Antibacterial activity and cytotoxicity of a novel bacteriocin isolated from Pseudomonas sp. strain 166

Abstract

Pseudomonas sp. strain 166 was isolated from soil samples from Changbai Mountains. A novel bacteriocin PA166 from Pseudomonas sp. 166 was purified using ammonium sulfate, dextran gel chromatography column and Q-Sepharose column chromatography successively. The molecular mass of bacteriocin PA166 was found to be 49.38 kDa by SDS-PAGE and liquid chromatography-mass spectrometry (MS)/MS. Bacteriocin PA166 showed stability at a wide range of pH (2-10), and thermal stability (40, 60, 80 and 100°C). The bacteriocin PA166 antimicrobial activity was slightly inhibited by Ca²⁺ , K⁺ and Mg²⁺ . The minimum bactericidal concentrations of bacteriocin PA166 against five Pasteurella multocida strains ranged from 2 to 8 μg ml^-1 . Bacteriocin PA166 showed low cytotoxicity and a higher treatment index (TI = 82.51). Fluorescence spectroscopy indicated that bacteriocin PA166 destroyed the cell membrane to exert antimicrobial activity. In summary, bacteriocin PA166 had strong antibacterial activity, high TI and low toxicity, and hence could serve as a potential clinical therapeutic drug.

Fig. 1

The phylogenetic tree of 16S rDNA gene sequences for isolated strain. The bootstrap values (%) are shown at the branches from 1000 replications.

Fig. 2

Molecular mass determination of bacteriocin PA166. M: low molecular marker; 1: bacteriocin PA166 loaded on the gel.

Fig. 3

Safety analysis of bacteriocin PA116. A. Hemolytic activity of bacteriocin PA116 against rabbit red blood cells, (B) cytotoxicity of the bacteriocin PA116 against Vero cells (blue) and NR8383 cells (red) and (C) zebrafish embryos were either treated with embryo medium as a negative control,bacteriocin PA166 (MIC, MBC) or sodium dehydroacetate (200 μg ml⁻¹) as a positive control.

Fig. 4

Therapy of P. multocid infection in mice (A) survival rates of mice (n = 10), PA166 treatment of lethal dose of P. multocida (1.12 × 10⁶ CFU ml⁻¹) improves the survival rate of mice. B. The amount of bacteria in the lungs of mice treated with antimicrobial substances (****P < 0.0001.) and (C) lung tissues with H&E staining (200x).

Fig. 5

Time‐killing curves of bacteriocin PA166 on different P. multocida.

Fig. 6

Mechanism of PA116. A. Decreased levels of intracellular ATP in P. multocida ATCC43137 by treatment with PA116. B. Total ROS accumulation in P. multocida ATCC43137 treatment using PA116. C. A gel retardation experiment was used to measure the DNA binding assay. M: λ/HindIII DNA Marker; 1: genomic DNA alone; 2–11: 0.5–256 μg ml⁻¹ PA116; 12: 256 μg ml⁻¹ PA116 alone. D. Inner membrane permeability of P. multocida after the treatment of PA116. E. Outer membrane permeability of P. multocida ATCC43137 treatment using PA116. F. Cytoplasmic membrane depolarization of P. multocida ATCC43137 by the PA116; g SEM images of P. multocida. H. TEM images of P. multocida. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.001.