A ketogenic diet enhances fluconazole efficacy in murine models of systemic fungal infection

Abstract

Invasive fungal infections are a significant public health concern, with mortality rates ranging from 20% to 85% despite current treatments. Therefore, we examined whether a ketogenic diet could serve as a successful treatment intervention in murine models of Cryptococcus neoformans and Candida albicans infection in combination with fluconazole-a low-cost, readily available antifungal therapy. The ketogenic diet is a high-fat, low-carbohydrate diet that promotes fatty acid oxidation as an alternative to glycolysis through the production of ketone bodies. In this series of experiments, mice fed a ketogenic diet prior to infection with C. neoformans and treated with fluconazole had a significant decrease in fungal burden in both the brain (mean 2.66 ± 0.289 log₁₀ reduction) and lung (mean 1.72 ± 0.399 log₁₀ reduction) compared to fluconazole treatment on a conventional diet. During C. albicans infection, kidney fungal burden of mice in the keto-fluconazole combination group was significantly decreased compared to fluconazole alone (2.37 ± 0.770 log₁₀-reduction). Along with higher concentrations of fluconazole in the plasma and brain tissue, fluconazole efficacy was maximized at a significantly lower concentration on a keto diet compared to a conventional diet, indicating a dramatic effect on fluconazole pharmacodynamics. Our findings indicate that a ketogenic diet potentiates the effect of fluconazole at multiple body sites during both C. neoformans and C. albicans infection and could have practical and promising treatment implications.IMPORTANCEInvasive fungal infections cause over 2.5 million deaths per year around the world. Treatments for fungal infections are limited, and there is a significant need to develop strategies to enhance antifungal efficacy, combat antifungal resistance, and mitigate treatment side effects. We determined that a high-fat, low-carbohydrate ketogenic diet significantly potentiated the therapeutic effect of fluconazole, which resulted in a substantial decrease in tissue fungal burden of both C. neoformans and C. albicans in experimental animal models. We believe this work is the first of its kind to demonstrate that diet can dramatically influence the treatment of fungal infections. These results highlight a novel strategy of antifungal drug enhancement and emphasize the need for future investigation into dietary effects on antifungal drug activity.

Keywords: Candida; Cryptococcus; cryptococcal meningitis; fluconazole; ketogenic diet.

Fig 1

Mice given a KD 10 days prior to inoculation with C. neoformans display significantly less brain and lung fungal burden after fluconazole treatment, compared to those fed a CD. (A) A 1.78 log₁₀ difference in fungal burden was observed between fluconazole and vehicle-treated groups on a CD (P < 0.0001), while fluconazole treatment in combination with a KD displayed a 4.24 log₁₀ reduction compared to the untreated KD cohort (P < 0.0001). While there was no difference between brain fungal burden in untreated KD or CD mice, there was a 2.61 log₁₀ decrease between fluconazole-treated dietary groups (P < 0.0001). (B) In the lung, fluconazole therapy resulted in a 1.46 log₁₀ reduction in fungal burden on a CD (P < 0.0001) and a 3.28 log₁₀ reduction on a KD (P < 0.0001) compared to vehicle. While there was no statistical difference between untreated KD and CD lung fungal burden, fluconazole treatment resulted in increased 1.18 log₁₀ reduction in fungal burden between CD and KD fluconazole-treated mice (P < 0.0001). Mean ± SEM reported for n ≥ 10 mice per group of one independent experiment. Two-way analysis of variance with post hoc Bonferroni multiple comparison test was used.

Fig 2

KD-induced potentiation of fluconazole therapy is effective in a C. albicans infection model. Fluconazole therapy on a KD resulted in a 2.37 log₁₀ reduction in kidney fungal burden compared to a CD (P = 0.0078). No difference was observed in kidney fungal burden between untreated CD and KD mice. Mean ± SEM reported for n ≥ 9 mice per group of one independent experiment. Two-way analysis of variance with post hoc Bonferroni test was used.

Fig 3

Serum and brain fluconazole levels are increased in mice given a ketogenic diet. (A) After a single dose of fluconazole, fluconazole serum exposure after 24 h (AUC_0–24) for a KD was 453 µg*h/mL, versus 340 µg*h/mL in the CD group. (B) At steady state after 4 days of dosing, brain exposure to fluconazole (AUC_120–144) was 310 µg*h/mL in the KD group and 185 µg*h/mL in the CD group. Fluconazole levels in both experiments were measured via liquid chromatography-mass spectrometry, and the mean of at least three mice was reported for each time point. AUC_0–24, area under the curve from 0 to 24 h; AUC_120–144, area under the curve from 120 to 144 h;PK, pharmacokinetic.

Fig 4

(A) Mice on a KD reach stasis at lower doses of fluconazole compared to mice on a conventional diet. Fungal burdens were equivalent for fluconazole doses of 80 and above on a KD, and all doses achieved fungal stasis. Mice on a CD displayed a stepwise reduction in yeasts as doses increased, only reaching stasis at 400 mg/kg. (B) The 50% efficacy level of fluconazole on a KD falls between 10 and 20 mg/kg. Mean ± SEM reported for n = 5 mice per group for panel A and n ≥ 3 mice per group for panel B. A and B panels represent independent experiments. Two-way analysis of variance with post hoc Bonferroni test.

Fig 5

Pharmacodynamic analysis of fluconazole exposure and C. neoformans fungal burden. An inhibitory sigmoid Emax model was used to estimate (A) plasma AUC versus effect relationship and (B) brain AUC versus effect relationship.

Fig 6

A delayed switch to a KD, 24 h post-inoculation, is just as effective as 10 days of dietary induction. Brain fungal burden in mice begun on a KD 24 h post-inoculation and treated with fluconazole (FCN) was statistically equivalent to those receiving a KD 10-day pre-inoculation. Mice switched to a ketogenic diet on day 3 after infection showed increased fungal burden compared to mice that started a ketogenic diet 24 h post-infection but had lower fungal burden than mice on a KD without fluconazole (P < 0.0001 for both). Mean ± SEM reported for ≥4 mice per group from one independent experiment. One-way analysis of variance with post hoc Tukey test was used.

Fig 7

Serum cytokine profiles of mice infected with C. neoformans, collected 6 days post-infection display several slight differences between dietary cohorts. (A) Mice on a KD showed an increased expression of IL-18 compared to a CD during fluconazole treatment (P = 0. 0.0243). (B) TNF-α expression was increased in vehicle-treated KD mice compared to CD (P = 0.0008). (C) IL-13 expression was enhanced in vehicle-treated KD mice compared to CD (P = 0.0021). (D) IL-2 expression was increased in vehicle-treated KD mice compared to CD (P = 0.0169). (E) IL-5 expression was significantly increased in vehicle-treated KD versus CD mice (P = 0.0014). Fluconazole treatment decreased IL-5 expression overall within both dietary groups (CD <0.0001, KD <0.0001). Cytokine concentrations were measured for at least nine mice per group and (log₁₀ +1) transformed. Two-way analysis of variance with post hoc Tukey test was used.