Unveiling the Synergistic Interaction Between Liposomal Amphotericin B and Colistin

Abstract

Patients with multiple comorbidities are often administered simultaneously or sequentially antifungals and antibacterial agents, without full knowledge of the consequences of drug interactions. Considering the clinical relevance of liposomal amphotericin B (L-AMB), the association between L-AMB and six antibacterial agents was evaluated against four clinical isolates and one type strain of Candida spp. and two clinical isolates and one type strain of Aspergillus fumigatus. In order to evaluate such combined effects, the minimal inhibitory concentration (MIC) of L-AMB was determined in the presence of 0.5-, 1-, 2-, and 4-fold peak plasma concentrations of each of the antibacterial drugs. Since the L-AMB/colistin (CST) association was the most synergic, viability assays were performed and the physiological status induced by this association was characterized. In addition, computational molecular dynamics studies were also performed in order to clarify the molecular interaction. The maximum synergistic effect with all antibacterial agents, except CST, was reached at fourfold the usual peak plasma concentrations, resulting in 2-to 8-fold L-AMB MIC reduction for Candida and 2-to 16-fold for Aspergillus. For CST, the greatest synergism was registered at peak plasma concentration (3 mg/L), with 4-to 8-fold L-AMB MIC reduction for Candida and 16-to 32-fold for Aspergillus. L-AMB at subinhibitory concentration (0.125 mg/L) combined with CST 3 mg/L resulted in: a decrease of fungal cell viability; an increase of cell membrane permeability; an increase of cellular metabolic activity soon after 1 h of exposure, which decreased until 24 h; and an increase of ROS production up to 24 h. From the molecular dynamics studies, AMB and CST molecules shown a propensity to form a stable molecular complex in solution, conferring a recognition and binding added value for membrane intercalation. Our results demonstrate that CST interacts synergistically with L-AMB, forming a stable complex, which promotes the fungicidal activity of L-AMB at low concentration.

Keywords: Aspergillus fumigatus; Candida species; colistin; liposomal amphotericin B; synergistic effect.

FIGURE 1

Effect of liposomal amphotericin B (L-AMB) in combination with colistin upon C. albicans (strain 596) physiological parameters. (A) Viability assessment by colony forming units (CFU) enumeration of C. albicans cells exposed to () L-AMB, () CST, and () L-AMB in association with CST. The CFU counts of cells non-treated are represented as (). (B) Cell membrane potential was evaluated using DiBAC₄(3) staining. (C) Cell membrane integrity was evaluated using propidium iodide (PI) staining. (D) Cell metabolic activity was evaluated using 5-CFDA staining. Data at respective time points corresponds to mean ± standard deviation. ^∗p-values < 0.05, significant differences between the treatments with L-AMB alone and CST alone vs. the control (non-treated cells); and between the association L-AMB/CST and L-AMB alone.

FIGURE 2

Effect of L-AMB in association with colistin upon endogenous ROS production. (A) Endogenous reactive oxygen species (ROS) production determined by DCFH-DA staining, assessed by flow cytometry. (B) Fluorescence microscopy imaging showing ROS-positive cells after treatment with (b) L-AMB and (c) CST alone and (d) in association after 24 h of exposure; (a) control, non-treated cells. ^∗p-values < 0.05, significant differences between the treatments with L-AMB alone and CST alone vs. the control (non-treated cells); and between the association L-AMB/CST and L-AMB alone.

FIGURE 3

Self-assembly of amphotericin B (AMB) and colistin (CST; represented in sticks) in water medium (represented in lines) through molecular dynamics (MD) simulation studies. (A) Complex conformation snapshot after 20 ns of simulation, including water molecules within 4 Å of the complex. (B) Time evolution of the h-bond distance between two molecules during the complex formation.