Tebuconazole Induced Medical Triazole-resistance of Aspergillus Fumigatus and Its Mechanisms

Author:Cui Ning

Supervisor:yu yun long


Degree Year:2018





Invasive aspergillosis(IA)is a fungal disease caused mainly by Aspergillus fumigatus,and results in a high morbidity and mortality in immunosuppressed patients.There medical triazoles,namely itraconazole,voriconazole and posaconazole are the first-line antifungal agents in the treatment and prophylaxis of aspergillosis.However,azole resistance in A.fumigatus isolates is increasingly reported with variable prevalence around the world,the source of azole resistance and mechanisms have been paid much attention.More and more studies have shown that the use of azole pesticides might be one of the sources of resistance of A.fumigatus.In this paper,the possibility and its mechanisms of induced A.fumigatus by tebuconazole were tested.1.Tebuconazole is a chiral pesticide and consists of two enantiomers with R-and S-configuration showing(-)and(+)rotation.Fungicidal activity to Botrytis cinerea of R-(-)-tebuconazole was 44 times higher than S-(+)-tebuconazole.The half-lives of tebuconazole in soil were from 32.2 d to 216.6 d.Enantioselective and rapid dissipation of tebuconazole was observed in soil HN and HJC,compared to the other soils.2.No significant difference was found in 48 h LC50 of rac-,S-and R-tebuconazole to E.fetida which were 10.78,10.84 and 10.48 μg/cm2,respectively.Enantioselective accumulation of tebuconazole in E.fetida was found with a preferential of S-(+)-tebuconazole.3.In liquid medium,a total of 10 wild type sensitive A.fumigatus were induced by enantiomers of tebuconazole.The results show that the toxicity of R-(-)-tebuconazole to A.fumigatus is higher than S-(+)-tebuconazole,the possibility of inducing triazole resistant A.fumigatus by R-(-)-tebuconazole was higher than that of S-(+)-tebuconazole.There are 5 resistant strains were induced by R-(-)-tebuconazole,but under the same conditions only one was induced by S-(+)-tebuconazole.4.The wild type sensitive A.fumigatus(S5)and a sensitive A.fumigatus with hygromycin marker gene(hph-1)were induced by tebuconazole under the filed residue concentrations.The results show that,4(S512,S552,S553 and S554)resistant strains were isolated after exposure of the strain S5 to tebuconazole at concentrations of 1 and 5 mg/1,3 resistant strains(H-5、H-8 and H-12)were isolated from hph-1 sensitive strain under the same conditions.Among the 7 resistant strains,only S554 contained mutations of TR46/Y121F/T289A in cyp51A.5.In the soil,the half-life of tebuconazole ranged from 43 d to 90 d,and tebuconazole might have an effect on triazole fungicide sensibility of A.fumigatus.With exposure of the strain hph-1 to tebuconazole at concentration of 0.5,1,2 and 5 mg/kg,1,3,2 and 3 resistant strains were isolated,respectively.The sequencing of cyp51A and its promoter region indicated that there is not any mutation in the target gene for all of the resistant strains isolated from tebuconazole treated soils.6.The resistance in HI-30 and HI-36 which were isolated from tested soils could be inherited stably to their generations,while the resistance in other four highly resistant strains(HI-35,HI-38,HI-39 and HI-41)were found to be disappeared after being transferred for 15 times.The results of RT-PCR indicated that the overexpression of target genes cyp51A,cyp51B and efflux pump AtrF,AfuMDR1 were involved in the voriconazole and itraconazole resistant HI-30,which were 5.76,2.64,1.73 and 7.58-fold more,respectively,than in the wild-type,respectively,than in the wild-type strain hph-1.The levels of constitutively expressed AtrF,AfuMDR1,AfuMDR2,cyp51A and cyp51B mRNA of strain HI-36 was 5.94,3.55,1.77,1.47 and 1.42-fold more copies of mRNA compared to the parental strain hph-1,respectively.The results of this study indicated clearly that there is a direct link between the using of agricultural triazoles and the appearance of the resistance in A.fumigatus to triazole medicals.The resistance of A.fumigatus could be induced by the applications of triazole fungicides with mutation(s)in cyp51A or overexpression of cyp51A,cyp51B,or efflux pump,respectively.