Determination of the presence of azole resistant Aspergillus fumigatus isolates in composters of horticultural plantations and organic farms

Plesni rodu Aspergillus so prisotne povsod v okolju. Posamezniki običajno vdihnejo stotine konidijev na dan. Pri večini ljudi so obrambni sistemi sposobni zlahka odstraniti glive iz pljuč. Če imunsko oslabljeni ljudje vdihnejo konidije vrste Aspergillus fumigatus, lahko pride do invazivne aspergiloze. Zdravila, ki se najpogosteje uporabljajo za zdravljenje aspergiloze, so azoli. Zaradi pogoste rabe azolov v kmetijstvu prihaja v naravi do večjega števila odpornih izolatov A. fumigatus proti azolom. Cilj magistrske naloge je bil opredeliti prevalenco proti azolom odpornih izolatov A. fumigatus v vzorcih zemlje iz področja ekoloških kmetij in hortikulturnih nasadov. Seve A. fumigatus sensu lato smo osamili na osnovnih mikoloških gojiščih. Občutljivost proti azolom smo opredelili s presejalnim testom VIPcheckTM. Odpornim sevom smo skušali molekularno dokazati prisotnost najpogosteje opisane mutacije TR34/L98H. Testirali smo 153 različnih vzorcev, od tega smo A. fumigatus izolirali iz 103 vzorcev. Od teh 103 vzorcev, smo s presejalno metodo VIPcheckTM našli tri izolate odporne proti azolom (dva proti itrakonazolu in enega proti vorikonazolu). Vsi trije izolati so izvirali iz Mozirskega gaja. Vsem trem domnevno odpornim izolatom smo testirali občutljivost z gradient difuzijsko metodo. Iz podatkov minimalnih inhibitornih koncentracij smo ugotovili, da je samo en od treh izolatov sodil med odporne, saj je izkazal visoke vrednosti zlasti za itrakonazol in tudi za posakonazol. Vse tri izolate smo opredelili tudi z molekularno metodo (PCR v realnem času). Izolat, ki je z gradient difuzijsko metodo izkazoval odpornost proti azolom nosi mutacijo na cyp51A genu, TR34/L98H.Molds of genus Aspergillus is ubiquitous in the environment. Individuals typically inhale hundreds of conidia per day. For the vast majority of people, their defense systems are able to easily remove fungi from lungs. If immunocompromised people inhale the conidia of Aspergillus fumigatus, invasive aspergillosis may occur. The most commonly used drugs to treat aspergillosis are azoles. Owing to the frequent use of azoles in agriculture, more resistant A. fumigatus isolates against azoles occur in nature. The aim of my master\u27s thesis is to determine the prevalence of azole-resistant A. fumigatus isolates in soil samples from organic farms and horticultural plantations. A. fumigatus sensu lato strains were isolated on basic mycological media. Sensitivity to azoles was determined by VIPcheckTM screening test. We were sought to molecularly demonstrate the presence of the TR34/L98H mutation on resistant strains. 153 different samples were tested, of which A. fumigatus was isolated from 103 samples. Of this 103 samples, we found 3 resistant isolates using the VIPcheckTM method (two against itraconazole and one against voriconazole). All three isolates came from horticultural plantation Mozirski gaj. All three supposedly resistant isolates were tested for sensitivity using a gradient diffusion method to obtain data on the minimum inhibitory concentration. With this method, only one sample proved to be resistant to itraconazole and posaconazole. We tested all three samples with a molecular methode (a real time PCR). An isolate, that showed azole resistance by a gradient diffusion method carries a mutation in the cyp51A gene, TR34/L98H