Molecular mechanisms of azole resistance in Candida bloodstream isolates

Background Antifungal resistance rates are increasing. We investigated the mechanisms of azole resistance of Candida spp. bloodstream isolates obtained from a surveillance study conducted between 2012 and 2015. Methods Twenty-six azole non-susceptible Candida spp. clinical isolates were investigated. Antifungal susceptibilities were determined using the Sensititre YeastOne® YO10 panel. The ERG11 gene was amplified and sequenced to identify amino acid polymorphisms, while real-time PCR was utilised to investigate the expression levels of ERG11, CDR1, CDR2 and MDR1. Results Azole cross-resistance was detected in all except two isolates. Amino acid substitutions (A114S, Y257H, E266D, and V488I) were observed in all four C. albicans tested. Of the 17 C. tropicalis isolates, eight (47%) had ERG11 substitutions, of which concurrent observation of Y132F and S154F was the most common. A novel substitution (I166S) was detected in two of the five C. glabrata isolates. Expression levels of the various genes differed between the species but CDR1 and CDR2 overexpression appeared to be more prominent in C. glabrata. Conclusions There was interplay of various different mechanisms, including mechanisms which were not studied here, responsible for azole resistance in Candida spp in our study.


Background
Candida bloodstream infections are an important healthcare issue known to be associated with high morbidity and mortality. There have been increasing reports of antifungal resistance. We have previously reported decreasing azole susceptibilities in our hospital, particular in Candida tropicalis. More than 20% of C. tropicalis were non-susceptible to fluconazole [1]. There are various mechanisms mediating azole resistance. It has been suggested that molecular mechanisms such as presence of mutations may be a predictive marker of clinical failure in Candida infections [2]. Whilst this has been established for echinocandin resistance, azole resistance mechanisms are not as well studied, particularly for non-albicans species. Elucidation of these mechanisms is crucial to make progress in understanding and treating invasive Candida infections.

Methods
In this study, we characterised the molecular mechanisms of azole resistance in 26 fluconazole non-susceptible Candida bloodstream isolates. These isolates were identified from a retrospective surveillance study conducted at a major regional tertiary referral hospital between 2012 and 2015 [1]. In brief, non-duplicate Candida bloodstream isolates from all adult inpatients (at least 21 years old) with temporally-related clinical signs and symptoms of infection admitted to the hospital during the study period were included. Antifungal susceptibility testing was performed using Sensititre YeastOne® YO10 panel (Trek Diagnostics System, West Sussex, England) according to manufacturer's recommendations. Minimum inhibitory concentrations were interpreted according to the current species-specific clinical breakpoints provided by the Clinical and Laboratory Standards Institute (CLSI) M27-S4 document or epidemiological cut-off values (ECV), where CLSI breakpoints were unavailable [3,4]. For Candida albicans and C. tropicalis, isolates meeting the susceptible-dose-dependent (SDD) and resistant criteria were included, whereas only resistant Candida glabrata were included in this study. A total of 26 fluconazole non-susceptible isolates [C. albicans -4/62 (6%); C. glabrata -5/82 (6%); C. tropicalis -17/78 (22%); C. parapsilosis -0/35 (0%)] were identified from 257 Candida spp. isolates included in the surveillance study.
ERG11, CDR1, CDR2 and MDR1 gene expression were quantified in triplicates using real-time reverse transcription-PCR (RTPCR) with total RNA extracted from exponential-phase yeast peptone dextrose broth cultures on a CFX96 Real-Time PCR Detection System (Bio-Rad, USA). The primers used were adopted from previous publications [5][6][7][8][9][10][11] Quantification of target genes was normalised to the level of ACT1, an endogenous reference gene. Relative gene expression was calculated as the fold change in expression of the isolates compared to the respective ATCC reference strains (C. albicans ATCC 90028, C. glabrata ATCC 2950, C. tropicalis ATCC 750). A fold increase of 3 times was considered to be an overexpression of the target gene. The ERG11 gene was amplified and sequenced to identify amino acid mutations by comparing with reference wild-type GenBank sequences (C. albicans -X13296; C. tropicalis -M23673; C. glabrata -L40389).

Results
The susceptibility profiles of the isolates are displayed in Table 1. Cross-resistance to all azoles was observed in all isolates except for one C. albicans (CW138) and two C. Among the different gene targets, it appeared that ERG11 expression levels were mostly similar compared to the respective wild-type reference strains. CDR2 expression was consistently elevated in fluconazole non-susceptible C. albicans. In the two resistant isolates with MIC 128 μg/mL, MDR1 was also up-regulated. CDR1 and CDR2 co-expression was observed in all C. glabrata isolates. Gene overexpression was not consistent among C. tropicalis isolatesthere were five isolates with CDR1 overexpression and six isolates with MDR1 overexpression. All C. tropicalis isolates only had overexpression of a single gene target. Interestingly, there were three C. tropicalis isolates with no ERG11 mutations or any gene up-regulation.

Discussion
In this study, we evaluated the molecular mechanisms associated with azole resistance in various Candida species in our institution. Identification of antifungal susceptibilities through phenotypic methods such as MIC testing is often limited by the length of time required. Furthermore, current fungal MIC breakpoint interpretations are not supported by robust clinical data and are not predictive of clinical success/failure. Therefore, there is interest in identifying genotypic markers which could be rapidly identified for use in clinical prediction. Various previous studies have investigated different mechanisms of azole resistance in Candida species [5,[12][13][14]. Some of these studies have identified key ERG11 substitutions which are associated with azole resistance e.g. Y132F, S154F [8,15] and suggested that these mutations could be potential predictive markers of azole resistance.
In our context, it appeared that there was an interplay of various different mechanisms, including mechanisms which were not studied here, responsible for azole resistance in Candida spp. ERG11 mutations were commonly detected in C. albicans, whereas the role of overexpression of azoles efflux pumps appeared to be more prominent in C. albicans (CDR1) and C. glabrata (CDR1, CDR2). In C. tropicalis, presence of Y132F and S154F substitutions was unable to explain the mechanisms of majority of our isolates. Less than half of the azole-resistant C. tropicalis harboured these amino acid substitutions. This was in contrast to the high frequency identified in another local study where > 90% of the isolates had Y132F and S154F substitutions [15]. Likewise, in another study, these mutations accounted for 95% of the fluconazole-resistant C. tropicalis [16].
Our study was limited by the small sample size although we had included all azole-resistant bloodstream isolates between 2012 and 2015. In addition, we did not perform further functional verification of the ERG11 mutations and homology modelling experiments, therefore the clinical significance of I166S amino acid substitution in C. glabrata remains to be validated.

Conclusions
In conclusion, our results indicated that the mechanisms mediating azole resistance in our isolates are heterogeneous. There were isolates with unidentified resistance mechanisms warranting further exploration. Moving ahead, the use of more advanced molecular technologies such as next-generation sequencing might be considered for an in-depth molecular characterisation of azole-resistant Candida spp to aid the identification of potential resistance markers.