TN-28 | MN964067 | nasal | 26/11/2018 | 0.25 | S | 0.125 | S | 1.73 | ±0,02 | 0.88 | ±0,02 | 1.95 | ±0,02 | 1.06 | ±0,02 | ND | ND | ND | ND | ||
TN-29 | MN964068 | sputum | 26/11/2018 | 0.5 | S | 0.094 | S | 2.11 | ±0,02 | 0.48 | ±0,02 | 4.39 | ±0,02 | 1.95 | ±0,02 | MN964022 | No mutation | MN964033 | No mutation | ||
Patient 13 | Invasive aspergillosis | TN-30 | MN964069 | sputum | 01/03/2017 | 0.5 | S | 0.094 | S | 2.37 | ±0,02 | 2.27 | ±0,08 | 0.68 | ±0,07 | 1.44 | ±0,03 | ND | ND | ND | ND |
TN-31 | MN964070 | lung biopsy | 17/04/2017 | 1.5 | R | 0.75 | R | 12.99 | ±0,02 | 11.32 | ±0,02 | 3.07 | ±0,04 | 2.71 | ±0,02 | MN964026 | C-T*(183) | MN964037 | A-G*(529) | ||
TN-32 | MN964071 | nasal | 01/03/2017 | 0.5 | S | 1 | R | 7.49 | ±0,02 | 3.51 | ±0,08 | 1.97 | ±0,02 | 0.77 | ±0,05 | MN964027 | C-T*(183) | MN964038 | No mutation | ||
Patient 14 | Invasive aspergillosis | TN-33 | MN964072 | nasal | 21/03/2018 | 1 | R | 0.75 | R | 16.89 | ±0,02 | 2.48 | ±0,02 | 2.57 | ±0,06 | 1.71 | ±0,02 | MN964028 | G-A*(6I6) | MN964039 | No mutation |
TN-34 | MN964073 | nasal | 28/03/2018 | 0.38 | S | 0.064 | S | 6.49 | ±0,02 | 1.52 | ±0,08 | 0.97 | ±0,02 | 0.77 | ±0,06 | ND | ND | ND | ND |
* – Punctual mutation, S – susceptible, R – resistant, VOR – voriconazole, AMB – amphotericin B, ND – not done
The crystal structures of sterol 14-alpha demethylase (CYP51B) from a pathogenic filamentous fungus
The molecular docking studies were performed using open-source software (PatchDock) (
The antifungal drug sensitivity profiles of the
Thirty-four clinical
So, the expression of
The RT-qPCR results are presented in Fig. 1 and 2 as well as in Table III. In the 28 IT/POS sensitive isolates,
Study of the level of expression of
*
Study of the level of expression of
*
The overexpression of the
The PCR sequencing showed the presence of three mutations for
The protein-coding regions of the
The sequence of the
Alignment of the DNA sequences of the
For
DNA sequences of the
Sequence alignment was performed using the Clustal Omega program for homology modeling, and open-source software (PatchDock) was used to generate the interaction between the structure of the protein and the drug automatically.
In addition, for all
Overall view of the 3D model of the
Overall view of the 3D model of the
Several antifungal drugs can be used against
The triazole antifungals present a broad spectrum for the management of invasive aspergillosis. The treatment of
Over the past two decades, many researchers have studied and identified that resistance mechanisms to azoles vary depending on the country and the studies of clinical and environmental strains of
However, there is minimal data on
Our findings showed overexpression in two IT/POS resistant
However, Sharma et al. (2018), found that
In this study, the option of overexpression in sensitive profile strains may be explained by the vast clinical use of this antifungal in our country, particularly in patients with chronic diseases. This suggests that the
Furthermore, the mechanism of azole resistance in most of the previously published studies is associated with changes that are often corresponding to mutations in the
Here, we described for
As stated previously, Krishnan-Natesan et al. (2008) were among the first to identify different mutations of
In previous studies, the crystallographic structures of the CYP51 proteins of
According to the models provided by the PatchDock software, three new mutations, a synonym and a nonsynonym in
Therefore, further examination of other resistance mechanisms is necessary, like the CCAAT-binding domain complex CBC (a heterotrimer comprising HapB, HapC, and HapE, which is a negative regulator of sterol biosynthesis directly binding the promoters of 14 ergosterol biosynthetic genes), overexpression of MFS (major facilitator superfamily) transporters or overexpression of ABC transporters (ATP binding cassette superfamily). A deeper understanding of azole resistance mechanisms will facilitate the development of new therapeutic drugs against azole-resistant strains of