Hyperthermia Sensitizes Rhizopus oryzae to Posaconazole and Itraconazole Action through Apoptosis

Abstract

The high mortality rate of mucormycosis with currently available monotherapy has created interest in studying novel strategies for antifungal agents. With the exception of amphotericin B (AMB), the triazoles (posaconazole [PCZ] and itraconazole [ICZ]) are fungistatic in vitro against Rhizopus oryzae. We hypothesized that growth at a high temperature (42°C) results in fungicidal activity of PCZ and ICZ that is mediated through apoptosis. R. oryzae had high MIC values for PCZ and ICZ (16 to 64 g/ml) at 25°C; in contrast, the MICs for PCZ and ICZ were significantly lower at 37°C (8 to 16 g/ml) and 42°C (0.25 to 1 g/ml). Furthermore, PCZ and ICZ dose-dependent inhibition of germination was more pronounced at 42°C than at 37°C. In addition, intracellular reactive oxygen species (ROS) increased significantly when fungi were exposed to antifungals at 42°C. Characteristic cellular changes of apoptosis in R. oryzae were induced by the accumulation of intracellular reactive oxygen species. Cells treated with PCZ or ICZ in combination with hyperthermia (42°C) exhibited characteristic markers of early apoptosis: phosphatidylserine externalization visualized by annexin V staining, membrane depolarization visualized by bis-[1,3-dibutylbarbituric acid] trimethine oxonol (DiBAC) staining, and increased metacaspase activity. Moreover, terminal deoxynucleotidyltransferasemediated dUTP-biotin nick end labeling (TUNEL) assay and DAPI (4=,6-diamidino-2-phenylindole) staining demonstrated DNA fragmentation and condensation, respectively. The addition of N-acetylcysteine increased fungal survival, prevented apoptosis, reduced ROS accumulation, and decreased metacaspase activation. We concluded that hyperthermia, either alone or in the presence of PCZ or ICZ, induces apoptosis in R. oryzae. Local thermal delivery could be a therapeutically useful adjunct strategy for these refractory infections.

Enhanced antifungal effects of amphotericin B-TPGS-b-(PCL-ran-PGA) nanoparticles in vitro and in vivo

“…In brief, 20 µL of annexin V-FITC (50 µg/mL) (Annexin V FITC Apoptosis Detection Kit; BD Pharmingen™, San Diego, CA, USA) and 20 µL of PI (200 µg/mL) were mixed with 1 mL of cell suspension, followed by incubation for 20 minutes in the dark, at room temperature. [13][14][15] The apoptotic cells showed green fluorescence and necrotic cells showed red fluorescence, as described by Khan et al and Shirazi and Kontoyiannis. 13,14 The samples were detected by fluorescence microscopy or flow cytometry (BD Biosciences).…”

“…The ZP distribution of AMB-NPs is shown in Table 1 and ranged from -18 mV to -19 mV. As shown in Table 1 were higher than those of previously reported NPs, [11][12][13][14][15] indicating a higher affinity between the star-shaped core region of PLGA and hydrophobic AMB. Moreover, the LC reached approximately 10.0%, and EE% reached approximately 85%, suggesting an ideal delivery vehicle for drugs.…”

Thermal Augmentation of Vancomycin Against Staphylococcal Biofilms

“…Quantitative assessment of gene expression showed increased presence of cell wall synthetic enzyme transcripts, corresponding to ultrastructural evidence of increased cell wall thickness and corroborating previous reports of increased muropeptide production during heat stress by S. aureus (25). These responses are recognized to have complex origins (13, 26, 27), to be tied to temperature-variant susceptibility to antibiotics (28), and, at least among isolates of S. aureus , to vary between isolates. From previous reports, as here, it is not clear whether these represent an adaptive or maladaptive response.…”

Update on mucormycosis pathogenesis

“…Azole activity is mainly characterized by a ‘static’ effect rather than cidality in vitro. However, emerging awareness that noxious environmental cues such as hyperthermia [54■], inhibition of homeostatic mitochondrial function [55] and inhibition of stress response pathways such as calcineurin pathway [56] render static azole drugs to be more potent and rapidly cidal in vitro through a mechanism that involves induction of Mucorales apoptosis (Fig. 2).…”