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

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

Table II

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’

Table III

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
		Strain	GenBank accession	Isolation site	Isolation date	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
Patient 3	Invasive aspergillosis	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
Patient 4	Invasive aspergillosis	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
Patient 5	Invasive aspergillosis	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
Patient 14	Invasive aspergillosis	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

1 * – Punctual mutation, S – susceptible, R – resistant, VOR – voriconazole, AMB – amphotericin B, ND – not done

Study of the level of expression of *cyp*51A 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 *cyp*51B 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

Alignment of the DNA sequences of the *cyp*51A gene fragments from *Aspergillus flavus*, was compared with wild type *A. flavus* strain (Genbank ID: XM_002375082.1).

DNA sequences of the *cyp*51B gene fragments from *Aspergillus flavus* were compared with wild type *A. flavus* strain (Genbank ID: XM_002379089.1).

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* 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.

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Articles in this issue

DOI: https://doi.org/10.33073/pjm-2024-001 | Journal eISSN: 2544-4646 | Journal ISSN: 1733-1331

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Language: English

Page range: 131 - 142

Submitted on: Aug 3, 2023

Accepted on: Dec 3, 2023

Published on: May 3, 2024

Published by: Polish Society of Microbiologists

In partnership with: Paradigm Publishing Services

Publication frequency: 4 issues per year

Keywords:

Aspergillus flavus,

antifungal susceptibility,

mechanisms of resistance,

cyp51A,

Related subjects:

Microbiology and virology

© 2024 Dhoha Ghorbel, Imen Amouri, Nahed Khemekhem, Sourour Neji, Houaida Trabelsi, Moez Elloumi, Hayet Sellami, Fattouma Makni, Ali Ayadi, Ines Hadrich, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Volume 73 (2024): Issue 2 (June 2024)

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

Figures & Tables

Table I

Table II

Table III

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

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