Mechanism of Fluconazole Resistance in Candida krusei

The mechanisms of fluconazole resistance in three clinical isolates of Candida krusei were investigated. Analysis of sterols of organisms grown in the absence and presence of fluconazole demonstrated that the predominant sterol of C. krusei is ergosterol and that fluconazole inhibits 14α-demethylase in this organism. The 14α-demethylase activity in cell extracts of C. krusei was 16- to 46-fold more resistant to inhibition by fluconazole than was 14α-demethylase activity in cell extracts of two fluconazole-susceptible strains of Candida albicans. Comparing the carbon monoxide difference spectra of microsomes from C. krusei with those of microsomes from C. albicans indicated that the total cytochrome P-450 content of C. krusei is similar to that of C. albicans. The Soret absorption maximum in these spectra was located at 448 nm for C. krusei and at 450 nm for C. albicans. Finally, the fluconazole accumulation of two of the C. krusei isolates was similar to if not greater than that of C. albicans. Thus, there are significant qualitative differences between the 14α-demethylase of C. albicans and C. krusei. In addition, fluconazole resistance in these strains of C. krusei appears to be mediated predominantly by a reduced susceptibility of 14α-demethylase to inhibition by this drug.     

Treatment of murine Candida krusei or Candida glabrata infection with L-743,872.

L-743,872 is a broad-spectrum pneumocandin antifungal drug developed by Merck Research Co., and in the present work it was evaluated in vivo in murine models of Candida krusei and Candida glabrata infection. Mice were infected intravenously with two isolates of C. krusei and treated with fluconazole or L-743,872. Fluconazole was beneficial only in immune-competent mice infected with isolate 94-2696. At > 0.5 mg/kg of body weight/day, L-743,872 was effective against both infecting isolates in immune-competent and immune-suppressed mice. Against C. glabrata, L-743,872 was effective, at doses > or = 0.5 mg/kg, in reducing fungal cell counts in the kidneys but not in the spleen. L-743,872 has significant potential for clinical development.     

Rapid Development of Candida krusei Echinocandin Resistance during Caspofungin Therapy

In invasive candidiasis, there has been an epidemiological shift from Candida albicans to non-albicans species infections, including infections with C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei. Although the prevalence of C. krusei remains low among yeast infections, its intrinsic resistance to fluconazole raises epidemiological and therapeutic concerns. Echinocandins have in vitro activity against most Candida spp. and are the first-line agents in the treatment of candidemia. Although resistance to echinocandin drugs is still rare, individual cases of C. krusei resistance have been reported in recent years, especially with strains that have been under selective pressure. A total of 15 C. krusei strains, isolated from the blood, urine, and soft tissue of an acute lymphocytic leukemia patient, were analyzed. Strains developed echinocandin resistance during 10 days of caspofungin therapy. The molecular epidemiology of the isolates was investigated using two different typing methods: PCR-based amplification of the species-specific repetitive polymorphic CKRS-1 sequence and multilocus sequence typing. All isolates were genetically related, and the mechanism involved in decreased echinocandin susceptibility was characterized. Clinical resistance was associated with an increase in echinocandin MICs in vitro and was related to three different mutations in hot spot 1 of the target enzyme Fks1p. Molecular evidence of the rapid acquisition of resistance by different mutations in FKS1 highlights the need to monitor the development of resistance in C. krusei infections treated with echinocandin drugs.     

Efficacy of Triazoles in a Murine Disseminated Infection by Candida krusei

We evaluated the efficacies of posaconazole and voriconazole in comparison with that of amphotericin B in a systemic murine infection by Candida krusei. Posaconazole at 50 mg/kg/day and voriconazole at 40 and 60 mg/kg/day prolonged survival and reduced the fungal tissue burden in the kidneys of mice similarly to amphotericin B at 1.5 mg/kg/day and liposomal amphotericin B at 10 mg/kg/day. None of the treatments tested completely resolved the infection.Candidiasis has become one of the most frequent causes of nosocomial infections. Fluconazole (FLC) is the recommended drug, but several non-albicans Candida species such as Candida krusei have an intrinsic resistance to FLC. This species is the fifth most common Candida species to cause candidemia. An ideal therapy does not yet exist for C. krusei infections, and a high mortality rate is reported (15). Currently recommended antifungals for the treatment of disseminated C. krusei infections are amphotericin B (AMB) and echinocandins, with voriconazole (VRC) being regarded as an alternative (11, 24). Posaconazole (PSC) is a promising drug, though not yet explored enough in vivo, that shows in vitro MICs against C. krusei similar to or lower than those of VRC (12). In this study, we have tested the triazoles VRC and PSC, comparing their efficacies with those of two different formulations of AMB, in an immunocompromised murine model of disseminated infection by C. krusei.Two clinical strains of C. krusei, FMR 9728 and FMR 9729, were used. The inocula containing ≥99% of the viable cells for both the in vitro and in vivo studies were adjusted to the desired concentration by counting them with a hemocytometer. The in vitro susceptibilities of both strains were determined using a reference method (7). The minimal fungicidal concentration (MFC) was defined as a 99.9% or greater reduction in the number of CFU/ml (2) (Table ​(Table11).

TABLE 1.

In vitro antifungal activity of AMB, VRC, and PSC against the two strains of C. krusei^a

Ergosterol biosynthesis inhibitors become fungicidal when combined with calcineurin inhibitors against Candida albicans,Candida glabrata,and Candida krusei

Azoles target the ergosterol biosynthetic enzyme lanosterol 14alpha-demethylase and are a widely applied class of antifungal agents because of their broad therapeutic window, wide spectrum of activity, and low toxicity. Unfortunately, azoles are generally fungistatic and resistance to fluconazole is emerging in several fungal pathogens. We recently established that the protein phosphatase calcineurin allows survival of Candida albicans during the membrane stress exerted by azoles. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) are dramatically synergistic with azoles, resulting in potent fungicidal activity, and mutant strains lacking calcineurin are markedly hypersensitive to azoles. Here we establish that drugs targeting other enzymes in the ergosterol biosynthetic pathway (terbinafine and fenpropimorph) also exhibit dramatic synergistic antifungal activity against wild-type C. albicans when used in conjunction with CsA and FK506. Similarly, C. albicans mutant strains lacking calcineurin B are markedly hypersensitive to terbinafine and fenpropimorph. The FK506 binding protein FKBP12 is required for FK506 synergism with ergosterol biosynthesis inhibitors, and a calcineurin mutation that confers FK506 resistance abolishes drug synergism. Additionally, we provide evidence of drug synergy between the nonimmunosuppressive FK506 analog L-685,818 and fenpropimorph or terbinafine against wild-type C. albicans. These drug combinations also exert synergistic effects against two other Candida species, C. glabrata and C. krusei, which are known for intrinsic or rapidly acquired resistance to azoles. These studies demonstrate that the activity of non-azole antifungal agents that target ergosterol biosynthesis can be enhanced by inhibition of the calcineurin signaling pathway, extending their spectrum of action and providing an alternative approach by which to overcome antifungal drug resistance.     

Reduced accumulation of drug in Candida krusei accounts for itraconazole resistance.

Due to intrinsic resistance Candida krusei is emerging as a systemic pathogen in AIDS patients undergoing fluconazole therapy, but acquired resistance to itraconazole has not been studied biochemically. We report here studies on the basis for azole resistance and sterol composition in C. krusei. An itraconazole-resistant isolate showed reduced susceptibility to azole drugs in in vitro growth inhibition studies. Accumulation of 14 alpha-methyl-3,6-diol under azole treatment was associated with growth arrest. In vitro ergosterol biosynthesis and type II binding studies suggested no alteration in the affinity to azole drugs of the target enzyme, the cytochrome P-450 sterol 14 alpha-demethylase, in the resistant isolate. Resistance was associated with a decreased intracellular content of drug in the resistant isolate.     

米卡芬净治疗光滑念珠菌和克柔念珠菌致念珠菌血症或侵袭性念珠菌病的转归

念珠菌属是医院获得性血流感染的常见病原菌之一,目前光滑念珠菌和克柔念珠菌约占念珠菌血症或侵袭性念珠菌病病原菌的25%。多项临床试验证实米卡芬净治疗念珠菌血症有效,但单个试验有一定局限性.光滑念珠菌和克柔念珠菌例数较少,本研究对2例评估米卡芬净治疗念珠菌血症及侵袭性念珠菌病的随机对照试验进行分析,评价米卡芬     

Comparison of posaconazole and voriconazole in vitro killing against Candida krusei

The in vitro activity of posaconazole and voriconazole was evaluated by MIC, minimum fungicidal concentration (MFC), and time-kill methods. MFCs were determined for 15 Candida krusei and time-killing curves for 5 of these isolates. MFCs were obtained transferring 100 microL from clear MIC wells onto Sabouraud dextrose agar. Time-kill studies were performed in RPMI 1640 medium (5 mL, inoculum approximately 10(5) colony-forming unit [CFU]/mL). Geometric mean (GM) MIC and GM-MFC were 0.2 and 0.72 mg/L for posaconazole and 0.4 and 2.64 mg/L for voriconazole. The killing rate was isolate and concentration dependent; reductions in CFUs start at >or=0.12 mg/L (posaconazole) and >or= 0.5 mg/L (voriconazole). The mean time to reach a 90% growth reduction was 41.5+/-14.2 h (8 mg/L posaconazole) and 48.7+/-61.3 h (32 mg/L voriconazole). By time kill, a 99% killing rate was not reached by either agent. Both methods demonstrated that posaconazole (more active and greater killing rate) and voriconazole have fungicidal activity against C. krusei.     

Prior antimicrobial therapy and risk for hospital-acquired Candida glabrata and Candida krusei fungemia: a case-case-control study

The incidence of infections caused by Candida glabrata and Candida krusei, which are generally more resistant to fluconazole than Candida albicans, is increasing in hospitalized patients. However, the extent to which prior exposure to specific antimicrobial agents increases the risk of subsequent C. glabrata or C. krusei candidemia has not been closely studied. A retrospective case-case-control study was performed at a university hospital. From 1998 to 2003, 60 patients were identified with hospital-acquired non-C. albicans candidemia (C. glabrata or C. krusei; case group 1). For comparison, 68 patients with C. albicans candidemia (case group 2) and a common control group of 121 patients without candidemia were studied. Models were adjusted for demographic and clinical risk factors, and the risk for candidemia associated with exposure to specific antimicrobial agents was assessed. After adjusting for both nonantimicrobial risk factors and receipt of other antimicrobial agents, piperacillin-tazobactam (odds ratio [OR], 4.15; 95% confidence interval [CI], 1.04 to 16.50) and vancomycin (OR, 6.48; CI, 2.20 to 19.13) were significant risk factors for C. glabrata or C. krusei candidemia. For C. albicans candidemia, no specific antibiotics remained a significant risk after adjusted analysis. Prior fluconazole use was not significantly associated with either C. albicans or non-C. albicans (C. glabrata or C. krusei) candidemia. In this single-center study, exposure to antibacterial agents, specifically vancomycin or piperacillin-tazobactam, but not fluconazole, was associated with subsequent hospital-acquired C. glabrata or C. krusei candidemia. Further studies are needed to prospectively analyze specific antimicrobial risks for nosocomial candidemia across multiple hospital centers.     

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

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 μg/ml of VRC in vitro. Interestingly, after 5 days of exposure to VRC, a MIC of 4 μg/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.     

Genetic basis for differential activities of fluconazole and voriconazole against Candida krusei

Invasive infections caused by Candida krusei are a significant concern because this organism is intrinsically resistant to fluconazole. Voriconazole is more active than fluconazole against C. krusei in vitro. One mechanism of fluconazole resistance in C. krusei is diminished sensitivity of the target enzyme, cytochrome P450 sterol 14alpha-demethylase (CYP51), to inhibition by this drug. We investigated the interactions of fluconazole and voriconazole with the CYP51s of C. krusei (ckCYP51) and fluconazole-susceptible Candida albicans (caCYP51). We found that voriconazole was a more potent inhibitor of both ckCYP51 and caCYP51 in cell extracts than was fluconazole. Also, the ckCYP51 was less sensitive to inhibition by both drugs than was caCYP51. These results were confirmed by expressing the CYP51 genes from C. krusei and C. albicans in Saccharomyces cerevisiae and determining the susceptibility of the transformants to voriconazole and fluconazole. We constructed homology models of the CYP51s of C. albicans and C. krusei based on the crystal structure of CYP51 from Mycobacterium tuberculosis. These models predicted that voriconazole is a more potent inhibitor of both caCYP51 and ckCYP51 than is fluconazole, because the extra methyl group of voriconazole results in a stronger hydrophobic interaction with the aromatic amino acids in the substrate binding site and more extensive filling of this site. Although there are multiple differences in the predicted amino acid sequence of caCYP51 and ckCYP51, the models of the two enzymes were quite similar and the mechanism for the relative resistance of ckCYP51 to the azoles was not apparent.     

A new triazole, voriconazole (UK-109,496), blocks sterol biosynthesis in Candida albicans and Candida krusei.

Voriconazole (UK-109,496) is a novel triazole derivative with potent broad-spectrum activity against various fungi, including some that are inherently resistant to fluconazole, such as Candida krusei. In this study we compared the effect of subinhibitory concentrations of voriconazole and fluconazole on sterol biosynthesis of fluconazole-resistant and -susceptible Candida albicans strains, as well as C. krusei, in an effort to delineate the precise mode of action of voriconazole. Voriconazole MICs ranged from 0.003 to 4 microg/ml, while fluconazole MICs ranged from 0.25 to >64 microg/ml. To investigate the effects of voriconazole and fluconazole on candidal sterols, yeast cells were grown in the absence and presence of antifungals. In untreated C. albicans controls, ergosterol was the major sterol (accounting for 53.6% +/- 2.2% to 71.7% +/- 7.8% of the total) in C. albicans and C. krusei strains. There was no significant difference between the sterol compositions of the fluconazole-susceptible and -resistant C. albicans isolates. Voriconazole treatment led to a decrease in the total sterol content of both C. albicans strains tested. In contrast, exposure to fluconazole did not result in a significant reduction in the total sterol content of the three candidal strains tested (P > 0.5). Gas-liquid chromatographic analysis revealed profound changes in the sterol profiles of both C. albicans strains and of C. krusei in response to voriconazole. This antifungal agent exerted a similar effect on the sterol compositions of both fluconazole-susceptible and -resistant C. albicans strains. Interestingly, a complete inhibition of ergosterol synthesis and accumulation of its biosynthetic precursors were observed in both strains treated with voriconazole. In contrast, fluconazole partially inhibited ergosterol synthesis. Analysis of sterols obtained from a fluconazole-resistant C. albicans strain grown in the presence of different concentrations of voriconazole showed that this agent inhibits ergosterol synthesis in a dose-dependent manner. In C. krusei, voriconazole significantly inhibited ergosterol synthesis (over 75% inhibition). C. krusei cells treated with voriconazole accumulated the following biosynthetic intermediates: squalene, 4,14-dimethylzymosterol, and 24-methylenedihydrolanosterol. Accumulation of these methylated sterols is consistent with the premise that this agent functions by inhibiting fungal P-450-dependent 14alpha-demethylase. As expected, treating C. krusei with fluconazole minimally inhibited ergosterol synthesis. Importantly, our data indicate that voriconazole is more effective than fluconazole in blocking candidal sterol biosynthesis, consistent with the different antifungal potencies of these compounds.     

Emergence of a Candida krusei isolate with reduced susceptibility to caspofungin during therapy

Clinical failure associated with reduced susceptibility to caspofungin has been described in Candida albicans and C. parapsilosis. We report a case of Candida krusei infection that progressed despite caspofungin therapy. Reduced microbial susceptibility to all three echinocandins (caspofungin, anidulafungin, and micafungin) was noted but was not associated with mutations in FKS1.     

Inhibitory effects of aminoglycoside antibiotics on reduction of cytochrome c from Candida krusei

Five aminoglycoside antibiotics (AGAs)--kanamycin (KM), bekamycin (AKM), dibekacin (DKB), ribostamycin (RSM) and paromomycin (PRM)--were studied for their effects on the nonenzymic reduction of cytochrome c by FeSO4 (Yamabe's system). Their inhibitory activity was in the order: DKB greater than AKM greater than KM greater than RSM greater than PRM. As this order correlated closely with that of the antibacterial activity of AGAs, Yamabe's system has proved useful in predicting the latter activity. Divalent metal ions other than Fe2+ enhanced the AGA-dependent inhibition of Yamabe's system in the order: Cu2+ greater than Mn2+ greater than Zn2+ greater than Co2+ greater than Ni2+ greater than. This order was similar to that of stability constants with general chelators except for the low positions of Ni2+ and Co2+. These findings suggested a metal chelation with free or bound Fe2+ for the action mechanism of AGAs on Yamabe's system and the bacterial growing system. The antagonistic effects of exogenous Fe2+ on the antibacterial activity against Staphylococcus aureus and Escherichia coli as measured by the agar dilution method supported this concept. A dual relationship of molecular structure with chelating and antibacterial activities demonstrated the importance of high molecular basicity in a potent AGA. However, the combination effect of pipemidic acid (stimulator on Yamabe's system) with KM was different from that with 1,10-phenanthroline (inhibitor on Yamabe's system) as measured by Dye's method using Pseudomonas aeruginosa.     

Successful treatment of Candida krusei infection with caspofungin acetate: a new antifungal agent

OBJECTIVE: Systemic fungal infections have high mortality, and therapy is often toxic. Caspofungin acetate, a new antifungal agent with minimal toxicity, may provide a better alternative to typical therapy for Candida krusei. DESIGN: Case report. SETTING: Multidisciplinary intensive care unit (ICU) of a community teaching hospital. PATIENT: A 22-yr-old male with acute lymphoblastic leukemia and Candida krusei fungemia failed therapy with fluconazole and amphotericin B. INTERVENTIONS: Caspofungin acetate given intravenously as a 70-mg loading dose, followed up by 50 mg daily along with standard ICU care. RESULTS: Survival without toxicity from therapy. CONCLUSION: Efficacy of caspofungin acetate in a patient with life-threatening Candida Krusei infection.     

Multilaboratory testing of antifungal combinations against a quality control isolate of Candida krusei

Candida krusei ATCC 6258 was tested by eight laboratories using 96-well plates containing checkerboard pairwise combinations of amphotericin B (AMB), posaconazole (PSC), caspofungin (CSP), and voriconazole (VRC). The methodology led to reproducible results across the laboratories. All drug combinations yielded MICs lower than the MICs of any two drugs tested singly, and combinations of AMB, PSC, CSP, and VRC were indifferent (no antagonism) by summations of fractional inhibitory concentration.     

Acquired echinocandin resistance in a Candida krusei isolate due to modification of glucan synthase

A Candida krusei strain from a patient with acute myelogenous leukemia that displayed reduced susceptibility to echinocandin drugs contained a heterozygous mutation, T2080K, in FKS1. The resulting Phe655-->Cys substitution altered the sensitivity of glucan synthase to echinocandin drugs, consistent with a common mechanism for echinocandin resistance in Candida spp.