Resistance And Its Mechanisms Of Aspergillus Fumigatus To Triazole Antifungal Agents Induced By Tebuconazole In Soil

Triazole resistance of Aspergillus fumigatus is the main reason for the failure of clinical treatment of invasive aspergillosis(IA).The sources of azole resistance have gained wide attention.The aim of this study is to explore the potential relationship between the usage of triazole fungicides in agriculture and the prevalence of azoleresistant A.fumigatus(ARAF).The study investigated whether the resistant A.fumigatus and its associated mechanisms exists in hospital surroundings and if the application of tebuconazole could induce cross-resistance in A.fumigatus against triazole drugs.In addition,we attempted to establish the relationship between residual levels of azole fungicides and the prevalence of ARAFs in agricultural soil through an unprecedented,large-scale survey.The results were as follows:(1)A total of 134 strains of A.fumigatus were isolated from 64 soil samples collected from the surroundings of hospitals in 2018.Among them,there were 10.4%(10/134)strains identified as azole resistant.The pan-azole resistant mutations TR46/Y121F/T289 A,TR34/L98H/S297T/F495 I and TR34/L98 H were found in 4,3and 3 strains,respectively.The triazole resistance of non-mutant strains H-237 and H-258 may be related to the overexpression of target gene cyp51 and efflux pump genes Afu MDR3 and Afu MDR4.By comparing the prevalence of ARAF collected in 2014 to2018,it was found that the proportion of ARAF in soil around hospitals in Hangzhou increased by 1.2-fold in five years.And the mutation mechanism of ARAF was also increasing year by year.(2)The hygromycin-labeled wild-type A.fumigatus(H-2002-2)was induced by tebuconazole in soil.And the effect of planting environments(Open environment,simulated open field,O;Closed,simulated greenhouse,SG),application interval(7 and14 d),and application dose(Recommended dose,R;Double recommended dose,DR)on the development of ARAF was explored under laboratory-simulated conditions.A total of 11 resistant strains of H-2002-2 were induced by tebuconazole,and there were2,4,2 and 3 resistant strains obtained from treatments of DRO-7,DRSG-7,DRO-14,and DRSG-14,respectively.No mutations existed in the cyp51A gene in all the ARAF strains.The 11 ARAF strains are all from the double dose treatments,suggesting that higher concentrations of tebuconazole lead to higher selective pressures once resistance mutations have arisen.Compared with open field conditions,application of tebuconazole in greenhouses may be more conducive to the development of ARAF.(3)Of the 11 resistant strains isolated from tebuconazole-contaminated soil,two strains(HP-139 and HP-155)remained resistant to triazole drugs after fifteen transfers.RT-q PCR results indicated that the resistance in HP-139 and HP-155 to triazole drugs may be related to the overexpression of cyp51 A,Afu MDR2,Afu MDR3,and Afu MDR4 genes.The remaining 9 strains recovered to the sensitivity level after being transferred for 15 times.(4)The effect of tebuconazole applied in traditional way(1.5-fold the recommended dose,with an interval of 14 d)on the sensitivity of A.fumigatus in the soil-crop system under open field and greenhouse conditions was explored.A total of18 ARAF strains were isolated from soil and leaves after treatment with tebuconazole.Out of these,4,2,2 and 2 ARAF were isolated from the tomato-plant soil,cherry tomato-plant soil,tomato leaves and cherry tomato leaf in open fields,respectively;There were 6 and 2 ARAF strains isolated from tomato-plant soil and cherry tomatoplant soil in greenhouses,respectively.The cyp51 A amplification results showed that there were three cyp51 A mutations in 8 strains,namely TR34/L98 H,TR34/L98H/S297T/F495 I and T289 A.There seem to be more ARAF strains isolated from soil in greenhouse than that in open field,which is consistent with the results observed in laboratory-simulation tests.The above results indicate that the use of tebuconazole,especially the use of tebuconazole under greenhouse conditions,promotes the evolution of ARAF.(5)A total of 1772 paddy soils after the harvest of rice grains was collected from the fields spanning 6 provinces in eastern China,and the potential link between residual concentration of the five representative azole fungicides(tebuconazole,propiconazole,difenoconazole,hexaconazole,and prochloraz)and prevalence of ARAF was analyzed.The results showed that 67.3%,15.2%,12.3%,2.9%,1.5%,0.4%,and 0.3% of the soil samples contained these five fungicides of 0?100,100-200,200-400,400?600,600?800,800?1000,and >1000 ng/g,respectively.The fractions of samples containing ARAF isolates were 2.4%,5.1%,6.4%,7.7%,7.4%,14.3% and 20.0% of the samples with total azole fungicide residues of 0?100,100?200,200?400,400?600,600?800,800?1000,and >1000 ng/g,respectively.The Poisson analysis indicated that prevalence of ARAF is significantly(P<0.0001)increased with azole fungicide residues in the fields.