The aim of our study was to investigate whether Prochloraz (PCZ), an azole extensively used in agriculture, could be associated with the development of cross-resistance to clinical azoles among A. fumigatus. Results and discussion The three isolates developed a progressive increment of PCZ minimal inhibitory concentrations (MIC) value. In addition, 17DMAG cost a concomitant increase of the MIC of VRC, POS and Itraconazole (ITZ) was also observed (Table 1). During the induction assay, MIC of PCZ increased 256 times from day 0 until day 30. Concerning the clinical azoles, cross-resistance was developed since all isolates changed from a susceptible to a resistant phenotype, according

to Meletiadis and colleagues [12]. Table 1 Susceptibility pattern of tested A. fumigatus isolates to Prochloraz and clinical azoles A. fumigatus isolate

Time of exposure (days)   MIC (mg/L) PCZ VRC POS ITZ FLC LMF05 0 0.125 0.125 0.25 2 >64 10 0.25 0.25 0.5 2 >64 20 8 2 1 4 >64 30 32 8 2 8 >64   Ø30 32 2 2 2 >64 LMF11 0 0.125 0.25 0.125 0.5 >64 10 0.125 2 0.25 1 >64 20 8 8 1 2 >64 30 32 >16 4 4 >64   Ø30 32 2 1 0.5 >64 LMN60 0 0.25 0.25 0.125 0.25 >64 10 4 8 0.25 1 >64 20 8 8 0.5 2 >64 30 64 >16 4 4 >64   Ø30 64 2 1 0.25 >64 PCZ, Prochloraz; VRC, Voriconazole; POS, Posaconazole; ACY-241 nmr ITZ, Itraconazole; FLC, Fluconazole; Ø, MIC after 30 days of culture Demeclocycline in the absence of PCZ. There are several studies that have characterized azole resistance in A. fumigatus, and most recently some addressed the possible cross-resistance between environmental and medical azoles [8–11]. Our study demonstrated the time frame between the introduction of a widely used agricultural antifungal and the emergence of cross-resistance to medical triazoles.

During the induction assay, we found that besides the emergence of cross-resistance, PCZ exposure caused marked morphological colony changes, both macroscopically and microscopically. Macroscopic modification of the pigmentation of A. fumigatus colonies, changing from the original green colour to white (Figure 1A, B and C) was remarkable at the beginning of the assay. With the increase of MIC values of PCZ the colonies became scarcer, GW 572016 smaller and totally white (Figure 1C). Microscopic examination showed a progressive absence of conidiation: the original strain (Figure 1A) showed normal microscopic features regarding conidiation (Figure 2A) while almost white colonies (Figure 1B) showed nearly complete absence of conidiation (Figure 2B). The totally white mycelia (Figure 1C) corresponded solely to hyphae and immature little conidiophore structures without conidia (Figure 2C). These changes in pigmentation and in conidiation as a consequence of exposure to azoles have already been reported.