MDR1 overexpression combined with ERG11 mutations induce high-level fluconazole resistance in Candida tropicalis clinical isolates

In recent years, Candida species have emerged as important causes of nosocomial infections leading to high morbidity and mortality, particularly in immunocompromised patients [1‐3]. In China, the prevalence of Candida tropicalis is similar to that of Candida parapsilosis, making it the second most common non-albicans Candida species [4].

Currently, azole antifungal agents are widely used in the battle against clinical Candida infections [5]. Due to the lower risk of side effects and low cost associated with fluconazole, it has become one of the most commonly used antifungals [6]. Until recently, C. tropicalis was susceptible to azole and other antifungal agents. However, azole resistance in C. tropicalis has been recently observed, possibly owing to the extensive use of antifungals in medical institutions [7]. Pan-azole-resistant C. tropicalis isolates, which pose serious clinical challenges, have also been found in multiple countries [8‐10]. Indeed, the widespread use of azoles has been deemed to be an independent risk factor for fluconazole resistance in Candida species [11]. In addition, C. tropicalis appears to develop fluconazole resistance much more rapidly than other Candida species under in vitro selection [12].

Azole resistance in Candida species has been associated with mutations in or overexpression of the ERG11 gene or with upregulation of the CDR1 and MDR1 genes, the products of which are active transport pumps [13]. ERG11 gene mutations and overexpression have been demonstrated to be responsible for azole resistance in C. tropicalis clinical isolates in China [14]. However, there is still insufficient evidence of the role of the MDR1 and CDR1 genes in azole resistance of C. tropicalis clinical isolates. Therefore, in order to better understand the molecular mechanisms underlying azole resistance in C. tropicalis, we obtained a collection of fluconazole-resistant C. tropicalis isolates from a tertiary care teaching hospital and evaluated them for efflux pump gene expression level as well as ERG11 missense mutations. Our findings suggest that ERG11 missense mutations and increased MDR1 gene expression are highly correlated with fluconazole resistance in C. tropicalis clinical isolates.

C. tropicalis has received widespread attention in recent years due to an increase in fluconazole resistance and the high mortality rate associated with infections [7, 18]. In a global surveillance study conducted in 2013, 31.8% of non-susceptible C. tropicalis isolates worldwide were obtained from mainland China [19]. Moreover, the rate of fluconazole resistance of C. tropicalis increased from 11.2 to 42.7% between 2011 and 2015 in China [7]. However, understanding of the mechanisms underlying azole resistance in clinical C. tropicalis isolates is insufficient compared to that of other Candida species.

Studies have demonstrated that Erg11p amino acid substitutions in the 14α-sterol demethylase involved in the biosynthesis of sterol ergosterol results in decreased fluconazole susceptibility in C. tropicalis clinical isolates [9, 20, 21]. Jiang et al. demonstrated that two mutations, Y132F and S154F, are responsible for azole resistance by inserting them into wild-type isolate [14]. Similarly, the amino acid substitutions Y132F and S154F in Erg11p were found in all fluconazole-resistant isolates except one (Ct02R: MIC, 8 mg/L) in our study.

A previous study confirmed that the UPC2 gene, which encodes a global transcriptional regulator of the ERG11 gene, was up-regulated in azole-resistant C. tropicalis isolates [17]. Therefore, we examined UPC2 gene expression levels in 12 fluconazole-resistant isolates. However, we found that there was no difference in the expression of the UPC2 gene between fluconazole-resistant and control isolates. These results suggest that ERG11 overexpression, which results in up-regulation of target enzymes, is not involved in azole resistance in fluconazole-resistant isolates.

In addition to the targeted mutations and overexpression of the enzyme Erg11p, we found that overexpression of the efflux pumps Mdr1p and Cdr1p, encoded by the MDR1 and CDR1 genes, are involved in C. tropicalis azole resistance [22]. Consistent with this finding, pan-azole resistant C. tropicalis isolate, which overexpresses the MDR1 gene and contains the Y132F and S154F amino acid substitutions in Erg11p, has reportedly been isolated from a patient with acute lymphoblastic leukemia [8]. In the present study, we found that fluconazole-resistant isolates had significantly higher expression levels of the MDR1 gene than control isolates. Furthermore, high-level fluconazole-resistant isolates had higher levels of MDR1 expression than low-level fluconazole-resistant isolates. Most importantly, among the 12 fluconazole-resistant isolates, MDR1 expression levels were highest in the three pan-azole-resistant isolates. Hence, we conclude that overexpression of the MDR1 gene is associated with high-level fluconazole resistance in C. tropicalis clinical isolates. Additionally, these results indicate that the combination of MDR1 gene overexpression and the amino acid substitutions Y132F and S154F in Erg11p is an important mechanism of pan-azole resistance in C. tropicalis clinical isolates.

Of the 12 fluconazole resistant isolates obtained, four belonged to DST225, including two pan-azole resistant isolates (Ct07R and Ct09R). Interestingly, human and fruit samples have recently been found to contain DST225 C. tropicalis in Taiwan and these isolates have been shown to be less susceptible to azole drugs [23]. This suggests that C. tropicalis fluconazole resistance is related to specific genotypes, such as DST225.

In conclusion, specific mutations in the ERG11 gene and upregulation of drug efflux pumps are the most common mechanisms underlying azole resistance in C. tropicalis. This study finds that the MDR1 gene may have a role in determining a high-level of fluconazole resistance and pan-azole resistance in C. tropicalis clinical isolates. To the best of our knowledge, this is the first study investigating the relationship between MDR1 gene overexpression and high-level fluconazole resistance. However, further studies are needed to identify the mechanisms by which MDR1 is overexpressed. Homologs of gain-of-function mutations in the C. parapsilosis MRR1 gene, a regulator of the MDR1 gene [24], also need to be further examined in the future.