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. 2015 Dec 31;60(1):229–238. doi: 10.1128/AAC.02157-15

Activity of Isavuconazole and Other Azoles against Candida Clinical Isolates and Yeast Model Systems with Known Azole Resistance Mechanisms

Dominique Sanglard 1,✉, Alix T Coste 1
PMCID: PMC4704203  PMID: 26482310

Abstract

Isavuconazole is a novel, broad-spectrum, antifungal azole. In order to evaluate its interactions with known azole resistance mechanisms, isavuconazole susceptibility among different yeast models and clinical isolates expressing characterized azole resistance mechanisms was tested and compared to those of fluconazole, itraconazole, posaconazole, and voriconazole. Saccharomyces cerevisiae expressing the Candida albicans and C. glabrata ATP binding cassette (ABC) transporters (CDR1, CDR2, and CgCDR1), major facilitator (MDR1), and lanosterol 14-α-sterol-demethylase (ERG11) alleles with mutations were used. In addition, pairs of C. albicans and C. glabrata strains from matched clinical isolates with known azole resistance mechanisms were investigated. The expression of ABC transporters increased all azole MICs, suggesting that all azoles tested were substrates of ABC transporters. The expression of MDR1 did not increase posaconazole, itraconazole, and isavuconazole MICs. Relative increases of azole MICs (from 4- to 32-fold) were observed for fluconazole, voriconazole, and isavuconazole when at least two mutations were present in the same ERG11 allele. Upon MIC testing of azoles with clinical C. albicans and C. glabrata isolates with known resistance mechanisms, the MIC90s of C. albicans for fluconazole, voriconazole, itraconazole, posaconazole, and isavuconazole were 128, 2, 1, 0.5, and 2 μg/ml, respectively, while in C. glabrata they were 128, 2, 4, 4, and 16 μg/ml, respectively. In conclusion, the effects of azole resistance mechanisms on isavuconazole did not differ significantly from those of other azoles. Resistance mechanisms in yeasts involving ABC transporters and ERG11 decreased the activity of isavuconazole, while MDR1 had limited effect.

INTRODUCTION

Invasive fungal infections (IFIs) caused by yeasts are an important cause of morbidity and mortality. Candida spp. are opportunistic fungal pathogens and a particular cause for concern in immunocompromised individuals (1). Candida infections may account for more than 70% of all IFIs (2). Candidemia is a significant problem in the intensive care unit, as it is associated with a crude mortality rate of 42.6% and high resource use (3).

Azoles constitute a class of drug commonly used for treating infections caused by Candida spp., and the Infectious Diseases Society of America recommends them as a primary treatment for candidemia in nonneutropenic patients (1). They are fungistatic in most cases and work by binding and inhibiting the enzyme lanosterol 14-α-sterol-demethylase (encoded by ERG11), which is involved in converting lanosterol into ergosterol (4, 5). As ergosterol is an essential component of the fungal cell membrane, disruption of its biosynthesis inhibits fungal growth.

Repeated exposure to azoles and drug pressure in Candida spp. can lead to the emergence of azole-resistant strains, thereby increasing the risk of treatment failure and breakthrough infections (6, 7). Fluconazole resistance is common in many isolates of Candida spp., but they can remain susceptible to other azoles; for example, C. krusei has intrinsic resistance to fluconazole but is susceptible to voriconazole (8).

Different mechanisms of azole resistance exist, and more than one mechanism can be present in azole-resistant strains (9, 10). A common mechanism of resistance is the increased expression of multiple drug resistance genes, which result in the overexpression of efflux pumps, consequently decreasing the drug concentration within the fungal cell (11–13).

ATP binding cassette (ABC) transporter overexpression is caused by the presence of gain-of-function (GOF) mutations in the transcriptional activator of CDR genes, TAC1 (14), while the overexpression of the major facilitator superfamily (MFS) transporter MDR1 involves GOF mutations in the multidrug resistance regulator gene MRR1 (15). Point mutations in ERG11 are also an important resistance mechanism, reducing the ability of azoles to interact with or bind at the enzyme's target site, thereby reducing the effectiveness of the drug (9, 16). ERG11 upregulation also can occur via mechanisms that include GOF mutations in transcription factors such as UPC2 (17), thereby decreasing the ability of azoles to inhibit its action.

Isavuconazonium sulfate is a water-soluble antifungal prodrug that is hydrolyzed rapidly into isavuconazole, an active triazole. The oral and intravenous (i.v.) formulations of the prodrug have been approved by the U.S. Food and Drug Administration for treating adults with invasive aspergillosis and invasive mucormycosis (18). In phase 2 studies, isavuconazonium was observed to have efficacy and safety comparable to that of once-daily fluconazole in the primary treatment of uncomplicated esophageal candidiasis (19). In vitro, isavuconazole is highly active against bloodstream isolates of Candida spp. and has demonstrated good efficacy in animal models of candidiasis (20–22). However, it is important to understand how isavuconazole interacts with known mechanisms of azole resistance and what patterns of cross-resistance it shares with other azoles. In this study, we assessed the possible role of isavuconazole as a substrate for yeast multidrug efflux transporters. We also evaluated the susceptibility profile of isavuconazole when cytochrome P450 proteins encoded by different ERG11 alleles are expressed in a heterologous yeast host. In addition, isavuconazole susceptibility of clinical strains of Candida spp. with different azole susceptibility profiles and known azole resistance mechanisms were evaluated and compared to other azoles (fluconazole, itraconazole, voriconazole, and posaconazole).

MATERIALS AND METHODS

Strains.

Saccharomyces cerevisiae isolates expressing Candida albicans ATP binding cassette (ABC) transporter genes CDR1 and CDR2, the C. albicans major facilitator BENr (MDR1), FLU1, and ERG11 alleles, and C. glabrata CDR1 and CDR2 (CgCDR1 and CgCDR2) were used in this study; these resistance-conferring alleles have been described elsewhere (23–25). The S. cerevisiae strain YKKB13 (MATa ura3-53 lys2-801amber ade2-101ochre trp1-Δ63 his3-Δ200 leu2-Δ1 pdr5Δ::TRP1) was used for all transporter and ERG11 allele expression experiments with YEp24- and YEp51-derived plasmids, respectively (23, 26, 27).

Antifungal drugs.

Azole antifungal drugs were obtained as pure substances from pharmaceutical companies (fluconazole and voriconazole, Pfizer Inc., Groton, CT; itraconazole, Janssen Pharmaceuticals, Inc., Titusville, NJ; posaconazole, Schering-Plough, Kenilworth, NJ; isavuconazole, Basilea Pharmaceutica International Ltd., Basel, Switzerland).

Isavuconazole susceptibility profile of S. cerevisiae strains expressing drug resistance genes.

Susceptibility testing with S. cerevisiae was performed with broth microdilutions based on protocol M27-A3 of the Clinical and Laboratory Standards Institute (CLSI) (28). This protocol uses selective yeast medium (yeast nitrogen base with 2% added glucose) containing doubling dilutions of azoles (fluconazole, 128 to 0.0625 μg/ml; itraconazole, voriconazole, posaconazole, and isavuconazole, 8 to 0.0039 μg/ml) at 30°C for 48 h. We grew S. cerevisiae isolates for C. albicans ERG11 alleles expressed from plasmid YEp51 in YKKB13 in medium containing 1% galactose and 1% raffinose to induce ERG11 expression as reported previously (23). MICs were obtained with a microplate reader at a wavelength of 540 nm. Normalization was performed by allowing growth to 100% in the absence of drugs. The MIC was defined as the concentration of the drug needed to decrease fungal growth by at least 50%.

Isavuconazole susceptibility of clinical Candida species isolates and C. albicans mutants.

Sequential C. albicans and C. glabrata clinical isolates with increasing MICs for several azoles were obtained from the University Hospital Centre (Lausanne, Switzerland) between 1993 and 2004; their azole resistance mechanisms have been described elsewhere (14, 15, 29–32), and their origins were summarized (14, 15, 25, 29, 31–36) (Table 1).

TABLE 1.

Clinical isolates used in the study

Clinical strain
 Species Parent strain Resistance mechanism/genotypea Reference or source
DSY no. Alternative no.
DSY281 C23 C. albicans Related to DSY284 WT 32
DSY284 C39 C. albicans ERG11 (S405F), TAC1 (G980E)b 32
DSY347 C33 C. albicans Related to DSY348 and DSY289 WT 25
DSY288 C34 C. albicans ERG11 (S405F) 25
DSY289 C26 C. albicans ERG11 (S405F, Y132H), TAC1 (A736V) 25, 29
DSY348 C82 C. albicans ERG11 (S405F), TAC1 (A736V) 25, 29
DSY290 C27 C. albicans Related to DSY291, DSY292 WT 25
DSY291 C37 C. albicans ERG11 (G464S) (R467K) 15, 25
DSY292 C40 C. albicans ERG11 (G464S) (R467K, Y132H),b MRR1 (P683H)b 15, 25
DSY294 C43 C. albicans Related to DSY296 WT 14
DSY296 C56 C. albicans ERG11 (G464S), TAC1 (N977D) 14
DSY2321 C. albicans Related to DSY2322 and DSY2323 ERG11 (S405F)b 29
DSY2322 C. albicans ERG11 (S405F), TAC1 (G980E) 29
DSY2323 C. albicans ERG11 (S405F), TAC1 (G980E) 29
DSY731 C. albicans Related to DSY732 and DSY735 WT 29
DSY732 C. albicans TAC1 (ΔM677)c 29
DSY735 C. albicans TAC1 (ΔM677), i (5L) 29
DSY544 C. albicans Related to DSY775 WT 32
DSY775 C. albicans ERG11 (G464S), TAC1 (G980W) 32
DSY2309 C. albicans Related to DSY750 and DSY751 WT 33
DSY750 C. albicans MRR1 (N378D)b 33
DSY751 C. albicans ERG11 (S405F), MRR1 (N378D)b 33
DSY2243 C. albicans Related to DSY2242 ERG11 (S442F, R467K) 32
DSY2242 C. albicans ERG11 (S442F, R467K), TAC1 (G980E) 32
DSY2284 C. albicans Related to DSY2285 WT 15, 36
DSY2285 C. albicans MRR1 (T896I), i (5L) 15, 36
DSY550 C. albicans Related to DSY551 WT 15
DSY551 C. albicans ERG11 (G464S, Y132H) 33
DSY520 C. albicans Related to DSY522 TAC1 (N972D) 32
DSY522 C. albicans ERG11 (G464S), TAC1 (N972D) 32
DSY2250 C. albicans Related to DSY2250 ERG11 (S442F, G465S) 32
DSY2251 C. albicans ERG11 (S442F, G465S), TAC1 (N972D) 32
DSY741 C. albicans Related to DSY742 WT 33
DSY742 C. albicans MRR1 (T360I) 33–35
DSY562 C. glabrata WT 33
DSY565 C. glabrata DSY562 CgPDR1 (I280F) 33–35
DSY1041 C. glabrata DSY1029 Cgcdr1Δ 34
DSY1612 C. glabrata DSY1029 Cgcdr2Δ 34
DSY1613 C. glabrata DSY1056 Cgcdr1Δ, Cgcdr2Δ 34
DSY529 C. glabrata WT 31
DSY530 C. glabrata DSY529 CgPDR1 (E1083Q) 31
DSY2324 C. glabrata WT 31
DSY2325 C. glabrata DSY2324 Mutation petite (loss of mitochondrial DNA) 31
DSY724 C. glabrata WT This study
DSY726 C. glabrata DSY724 WT 31
DSY727 C. glabrata DSY726 CgPDR1 (D876Y) 31
DSY2270 C. glabrata WT 31
DSY2271 C. glabrata DSY2270 CgPDR1 (D261G) 31
DSY759 C. glabrata WT 31
DSY2268 C. glabrata DSY759 CgPDR1 (S316I) 31
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a

Mutations are indicated in parentheses. Amino acid changes at given codon numbers of the corresponding gene are given. i (5L), isochromosome 5L.

b

Heterozygous state.

c

Deletion of methionine at position 677 in TAC1.

Several C. albicans mutants lacking the major multidrug transporters involved in azole resistance were used in this study (37). DSY1054 (flu1Δ/Δ), DSY1021 (cdr1Δ/Δ, cdr2Δ/Δ, flu1Δ/Δ), and DSY1055 (flu1Δ/Δ, mdr1Δ/Δ) are equivalent to DCY2, DCY20, and DCY7, respectively, and have been described by Calabrese et al. (38). DSY1050 (cdr1Δ/Δ, cdr2Δ/Δ, mdr1Δ/Δ) is the parent of DSY1024 (cdr1Δ/Δ, cdr2Δ/Δ, flu1Δ/Δ, mdr1Δ/Δ) and has been described previously (39).

C. albicans mutants with the inactivation of known resistance mechanisms also were used in this study; they originated from strain DSY296 (12). This strain originally had a GOF mutation in TAC1 and a G464S mutation in ERG11, and both are homozygous in DSY296. The MICs for Candida clinical isolates and mutants were determined using the standard protocol (CLSI M27-A3) (28). Isolates were grown at 35°C for 48 h in RPMI 1640 medium (Sigma-Aldrich, Switzerland) with azole doubling dilutions, as described previously in this report. Quality controls included C. albicans ATCC 928, C. krusei ATCC 6258, C. tropicalis ATCC 750, C. parapsilosis ATCC 22019, and C. glabrata ATCC 930 strains, and these were tested with all azoles used in the study.

RESULTS

Susceptibility testing of S. cerevisiae containing drug resistance genes.

In order to test whether isavuconazole is a potential substrate for ABC and MFS transporters, drug susceptibility testing was carried out with S. cerevisiae containing specific transporter genes associated with drug resistance. Changes to the MICs of isavuconazole upon expression of a given transporter indicated whether this azole was a substrate for the expressed transporter.

The expression of ABC transporters, including CDR1, CDR2, CgCDR1, and CgCDR2, elevated the MICs of isavuconazole from 2- to 32-fold compared with the MICs of the recipient S. cerevisiae strain (Table 2). These values depended on the expressed ABC transporters. CDR1 expression had the greatest impact on MICs (32-fold increase), while CgCDR2 expression had the least impact (2-fold increase). Fluconazole and voriconazole showed the largest increases in MICs, ranging from 16- to 128-fold and 8- to 56-fold, respectively.

TABLE 2.

Activity of azoles in S. cerevisiae containing several Candida multidrug transporters

S. cerevisiae strain Resistance mechanism/genotype MIC (μg/ml [fold differencea])
Fluconazole Itraconazole Posaconazole Voriconazole Isavuconazole
DSY390 YEp24b in YKKB13 1 (1) 0.0625 (1) 0.25 (1) 0.0156 (1) 0.25 (1)
DSY415 CDR1 in YKKB13 128 (128) 8 (128) 2 (8) 4 (256) 8 (32)
DSY417 CDR2 in YKKB13 16 (16) 0.5 (8) 1 (4) 0.125 (8) 1 (4)
DSY416 MDR1 in YKKB13 128 (128) 0.0625 (1) 0.0625 (0) 0.5 (32) 0.25 (1)
DSY426 FLU1 in YKKB13 16 (16) 0.0625 (1) 0.125 (1) 0.125 (8) 0.125 (1)
DSY691 CgCDR1 in YKKB13 64 (64) 1 (16) 1 (4) 0.5 (32) 2 (8)
DSY687 CgCDR2 in YKKB13 128 (128) 0.0625 (1) 0.125 (1) 0.5 (32) 0.5 (2)
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a

Fold difference is relative to the level for DSY390 for each azole.

b

Yeast replicating vector (parent vector) into which drug resistance genes were cloned.

Expression of MFS transporters MDR1 and FLU1 did not increase the MICs of isavuconazole, which was a characteristic shared with itraconazole and posaconazole (Table 2). In contrast, MDR1 and FLU1 expression increased the MICs of fluconazole by 128-fold and 16-fold, respectively, and those of voriconazole by 32-fold and 8-fold, respectively. These results indicated that, in contrast to isavuconazole, fluconazole and voriconazole were substrates for these MFS transporters.

Ten S. cerevisiae strains, each expressing an ERG11 allele from different C. albicans clinical isolates, were tested for their susceptibility to isavuconazole and other azoles and were compared to a strain carrying a wild-type (WT) allele. The ERG11 alleles used in this study carried nine distinct single or combined mutations that are known to be involved in azole resistance (23). Isavuconazole MICs ranged from 0.125 to 1 μg/ml. The highest increase for isavuconazole was between 4- and 8-fold and was associated with the substitution Y132H alone or combined with the substitution S405F or G464S.

When C. albicans ERG11 mutant alleles were expressed in S. cerevisiae, increases in azole MICs relative to those of the control strain were observed for fluconazole (4- to 32-fold) and voriconazole (1- to 16-fold) when at least two mutations were present in the same ERG11 allele (Table 3). Correlation coefficient analyses between the different azoles demonstrated that isavuconazole was more closely related to voriconazole and itraconazole than to fluconazole (see Fig. SA1 in the supplemental material). Interestingly, fluconazole MICs in S. cerevisiae showed the highest variations, followed by voriconazole MICs. No changes in posaconazole MICs were observed with any of the ERG11 mutant alleles expressed in S. cerevisiae.

TABLE 3.

Activity of azoles in S. cerevisiae containing C. albicans ERG11 alleles

S. cerevisiae Resistance mechanism/genotype MIC (μg/ml [fold differencea])
Fluconazole Itraconazole Posaconazole Voriconazole Isavuconazole
DSY595b YKKB13 and C. albicans ERG11 4 (1) 0.25 (1) 0.5 (1) 0.125 (1) 0.125 (1)
DSY1109 YKKB13 and C. albicans ERG11 (G129A) 4 (1) 0.25 (1) 0.5 (1) 0.0156 (1) 0.125 (1)
DSY949 YKKB13 and C. albicans ERG11 (S405F) 8 (2) 0.25 (1) 0.5 (1) 0.125 (1) 0.25 (2)
DSY952 YKKB13 and C. albicans ERG11 (Y132H) 8 (2) 0.5 (2) 0.25 (1) 0.25 (2) 0.5 (4)
DSY950 YKKB13 and C. albicans ERG11 (G464S) 8 (2) 0.25 (1) 0.5 (1) 0.125 (1) 0.125 (1)
DSY951 YKKB13 and C. albicans ERG11 (R467K) 8 (2) 0.25 (1) 0.5 (1) 0.125 (1) 0.25 (2)
DSY963 YKKB13 and C. albicans ERG11 (S405F/Y132H) 128 (32) 1 (4) 0.5 (1) 2 (16) 1 (8)
DSY965 YKKB13 and C. albicans ERG11 (G464S/Y132H) 32 (8) 0.5 (2) 0.5 (1) 2 (16) 1 (8)
DSY1107 YKKB13 and C. albicans ERG11 (G464S/R467K) 32 (8) 0.25 (1) 0.5 (1) 0.25 (2) 8 (4)
DSY1108 YKKB13 and C. albicans ERG11 (G464S/G129A) 15 (4) 0.25 (1) 0.5 (1) 0.125 (1) 0.25 (2)
DSY612 YKKB13 and YEp51c 1 (0.25) 0.0625 (0.25) 0.25 (0.5) 0.0078 (0.0625) 0.0625 (0.5)
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a

Fold difference is relative to the level for DSY595 for each azole.

b

Baseline strain.

c

Yeast replicating vector (parent vector) into which ERG11 alleles were cloned.

Susceptibility testing of Candida species isolates with known azole resistance mechanisms.

Our strain collection included a number of clinical isolates of C. albicans and C. glabrata with known and diverse resistance mechanisms. Paired isolates exhibited closely related genotypic patterns deduced from multilocus sequence typing (31, 40) (see Table SA1 in the supplemental material). Therefore, it was possible to compare azole MICs from the most susceptible isolates to those of the most resistant isolates. All quality controls were observed to have azole MICs within the accepted range (20, 41) (Table 4).

TABLE 4.

Azole MIC quality controls

Reference strain MIC (μg/ml)
Fluconazole Itraconazole Posaconazole Voriconazole Isavuconazole
C. albicans ATCC 928 0.125 0.0625 0.0078 <0.0039 0.0078
C. krusei ATCC 6258 32 0.125 0.125 0.125 0.5
C. tropicalis ATCC 750 0.5 0.0078 0.0156 0.0078 0.0312
C. parapsilosis ATCC 22019 2 0.0625 0.0625 0.0156 0.125
C. glabrata ATCC 930 4 0.125 0.125 0.0312 0.125
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Susceptibility testing of C. albicans clinical isolates.

The MIC range for isavuconazole (0.004 to 8 μg/ml) was similar to that for voriconazole (0.004 to 4 μg/ml) (Table 5). The MICs for fluconazole, itraconazole, and posaconazole ranged from 0.125 to 128, 0.016 to 2, and 0.016 to 4 μg/ml, respectively. The relative increase in MICs for each azole compared to that of a susceptible control ranged from 32- to 512-fold for fluconazole, voriconazole, and isavuconazole but from only 4- to 32-fold for itraconazole and 2- to 128-fold for posaconazole. The MIC90s for the matched clinical C. albicans isolates for fluconazole, voriconazole, itraconazole, posaconazole, and isavuconazole were 128, 2, 1, 0.5, and 2 μg/ml, respectively. This suggests that isavuconazole had activity similar to those of itraconazole, posaconazole, and voriconazole.

TABLE 5.

Activity of azoles in C. albicans clinical isolates with known resistance mechanisms

C. albicans clinical strain Resistance mechanism/genotype MIC (μg/ml [fold differenceb])
Fluconazole Itraconazole Posaconazole Voriconazole Isavuconazole
DSY281 WT 0.250 (1) 0.125 (1) 0.031 (1) 0.004 (1) 0.016 (1)
DSY284 ERG11 (S405F), TAC1 (G980E)a 8 (32) 0.25 (2) 0.25 (8) 0.063 (16) 0.25 (16)
DSY347 WT 0.250 (1) 0.125 (1) 0.125 (1) 0.004 (1) 0.031 (1)
DSY288 ERG11 (S405F) 0.5 (2) 0.125 (1) 0.125 (1) 0.031 (8) 0.063 (2)
DSY289 ERG11 (S405F, Y132H), TAC1 (A736V) >128 (512) 2 (16) 0.5 (4) 4 (1026) 8 (256)
DSY348 ERG11 (S405F), TAC1 (A736V) 8 (32) 1 (8) 0.5 (4) 0.063 (16) 1 (32)
DSY290 WT 0.500(1) 0.063 (1) 0.063 (1) 0.004 (1) 0.008 (1)
DSY291 ERG11 (G464S, R467K) 2 (1) 0.25 (4) 0.063 (1) 0.031 (8) 0.031 (4)
DSY292 ERG11 (G464S, R467K, Y132H),a MRR1 (P683H)a 128 (256) 1 (16) 0.25 (4) 0.5 (128) 2 (256)
DSY294 WT 0.5 (1) 0.016 (1) 0.063 (1) 0.004 (1) 0.016 (1)
DSY296 ERG11 (G464S), TAC1 (N977D) 64 (128) 0.125 (8) 0.25 (4) 0.5 (128) 2 (128)
DSY2321 ERG11 (S405F)a <0.125 (1) 0.031 (1) 0.063 (1) 0.004 (1) <0.0039 (1)
DSY2322 ERG11 (S405F), TAC1 (G980E) 16 (128) 1 (32) 0.5 (8) 0.125 (32) 2 (513)
DSY2323 ERG11 (S405F), TAC1 (G980E) 32 (256) 0.5 (16) 0.5 (8) 0.125 (32) 2 (513)
DSY731 WT 0.250 (1) 0.063 (1) 0.031 (1) 0.004 (1) 0.008 (1)
DSY732 TAC1 (ΔM677) 16 (64) 1 (16) 0.5 (16) 0.25 (64) 4 (513)
DSY735 TAC1 (ΔM677), i (5L) 64 (256) 0.5 (8) 1 (32) 0.25 (64) 2 (256)
DSY544 WT 0.125 (1) 0.063 (1) 0.063 (1) <0.0039 (1) <0.0039 (1)
DSY775 ERG11 (G464S), TAC1 (G980W) 64 (512) 1 (16) 0.5 (8) 0.5 (128) 2 (513)
DSY2309 WT 2 (1) 0.125 (1) 0.125 (1) 0.004 (1) <0.0039 (1)
DSY750 MMR1 (N378D)a 16 (8) 0.25 (2) 0.125 (1) 0.063 (16) 0.063 (16)
DSY751 ERG11 (S405F), MMR1 (N378D)a 128 (64) 0.5 (4) 0.25 (2) 0.125 (32) 1 (256)
DSY2243 ERG11 (S442F, R467K) 0.25 (1) 0.125 (1) 0.063 (1) 0.063 (1) <0.0039 (1)
DSY2242 ERG11 (S442F, R467K), TAC1 (G980E) 8 (32) 0.5 (4) 0.5 (8) 1 (16) 1 (256)
DSY2284 WT 0.125 (1) 0.063 (1) 0.063 (1) 0.004 (1) 0.004 (1)
DSY2285 MRR1 (T896I), i (5L) 64 (512) 0.25 (4) 0.25 (4) 0.125 (32) 0.25 (64)
DSY550 WT 0.125 (1) 0.016 (1) 0.016 (1) 0.004 (1) 0.008 (1)
DSY551 ERG11 (G464S, Y132H) 64 (512) 0.5 (32) 0.5 (32) 2 (513) 2 (256)
DSY520 TAC1 (N972D) 16 (1) 0.063 (1) 0.5 (1) 1 (1) 0.250 (1)
DSY522 ERG11 (G464S), TAC1 (N972D) 128 (8) 2 (32) 1 (2) 2 (2) 2 (8)
DSY2250 ERG11 (S442F, G465S) 1 (1) 0.125 (1) 0.031 (1) 0.004 (1) 0.008 (1)
DSY2251 ERG11 (S442F, G465S), TAC1 (N972D) 32 (32) 1 (8) 4 (128) 0.25 (64) 4 (513)
DSY741 WT 0.125 (1) 0.063 (1) 0.125 (1) 0.008 (1) 0.008 (1)
DSY742 MRR1 (T360I) 16 (128) 0.250 (4) 0.125 (1) 0.031 (4) 0.125 (16)
DSY757 WT 0.5 (1) 0.031 (1) 0.063 (1) <0.0039 (1) <0.0039 (1)
DSY758 ERG11 (G464S, F145L),a TAC1 (A736V) 32 (64) 0.5 (16) 0.25 (4) 0.125 (32) 1 (256)
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a

Heterozygous state.

b

Fold difference is relative to the WT or to the most susceptible isolate for each related isolate group.

Isavuconazole MICs for each isolate were compared to those of other azoles, separating non-WT isolates from WT isolates, i.e., those with and without known azole resistance mechanisms (Fig. 1A). The upper isavuconazole MIC value for WT isolates was 0.031 μg/ml. This value is in agreement with another study which reported that 90% of C. albicans isolates exhibit isavuconazole MICs of ≤0.03 μg/ml (42). Correlation curves between isavuconazole MICs of WT and non-WT isolates and other azoles were established (Fig. 1). Isavuconazole MICs correlated with other azoles MICs in the following order of correlation strength: itraconazole < voriconazole < fluconazole < posaconazole. However, the analysis of relative MIC increases of matched isolates for each azole correlation coefficient demonstrated that the MIC profile of isavuconazole was related significantly to those of posaconazole and itraconazole, while profiles of fluconazole and voriconazole were significantly more similar to each other (see Fig. SA2 in the supplemental material).

FIG 1.

FIG 1

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Relationship of isavuconazole MICs to those of other azoles for C. albicans isolates (MICs are from Tables 5, 7, and 8) (A) and C. glabrata isolates (MICs are from Table 6) (B). Correlations were calculated with nonlinear regression fits and log-log lines as an option using GraphPad Prism 6. The R2 values are indicated within figure captions. Wild-type (WT) isolates and non-WT isolates with resistance mechanisms are shown with empty and filled symbols, respectively.

Susceptibility testing of C. glabrata isolates.

The MIC range for isavuconazole (0.25 to 16 μg/ml) was similar to the range for posaconazole (0.125 to 16 μg/ml) (Table 6). The MIC ranges for fluconazole, itraconazole, and voriconazole were 1 to 128, 0.0312 to 16, and 0.0156 to 2 μg/ml, respectively. MIC90s of clinical isolates for fluconazole, voriconazole, itraconazole, posaconazole, and isavuconazole were 128, 2, 4, 4, and 16 μg/ml, respectively. These values were higher than those observed for the C. albicans isolates DSY565 and DSY2325 and were associated with the highest relative increase in MICs for isavuconazole (32-fold). Comparison of isavuconazole MICs from WT isolates and non-WT isolates revealed that the upper isavuconazole MIC value of WT isolates was 1.0 μg/ml (Fig. 1B). This value was equivalent to that observed in another study (42). Correlation curves between isavuconazole MICs of WT and non-WT isolates and other azoles highlighted that isavuconazole MICs were correlated with MICs of other azoles in the following order: voriconazole < fluconazole < itraconazole < posaconazole. When we analyzed correlation coefficients between relative MIC increases of matched isolates for each azole in C. glabrata, we observed that the profile of isavuconazole was related significantly only to that of fluconazole, in contrast to the case for C. albicans (see Fig. SA3 in the supplemental material).

TABLE 6.

Activity of azoles in C. glabrata clinical isolates with known resistance mechanisms

C. glabrata clinical strain Resistance mechanism/genotype MIC (μg/ml [fold differencea])
Fluconazole Itraconazole Posaconazole Voriconazole Isavuconazole
DSY562 WT 2 (1) 1 (1) 0.5 (1) 0.25 (1) 0.5 (1)
DSY565 CgPDR1 (I280F) 64 (32) 4 (4) 2 (4) 1 (4) 16 (32)
DSY1041 Cgcdr1Δ 2 (1) 1 (1) 0.5 (1) 0.0312 (0.12) 1 (2)
DSY1612 Cgcdr2Δ 32 (16) 1 (1) 1 (2) 0.5 (2) 8 (16)
DSY1613 Cgcdr1Δ, Cgcdr2Δ 1 (0.5) 0.0312 (0.03) 0.125 (0.25) 0.0156 (0.06) 0.25 (0.5)
DSY529 WT 4 (1) 1 (1) 0.5 (1) 0.0312 (1) 0.5 (1)
DSY530 CgPDR1 (E1083Q) 64 (16) 2 (2) 2 (4) 0.5 (32) 8 (16)
DSY2324 WT 2 (1) 1 (1) 0.25 (1) 0.0312 (1) 0.25 (1)
DSY2325 Mutation petite 128 (64) 2 (0.5) 2 (8) 1 (32) 8 (32)
DSY724 WT 4 (1) 0.5 (1) 1 (1) 0.0625 (1) 1 (1)
DSY726 WT 2 (0.5) 0.25 (0.5) 0.5 (0.5) 0.0312 (0.5) 0.25 (0.25)
DSY727 CgPDR1 (D876Y) 128 (32) 8 (16) 4 (4) 1 (16) 16 (16)
DSY2270 WT 4 (1) 1 (1) 1 (1) 0.0625 (1) 1 (1)
DSY2271 CgPDR1 (D261G) 128 (32) 16 (16) 16 (16) 2 (32) 16 (16)
DSY759 WT 4 (1) 1 (1) 1 (1) 0.0312 (1) 1 (1)
DSY2268 CgPDR1 (S316I) 64 (16) 4 (4) 4 (4) 0.5 (16) 8 (8)
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a

Fold difference relative to the level for the WT for each related isolate group.

Susceptibility testing of C. albicans multidrug transporter mutants.

MICs for isavuconazole and other azoles were determined for C. albicans mutants lacking the major multidrug transporters involved in azole resistance in order to test their impact on drug resistance. The absence of CDR1 had the largest impact on azole MICs (Table 7). For isavuconazole, the MIC decreased from 0.0078 μg/ml in WT CAF2-1 to <0.001 μg/ml in the cdr1Δ/Δ mutant. The fluconazole MIC decreased from 0.25 to <0.0625 μg/ml in the absence of CDR1. MIC decreases in itraconazole and posaconazole were less pronounced, 0.0625 to 0.0156 μg/ml, when multiple transporters were deleted.

TABLE 7.

Activity of azoles in C. albicans multidrug transporter mutants

C. albicans transporter mutant Resistance mechanism/genotype MIC (μg/ml)
Fluconazole Itraconazole Posaconazole Voriconazole Isavuconazole
CAF2-1 URA3/ura3Δ 0.25 0.0625 0.0625 <0.0039 0.0078
DSY448 cdr1Δ/Δ <0.0625 0.0625 0.0625 <0.0039 <0.001
DSY465 mdr1Δ/Δ 0.25 0.0625 0.0625 <0.0039 0.0078
DSY468 cdr1Δ/Δ mdr1Δ/Δ <0.0625 0.0625 0.0156 <0.0039 <0.001
DSY653 cdr2Δ/Δ 0.125 0.0625 0.0156 <0.0039 0.0078
DSY654 cdr1Δ/Δ cdr2Δ/Δ <0.0625 0.0156 0.0156 <0.0039 0.002
DSY1054 flu1Δ/Δ 0.25 0.0625 0.0625 <0.0039 0.0156
DSY1021 cdr1Δ/Δ cdr2Δ/Δ flu1Δ/Δ <0.0625 0.0625 0.0625 <0.0039 <0.001
DSY1024 cdr1Δ/Δ cdr2Δ/Δ flu1Δ/Δ mdr1Δ/Δ <0.0625 0.0156 0.125 <0.0039 <0.001
DSY1050 cdr1Δ/Δ cdr2Δ/Δ mdr1Δ/Δ <0.0625 0.0156 0.0312 <0.0039 0.002
DSY1055 flu1Δ/Δ mdr1Δ/Δ 0.25 0.0156 0.125 <0.0039 0.0156
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Susceptibility testing of C. albicans mutants with inactivation of known azole resistance mechanisms.

The DSY3706 derivative, which lacks TAC1 (the transcriptional activator of CDR1 and CDR2) and contains WT ERG11, had MICs for all azoles tested that were similar to those of DSY294, which is the susceptible parent strain (Table 8). Reintroduction of the TAC1 mutation in DSY3606-1 increased the MIC of fluconazole by approximately 32-fold. The largest increases in MICs for isavuconazole were observed in the DSY3606-1 (64-fold) and DSY296 (128-fold) strains. These strains contain TAC1 GOF mutations, suggesting that increases in CDR1 and CDR2 are associated with reduced susceptibility to isavuconazole. The G464S mutation in DSY296 resulted in the largest increases in MICs (128-fold) for fluconazole, voriconazole, and isavuconazole. The reintroduction of ERG11 WT alleles into the TAC1 deletion mutant DSY3083 resulted in MICs that were decreased by 16-fold (fluconazole) and 4-fold (voriconazole) in strain DSY3706.

TABLE 8.

Activity of azoles in C. albicans mutants reconstituting the azole-susceptible parent

C. albicans ERG11/TAC1 mutant Resistance mechanism/genotype MIC (μg/ml [fold differencea])
Fluconazole Itraconazole Posaconazole Voriconazole Isavuconazole
DSY294 WT 0.5 (1) 0.0156 (1) 0.0156 (1) <0.0039 (1) 0.0156 (1)
DSY296 ERG11 (G464S), TAC1 (N977D) 64 (128) 0.125 (8) 0.25 (16) 0.5 (128) 2 (128)
DSY3082 ERG11, tac1Δ/Δ 0.5 (1) 0.0156 (1) 0.0156 (1) 0.0078 (2) 0.0312 (2)
DSY3083 ERG11 (G464S), tac1Δ/Δ 4 (8) 0.0078 (0.5) 0.0312 (2) 0.0156 (4) 0.0312 (2)
DSY3706 tac1Δ/Δ, ERG11 0.25 (0.5) 0.0156 (1) 0.0156 (1) <0.0039 (1) 0.0156 (1)
DSY3606-1 tac1Δ/Δ, ERG11, TAC1-5 (N977D) 8 (32) 0.125 (4) 0.0625 (4) 0.0625 (16) 0.5 (64)
DSY3608-2 tac1Δ/Δ, ERG11, TAC1-1 0.125 (0.5) 0.0312 (1) 0.0156 (1) <0.0039 (1) 0.0156 (2)
DSY3604 tac1Δ/Δ, ERG11 (G464S)b 0.5 (1) 0.0312 (2) 0.0312 (2) 0.0078 (2) 0.0078 (0.5)
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a

Fold difference relative to the level of the WT for each azole.

b

Heterozygous state.

DISCUSSION

This study assessed the mechanisms of isavuconazole resistance in Candida species using either S. cerevisiae as a vector for expressing specific azole resistance genes or various C. albicans and C. glabrata isolates with known resistance mechanisms to determine azole susceptibility. Isavuconazole had mechanisms of resistance similar to those of other azoles. Its range of activity was comparable to that of voriconazole in the Candida strains tested in this study.

The presence or absence of ABC transporters had the greatest effect on the MICs of isavuconazole as well as those of voriconazole, posaconazole, and itraconazole. The expression of CDR1, CDR2, CgCDR1, and CgCDR2 in S. cerevisiae resulted in elevated isavuconazole MICs compared to those of the controls, indicating that isavuconazole is a substrate for these transporters. This is particularly the case with the CDR1 and CgCDR1 transporters of C. albicans and C. glabrata, respectively. The expression of these transporters resulted in the largest increases in MICs for isavuconazole. Changes in MICs due to the expression of CDR genes were similar to those for isavuconazole, voriconazole, and itraconazole, although isavuconazole exhibited average MIC increases compared to those of other drugs.

MDR1 and FLU1 expression did not result in increases in MIC levels for isavuconazole, demonstrating that isavuconazole is not a substrate for MFS transporters. The opposite was observed for fluconazole and voriconazole, for which increases in MICs were linked to the expression of these transporters. This is supported in a study by Cheng et al. (43) in which MDR1 overexpression in a C. albicans petite mutant was associated with increased resistance to fluconazole and voriconazole, but the strain remained susceptible to itraconazole and ketoconazole.

The MIC increases for isavuconazole were moderate when associated with ERG11 mutations in the S. cerevisiae model. The MICs for fluconazole demonstrated the greatest variations, followed by those for voriconazole. A closer look at all azole susceptibility profiles indicated that the profile of fluconazole was distinct from those of isavuconazole and itraconazole. Interestingly, posaconazole MICs exhibited no changes with any of the mutant alleles expressed in S. cerevisiae. The largest effect on the MIC for isavuconazole was with S. cerevisiae strains expressing ERG11 alleles with the Y132H mutation either alone or combined with mutation S405F or G464S. These results are consistent with recent Erg11p crystallographic data published by Monk et al. (44), who posited that individual azole molecules interact with specific Erg11p residues depending on their structure. Therefore, we expect that ERG11 mutations do not have the same effect on the binding efficiency of specific azoles.

When testing C. albicans and C glabrata isolates, MIC changes for isavuconazole were similar to those observed for fluconazole, voriconazole, itraconazole, and posaconazole. In general, isavuconazole was more active in C. albicans than in C. glabrata (MIC ranges of 0.004 to 8 μg/ml and 0.25 to 16 μg/ml, respectively). This difference also has been observed in other studies (20, 45). C. albicans clinical isolate DSY289 had the highest isavuconazole MIC (8 μg/ml). This strain originally was isolated from an HIV-positive patient who had oropharyngeal candidiasis treated with fluconazole (25); this strain is associated with the ERG11 mutation S405F/Y132H and with the A736V mutation in TAC1. The TAC1 mutation is associated with increased CDR1 and CDR2 levels (29). This suggests that combined resistance mechanisms result in high resistance levels against isavuconazole and other azoles. This is consistent with data in the present study, in which the MICs of non-WT isolates were shifted to higher values than those of WT isolates. However, comparison of relative increases in MICs for fluconazole (512-fold) and voriconazole (1,026-fold) to that of isavuconazole (256-fold) indicates that the effect of a combined azole resistance mechanism was reduced on isavuconazole.

Increases in MICs for isavuconazole in C. glabrata clinical isolates were observed when the CgPDR1 GOF mutation was present in non-WT isolates. These mutations cause the upregulation of CgCDR1 and CgCDR2 (46, 47). Relative increases for isavuconazole ranged from 8- to 32-fold, similar to values reached by other azoles. However, the analysis of correlation coefficients between relative MIC increases by each azole in specific isolates highlighted that the susceptibility profile of isavuconazole was closely related to those of posaconazole and itraconazole in C. albicans but distinct from that of fluconazole. In C. glabrata this tendency was not verified, since the profile of isavuconazole was more closely related to that of fluconazole. The present study also reflects these features, as the MIC profile for isavuconazole was similar to the MIC profile for itraconazole in C. albicans and for the fluconazole profile in C. glabrata. Azole resistance mechanisms in C. albicans and C. glabrata are not equally distributed between the two species and therefore may have contributed to the observed susceptibility profile differences. On the other hand, isavuconazole exhibits a chemical structure that is different from those of the two structurally related groups of fluconazole/voriconazole and posaconazole/itraconazole (48). Therefore, isavuconazole might adopt susceptibility profiles that cannot be predicted simply from structural resemblance to the two known azole groups.

The absence of CDR1 from C. albicans mutants had the greatest effect on the MICs of azoles. The lowest MICs for both fluconazole and isavuconazole were associated with CDR1 knockout either on its own or in association with the deletion of other transporters. Isavuconazole and fluconazole had similar MIC profiles in these mutants, while decreases in MICs were less pronounced for itraconazole and posaconazole. The inactivation of known resistance mechanisms demonstrated that TAC1 had the greatest effect on isavuconazole, with its deletion resulting in decreases in MICs and its reintroduction increasing them.

This study suggests that isavuconazole follows the resistance patterns observed in other azoles, with slight variations depending on the investigated fungal species. Currently, there is a lack of data on isavuconazole susceptibility patterns among clinical Candida isolates. In an Egyptian epidemiology study, all strains of Candida isolated from 187 patients were susceptible to isavuconazole (range, < 0.016 to 1 μg/ml), while overall resistance to voriconazole was 2.5% in all strains (49). Another study demonstrated isavuconazole to be highly active against 296 Candida bloodstream isolates; the activity of isavuconazole was more potent than that of fluconazole against all organisms tested, and often it was more potent than that of itraconazole or voriconazole (20). It also was noted that only two isolates, both C. glabrata, had a MIC for isavuconazole of >0.5 μg/ml. In a study investigating approximately 1,400 Candida species, isavuconazole exhibited high activity. A few isolates with high isavuconazole MICs in this study (1 to 8 μg/ml) could not be analyzed for their cross-resistance to other azoles (42). C. albicans and C. glabrata isolates were directly compared by using isavuconazole MICs. Fluconazole- and voriconazole-resistant isolates corresponded to high isavuconazole MICs in C. albicans (0.12 to 1 μg/ml) and C. glabrata (1 to 8 μg/ml), which overlap values obtained in the study reported here (42).

Isavuconazole activity has been investigated in other fungal species with respect to a possible correlation between azole resistance and levels of isavuconazole MICs. One study on Aspergillus fumigatus highlighted that the acquisition of resistance to voriconazole and itraconazole due to Cyp51 mutations was followed by an increase of isavuconazole MICs compared to those of susceptible isolates (50). Thus, isavuconazole does not deviate from other azoles in the characteristic decrease of activity in the presence of azole resistance mechanisms.

In conclusion, isavuconazole, as a substrate of multidrug ABC transporters, has properties in common with other azoles. However, in contrast to fluconazole and voriconazole, isavuconazole is not a substrate for the MFS transporter MDR1 or FLU1. Isavuconazole was sensitive to mutations in ERG11, but these mutations had less impact on MIC increases in isavuconazole than they did on MIC increases for fluconazole and voriconazole. These mutations had minimal effect on MICs of posaconazole. Isavuconazole had an activity range similar to that of voriconazole in the Candida strains tested in this study.

Supplementary Material

Supplemental material
supp_60_1_229__index.html (965B, html)

ACKNOWLEDGMENTS

This study was supported by an investigation grant from Basilea Pharmaceutica AG, Basel, Switzerland, and partially by the Swiss National Research Foundation (31003A_146936/1).

Technical support was provided by Francoise Ischer. Editorial support was provided by Barrie J. Anthony (Envision Scientific Solutions), who is funded by Astellas Pharma Global Development, Inc.

Footnotes

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.02157-15.

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