Effect Of Paclobutrazol On Susceptibility Of Aspergillus Fumigatus To Triazole Antifungals

Aspergillus fumigatus is the main pathogen of invasive aspergillosis(IA),and azole antifungals play an important role in the treatment of IA.However,there are more and more reports about the resistance of A.fumigatus to azole antifungals.Studies have shown that the application of triazole fungicides may be related to the prevalence of resistant A.fumigatus.Paclobutrazol is one of the triazole compounds,which can be used as plant growth regulator and fungicide.The aim of this study is to explore whether if the application of paclobutrazol could affect the sensitivity of A.fumigatus to azole antifungals in liquid medium and soil.The results could be useful for verifying the possibility of triazole fungicides for inducing resistant A.fumigatus strains.The results were as follows:(1)In the liquid medium,the sensitive A.fumigatus strain P3 labeled with hygromycin was induced by paclobutrazol,and five resistant strains(Y-1,Y-3,Y-6,Y-8,Y-9)were isolated.Among them,4 strains(Y-1,Y-3,Y-6,Y-9)showed high resistance to itraconazole(MIC≥16 mg/L),but none of them had mutations in the target gene cyp51A.(2)The half-life of paclobutrazol with the concentration of 1 mg/kg,2 mg/kg and 5 mg/kg in the soil were 31.5 days to 49.5 days.The residual paclobutrazol in the soil provided selective pressure for the changes in sensitivity against triazole drugs in A.fumigatus.There were two(S28-1,S42-1-2),three(S42-2-1,S42-2-2,S84-2),two(S42-5,S84-5-2)resistant P3 strains isolated from the soil treated with 1 mg/kg,2 mg/kg,5 mg/kg paclobutrazol,respectively.Among them,five strains(S42-1-2,S42-2-1,S42-2-2,S84-2,S84-5-2)showed high resistance to itraconazole.The sequencing results showed that none of the seven resistant strains has cyp51A mutation.(3)Susceptibility recovery experiments were conducted on nine highly itraconazole resistant strains isolated from liquid medium and soil.After fifteen transfers,among the four strains induced in liquid medium,two strains(Y-3,Y-6)recovered to the susceptibility level,and two strains(Y-1,Y-9)still maintained resistance to triazoles.Among the five strains isolated in soil,three strains(S42-1-2,S42-2-1,S42-2-2)recovered to the susceptibility level,and two strains(S84-2,S84-5-2)could stably inherit the resistance to azoles.(4)Four strains(Y-1,Y-9,S84-2,S84-5-2)which resistance could be stably inherited were performed by RT-qPCR analysis.The results showed that the resistance of strain Y-1 to itraconazole and posaconazole might be related to the overexpression of the target genes cyp51A,cyp51B and the efflux pump genes AtrF and AfuMDRl,and the expression of the four genes was 2.15,2.21,2.04 and 3.48-fold higher than that of the original sensitive strain,respectively.The expression of AtrF,AfuMDRl,AfuMDR2,cyp51A and cyp51B of the strain Y-9 were 2.88,5.49,2.43,2.50 and 2.92-fold of the original strain,respectively.The expression of AfuMD2,AfuMDR4,cyp51A and cyp51B of S84-2 strain were 1.58,7.24,2.16 and 2.11-fold of the original strain,respectively.The expression of AfuMDRl,AfuMDR4,cyp51A and cyp51B of S84-5-2 were 5.84,3.15,2.17 and 2.50-fold of the original strain,respectively.These results showed that the use of paclobutrazol can induce cross-resistance in A.fumigatus to azole antifungals,and its resistance is related to the overexpression of cyp51A,cyp51B and efflux pump related genes.The application of paclobutrazol in fields may lead to increasing risk of resistance in A.fumigatus.