Changes in susceptibility to posaconazole in clinical isolates of Candida albicans

Xin Li1, Nathaniel Brown1, Andrew S. Chau1, José L. López-Ribot2, Maria T. Ruesga3, Guillermo Quindos3, Cara A. Mendrick1, Roberta S. Hare1, David Loebenberg1, Beth DiDomenico1 and Paul M. McNicholas1,*

1 Schering-Plough Research Institute, 2015 Galloping Hill Road, 4700 Kenilworth, NJ 07033; 2 Department of Medicine and Division of Infectious Diseases, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA; 3 Departamento de Immunologia, Microbiologia y Parasitologia, Facultad de Medicina y Odontologia, Universidad del Pais Vasco, Bilbao, Vizcaya, Spain

Received 24 July 2003; returned 15 September 2003; revised 13 October 2003; accepted 19 October 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To characterize the molecular mechanisms responsible for reduced susceptibility to azoles in Candida albicans clinical isolates.

Materials and methods: Seven sequential C. albicans isolates were cultured from an AIDS patient treated with posaconazole for refractory oropharyngeal candidiasis. Expression levels of the CDR1, CDR2 and MDR1 genes, encoding efflux pumps previously implicated in azole resistance, and ERG11, encoding the azole target site, were monitored using northern blot and real-time PCR. The ERG11 genes from all seven isolates were sequenced.

Results: The seven closely related isolates exhibited significant decreases in susceptibility to fluconazole (MIC >= 32 mg/L) and voriconazole (MIC >= 2 mg/L) and progressive decreases in susceptibility to both posaconazole (isolates 1–4 MIC 0.25 mg/L, isolates 5–7 MIC 2 mg/L) and itraconazole (isolates 1–4 MIC 1 mg/L, isolates 5–7 MIC > 8 mg/L). None of the isolates exhibited any significant changes in the expression levels of ERG11 or the efflux pump genes. All seven isolates had multiple mutations in ERG11; isolates one through four each had five missense mutations; four of the resultant amino acid changes were previously associated with azole resistance. The fifth isolate had an additional novel mutation in one copy of ERG11, resulting in a Pro-230 to Leu substitution. This mutation was present in both ERG11 genes in the last two isolates. Select ERG11 genes were expressed in Saccharomyces cerevisiae, the ERG11 allele with all six mutations conferred the highest level of posaconazole resistance.

Conclusions: Multiple mutations in ERG11 are required to confer decreased susceptibility to posaconazole.

Keywords: azoles, drug resistance, ERG11, efflux pumps


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Candida species are the fourth most common nosocomial bloodstream pathogen in the United States.1 Immunocompromised individuals are at particular risk from fungal infections caused by yeast pathogens. Consequently, the AIDS epidemic has led to a dramatic increase in the number of patients requiring antifungal agents. Not surprisingly, this increase in drug usage has been accompanied by an increase in drug-resistant isolates. The problem of resistance has been exacerbated by the limited antifungal armamentarium. Resistance to flucytosine is the major cause of treatment failure with this agent.2 Amphotericin B resistance in Candida albicans remains rare but problems with toxicity have limited the usefulness of this drug.2,3 Azole resistance in C. albicans, particularly to fluconazole, has been well documented.2,4,5 Resistance mechanisms have fallen into three main categories, the most prevalent appears to be decreased intracellular drug accumulation resulting from increased expression of efflux pumps, either the major facilitators such as MDR1 or the ATP-dependent pumps CDR1 and CDR2.5,6 In addition, alterations in the expression level or sequence of the target enzyme, 14{alpha}-demethylase (encoded by ERG11) have resulted in reduced azole binding.79 Finally, inactivation of ERG3, encoding the sterol {Delta}5,6 desaturase, abrogates the toxicity of the methylated sterols and thereby confers azole resistance in C. albicans10 but not in C. glabrata.11

Posaconazole (SCH56592) is a potent, novel, broad-spectrum triazole in Phase III trials. Both in vitro and in vivo testing have demonstrated that posaconazole is more effective than fluconazole against Candida spp. and Aspergillus spp.12 In this study we analysed a collection of related C. albicans isolates from a patient undergoing azole therapy, to better understand the molecular basis of resistance to posaconazole. We identified specific amino acid substitutions in Erg11p, including a novel substitution, and correlated changes in susceptibility with the occurrence of specific mutations (part of this work was presented at the Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, USA, September 2002).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strains and plasmids

Clinical C. albicans isolates were obtained by oral saline rinses. Saccharomyces cerevisiae strain YKKB-13 and plasmid YEp51 were obtained from D. Sanglard, Institut de Microbiologie, Universitaire Vaudois. C. albicans strain C43 is an azole-susceptible clinical isolate from the Schering-Plough Research Institute (SPRI; Kenilworth, NJ, USA) culture collection.

Antifungal agents

Posaconazole was prepared at SPRI as a micronized powder. Fluconazole and the parenteral formulation of voriconazole were obtained from Pfizer Inc. (New York, NY, USA). Itraconazole and amphotericin B powders were obtained from Janssen Pharmaceutica Inc. (Beerse, Belgium) and Sigma Chemical Co. (St Louis, MO, USA), respectively. All drugs, except voriconazole (which was dissolved in water) were dissolved in DMSO, dilutions were made in RPMI 1640 media (BioWhittaker, Walkersville, MD, USA).

Antifungal drug susceptibility testing

The MICs for C. albicans strains were determined according to the procedures of the NCCLS.13

Strain identification

The identity of the clinical isolates was confirmed using the following tests: the Vitek Identification System with the Yeast Biochemical Card (bioMérieux Vitec Inc, Hazelwood, MO, USA), germ tube formation in serum-containing medium and colour of colonies on CHROMagar Candida medium (CHROMagar, Paris, France). Karyotyping, restriction fragment length polymorphism (RFLP) analysis using SfiI, and subsequent probing of SfiI digests by Southern blotting using the Ca3 probe, were all carried out as described previously.6 Repetitive element PCR (Rep-PCR) analysis was carried out using the DiversiLab Candida kit as directed by the manufacturer (Bacterial BarCodes, Houston, TX, USA). The 10 C. albicans isolates used for comparative purposes were randomly chosen from the SPRI culture collection.

Measurement of transcript levels

Strains were propagated in yeast peptone dextrose (YPD, Qbiogene) medium and harvested at mid-logarithmic growth phase. Total RNA was extracted using the RNAeasy mini kit (Qiagen Inc., Valencia, CA, USA). Probe preparation, northern blots and quantification of signals were all carried out as described previously.6 Real-time PCR analysis was carried out as described previously.14 Reactions were run in duplicate and repeated at least once, relative gene expression levels ({Delta}CT) were calculated using an 18S rRNA internal control.

PCR amplification and sequencing of ERG11

The ERG11 alleles were PCR amplified in overlapping 600 base pair (bp) segments from total genomic DNA. Both strands of the PCR fragments were sequenced directly by MWG-Biotech Inc. (High Point, NC, USA) and the sequences were compared with the published sequence (GenBank accession number X13296).

Cloning of ERG11 alleles

The ERG11 alleles were PCR amplified from chromosomal DNA with the following oligonucleotides to generate fragments with flanking SalI and BamHI restriction sites; 5'-ACGCGTCGACAATATGGCTATTGTTGAAACTGTC-3' and 5'-GCGGATCCTTAAAACATACAAGTTTCTCTTTT-3'. To mutate codon 230, the 5'-region and 3'-regions of ERG11 were separately PCR amplified (oligonucleotides: 5'-region, 5'-TGTAAAACGACGGCCAGTATGATTATTAAACTTCTTTGCGTCCATCCA-3' and 5'-AACAAAATTAATAAGGGTAAAACCTTTATC-3'; 3'-region, 5'-GATAAAGGTTTTACCCTTATTAATTTTGTT-3' and 5'-CAGGAAACAGCTATGACCTCATGGCGACCACACCCGTCCT-3') to introduce the required nucleotide substitution at codon 230. Using fusion PCR, the two halves were fused together using the same oligonucleotides used to clone the full-length genes. In all cases, the PCR products were cloned into the multicopy plasmid YEp51, sequenced to verify amplification, and then used to transform S. cerevisiae strain YKKB-13 to leucine prototrophy. We note that compared to the sequence published in GenBank, the ERG11 genes from the azole-susceptible C. albicans strains C43 encode a E266D substitution.

Spotting tests

S. cerevisiae transformants, expressing various ERG11 alleles, were suspended in YNB broth (Qbiogene, Carlsbad, CA, USA) supplemented with 2% raffinose and 2% galactose to achieve an OD530 of 0.1. Serial 10-fold dilutions of this inoculum were spotted on YNB plates supplemented with 2% raffinose, 2% galactose and azoles at various concentrations. The plates were incubated at 35°C for 72 h.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient case history

An AIDS patient was diagnosed with oropharyngeal candidiasis (OPC) and was treated initially with amphotericin B (Abelcet) for 2 months and then with itraconazole for 5 months. At the end of this time a C. albicans isolate (C369, Table 1) with reduced susceptibility to fluconazole, voriconazole and itraconazole was cultured from the patient. The patient was switched to posaconazole therapy and received 400 mg posaconazole twice a day for approximately 6 months (November 1998 to June 1999). During this time, three C. albicans isolates (C378, C371 and C372) were cultured from the patient. Less than 1 month after discontinuation of posaconazole therapy, the patient experienced a recurrence of OPC and was treated with both low and high doses of voriconazole. There was no clinical improvement and in August 1999, the patient was enrolled in a long-term clinical study and treated with posaconazole as above. The patient again responded positively to drug therapy but after 9 months (May 2000), the patient experienced a relapse, therapy was discontinued and the patient was diagnosed as a treatment failure. Three additional C. albicans isolates (C373, C376 and C375) were obtained during this second course of posaconazole therapy.


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Table 1. Drug susceptibilities of the clinical C. albicans isolates
 
Isolation and identification of C. albicans isolates

Isolate C369 was obtained before posaconazole treatment. Five additional isolates were cultured from the patient over the course of the two periods of posaconazole therapy (see above) and a final isolate (C375) was obtained immediately after discontinuation of posaconazole therapy. All seven isolates were identified as C. albicans. Five of the seven isolates were tested using karyotyping and RFLP mapping using SfiI. All five yielded nearly identical banding patterns suggesting that they are closely related (Figure 1a and b). In addition, Rep-PCR analysis of all seven strains yielded nearly identical profiles (Figure 1c). When these profiles were compared with those obtained from 10 randomly chosen C. albicans clinical isolates, the isolates from this study clearly grouped together. Finally, a further indication of relatedness came from the finding that the seven isolates had acquired 12 identical mutations (seven silent and five missense, see below) in the ERG11 genes.



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Figure 1. Confirmation of relatedness among the sequential C. albicans clinical isolates. Five of the seven sequential C. albicans clinical isolates were analysed by karyotyping (a) and RFLP of chromosomal DNA restricted with SfiI (b); shown are the ethidium bromide-stained gels. Rep-PCR was carried out on all seven sequential isolates plus 10 randomly chosen C. albicans clinical isolates from the SPRI culture collection. The dendrogram illustrates strain relatedness (c).

 
Antifungal susceptibilities

All of the isolates were susceptible to amphotericin B (Table 1). However, all seven isolates exhibited marked reductions in susceptibilities to azoles. According to NCCLS guidelines,13 all seven isolates were itraconazole-resistant (MIC >= 1 mg/L), the middle three isolates were fluconazole-resistant (MIC >= 64 mg/L) and the remainder were susceptible dose-dependent (SDD, MIC 16–32 mg/L). There are no breakpoints established for either voriconazole or posaconazole. However, based on a sample size of 4195 isolates, the MIC at which 90% of clinical C. albicans isolates were inhibited (MIC90) by voriconazole was 0.03 mg/L.15 Compared to this baseline value, the seven isolates exhibited on average a >100-fold decrease in susceptibility to voriconazole. Based on a sample size of 1900 strains, the MIC90 for posaconazole was 0.06 mg/L.16 Compared to this baseline value, the first four and the last three isolates exhibited four-fold and 30-fold decreases in posaconazole susceptibility, respectively.

Expression levels of efflux pump and ERG11 genes

Azole resistance has been associated with up-regulation of genes encoding efflux pumps and/or ERG11. We therefore quantified transcript levels of CDR1, CDR2, MDR1 and ERG11 in exponentially growing cultures using both northern blot analysis and real-time PCR. Northern blot analysis of five of the isolates revealed no significant changes in ERG11, MDR1 and CDR1 transcript levels (Figure 2a). However, the last two strains in the series, C376 and C375, exhibited minor (approximately three-fold) increases in CDR2 transcript levels. Real-time PCR was used to compare expression levels in the baseline strain, C369, with those in strains C373 and C375. Consistent with the northern blot analysis, the only changes in expression were minor (two- to three-fold) increases in expression of CDR2 in C373 and C375 (Figure 2b).



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Figure 2. Measurement of the expression levels of the ERG11, CDR1, CDR2 and MDR1 genes in seven sequential C. albicans clinical isolates. (a) Northern blot of total RNA from selected isolates analysed using a probe that hybridized non-specifically to both CDR1 and CDR2 (labelled CDR) and probes specific for ERG11, CDR1, CDR2 and MDR1. The loading of equal amounts of total RNA was confirmed using a probe specific for 18S rRNA. (b) Real-time PCR analysis of gene expression levels in isolates C369, C373 and C375. For each target gene, expression levels in C373 and C375 were compared with the expression level of the same gene measured in the baseline strain C369. ERG3, ERG25 and ACT1 were included as controls.

 
Nucleotide sequence of the ERG11 genes

The ERG11 genes from the seven isolates were sequenced. Compared to the published sequence from an azole-susceptible strain, the seven isolates all had the same seven silent mutations scattered throughout ERG11 (data not shown). In addition, all seven isolates had the same five missense mutations; the resultant amino acid substitutions are shown in Table 2. The fifth isolate in the series, C373, had acquired an additional mutation in one copy of the ERG11 gene resulting in a proline to leucine substitution at residue 230 (P230L). The last two isolates, C376 and C375, were homozygous for this mutation.


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Table 2. Amino acid substitutions in Erg11p in clinical C. albicans isolates
 
Expression of ERG11 alleles in an azole-susceptible S. cerevisiae strain

To verify that the amino acid substitutions in Erg11p were responsible for changes in azole susceptibility, the ERG11 alleles from an azole-susceptible strain, C43, along with those from isolates C369 and C375, were expressed in S. cerevisiae. The pattern of azole resistance in S. cerevisiae mirrored that seen in the original C. albicans isolates: the ERG11 alleles from isolates C369 and C375 conferred reduced susceptibility to posaconazole (Figure 3), itraconazole, fluconazole and voriconazole (data not shown). The ERG11 allele from isolate C375, which encodes the P230L substitution, conferred the highest levels of posaconazole (Figure 3) and itraconazole resistance (data not shown). However, the P230L substitution did not appear to impact resistance to voriconazole as the ERG11 alleles from C369 and C375 conferred the same levels of resistance to voriconazole (data not shown). The P230L substitution was introduced into an otherwise wild-type ERG11 gene and expressed in S. cerevisiae as above; by itself, the P230L substitution did not cause any change in the strain’s susceptibility to any of the azoles tested above (data not shown).



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Figure 3. Expression of C. albicans ERG11 alleles in S. cerevisiae. The ERG11 alleles from an azole-susceptible strain (C43) and from the first (C369) and last (C375) isolates in the series of azole-resistant C. albicans isolates were cloned in front of the GAL10 promoter on the multicopy plasmid YEp51. For each ERG11 allele, two independent clones were transformed into an azole-susceptible S. cerevisiae strain YKKB-13. Serial 10-fold dilutions of the transformants were spotted on YNB plates supplemented with 2% raffinose, 2% galactose, to induce expression from the GAL10 promoter, and posaconazole (POS) at the indicated concentrations.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The goal of this study was to characterize the molecular mechanisms of azole resistance in seven sequential C. albicans isolates from an AIDS patient receiving an extended course of posaconazole therapy. Of particular interest was the subset of isolates that exhibited significant decreases in susceptibility to azoles in current clinical use (voriconazole, fluconazole and itraconazole) yet remained susceptible to posaconazole. The baseline isolate, obtained before posaconazole therapy, was itraconazole-resistant, SDD to fluconazole and 140-fold less susceptible to voriconazole than wild-type C. albicans strains. The isolate also exhibited a minor (four-fold) decrease in susceptibility to posaconazole. Over the course of 6 months of posaconazole therapy, three additional isolates were cultured from the patient. All four isolates exhibited the same drug susceptibility patterns and molecular typing techniques confirmed that they were all closely related. None of the isolates exhibited any significant changes in the expression levels of genes encoding efflux pumps (CDR1, CDR2, and MDR1) or ERG11. However, since the baseline isolate exhibited significant decreases in susceptibility to fluconazole, itraconazole and voriconazole, it was conceivable that the isolate already had elevated pump/ERG11 expression levels. Previously, we used real-time PCR to measure CDR1, CDR2, MDR1 and ERG11 expression levels in 14 azole-susceptible (i.e. fluconazole MICs < 0.5 mg/L) clinical isolates. For each test gene, variation in expression levels (i.e. the range) and population averages were calculated (A. Chau et al., unpublished data). The expression levels of all four test genes in the baseline isolate analysed in this study (C369) fell within the same range calculated above suggesting that over-expression of either ERG11 or efflux pump genes was not responsible for the observed changes in azole susceptibility in this isolate (data not shown). The ERG11 genes were sequenced to determine whether the isolates had acquired mutations that reduced binding of the drugs to the 14{alpha}-demethylase enzyme. Each isolate had five missense mutations; three of the resulting substitutions (K128T, Y132H and G464S) were previously associated with azole resistance in C. albicans.1719 Furthermore, Y132H was shown to reduce fluconazole binding in vesicle preparations.7 Although the substitution D278N appears to be novel, D278E was identified in a fluconazole-resistant isolate.18 The substitution D116E has been identified in numerous azole-susceptible isolates and probably does not contribute to resistance.6,19,20

Three additional isolates were cultured from the patient after resumption of posaconazole therapy to treat a recurrence of refractory OPC. The first isolate was obtained after 5 months of therapy, a second isolate was obtained 3 months later and the final isolate was collected a month later, immediately after the termination of posaconazole therapy. All three isolates exhibited additional reductions in susceptibility to posaconazole and itraconazole, but no further changes in susceptibility to voriconazole or fluconazole. Molecular typing techniques (and the sequence of the ERG11 gene) confirmed that the three isolates were closely related to the prior isolates suggesting this was a breakthrough infection, rather than a new one. Compared to the baseline isolate, there were no large increases in either pump or ERG11 gene expression. Sequence analysis of ERG11 identified a sixth mutation in one copy of ERG11 in the first of these additional isolates, the next two isolates were homozygous for this mutation. The resulting substitution, P230L, appears to be novel. We confirmed that the P230L substitution was responsible for the additional decrease in posaconazole susceptibility by demonstrating that expression of this allele in S. cerevisiae conferred an elevated level of posaconazole resistance.

The first four isolates of the series had mutations in the C. albicans ERG11 gene that caused dramatic changes in their susceptibility to fluconazole and voriconazole, without significantly affecting posaconazole susceptibility. This is consistent with previous studies looking at azole resistance in both C. albicans21 and in Aspergillus fumigatus.22 These effects presumably result from differences in the way the azoles interact with Erg11p. All azoles interact with the haem cofactor of Erg11p via coordination between the free nitrogen on the triazole ring and the haem iron in the active site cavity of the enzyme. The Erg11p substitutions that specifically reduced binding of fluconazole and voriconazole are proposed to interfere with either the entry into and/or binding to the active site (K128T and Y132H), or are located close to the key cysteine residue involved in haem coordination (G464S).23 The fact that these mutations have relatively minor effects on posaconazole and itraconazole binding suggests that these azoles make additional contacts elsewhere in Erg11p. In a three-dimensional model of Erg11p constructed in-house,24 the long side chains of posaconazole and itraconazole (Figure 4) are enclosed within a channel and are predicted to make extensive hydrophobic contacts along their entire lengths. In the same model, Pro-230 is in close contact with the distal end of the posaconazole/itraconazole side chains and presumably the P230L substitution acts by disrupting binding of this segment of the drugs. However, it is important to note that the P230L substitution alone is not sufficient to disrupt binding of posaconazole and itraconazole; the presence of some, or all, of the additional substitutions is required to cause a shift in the posaconazole MIC.



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Figure 4. Structures of triazoles. (a) Posaconazole, (b) itraconazole, (c) voriconazole, (d) fluconazole.

 
In summary, we demonstrated that in a series of clinical C. albicans isolates, significant decreases in posaconazole susceptibility require multiple ERG11 mutations. Furthermore, posaconazole retained activity against isolates that exhibited reduced susceptibility to other azoles currently used in the treatment of mycoses. These findings indicate that posaconazole will be a valuable addition to the antifungal armamentarium and may have particular utility in the treatment of refractory OPC.


    Acknowledgements
 
Xin Li, Nathaniel Brown and Andrew Chau contributed equally to this work. We thank Robyn Hawkinson (SPRI) for technical help, Robert Palermo (SPRI), Michelle Treitel (SPRI) and Gavin Corcoran (SPRI) for helpful comments.


    Footnotes
 
* Corresponding author. Tel: +1-908-740-7644; Fax: +1-908-740-3918; E-mail: paul.mcnicholas{at}spcorp.com Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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