| Aspergillus fumigatus is a pathogenic fungus that can infect immunodeficiency people and cause invasive aspergillosis with high morbidity and mortality.Since the first isolation of resistant A.fumigatus in 1997,the problem of triazole-resistance in A.fumigatus has become increasingly serious.The origin of triazole-resistance in A.fumigatus was initially thought to be attributed to the application of triazole medicines in clinical.However,resistant strains were frequently isolated from both triazole naive patients and field samples where triazole fungicides had been used.Therefore,many studies proposed that triazole-resistance in A.fumigatus was associated with the use of triazole fungicides in the field.Difenoconazole and propiconazole,which have a similar structure with antifungal triazole medicines,are wide applicated.There are many preparations for the combination of these 2 fungicides.The possibility of inducing triazole-resistance in A.fumigatus was conducted in both liquid medium and soil.The aim of this study was to determine the relationship between the resistance in A.fumigatus and the residual levels of propiconazole,difenoconazole and their combinations after application.The results are shown as follows:(1)Standard difenoconazole(SDFC),standard propiconazole(SPPC),formulated difenoconazole(FDFC),formulated propiconazole(FPPC),a combination of SDFC and SPPC(S-CDP),and a combination of FDFC and FPPC(F-CDP)were set as 6 different substrates,while 4 wild-type susceptible A.fumigatus(S17,S29,S82,and SX)and a susceptible strain marked with hygromycin resistance gene(P3)were selected as parental strains.The concentration of each substrate was gradually increased in Sabouraud's dextrose broth medium to induce these 6 susceptible strains.The results show that all 6 different substrates could induce the cross-resistance against triazole fungicides and triazole medicines in parental strains,and 4,6,6,5,5,and 9 resistant strains were induced by SPPC,SDFC,FPPC,FDFC,S-CDP,and F-CDP,respectively.Among the 35 induced resistant strains,the 6 strains S17-4,SX-15,S82-23,SX-26,S17-25,and P3-26 harbored with H147Y,D430N,I246M,G138S,G138D,and M263I mutations in cyp51A gene,respectively,while the remaining 29 resistant strains had no cyp51A mutations.(2)The correlation between the 5 new mutations of H147Y,D430N,I246M,G138D,and M263I in cyp51A and triazole-resistance in A.fumigatus was explored.The results show that H147Y and M263I mutations were responsible to VRC-,ITZ-,and POS-resistance in A.fumigatus,I246M mutation was related to ITZ-resistance,but G138D and D430N mutations could not cause triazole-resistance in A.fumigatus.(3)Hereditary stability of induced triazole-resistance in A.fumigatus was studied.The results show that after the subculture of 31 resistant strains for 15 generations,there were 8 strains(S17-2,SX-15,SX-16,P3-6,S82-24,S17-25,S29-24,and S29-22)retained resistance against at least one medical triazole,and the remaining 23 induced strains recovered to sensitivity or intermediate level to medical triazoles.The RT-qPCR was conducted for the strains(SX-16,S29-22,and S82-24)without mutations but retaining high ITZ-resistance and 2 strains(SX-15 and S17-25)harboring nonsense mutations.The results show that the expression levels of cyp51A and AfuMDR4 genes in the 5 resistant strains were 3.21?8.35 and 2.35?10.20 times higher compared to their parental strains,respectively.The level of AtrF gene in resistant strains SX-15 and SX-16,the AfuMDRl gene in strains SX-16 and S82-24,and the cyp51B gene in the strain S17-25 were 2.25?3.56,3.88?5.01,and 2.84 times higher than their original strains,respectively.(4)FPPC,FDFC,and F-CDP were conducted to induce triazole-resistance in A.fumigatus with 5 residual concentrations(0.004,0.02,0.1,0.5,and 2.5 mg/L).The results show that 0.004 and 0.02 mg/L of triazole fungicides had no effect on the growth of the strain P3,while 0.1,0.5,and 2.5 mg/L of triazole fungicides could inhibit mycelial growth and sporulation ability of P3 with different degrees.At the concentration of 2.5 mg/L,2 resistant strains were isolated in FPPC treatment,while 1,1,and 2 resistant strains were isolated under the selective pressure of 0.1,0.5,and 2.5 mg/L of FDFC,respectively.In addition,2 and 5 resistant strains were induced by 0.5 and 2.5 mg/L of F-CDP,respectively.These induced strains showed cross-resistance to triazole fungicides and triazole drugs,but there were no mutations in their cyp51A gene.(5)The soil was treated with FPPC,FDFC,and F-CDP at a total concentration of 2 mg/kg to study the effects of triazole fungicides on the sensitivity of the parental strain P3 in soil-earthworm system.During the 56-day assay,propiconazole and difenoconazole residues were detected from soil and earthworms in each treatment.Meanwhile,1,3,and 2 triazole-resistant A.fumigatus strains were isolated from the soil samples in FPPC,FDFC,and F-CDP treatment,respectively.The earthworm guts from FDFC and F-CDP treatment were detected with 2 and 2 triazole resistant strains,respectively,but no resistant strains were isolated from earthworm guts in FPPC treatment.All these 10 resistant strains had no mutations in cyp51A gene.After the subculture of strains with high ITZ-resistance,3 strains still maintained triazole-resistance in their generations attributed to the overexpression of cyp51A,AfuMDR1,and AtrF genes.The results of this study show that the fungicide residues after the application of propiconazole,difenoconazole and their combinations could induce triazole-resistance in A.fumigatus in soil,and resistant strains could be isolated from earthworm guts.This indicates the use of triazole fungicides such as propiconazole and difenoconazole has the risk to causing resistance in human pathogen A.fumigatus. |
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