Transmission electron microscopic morphological study and flow cytometric viability assessment of Acinetobacter baumannii susceptible to Musca domestica cecropin

Abstract

Multidrug-resistant (MDR) Acinetobacter baumannii infections are difficult to treat owing to the extremely limited armamentarium. Expectations about antimicrobial peptides' use as new powerful antibacterial agents have been raised on the basis of their unique mechanism of action. Musca domestica cecropin (Mdc), a novel antimicrobial peptide from the larvae of Housefly (Musca domestica), has potently active against Gram-positive and Gram-negative bacteria standard strain. Here we evaluated the antibacterial activity of Mdc against clinical isolates of MDR-A. baumannii and elucidate the related antibacterial mechanisms. The minimal inhibitory concentration (MIC) of Mdc was 4 μg/mL. Bactericidal kinetics of Mdc revealed rapid killing of A. baumannii (30 min). Flow cytometry using viability stain demonstrated that Mdc causes A. baumannii membrane permeabilization in a concentration- and time-dependent process, which correlates with the bactericidal action. Moreover, transmission electron microscopic (TEM) examination showed that Mdc is capable of disrupting the membrane of bacterial cells, resulting in efflux of essential cytoplasmic components. Overall, Mdc could be a promising antibacterial agent for MDR-A. baumannii infections.

Figure 1

Growth inhibitory activity of Mdc against A. baumannii GIM1.650. The experiment was carried out in triplicate, and plots represent the average values. Error bars refer to the standard deviations.

Figure 2

Bactericidal kinetics of Mdc against A. baumannii GIM1.650 after incubation with different concentration of Mdc. A 5-log reduction occurs after 30 min at the MIC (4 μg/mL). The experiment was carried out in triplicate, and average values are reported (SD ⩽10%).

Figure 3

Effect of Mdc on the membrane permeability of A. baumannii GIM1.650 analyzed by the flow cytometer. The relative fluorescence intensities within the P3 regions were taken as PI-positive cells. (a) Untreated cells control, (b) cells treated with 4 μg/mL Mdc at 37°C for 1 hour, (c) dead control (cells heated at 70°C), (d) percentage of cells stained with propidium iodide (PI), and (e) kinetics of Mdc membrane permeability against A. baumannii.

Figure 4

Ultrastructural damages in A. baumannii GIM1.650 treated with Mdc. Bacteria were untreated (a) or treated with Mdc (b) at a concentration of 8 μg/mL. Cells were processed to be analyzed under TEM.