In Vivo and In Vitro Acquisition of Resistance to Voriconazole by Candida krusei

Abstract

e Candida krusei is an important agent of opportunistic infections that often displays resistance to several antifungals. We describe here the in vivo acquisition of resistance to voriconazole (VRC) by C. krusei isolates recovered from a leukemia patient during a long period of VRC therapy. In order to mimic the in vivo development of VRC resistance, a susceptible C. krusei isolate was exposed daily to 1 g/ml of VRC in vitro. Interestingly, after 5 days of exposure to VRC, a MIC of 4 g/ml was achieved; this value remained constant after 25 additional days of treatment with VRC and also after 30 consecutive days of incubation in VRC-free medium. Our objective was to determine the associated molecular resistance mechanisms, such as expression of efflux pump genes and ERG11 gene mutations, among the resistant strains. Synergistic effects between the efflux blocker tacrolimus (FK506) and VRC were found in all of the resistant strains. Moreover, ABC1 gene expression increased over time in both the in vivo-and in vitro-induced resistant strains, in contrast to the ABC2 and ERG11 genes, whose expression was invariably lower and constant. ERG11 gene sequencing showed two different types of mutations, i.e., heterozygosity at T1389T/C, corresponding to synonymous mutations, in C. krusei strains and a missense mutation at position T418C, resulting in a change from Tyr to His, among resistant C. krusei clinical isolates. This study highlights the relevance of ATP-dependent efflux pump (namely, Abc1p) activity in VRC resistance and describes new mutations in the ERG11 gene among resistant C. krusei clinical isolates.

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

“…However, more recently it has been suggested that overexpression of genes encoding both Erg11p and the efflux pump Abc2p may also play a role with itraconazole resistance (Tavakoli et al, 2010; He et al, 2015). Despite its fungicidal activity in C. krusei (Rubio et al, 2005), resistance to voriconazole has also emerged, and current research supports a theory where overexpression of the genes encoding the efflux pump Abc2 and Erg11 impart more transient resistance properties, while increased expression of Abc1p and point mutations in ERG11 predominate as time progresses to yield a stably resistant pathogen in the prolonged presence of voriconazole (Ricardo et al, 2014). Erg11p amino acid substitutions have been observed in azole-resistant C. krusei and, in the case of Y166S, have been predicted to interfere with Erg11p function (Ricardo et al, 2014; Silva et al, 2016).…”

“…Despite its fungicidal activity in C. krusei (Rubio et al, 2005), resistance to voriconazole has also emerged, and current research supports a theory where overexpression of the genes encoding the efflux pump Abc2 and Erg11 impart more transient resistance properties, while increased expression of Abc1p and point mutations in ERG11 predominate as time progresses to yield a stably resistant pathogen in the prolonged presence of voriconazole (Ricardo et al, 2014). Erg11p amino acid substitutions have been observed in azole-resistant C. krusei and, in the case of Y166S, have been predicted to interfere with Erg11p function (Ricardo et al, 2014; Silva et al, 2016). While the newer antifungal agents posaconazole and isavuconazole have shown good activity against C. krusei (Lee et al, 2000; Rybak et al, 2015), reports of resistance against these agents are relatively sparse (Espinel-Ingroff et al, 2014; Pfaller et al, 2015).…”

Fluconazole resistance in <em>Candida</em> species: a current perspective

“…Other studies have detected point mutations in ERG11 and linked them to reduced susceptibility, but these studies are in relation to azoles other than fluconazole. 56 , 57 …”

Antifungal Therapy: New Advances in the Understanding and Treatment of Mycosis

“…The substitutions R467K, I471T, G464S, S405F, and K143R were only related to azole-resistant C. albicans isolates (Sanglard et al, 1998; Lamb et al, 2000; Morio et al, 2010), with the most common being R467K and G464S (Morio et al, 2010). ERG11 mutations have been described for other azole-resistant clinical Candida isolates, including C. dubliniensis (Perea et al, 2002), C. krusei (Ricardo et al, 2014), C. tropicalis (Vandeputte et al, 2005; Jiang et al, 2013) and more recently C. parapsilosis (Grossman et al, 2015). However, the same finding has not yet described for C. glabrata (Gonçalves et al, 2016).…”