Antibacterial action of a novel functionalized chitosan-arginine against Gram-negative bacteria

Abstract

The antimicrobial activity of chitosan and chitosan derivatives has been well established. However, although several mechanisms have been proposed, the exact mode of action is still unclear. Here we report on the investigation of antibacterial activity and the antibacterial mode of action of a novel water-soluble chitosan derivative, arginine-functionalized chitosan, on the Gram-negative bacteria Pseudomonas fluorescens and Escherichia coli. Two different arginine-functionalized chitosans (6% arginine-substituted and 30% arginine-substituted) each strongly inhibited P. fluorescens and E. coli growth. Time-dependent killing efficacy experiments showed that 5000 mg l(-1) of 6%- and 30%-substituted chitosan-arginine killed 2.7 logs and 4.5 logs of P. fluorescens, and 4.8 logs and 4.6 logs of E. coli in 4h, respectively. At low concentrations, the 6%-substituted chitosan-arginine was more effective in inhibiting cell growth even though the 30%-substituted chitosan-arginine appeared to be more effective in permeabilizing the cell membranes of both P. fluorescens and E. coli. Studies using fluorescent probes, 1-N-phenyl-naphthylamine (NPN), nile red (NR) and propidium iodide (PI), and field emission scanning electron microscopy (FESEM) suggest that chitosan-arginine's antibacterial activity is, at least in part, due to its interaction with the cell membrane, in which it increases membrane permeability.

Fig 1

Fig 1 (A-B) – The growth of P. fluorescens (A) and E. coli (B) in the presence of a series of concentrations of 6%-substituted and 30%-substituted chitosan-arginine, after 24 h incubation.

Fig 2

Time-dependent killing efficacy of 5000 mg L-1 of 6% substituted and 30% substituted chitosan-arginine against P. fluorescens (A) and E. coli (B). Each data point is the average of two independent experiments and bar is the standard deviation.

Fig 3

Fig 3 (A-B) – The uptake of NPN probe by P. fluorescens (A) and E. coli (B) over time, with the addition of 50 mg L^-1 6%- and 30%- substituted chitosan-arginine.

Fig 4

Fig 4 (A-D) – The fluorescence spectra of (Nile Red) NR before and after the addition of 50 mg L^-1 6%- substituted chitosan-arginine to P. fluorescens (A) or E. coli (B). The uptake of Nile Red (NR) probe by P. fluorescens (C) or E. coli (D) over time with the addition of 50 mg L^-1 6%- and 30%- substituted chitosan-arginine.

Fig 5

The uptake of PI probe by P. fluorescens with the addition of 50 mg L^-1 6%- and 30%- substituted chitosan-arginine.

Fig 6

Fig 6 (A-C) – The fluorescence spectra of PI before and after the addition of 50 mg L^-1 6%- substituted chitosan-arginine to E. coli (A). The uptake of PI probe by E. coli in a period of 2 h (B) and up to 24 h (C) with the addition of 50 mg L^-1 6%- and 30%- substituted chitosan-arginine.

Fig 7

Fig 7 (A-F) – SEM of E. coli after incubation with 100 mg L^-1 chitosan-arginine for 3 h. Controls (A and D), cells treated with 6%-substituted chitosan-arginine (B and E), and cells treated with 30%-substituted chitosan-arginine (C and F).

Fig 8

Fig 8 (A-F) – SEM of P. fluorescens after incubation with 100 mg L^-1 chitosan-arginine for 3 h. Controls (A and D), cells treated with 6%-substituted chitosan-arginine (B and E), and cells treated with 30%-substituted chitosan-arginine (C and F).