Both human and agricultural diseases are treated with azole antifungals. These compounds target 14 sterol demethylase and interfere with ergosterol production in the fungus (Yoshida & Aoyama, 1984, 1987; Wright et al., 1990). Recent testing of A. fumigatus clinical isolates has identified cases of azole resistance (Sanglard et al., 1995, 1997; Kelly et al., 1997; Nolte et al., 1997; MG-132 mouse Franz et al.,

1998; Gupta et al., 1998; Schaller et al., 1998; Marichal et al., 1999; Calabrese et al., 2000; Moore et al., 2000; Yang & Lo, 2001; Verweij et al., 2002; Gomez-Lopez et al., 2003; Drago et al., 2004; Hsueh et al., 2005; Pfaller et al., 2006) some of which have been shown to be resistant to treatment with itraconazole (ITR) in murine models of infection (Denning et al., 1997a; Dannaoui et al., 1999, 2001). Long-term

treatment for patients with ITR (Chryssanthou, 1997; Denning et al., 1997a; Dannaoui GSK3235025 et al., 2001; Warris et al., 2002; Chen et al., 2005) appears to lead to azole resistance in infecting fungi, although these cases were not always associated with ITR treatment failure. Two per cent of 913 A. fumigatus isolates in the literature published before 2000 were found to be resistant (Dannaoui et al., 2001). Recent surveys have reported frequencies ranging from 1% to 6% (total number of isolates surveyed was 357; Verweij et al., 2002; Gomez-Lopez et al., 2003; Drago et al., 2004; Hsueh et al., 2005), although other surveys have reported no resistant isolates out of a total of 2100 isolates (Verweij et al., 1998; Chandrasekar et al., 2001; Diekema et al., 2003; Balajee et al., 2004; Dannaoui et al., 2004; Kauffmann-Lacroix

et al., 2004; Guinea et al., 2005; Pfaller et al., 2005). Recent estimates from our laboratory suggest higher levels of azole resistance (Howard et al., 2009) and similar studies in the Netherlands have also shown high levels of azole resistance. A number of mutations in the cyp51A lanosterol 14α-demethylase gene have been associated with azole resistance (Denning et al., 1997a; Diaz-Guerra et al., 2003, 2004; Ferreira et al., 2004; Mellado et al., 2004, 2005; Chen et al., 2005). Other mechanisms for azole resistance include before increased expression of efflux pumps. The increased expression of an ATP-binding cassette (ABC) transporter (AtrF) in the presence of ITR has been shown in a clinical isolate with reduced drug accumulation (Slaven et al., 2002), and possible transporters have been implicated in azole resistance of laboratory selected mutants (Denning et al., 1997a; Nascimento et al., 2003; Ferreira et al., 2004). Restriction enzyme-mediated integration (REMI) was employed, as it increases the transformation efficiency and promotes single-copy, non-rearranged integrations of the transforming DNA (Lu et al., 1994; Bolker et al., 1995; Brown et al., 1998; de Souza et al., 2000).