Project Number: 2030-42000-039-11-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jul 1, 2018
End Date: Jul 29, 2019
Control of fungal pathogens is increasingly problematic due to the small number of effective drugs or fungicides available for treatment. Moreover, the increased incidences of fungal resistance to a class of azoles, commonly used fungicides, make this problem a global human health issue. Certain azole fungicides such as propiconazole or tebuconazole that are applied to agricultural fields have the same mode of antifungal action as clinical azole drugs. Such long-term application of azole fungicides to fields could provide selection pressure for the emergence of pan-azole-resistant strains such as the A. fumigatus TR34/L98H mutant. While first identified in European countries, the A. fumigatus TR34 L98H mutant have recently been detected in the United States. To better understand the prevalence of azole-resistant A. fumigatus at the human-animal-environment interface, we will examine triazole resistance and resistance mechanisms of A. fumigatus and also aflatoxin-producing Aspergillus flavus in California farms where azole fungicides are routinely applied for crop protection.
Environmental samples: Soil samples will be collected from different environments (agricultural farms) in California (two different time points: pre- and post-harvest time points). Types of registered and experimental azoles (DMI inhibitors) used on deciduous tree fruit, nut (almond, etc.), strawberry, and vine crops in California include difenoconazole, fenbuconazole, flutriafol, metconazole, myclobutanil, propiconazole, tebuconazole, tetraconazole and triadimefon [https://cloudfront.escholarship.org/dist/prd/content/qt05b5z3vs/qt05b5z3vs.pdf?t=lmwp9p]. For example, the California statewide integrated pest management guidelines recommends the use of tebuconazole, propiconazole or tebuconazole in onion/garlic, celery or peach farms, respectively. Soil samples will be collected from at least four different crop fields during pre- and post-harvest seasons and at animal farm environments. Sample analysis for azole resistance: Soil samples will be positively screened for A. fumigatus/A. flavus itraconazole (ITZ) resistance on Dextrose Sabouraud agar plate incorporated with ITZ at 4 µg/ml (Sab-ITZ). Soil samples will be suspended in a solution (8 mL; sterile water, 1 % tween 20, 0.5 g/L chloramphenicol). Each sample will be thoroughly mixed using vortex (1 min), and then sedimented (3 hours, room temperature). One hundred µl supernatant from the sedimented solution will be placed on two Sabouraud dextrose agar plates (without ITZ) and also on two Sab-ITZ plates. These plates will be incubated at 37°C for 72 hours, and colony appearance will be monitored. Colony identification will include colony characterization, microscopic examination, and DNA sequence analysis of 26S to 28S rDNA and internal transcribed regions. Colonies growing on Sab-ITZ will be screened further for resistance using the VIP-check test, which consists of a 4-well agar plate supplemented with 3 azole drugs (Voriconazole at 2 µg/ml, Itraconazole at 4 µg/ml and Posaconazole at 0.5 µg/ml) and a growth control. The plates will be incubated at 37°C for 72 hours. A. fumigatus isolates with a growth on Sab-ITZ and the VIP-check test will be examined for their susceptibility profiles. The susceptibility/resistance of A. fumigatus/A. flavus isolates to azoles will also be performed according to the CLSI M38-A2 protocol outlined by the Clinical Laboratory Standards Institute (CLSI) as performed previously. The minimum inhibitory concentration (MIC) will be defined as the azole concentration at which no visual growth is detected. Comparative analysis for azole susceptibility/resistance between A. fumigatus and A. flavus: In California, A. flavus is also a serious foodborne fungal pathogen, which produces toxic secondary metabolites such as aflatoxin in contaminating tree nus or various food sources, imposing serious health concerns such as hepatocarcinogenesis, suppression of immune systems, inhibition of embryo and fetal development, etc. During soil sampling, environmental A. flavus will also be collected, and azole susceptibility/resistance will be compared between A. fumigatus and A. flavus.