Resistance Mechanisms and Clinical Features of Fluconazole-Nonsusceptible Candida tropicalis Isolates Compared with Fluconazole-Less-Susceptible Isolates

Abstract

eWe investigated the azole resistance mechanisms and clinical features of fluconazole-nonsusceptible (FNS) isolates of Candida tropicalis recovered from Korean surveillance cultures in comparison with fluconazole-less-susceptible (FLS) isolates. Thirtyfive clinical isolates of C. tropicalis, comprising 9 FNS (fluconazole MIC, 4 to 64 g/ml), 12 FLS (MIC, 1 to 2 g/ml), and 14 control (MIC, 0.125 to 0.5 g/ml) isolates, were assessed. CDR1, MDR1, and ERG11 expression was quantified, and the ERG11 and UPC2 genes were sequenced. Clinical features of 16 patients with FNS or FLS bloodstream isolates were analyzed. Both FNS and FLS isolates had >10-fold higher mean expression levels of CDR1, MDR1, and ERG11 genes than control isolates (P values of <0.02 for all). When FNS and FLS isolates were compared, FNS isolates had 3.4-fold higher mean ERG11 expression levels than FLS isolates (P ‫؍‬ 0.004), but there were no differences in those of CDR1 or MDR1. Of all 35 isolates, 4 (2 FNS and 2 FLS) and 28 (8 FNS, 11 FLS, and 9 control) isolates exhibited amino acid substitutions in Erg11p and Upc2p, respectively. Both FNS and FLS bloodstream isolates were associated with azole therapeutic failure (3/4 versus 4/7) or uncleared fungemia (4/6 versus 4/10), but FNS isolates were identified more frequently from patients with previous azole exposure (6/6 versus 3/10; P ‫؍‬ 0.011) and immunosuppression (6/6 versus 3/10; P ‫؍‬ 0.011). These results reveal that the majority of FNS C. tropicalis isolates show overexpression of CDR1, MDR1, and ERG11 genes, and fungemia develops after azole exposure in patients with immunosuppression. Candida tropicalis has become an important cause of bloodstream infections (BSIs) in seriously ill patients (1-3). Although C. tropicalis is usually susceptible to azole antifungals, 7 to 40% of clinical isolates have recently been reported to be resistant to azoles, particularly fluconazole (2-5). The mechanisms responsible for acquired azole resistance are well-characterized in Candida albicans and include mutations in the ERG11 gene, which encodes the drug target enzyme (lanosterol 14␣-demethylase), overexpression of ERG11, and overexpression of genes encoding efflux pumps (6). In C. tropicalis, overexpression of ERG11 associated with missense mutations has been described as the most frequent azole resistance mechanism in clinical isolates (7,8). However, the azole resistance mechanisms of C. tropicalis remain a matter of debate, and only five studies have been reported to date (7-11). In addition, there is a lack of substantial research on mutations in the transcription factor Upc2p of C. tropicalis, which can induce ERG11 overexpression and contribute to the development of fluconazole resistance in C. albicans (12)(13)(14).Fluconazole MIC distributions of wild-type C. tropicalis ranged from 0.125 to 64 g/ml following 24 h of incubation using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) methods, and their modal MIC values were 0.125 to 0.5 g/ml (6). Recently, the CLSI es...

Azole Antifungal Resistance in Candida albicans and Emerging Non-albicans Candida Species

“…Moreover, ERG11 expression was even higher among a subset of fluconazole-resistant isolates also resistant to itraconazole and voriconazole. These results were recently echoed by a similar study characterizing 35 C. tropicalis isolates from Korean university hospitals, nine of which were fluconazole-non-susceptible (Choi et al, 2016). While considerable variability in ERG11 expression (~150-fold) was observed in the highly fluconazole-susceptible group, ERG11 expression was significantly higher among both less fluconazole-susceptible (MIC 1–2 μg/ml) and fluconazole-non-susceptible (MIC ≥ 4 μg/ml) isolates.…”

“…Molecular characterization of azole-resistant clinical C. tropicalis isolates has also revealed alterations in the ergosterol biosynthetic pathway (Vandeputte et al, 2005; Eddouzi et al, 2013; Jiang et al, 2013; Choi et al, 2016). A fluconazole-resistant C. tropicalis isolate recovered from a clinical blood specimen from Tunisia was found to have mutations in both ERG3 and ERG11 which were individually observed to be detrimental to ergosterol biosynthesis when heterologously expressed in S. cerevisiae (Eddouzi et al, 2013).…”

Implications of the EUCAST Trailing Phenomenon in Candida tropicalis for the In Vivo Susceptibility in Invertebrate and Murine Models

“…Methodological issues, including pH value and incubation time, were shown to impact the degree of resistance (15)(16)(17), and some authors report more trailing with the EUCAST method than with CLSI at 24 h (22% versus 1.7%) (18). In Candida albicans, variable regulation of drug resistance genes was observed for isolates trailing by the 48-h CLSI method (19), and various drug resistance mechanisms have been implicated in azole resistance in C. tropicalis (20)(21)(22). Yet, it is unknown if trailing is related to the variable regulation of epigenetic traits or resistance mechanisms.…”

Synergistic Interaction of Fluconazole/Amphotericin B on Inhibition of Enzymes Contributes to the Pathogenesis of Candida Tropicalis

“…Significant up-regulation of SKN1 expression and to a lesser extent KRE1 was observed in Candida biofilms treated with amphotericin B alone or in combination. Choi et al 38 revealed up-regulation of CDR1, MDR1, and ERG11 genes in fluconazole-nonsusceptible C. tropicalis isolates. Fernandes et al 39 showed that C. tropicalis resistance to voriconazole is unable to control biofilms, and the up-regulation of ERG genes is likely to be probable molecular mechanism of Candida biofilm resistance.…”