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Investigation of Azole Resistance Involving cyp51A and cyp51B Genes in Clinical Aspergillus flavus Isolates

Dhoha Ghorbel
Dhoha Ghorbel
, 
Imen Amouri
Imen Amouri
, 
Nahed Khemekhem
Nahed Khemekhem
, 
Sourour Neji
Sourour Neji
, 
Houaida Trabelsi
Houaida Trabelsi
, 
Moez Elloumi
Moez Elloumi
, 
Hayet Sellami
Hayet Sellami
, 
Fattouma Makni
Fattouma Makni
, 
Ali Ayadi
Ali Ayadi
 and 
Ines Hadrich
Ines Hadrich
   | May 03, 2024
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Article Category: Original Paper
Published Online: May 03, 2024
Page range: 131 - 142
Received: Aug 03, 2023
Accepted: Dec 03, 2023
DOI: https://doi.org/10.33073/pjm-2024-001
Keywords
© 2024 Dhoha Ghorbel et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

Study of the level of expression of cyp51A genes by relative quantification with RT-qPCR in Aspergillus flavus: 28 susceptible strains (striped bars) and 6 IT-POS resistant strains (white bars). Gene expression values are represented as bar plots with mean + SD. p-Values were calculated using the Mann-Whitney U test, p-values of statistical tests are shown within the graphs. *p < 0.005
Study of the level of expression of cyp51A genes by relative quantification with RT-qPCR in Aspergillus flavus: 28 susceptible strains (striped bars) and 6 IT-POS resistant strains (white bars). Gene expression values are represented as bar plots with mean + SD. p-Values were calculated using the Mann-Whitney U test, p-values of statistical tests are shown within the graphs. *p < 0.005

Fig. 2.

Study of the level of expression of cyp51B genes by relative quantification with RT-qPCR in Aspergillus flavus: 28 susceptible strains (striped bars) and 6 IT-POS resistant strains (white bars). Gene expression values are represented as bar plots with mean + SD. p-Values were calculated using the Mann-Whitney U test, p-values of statistical tests are shown within the graphs. *p < 0.005
Study of the level of expression of cyp51B genes by relative quantification with RT-qPCR in Aspergillus flavus: 28 susceptible strains (striped bars) and 6 IT-POS resistant strains (white bars). Gene expression values are represented as bar plots with mean + SD. p-Values were calculated using the Mann-Whitney U test, p-values of statistical tests are shown within the graphs. *p < 0.005

Fig. 3.

Alignment of the DNA sequences of the cyp51A gene fragments from Aspergillus flavus, was compared with wild type A. flavus strain (Genbank ID: XM_002375082.1).
Alignment of the DNA sequences of the cyp51A gene fragments from Aspergillus flavus, was compared with wild type A. flavus strain (Genbank ID: XM_002375082.1).

Fig. 4.

DNA sequences of the cyp51B gene fragments from Aspergillus flavus were compared with wild type A. flavus strain (Genbank ID: XM_002379089.1).
DNA sequences of the cyp51B gene fragments from Aspergillus flavus were compared with wild type A. flavus strain (Genbank ID: XM_002379089.1).

Fig. 5.

Overall view of the 3D model of the Aspergillus flavus CYP51A protein in complex with three antifungal (IT, POS and VOR). Only TN33 strain presented the structure of CYP51A with the substitution of lysine for a glycine in the position 206 of the protein.
Overall view of the 3D model of the Aspergillus flavus CYP51A protein in complex with three antifungal (IT, POS and VOR). Only TN33 strain presented the structure of CYP51A with the substitution of lysine for a glycine in the position 206 of the protein.

Fig. 6.

Overall view of the 3D model of the Aspergillus flavus CYP51B protein in complex with three antifungal (IT, POS and VOR). Only TN31 strain presented the structure of CYP51B with the substitution of glycine for a lysine in the position 177 of the protein.
Overall view of the 3D model of the Aspergillus flavus CYP51B protein in complex with three antifungal (IT, POS and VOR). Only TN31 strain presented the structure of CYP51B with the substitution of glycine for a lysine in the position 177 of the protein.

The sequences of primers and probes used in RT-qPCR.

Gene Primers and probes
cyp51A F 5’-GCGCGCATGAGGGAGAT-3’
R 5’-CAATGCATGAGGTTCCAGATCA-3’
Probe HEX-TCATTAACGAGCGCCGCAAGAACC-MGB
cyp51B 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 sequences of primers used in PCR sequencing.

Gene Primers
cyp51A 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’
cyp51B BP1F 5’-CTTTTATCGGAAGTACCATC-3’
BP1R 5’-CTTTATCAGGAACAACTTCG-3’
BP2F 5’-ACTCTTCGCATACATGCACC-3’
BP2R 5’-CCAACGTGCATGATATTGCC-3’
BP3F 5’-CGACTCGACATTTGCTGAAC-3’
BP3R 5’-CTCTTCGCATACATGCACCA-3’

Antifungal susceptibility to the azoles, relative quantification of gene expression, gene copy number of cyp51A and cyp51B genes, and mutation in Aspergillus flavus.

Strain GenBank accession Isolation site Isolation date Itraconazole Posaconazole RNA relative quantification DNA relative quantification Cyp51A Cyp51B
MIC (μg/ml) R/S MIC (μg/ml) R/S cyp51A ±SD cyp51B ± SD cyp51A ±SD cyp51B ± SD GenBank accession Punctual mutation GenBank accession Punctual mutation
Patient 1 Invasive aspergillosis TN-1 MN964040 nasal 15/10/2018 0.125 S 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
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