Drosophila melanogaster as a model to study virulence and azole treatment of the emerging pathogen Candida auris.

OBJECTIVES: Candida auris is an emerging, often MDR, yeast pathogen. Efficient animal models are needed to study its pathogenicity and treatment. Therefore, we developed a C. auris fruit fly infection model. METHODS: TollI-RXA/Tollr632 female flies were infected with 10 different C. auris strains from the CDC Antimicrobial Resistance bank panel. We used three clinical Candida albicans strains as controls. For drug protection assays, fly survival was assessed along with measurement of fungal burden (cfu/g tissue) and histopathology in C. auris-infected flies fed with fluconazole- or posaconazole-containing food. RESULTS: Despite slower in vitro growth, all 10 C. auris isolates caused significantly greater mortality than C. albicans in infected flies, with >80% of C. auris-infected flies dying by day 7 post-infection (versus 67% with C. albicans, P < 0.001-0.005). Comparison of C. auris isolates from different geographical clades revealed more rapid in vitro growth of South American isolates and greater virulence in infected flies, whereas the aggregative capacity of C. auris strains had minimal impact on their growth and pathogenicity. Survival protection and decreased fungal burden of fluconazole- or posaconazole-fed flies infected with two C. auris strains were in line with the isolates' disparate in vitro azole susceptibility. High reproducibility of survival curves for both non-treated and antifungal-treated infected flies was seen, with coefficients of variation of 0.00-0.31 for 7 day mortality. CONCLUSIONS: Toll-deficient flies could provide a fast, reliable and inexpensive model to study pathogenesis and drug activity in C. auris candidiasis.

A murine model of cutaneous aspergillosis for evaluation of biomaterials-based local delivery therapies.

Cutaneous fungal infection is a challenging condition to treat that primarily afflicts immunocompromised patients. Local antifungal therapy may permit the delivery of high concentrations of antifungals directly to wounds while minimizing systemic toxicities. However, the field currently lacks suitable in vivo models. Therefore, a large cutaneous wound was created in immunosuppressed mice and inoculated with Aspergillus fumigatus. We fabricated biodegradable polymer microparticles (MPs) that were capable of locally delivering antifungal and characterized in vitro release kinetics. We compared wound bed size, fungal burden, and histological presence of fungi in mice treated with antifungal-loaded MPs. Mice with a cutaneous defect but no infection, mice with infected cutaneous defect but no treatment, and infected mice treated with blank MPs were used as controls. Infection of large wounds inhibited healing and resulted in tissue invasion in an inoculum-dependent manner. MPs were capable of releasing antifungals at concentrations above A. fumigatus Minimum Inhibitory Concentration (MIC) for at least 6 days. Wounds treated with MPs had significantly decreased size compared with no treatment (64.2% vs. 19.4% wound reduction, p = 0.002) and were not significantly different from uninfected controls (64.2% vs. 58.1%, p = 0.497). This murine model may serve to better understand cutaneous fungal infection and evaluate local biomaterials-based therapies. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1867-1874, 2019.

Statin Concentrations Below the Minimum Inhibitory Concentration Attenuate the Virulence of Rhizopus oryzae.

BACKGROUND: Mucormycosis is a destructive invasive mold infection afflicting patients with diabetes and hematologic malignancies. Patients with diabetes are often treated with statins, which have been shown to have antifungal properties. We sought to examine the effects of statins on Rhizopus oryzae, a common cause of mucormycosis. METHODS: Clinical strains of R. oryzae were exposed to lovastatin, atorvastatin, and simvastatin and the minimum inhibitory concentrations (MICs) were determined. R. oryzae germination, DNA fragmentation, susceptibility to oxidative stress, and ability to damage endothelial cells were assessed. We further investigated the impact of exposure to lovastatin on the virulence of R. oryzae RESULTS: All statins had MICs of >64 µg/mL against R. oryzae Exposure of R. oryzae to statins decreased germling formation, induced DNA fragmentation, and attenuated damage to endothelial cells independently of the expression of GRP78 and CotH. Additionally, R. oryzae exposed to lovastatin showed macroscopic loss of melanin, yielded increased susceptibility to the oxidative agent peroxide, and had attenuated virulence in both fly and mouse models of mucormycosis. CONCLUSIONS: Exposure of R. oryzae to statins at concentrations below their MICs decreased virulence both in vitro and in vivo. Further investigation is warranted into the use of statins as adjunctive therapy in mucormycosis.

Comparative pharmacodynamics of posaconazole in neutropenic murine models of invasive pulmonary aspergillosis and mucormycosis.

We used two established neutropenic murine models of pulmonary aspergillosis and mucormycosis to explore the association between the posaconazole area under the concentration-time curve (AUC)-to-MIC ratio (AUC/MIC) and treatment outcome. Posaconazole serum pharmacokinetics were verified in infected mice to ensure that the studied doses reflected human exposures with the oral suspension, delayed-release tablet, and intravenous formulations of posaconazole. Sinopulmonary infections were then induced in groups of neutropenic mice with Aspergillus fumigatus strain 293 (posaconazole MIC, 0.5 mg/liter) or Rhizopus oryzae strain 969 (posaconazole MIC, 2 mg/liter) and treated with escalating daily dosages of oral posaconazole, which was designed to achieve AUCs ranging from 1.10 to 392 mg · h/liter. After 5 days of treatment, lung fungal burden was analyzed by quantitative real-time PCR. The relationships of the total drug AUC/MIC and the treatment response were similar in both models, with 90% effective concentrations (EC90s) corresponding to an AUC/MIC threshold of 76 (95% confidence interval [CI], 46 to 102) for strain 293 versus 87 (95% CI, 66 to 101) for strain 969. Using a provisional AUC/MIC target of >100, these exposures correlated with minimum serum posaconazole concentrations (Cmins) of 1.25 mg/liter for strain 293 and 4.0 mg/liter for strain 969. The addition of deferasirox, but not liposomal amphotericin or caspofungin, improved the activity of a suboptimal posaconazole regimen (AUC/MIC, 33) in animals with pulmonary mucormycosis. However, no combination was as effective as the high-dose posaconazole monotherapy regimen (AUC/MIC, 184). Our analysis suggests that posaconazole pharmacodynamics are similar for A. fumigatus and R. oryzae when indexed to pathogen MICs.

Proangiogenic growth factors potentiate in situ angiogenesis and enhance antifungal drug activity in murine invasive aspergillosis.

In invasive pulmonary aspergillosis, direct invasion and occlusion of pulmonary vasculature by Aspergillus hyphae causes tissue hypoxia, which is enhanced by secreted fungal metabolites that downregulate compensatory angiogenic signaling pathways. We assessed the effects of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) on survival rates, fungal burden, and in situ angiogenesis in a murine invasive pulmonary aspergillosis model. bFGF and VEGF monotherapy significantly increased survival rates and potentiated the activity of amphotericin B. bFGF-containing regimens were associated with reduced tissue fungal burdens. bFGF and VEGF reversed the antiangiogenic activity of Aspergillus fumigatus; however, VEGF induced the formation of immature neovessels, providing an explanation for its lesser efficacy. Treatment with bFGF plus amphotericin B was associated with neutrophil influx into Aspergillus-infected pulmonary tissue, suggesting that this combination limits fungal growth through neutrophil trafficking. Vasculogenic pathways are unexplored targets for the treatment of invasive pulmonary aspergillosis and may potentiate both innate immunity and antifungal drug activity against A. fumigatus.

Increased frequency of non-fumigatus Aspergillus species in amphotericin B- or triazole-pre-exposed cancer patients with positive cultures for aspergilli.

Invasive aspergillosis (IA) can occur despite prior prophylactic or empiric use of triazoles or amphotericin B (AMB). Although profound immunosuppression may account for breakthrough IA, resistance of Aspergillus to antifungals may also play a role. To examine this question, we measured the minimal inhibitory concentration of 105 Aspergillus isolates recovered from 105 cancer patients (64 with IA, 41 with Aspergillus colonization) to AMB, itraconazole (ITC), and voriconazole (VRC) using the National Committee for Clinical Laboratory Standards (NCCLS) M38-A microdilution and E-test methods. We also determined the minimal fungicidal concentration (MFC) of these agents and the minimal effective concentration (MEC) of caspofungin (CAS) using standardized methods. We then collected information regarding pre-exposure to AMB or triazoles (fluconazole, ITC, VRC) within 3 months before Aspergillus isolation. Pre-exposure of cancer patients to AMB or triazoles was associated with increased frequency of non-fumigatus Aspergillus species. Aspergillus isolates recovered from patients who previously received AMB exhibited higher E-test AMB MICs compared with isolates from patients without prior AMB exposure (P = 0.01). In addition, the AMB MICs by E-test were higher in triazole-pre-exposed patients compared with those not exposed to triazoles (P = 0.001). The ITC and VRC MICs by E-test were not affected by prior AMB or triazole exposure. Finally, neither the AMB, ITC, and VRC MICs and MFCs by NCCLS method nor CAS MECs showed such changes. In conclusion, cancer patients with positive Aspergillus cultures who are pre-exposed to AMB or triazoles have high frequency of non-fumigatus Aspergillus species. These Aspergillus isolates were found to be AMB-resistant by the more sensitive E-test method.

Toll-deficient Drosophila flies as a fast, high-throughput model for the study of antifungal drug efficacy against invasive aspergillosis and Aspergillus virulence.

Invasive aspergillosis (IA) is the most important opportunistic mycosis in immunosuppressed patients. The lack of a sufficient number of effective antifungals and our incomplete understanding of the pathogenesis of IA contribute to its overall unfavorable prognosis. Studies of drug efficacy against IA and Aspergillus virulence rely on conventional animal models that are laborious and use limited numbers of animals; alternative, less cumbersome in vivo models are desirable. Using different inoculation models of IA, we found that Toll-deficient Drosophila flies exposed to voriconazole (VRC), the preferred drug for the treatment of IA in humans, had significantly better survival rates and lower tissue fungal burdens than did those not exposed to VRC. Furthermore, Toll-deficient Drosophila flies infected with an alb1-deleted hypovirulent Aspergillus mutant had significantly better survival rates than did those infected with a wild-type Aspergillus strain. Therefore, the Drosophila fly is a fast, high-throughput in vivo model for the study of drug efficacy against IA and Aspergillus virulence.