Time-kill kinetic assay and bacterial viability of A. baumannii upon treatment with Octominin. (A) The time-kill kinetics of A. baumannii treated with Octominin (1.0, 2.5, 5.0, and 7.5 µg/mL) were assessed at every 3 h intervals by measuring the optical density at 595 nm (OD595). Phosphate-buffered saline (PBS) and 50 µg/mL chloramphenicol were used as the negative and positive controls, respectively. The bars indicate the mean ± standard deviation (n = 3). (B) A. baumannii viability upon Octominin treatment. A. baumannii was treated with Octominin at increasing concentrations (0–10 µg/mL) and positive-control chloramphenicol. The samples were incubated for 24 h. Later, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed, and the OD595 was measured. * p < 0.05 compared to the control (0) group. The error bars indicate the mean ± standard deviation (n = 3). PC = Positive control (chloramphenicol).

Effect of Octominin on the morphological and structural changes in A. baumannii. Bacteria, which were (A) untreated, (B) treated at MIC (5 μg/mL) or (C) at MBC (10 μg/mL) of Octominin, or (D) treated with chloramphenicol (50 μg/mL), assessed under a field-emission scanning electron microscope (FE-SEM). Scale bar represents 200 nm.

Effect of Octominin on membrane permeability alteration in A. baumannii. PBS-treated A. baumannii, Octominin-treated A. baumannii at MIC (5 μg/mL) and MBC (10 μg/mL), and chloramphenicol-treated A. baumannii were incubated for 10 h and stained with Propidium iodide (PI) and Fluorescein diacetate (FDA). The cells were visualized through confocal microscopy (CFMC) for red fluorescence (PI) and green fluorescence (FDA) at excitation and emission wavelengths of 535 nm and 617 nm, respectively, and 488 nm and 535 nm, respectively. Scale bar represent 5 µm.

Effect of Octominin on the reactive oxygen species (ROS) generation in A. baumannii. PBS-treated A. baumannii, Octominin-treated A. baumannii at MIC (5 μg/mL) and MBC (10 μg/mL), and chloramphenicol-treated A. baumannii were incubated for 10 h and stained with 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA). The cells were visualized through confocal microscopy (CFMC) for green fluorescence at excitation and emission wavelengths of 488 nm and 535 nm, respectively. Scale bar represents 5 µm.

Lipopolysaccharide (LPS) neutralization activity of Octominin. LPS (0.5 EU/mL) was reacted with Octominin (0–100 µg/mL) and incubated for 30 min at 37 °C. The remaining LPS after the neutralization was quantified by the LAL chromogenic endotoxin quantitation kit, with an absorption measurement of colored substrate at 410 nm. * p < 0.05, compared to the negative control (0-Octominin) group. The error bars indicate the mean ± standard deviation (n = 3).

Anti-biofilm effect of Octominin against A. baumannii. (A) Quantitative measurement of the biofilm formation inhibition by Octominin. A. baumannii was treated with increasing concentrations of Octominin (0–12.5 µg/mL) and chloramphenicol (10 µg/mL and 50 µg/mL). After 24 h of treatment, the remaining biofilm was stained with crystal violet (CV), and the OD was measured at 595 nm (OD595); (B) Quantitative measurement of A. baumannii biofilm eradiation by Octominin. A. baumannii was allowed to form biofilm for 24 h and then the biofilm was treated with increasing concentrations of Octominin (0–12.5 µg/mL) and chloramphenicol (10 µg/mL and 50 µg/mL). After 24 h of treatment, the remaining biofilm was stained with CV, and the OD595 was measured. * p < 0.05, compared to the control (0-Octominin) group. The error bar indicates the mean ± standard deviation (n = 3). PC = Positive control (chloramphenicol).

Hemolytic activity and cytotoxicity of Octominin. (A) Mouse red blood cells (RBC) were treated with Octominin (0–500 µg/mL) for the determination of the hemolytic activity. Nuclease-free water and Triton-X (10 µg/mL) were used as the negative and positive controls, respectively. The samples were incubated for 1 h, and the RBCs were removed by centrifugation. The absorbance of the supernatant was measured at 414 nm. * p < 0.05, compared to the positive control (Triton-X, 10 µg/mL) group. The error bars indicate the mean ± standard deviation (n = 3). (B) Cytotoxicity of Octominin against murine Raw 264.7 macrophage cells using an MTT assay. The cells (2.0 × 105 cells/mL) were seeded onto 96-well plates (100 µL/well) and incubated for 12 h. Octominin was added to each well at 0–500 µg/mL, and the plate was incubated at 37 °C for 24 h in a 5% CO2 incubator. The supernatant was replaced with fresh media and treated with the MTT reagent and incubated for 4 h. Then, dimethyl sulfoxide (DMSO) (50 µL) was added and incubated for 30 min with constant shaking on a plate shaker. The absorbance was measured at 595 nm. * p < 0.05, compared to the negative control (0-Octominin) group. The error bars indicate the mean ± standard deviation (n = 3).

In vivo analysis of Octominin in A. baumannii-challenged adult zebrafish. (A) Schematic diagram of the experimental process. Adult zebrafish were challenged with 20 µL of A. baumannii culture (2.1 × 10¹¹ CFU/mL) intraperitonially (i.p.) and simultaneously treated with 10 µL of Octominin (10 mg/mL) and PBS as the control. Uninfected fish were maintained as the negative control. The fish were maintained at 28 °C. (B) RPS after treatment over 42 h. The rate of surviving zebrafish in each group is depicted (n = 24/group). (C) The microbial count in the spleen and kidney with A. baumannii infection. The spleen and kidney were isolated after 42 hpi, homogenized in PBS, plated on trypticase soy agar (TSA), and incubated. The CFU were counted, and the average CFU/mg was derived. (D) Pathological clinical signs of A. baumannii infection in zebrafish. The abdomen and gill area of the zebrafish (marked in red frames) were observed under a light microscope to detect clinical signs of infection. * p < 0.05, compared to the PBS-treated negative control group. The error bars indicate the mean ± standard deviation (n = 3).

Histological analysis of zebrafish spleen, kidney, and gill in the negative control, A. baumannii-infected, and PBS-treated, A. baumannii-infected, and Octominin-treated groups. The uninfected groups showed no deviation from the normal structure in all three tissue samples. However, A. baumannii-infected zebrafish spleen had a relatively higher amount of red pulp (RP) than white pulp (WP), while the Octominin-treated sample showed a regular arrangement of the spleen tissue. Kidney samples of A. baumannii-infected and PBS-treated zebrafish had shrunken glomeruli (G), with an increased Bowman space (arrowhead), macrophage infiltration (thin arrow), and cellular necrosis (thick arrow). However, the Octominin-treated group showed fewer deviations, with regular glomeruli, proximal tubule (PT), distal tubule (DT) and a few macrophages. The gill tissue showed erythrocyte infiltration (thick arrow), thickening in primary lamella (PL) (thin arrow), and secondary lamella (SL) clubbing (arrowhead) in A. baumannii-infected and PBS-treated zebrafish, while the Octominin-treated zebrafish showed comparatively fewer deviations. Scale bar represents 12.5 µm in spleen and kidney tissues and 25 µm in gill tissues.