Introduction

Fungi have become major human pathogens, and the isolation of Candida species less susceptible to current therapies and the recovery of increasingly resistant isolates are growing problems [1•]. Several factors contribute to this epidemiological situation with advances made in medical care resulting in sicker patients being susceptible to fungi [2]. Candida albicans still remains most important, but other non-C. albicans species may result from selective pressures associated with the increased administration of antifungal agents [3••]. Antifungal drug resistance is characterized as microbiological or clinical. Microbiological resistance displays the non-susceptibility of a fungal pathogen to an antifungal agent determined by in vitro susceptibility testing when compared with isolates of the same species. Primary, or intrinsic, resistance refers to an organism’s natural susceptibility to an antimicrobial and reflects to be a predictable trait. This innate level of susceptibility is thought to be a drug-organism characteristic and independent of drug exposure such as given for Candida krusei and fluconazole [4, 5]. Secondary or acquired resistance is much less predictable and potentially more problematic. Under the exposure of antifungal agents, a fungal population initially susceptible may begin to express resistance. It is likely that resistance occurs as the result of several processes, including the emergence of a resistant variant from a common genotype [6], the selection of resistant strains from a mixed population [7], and reinfection with a new resistant strain [8]. The antifungal susceptibility patterns and frequencies of various Candida species isolated vary considerably among institutions and even among units in the same institution. In southern countries such as Italy, Spain, and South America, Candida parapsilosis [9] ranks second, while in northern countries, Candida glabrata takes this position [10]. Clinical resistance refers to infection persistence despite treatment with adequate therapy. Although microbiological resistance can contribute to the development of clinical resistance, other factors may also be involved, such as impaired immune function, underlying disease, reduced drug bioavailability, biofilm formation, and increased drug metabolism [11]. Hence, microbiological resistance is one of the factors underlying clinical resistance but not the most important one. The controlled studies of clinical importance of cross-resistance (CR) and multidrug resistance (MR) are lacking, but irrespective of the pathogen, the issue of CR and MR is most likely to be considered in seriously compromised individuals with invasive fungal infection and extensive exposure to antifungal drugs. This review will focus on clinical relevant ascomycete yeasts and their tendencies to develop antifungal resistance against commonly used compounds for systemic therapy. The term Candida is used in this review in its “applied clinical sense” (were all ascomycete yeasts are pooled in an artificial genus called Candida). The authors are aware that the genus Candida (based on morphological features only) was split up in several taxonomical valid genera which are distantly related [12]. Giving some examples, Candida lusitaniae was renamed to Clavispora lusitaniae and C. krusei to Issatchenkia orientalis, respectively. Further information on up-to-date nomenclature of yeast can be found in a recent publication by Schmalreck et al. [3••]. In the current review, we will mainly focus on MR and CR of the two major species C. albicans and C. glabrata. It is important for clinicians to be aware of trends and mechanisms responsible for the expression of resistance to incorporate this knowledge into up-to-date patient management.

Figure 1 gives an overview on antimycotics, their sites of action, and mechanism of resistance.

Fig. 1
figure 1

Primary targets and resistance mechanisms of major systemic antifungal drugs

Full size image

Microbial Resistance—a Clinical Issue?

From a clinical point of view, it is of interest to give answers on how commonly resistance occurs, how easy it is induced through therapy, and how often it results in the clinical failure of treatment? The answer differs within the clinical setting [9, 13, 14], type of fungal disease [15, 16], class of antifungal agents [17], and treatment duration [18, 19]. Clearly, we know that exposure to azoles is a significant risk factor for resistance development and that azole treatment leads to the selection of less susceptible species such as C. glabrata and C. krusei; in the past, the latter species predominated superficial infections [20] whereas in these days shift to blood stream infections [10, 21••]. In addition, appearance or disappearance of azole resistance depends on the rate of fungal growth, the number of mutations, or phenotypic changes necessary for resistance [6]. Fluconazole resistance remains uncommon in C. albicans (<5 %) but is more prevalent in C. parapsilosis (4–10 %) and Candida tropicalis (4–9 %) [22]. C. glabrata is a haploid species of Candida that has emerged as the second most common Candida organism associated with fungemia [23]. A likely contributing factor to the rapid growth (2 % in the 1970s to 20 % now) is the robust ability of C. glabrata to acquire tolerance to commonly deployed antifungal agents. Breakthrough fungal infections in bone marrow transplant patients receiving fluconazole prophylaxis [24] were attributed to C. glabrata displaying CR to fluconazole, voriconazole, itraconazole, and posaconazole [8]. Not only does C. glabrata relatively easily converts to an azole-resistant pathogen, but also becomes simultaneously MR.

Despite more than 30 years of clinical use, minimal resistance has developed to amphotericin B, and the drug continues to be important in the treatment of a variety of fungal pathogens. This may be due to its inherently fungicidal effect, limiting the selection of mutants. However, some Candida species including C. lusitaniae, C. glabrata, and Candida guilliermondii are capable of expressing resistance to amphotericin B [1•].

Echinocandins have established themselves as valuable agents for the treatment of candidiasis, and data show resistance to occur primary and secondary to mutation of the FKS1 gene [25]. One survey showed the frequency of C. parapsilosis, a species known for its reduced susceptibility to the echinocandins, to be increased after treatment with caspofungin (13 to 31 %) [13]. Clinical studies display Candida species less susceptible or resistant to caspofungin being more prevalent following treatment (30 days) with the drug (P < 0.001) in the ICU setting [14] and in patients suffering from hematological malignancies [13]; 7 days of exposure to echinocandin is sufficient to induce FKS mutations in C. glabrata [26], whereby the nature and/or the number of FKS mutations in C. glabrata and C. albicans influences in vivo resistance [27••]. FKS mutations were found in 7.9 % of 313 C. glabrata isolates from blood samples, and up to 80 % of patients infected with strains with both FKS mutations and high minimum inhibitory concentrations (MICs) for caspofungin experienced clinical failure or recurrent infection [28].

Epidemiological Cut-off Values and Clinical Breakpoints for Candida Species

The Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have developed standard susceptibility testing methods for Candida species based on broth microdilution [29–31]. Epidemiological cut-off values (ECOFF) and clinical breakpoints (CBPs) have been developed for MIC interpretation. ECOFF is defined as the upper limit of the wild-type population, thereof discriminates wild-type from resistant strains and is useful to monitor MIC trends. CLSI and EUCAST introduced species-specific clinical breakpoints for azoles and echinocandins, and the classification covers strains being susceptible, susceptible dose dependent, or resistant [32]. However, in vivo and in vitro outcome still is not perfect [33], which in turn renders CBP setting into to a permanent changing process taking into account latest news on MICs and clinical outcome data to optimize patient management. So far, the last correction of CLSI CBPs [5, 4] caused an increase of micafungin-resistant C. glabrata isolates from 0.8 to 7.6 % and of voriconazole-resistant isolates from 6.1 to 18.4 % [34]. Overall, these changes resulted in 5.7 % instead of 2.1 % of all isolates being resistant [34]. Whether the use of revised CBPs may improve the clinical predictive value of in vitro susceptibility tests needs to be validated in more detail.

Cross-Resistance and Multidrug Resistance in Candida Species

CR is defined as resistance that occurs for two or more antifungal substances of one similar chemical class with a similar mode of action, e.g., resistance against ≥two azoles or echinocandins [6]. CR might develop in organisms which have been exposed to the same or similar substance. MR is defined as resistance against structurally unrelated antifungal agents with different cellular targets. MR may emerge by the long-term exposure of structurally unrelated antifungals (e.g., simultaneous azole and echinocandin resistance) or by the interaction of two structurally unrelated agents with linked cellular mechanism (e.g., simultaneous azole and amphotericin B resistance) [6]. CR and MR both are specified on in vitro phenotypes and may be associated with in vivo outcome or therapeutic failures. The terms pan-azole and pan-echinocandin cover resistance against all systemically applied azoles and echinocandins [27••]. Unfortunately, the clinical impact of all various drug-bug profiles is not known due to limited data available.

Cross-Resistance Among Azoles and Echinocandins

CR against the various systemically applied triazole agents such as fluconazole, itraconazole, posaconazole, and voriconazole is well known for Candida species [35] and has been described for anidulafungin, caspofungin, and micafungin [36]. CR was found to be associated with various molecular mechanisms, and most frequently, triazole resistance is associated with point mutations (single nucleotide polymorphisms (SNPs)) in the ERG11 gene, while echinocandin resistance is frequently connected with SNPs in the FKS1p gene [37–39]. Whether SNPs cause silent or missense amino acid mutations is essential for resistance characteristics, as silent mutations to not lead to a change in the amino acid (aa) substitution, while missense mutations do. Also, the differentiation between haploid and diploid yeasts is key for the interpretation of SNPs; as for diploid organisms such as C. albicans, mutations are either heterozygous (affecting only one of both alleles) or homozygous (affecting both alleles). Haploid Candida species (e.g., C. glabrata) mutate more frequently than diploid Candida species (e.g., C. albicans). In practice, a diploid strain carrying a heterozygous mutation still has the capacity to produce the wild-type (WT) protein, while strains with homozygous mutations exclusively produce the mutated protein [27••].

Fluconazole is frequently used for the treatment of invasive candidiasis and candidemia and for prophylaxis in non-neutropenic patients [18]. An intrinsic resistance against fluconazole, e.g., for C. krusei, does not necessarily result in CR against other triazoles, as the majority of this species are susceptible to voriconazole. In contrast, acquired resistance is to a greater extent associated with CR. Pfaller et al. [10] reports of approximately 9.5 % of C. glabrata causing blood stream infections being resistant against fluconazole and voriconazole. Such findings are proved by numerous cases of breakthrough infections and therapeutic failures under azoles [40–44], and a switch to echinocandins was found to be successful in several cases [41, 42]. Resistance against azoles and echinocandins is rare and so far only observed in chronically infected patients receiving antifungal long-term treatment [27••]. A switch to amphotericin B might be less successful as azole-resistant isolates may carry simultaneously resistance against amphotericin B (see chapter azole-amphotericin B MR) [45–48].

For C. albicans, various combinations of CR exist, and among them, ketoconazole/fluconazole, itraconazole/miconazole, fluconazole/clotrimazole/itraconazole, and itraconazole/ketoconazole are most important [25]. Pan-azole resistance is associated with CDR1 and CDR2 overexpression and SNPs in the ERG11 gene encoding for the 14α-sterol demethylase [49, 50]. Other mechanisms are ERG11 overexpression, upregulation of multidrug efflux transporters (including ATP-binding cassette and major facilitator superfamily (MFS) transporters), and a bypass of the ergosterol pathway via accompanying mutations in the Δ5,6-desaturase gene (erg3). Prasad and Singh [51] reviewed the role of lipids involved in cross talks between different cellular circuits that influence the acquisition of multidrug resistance in Candida species. They summarized how the lipid composition of the cell membrane impacts on the localization and function of multidrug transporter proteins (CDR1). Notable is the fact that not all multidrug resistance transporters are affected by cell membrane lipid imbalance; MDR1 remains correctly localized and shows no functional lost. New regulatory circuitries potential impacting the development of multidrug resistance are identified by Dhamgaye et al. [52] using gene profiling and RNA-Seq data.

Only limited information is known for C. tropicalis, but it is speculated that the molecular mechanisms of azole CR are highly similar to those described for C. albicans. Forastiero et al. [46] demonstrated azole CR being related to coding mutations in the ERG11p with or without alternations in the ergosterol biosynthesis pathway. In a case report, Couzigou et al. [37] describes a pan-azole-resistant isolate of Candida kefyr which was found to carry two coding mutations in the ERG11. An overview of ERG11 point mutations that cause pan-azole resistance in Candida isolates is given in Table 1. Pan-azole resistance is not rare to see accompanied by amphotericin B resistance (see chapter azole-amphotericin B multi-resistances).

Table 1 Overview on ERG11 amino acid (aa) substitutions that lead to azole cross-resistance in Candida species
Full size table

Breakthrough fungal infections during echinocandin treatment are mainly caused by C. albicans, C glabrata, and C. parapsilosis [19, 21••, 53–57]. An overview of echinocandin resistance and potential treatment strategies was recently published by Beyda et al. [20]. Herein, the authors provide a comprehensive overview on species involved, underlying molecular resistance mechanisms being present and patient’s related outcome [20]. Point mutations in the FKS1p (encoded by the genes FKS1, FKS2, and FKS3) are mainly responsible for echinocandin resistance. Together with the regulatory protein RHO1p, FKS1p (catalytic subunit) forms the 1,3-β-d-glucan synthase [58]. In C. albicans, C. tropicalis, and C. krusei, echinocandin resistance is associated with mutations in the two FKS1 hot spot (hs) regions, hs 1 stretching from aa 641 to 649 and hs 2 stretching from aa 1345 to 1365 [59]. While for C. glabrata in addition to FKS1, also FKS2 hs 1 (aa 659–667) and hs 2 (1374–1381) are involved [59, 54, 60]. One of the most commonly found FKS1p mutation that leads to pan-echinocandin resistance is S645P; this mutation was reported from C. albicans [16] and C. kefyr [61]. In addition, substitutions of F641 [62, 57] or a loss of aa F641 (F641Δ) [61] are among the top mutations in the FKS1p in C. albicans, C. glabrata, and C. kefyr. Heat shock protein 90 upregulation was associated with enhanced echinocandin resistance, especially when accompanied with FKS1 mutation [20]. An overview on currently known FKS1 and FKS2 aa substitutions and their impact on echinocandin resistance is given in Table 2.

Table 2 Overview on FKS amino acid (aa) substitutions that lead to echinocandin cross-resistance in Candida species
Full size table

Multidrug Resistance Against Azoles and Echinocandins

The most commonly found MR is fluconazole resistance occurring simultaneously with echinocandin resistance. Pfaller et al. [10] found that about 11 % of all fluconazole-resistant C. glabrata isolates were also resistant against echinocandins, all of these carried FKS1 (S629P, R631G, D632Y, or D648E) or FKS2 (F659V, F659Y, S663P, or S663F) mutations. Bizerra et al. [54] reported an isolate being pan-echinocandin, fluconazole, and voriconazole resistant after approximately 3 weeks of exposure to fluconazole and micafungin. Lackner et al. [27••] detected a pan-azole and pan-echinocandin resistant C. albicans from a patient suffering from CMC and receiving azole and echinocandin therapy for longer than 1 year. C. glabrata collected from patients receiving multiple antifungal treatment regimens display major therapeutic challenges [15, 28, 63, 64]. To the best of our knowledge so far, no Candida isolates were described that exhibit both amphotericin B and echinocandin resistance.

Multiresistance Against Azoles and Amphotericin B

In contrast to azoles, amphotericin B targets membrane-bound ergosterol; its high affinity to ergosterol, but low affinity to ergosterol’s precursors (such as lanosterol, fecosterol, lichesterol, and episterol) favors the replacement of ergosterol in the fungal cell membrane by its precursors and thus led to the development of polyene resistance [26]. Major resistance mechanisms of amphotericin B are quantitative and qualitative changes of the ergosterol cell membrane composition; enzyme activity of ERG2, ERG3, and ERG5, or mutations in these ERG2, ERG3, and ERG5, respectively, regulate the ergosterol content [65]. The potential to develop amphotericin B resistance depends on the species but is higher for C. glabrata and C. parapsilosis [26]. In contrast to amphotericin B, azoles inhibit a key enzyme in the biosynthetic pathway of ergosterol, namely lanosterol 14-α demethylase. This enzyme belongs to the P-450 cytochromes, and its catalytic site is the primary target of azoles. The inhibition of this enzyme results in the accumulation of ergosterol precursors in the plasma membrane with the subsequent hampering of the integrity and cellular processes. This azole and amphotericin B action results in reduced cell membrane ergosterol, which in turn explains at least partially MR of Candida to structurally unrelated substances. Other MR mechanisms found to be the upregulation of stress response and transporter and efflux pumps.

The first clinical pan-azole- and polyene-resistant C. albicans with mutations in ERG5 and ERG11 was reported in 2010 by Martel et al. [65]. The isolate showed an aa substitution in ERG11p gene at position A114S and a sequence repetition of 10 nucleotides in ERG5p gene. A nucleotide repetition in ERG5p led to nullified C22 desaturase; as a consequence, fungal cell membrane contained no ergosterol but >80 % of total sterol fraction consisted of ergosta-5,7-dienol [65]. In 2012, a clinical C. glabrata was identified with a missense mutation in ERG11p which leads to CR against fluconazole and voriconazole; in addition, a shift in the sterol composition favored accompanying amphotericin B resistance [47]. C. albicans and C. tropicalis clinical isolates with resistance against amphotericin B and azoles were discovered during a screening study by Eddouzi et al. [66]. The underlying resistance mechanism of C. albicans was explained by the overexpression of a multidrug efflux pump of the major facilitator superfamily Mdr1. C. tropicalis lacked ergosterol in its cell membrane, instead 14α-methyl-fecosterol was accumulated which indicates the functional perturbation of at least two main ergosterol biosynthesis proteins (ERG11 and ERG3).

Multi-Resistance Against Azoles and 5-Fluorouracil

Gabriel et al. [67] showed that the simultaneous application of fluorinated nucleotides (e.g., 5-fluorouracil) at subinhibitory doses and fluconazole triggers resistance against fluconazole in vitro. The authors speculated that intracellular fluorinated nucleotides may play a role in azole resistance by either preventing azoles to target the lanosterol 14-alpha-demethylase, or by preventing azoles to bind to the lanosterol 14-alpha-demethylase catalytic site, or by acting as molecular switch for triggering the efflux transport. These data remain to be verified in greater detail to finally state the molecular mechanism behind. Moreover, these findings need to be validated in murine studies.

Conclusion

Reports of antifungal drug resistance are emerging and a matter of serious concern (see Fig. 2). Positive is the fact that antifungal treatment is still successful, as 80 % of C. albicans infections in ICUs are cleared with echinocandins [9]. However, CR as well as MR may be associated with worse clinical outcome, breakthrough fungal infections [68], multiple changes of treatment regimens and increased health care costs. Clinical improvement failed in patients infected with fluconazole and voriconazole-resistant Candida isolates when compared to susceptible strains [8]; similar findings are valid for resistance to echinocandins among various Candida species [69–71]. Most worrying is the emergence of acquired resistance of C. glabrata against the azoles and echinocandins; the limited number of antifungals renders these phenotypes to an emerging pathogen.

Fig. 2
figure 2

Common problems associated with antifungal drug resistant pathogens

Full size image

Limited data are available on the economic impact of resistant Candida infections. However, it has been calculated that fungal infections add a total of US$8 billion to annual health care costs [72]. Resistant infections are thought to substantially increase these expenditures because of reinforced patients’ management consisting of a prolonged therapy, change of drug regimen applied, or rather using a combination, intense diagnostic procedures such as biopsies, as well as isolation procedures. Strategies for preventing the emergence and spread of antifungal drug resistance include the implementation of Antimicrobial Stewardship Programs covering (i) local fungal epidemiology and antifungal resistance rates, (ii) establishing therapeutic guidelines, (iii) implementation of treatment strategies for empirical and preemptive therapy including PK/PD data, (iv) catheter management, and (v) selection of adequate diagnostic assays.