Reports of Antifungal Resistance in Different Animal Species
Several studies have analyzed fungal isolates from different animals for resistance to antimycotic agents, and many of them reported surprisingly high levels of azole resistance in yeasts and molds.
In a retrospective study, Beltaire et al. (2012) analyzed fungal strains isolated from equine uterine collected between 1999 and 2011 and showed resistance rates of 19% and 2% for itraconazole and fluconazole, respectively. Cordeiro Rde et al. (2015) investigated 59 C. tropicalis isolates predominantly derived from healthy animals and found resistance to fluconazole and/or itraconazole in 50%, whereas all isolates were susceptible to caspofungin and amphotericin B. Using the same microbroth dilution assay, Brilhante et al. (2016) analyzed Candida isolates from the nasolacrimal duct of healthy horses and found that 40% of the C. tropicalis isolates were resistant to fluconazole and itraconazole (Brilhante et al. 2016). The same group also found high rates of fluconazole and itraconazole resistance also for Candida isolates from rheas and cockatiels (Sidrim et al. 2010; Brilhante et al. 2013), and efflux pumps were a major resistance mechanism (Rocha et al. 2017). Using a commercial kit covering 11 commonly used agents, Lord et al. (2010) tested 144 Candida, Cryptococcus, Rhodotorula, and Trichosporon isolates from bird feces for antifungal resistance. They reported that 45.8% of the strains were resistant to at least 4 of the 11 drugs, and 18.1% were resistant to all antifungals tested. A recent study found similar resistant levels for 111 C. glabrata isolates from the feces of sea gulls and 79 C. glabrata isolates from human patients, while other have reported only moderate azole resistance in Candida strains isolated from raptors (Brilhante et al. 2012; Al-Yasiri et al. 2016). Antifungal drug susceptibility of Cryptococcus neoformans/Cryptococcus gattii species complex isolated from dogs and cats in North America showed that C.
neoformans strains had higher MICs for flucytosine and itraconazole. However, C. gattii isolates exhibited a wider range of MICs than C. neoformans (Singer et al. 2014). Fluconazole resistance has also been reported in Cryptococcus neoformans var. grubii obtained from a case of feline cryptococcosis, due to overexpression of the ERG11 and ABC transporter (Kano et al. 2015).In another study, Cafarchia et al. determined the MIC distribution and the epidemiological cutoff values (ECVs) of 62 Malassezia pachydermatis from dogs with dermatitis and 78 M. furfur isolates from humans with bloodstream infections, using Clinical and Laboratory Standards Institute (CLSI) methodology. Overall, MIC data for azoles of M. pachydermatis were four twofold dilutions lower than those of M. furfur. Itraconazole and posaconazole displayed lower MICs than voriconazole, regardless of the Malassezia species. In addition, fluconazole resistance was detected only in M. pachydermatis isolates (Cafarchia et al. 2015). These studies indicate that resistance to certain azoles is a common phenomenon in pathogenic yeasts isolated from some animals. Strikingly, the azole resistance rates of C. albicans and C. tropicalis isolated from healthy animals are higher than those reported in some human studies (Pfaller et al. 2013; Goncalves et al. 2016). This indicates that the elevated resistance levels found in animals may not simply reflect a natural resistance of the respective species. However, differences in the methodology and breakpoints used, as well as the limited number of isolates included in several animal studies, make it difficult to directly compare data obtained for animal and human isolates.
Recent changes in the taxonomy of Aspergillus have major implications for our understanding of drug susceptibility profiles (Van Der Linden et al. 2011). New sibling species of A. fumigatus exhibit in vitro susceptibility profiles that differ significantly from that of A. fumigatus. While acquired resistance is an emerging problem in A.
fumigatus (Verweij 2007; Mellado et al. 2007; Verweij 2009a), other Aspergillus species may be intrinsically resistant to, e.g., amphotericin B and azoles (Van Der Linden et al. 2011). MICs of A. flavus clinical isolates to amphotericin B are consistently twofold dilution steps higher than those of A. fumigatus (Gomez- Lopez et al. 2003b; Garcia-Effron et al. 2003b). Using CLSI methodology (CLSI 2008), A. nidulans was shown to have MIC values of 1-2 mg/L for amphotericin B, which is higher than commonly observed with A. fumigatus (Kontoyiannis et al. 2001). Itraconazole and voriconazole cross-resistance and variable susceptibilities for caspofungin were observed in vitro against A. felis, another possibly intrinsically resistant sibling of the A. fumigatus species complex (Coelho et al. 2011; Barrs et al. 2013; Pelaez et al. 2013). In the section Usti, azoles are not active against A. calidoustus with MICs of ≥8 mg/L, while also other classes of antifungal drugs also appear less active (Varga et al. 2008). Resistance of A. terreus to amphotericin B is well known (Lass-Florl et al. 2009). Based on susceptibility to azoles, three different susceptibility patterns were distinguished in the black aspergilli (section Nigri). Some isolates showed low azole MICs, others showed high MICs, and a third group showed an uncommon paradoxical effect. However, these groups did not coincide with species boundaries, making it difficult to interpret as an intrinsic or acquired property (Alcazar-Fuoli et al. 2009).There is no evidence of emerging azole resistance among A. fumigatus isolates from dogs and cats, and topical azole therapy should be effective against most isolates (Talbot et al. 2015). However, acquired resistance to itraconazole and voriconazole has been reported for avian A. fumigatus strains obtained from domestic and wild birds in Belgium and the Netherlands (Beernaert et al. 2009a), where azole resistance is widespread both in clinical and environmental isolates (Beernaert et al.
2009b). The source of these resistant isolates is unclear. However, two of the four resistant strains were isolated from birds that received itraconazole. This is important, and a fungicide-driven route of resistance selection in A. fumigatus may have implications for the management of aspergillosis in animals. Another possibility in these birds can also be considered an indication of the presence of acquired resistance in the surrounding environment (Bromley et al. 2014). Of note, resistance to medical triazoles may be associated with resistance selection to azole fungicides in the environment (Verweij et al. 2009a, b). In humans azole-resistant Aspergillus disease can be observed in patients without previous azole therapy, indicating that hosts inhale both azole-susceptible and azole-resistant A. fumigatus conidia (ECDC Technical Report 2013).In another study, Ziolkowska et al. investigated the in vitro susceptibility of A. fumigatus strains isolates obtained from the oral cavity, lungs, and air sacs of healthy domestic geese, birds with aspergillosis, and from their environment. All of the strains were susceptible to enilconazole, itraconazole, and voriconazole, but, irrespective of source, showed various degree of resistance to miconazole, clotrimazole (MIC90 = 16 μg∕mL), and amphotericin B (MIC90 = 16 μg∕mL) (Ziolkowska et al. 2014). To assess the potential risk of azole resistance emergence in avian farms where azole compounds were used for the control of avian mycoses, a drug susceptibility study including A. fumigatus isolates from birds and avian farms was also conducted in France and Southern China (Wang et al. 2013). A total number of 175 A. fumigatus isolates were analyzed. No resistant isolate was detected, and the distribution of MICs was similar for isolates collected in farms with or without azole chemoprophylaxis. For 61 randomly selected isolates, the full coding sequence of Cyp51A gene was determined to detect mutations.
Nine amino acid alterations were found in the target enzyme, three of which were new mutations.Of note, invasive infections caused by azole-resistant A. fumigatus are challenging to treat due to the lack of therapeutic options. In humans, combination of an azole with echinocandins or lipid formulations of amphotericin B can be used, and 5-flucytosine has also been recommended to be added to other therapies in patients with central nervous system infections caused by resistant isolates (Verweij et al. 2015). However, both antifungals have limitations, including toxicities, which may prohibit their long-term use in both humans and animals. Depending on the mechanism of resistance, higher doses of certain triazoles may be attempted, and there is a recent report of the successful treatment of invasive aspergillosis caused by an A. fumigatus isolate harboring a TR46∕Y121F∕T289A mutation in a bottlenose dolphin with high-dose posaconazole (Bunskoek et al. 2017). Here, the oral solution of posaconazole was incorporated into gelatin capsules and administered with a goal of achieving trough concentrations of >3 mg∕L, which was achieved after prolonged administration and resulted in clinical improvement.
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