Bactericidal Activity of Usnic Acid-Chitosan Nanoparticles against Persister Cells of Biofilm-Forming Pathogenic Bacteria

Abstract

The present study aimed to prepare usnic acid (UA)-loaded chitosan (CS) nanoparticles (UA-CS NPs) and evaluate its antibacterial activity against biofilm-forming pathogenic bacteria. UA-CS NPs were prepared through simple ionic gelification of UA with CS, and further characterized using Fourier transform infrared spectroscopy, X-ray diffraction, and field-emission transmission electron microscopy. The UA-CS NPs presented a loading capacity (LC) of 5.2%, encapsulation efficiency (EE) of 24%, and a spherical shape and rough surface. The maximum release of UA was higher in pH 1.2 buffer solution as compared to that in pH 6.8 and 7.4 buffer solution. The average size and zeta potential of the UA-CS NPs was 311.5 ± 49.9 nm in diameter and +27.3 ± 0.8 mV, respectively. The newly prepared UA-CS NPs exhibited antibacterial activity against persister cells obtained from the stationary phase in batch culture, mature biofilms, and antibiotic-induced gram-positive and gram-negative pathogenic bacteria. Exposure of sub-inhibitory concentrations of UA-CS NPs to the bacterial cells resulted in a change in morphology. The present study suggests an alternative method for the application of UA into nanoparticles. Furthermore, the anti-persister activity of UA-CS NPs may be another possible strategy for the treatment of infections caused by biofilm-forming pathogenic bacteria.

Keywords: UA-CS NPs; anti-persister; bacteria; chitosan; nanoparticles; pathogens; usnic acid.

Figure 1

Diagrammatic representation of preparation of the usnic acid-loaded chitosan nanoparticles (UA-CS NPs).

Figure 2

(A) Time-dependent UV-Vis absorption spectrum of UA released in phosphate-buffered saline (PBS) (pH 7.4) from CS NPs. (a) CS NPs at 22 h, (b) UA-CS NPs at 0 h, (c) UA-CS NPs at 2 h, (d) UA-CS NPs at 4 h, (e) UA-CS NPs at 8 h, (f) UA-CS NPs at 10 h, (g) UA-CS NPs at 22 h, and (h) UA standard (512 µg/mL). The inset is UV-Vis absorption spectra of UA showing two maximum absorption peaks (230 and 270 nm) and prominent shoulder at 337 nm. (B) Cumulative release of UA from CS NPs, which was prepared from 1.2% chitosan and 0.5% UA in the presence of 0.5% tripolyphosphate (TPP). The release of UA from CS NPs was conducted in PBS at pH 1.2, 6.8, and 7.4 and the amount of UA released was quantified by measuring the absorbance at 337 nm.

Figure 3

(A) FTIR spectra of UA-CS NPs prepared from 1.2% chitosan and 0.5% UA in the presence of 0.5% TPP and (B) XRD-spectra of UA (a), UA-CS NPs (b) and CS NPs (c).

Figure 4

(A) Dynamic light scattering (DLS) particle size distribution of UA-CS NPs prepared from 1.2% chitosan and 0.5% UA in the presence of 0.5% TPP, (B) Zeta potential of UA-CS NPs prepared from 1.2% chitosan and 0.5% UA in the presence of 0.5% TPP, (C) FE-TEM micrograph of CS NPs prepared from 1.2% chitosan in the presence of 0.5% TPP, and (D) FE-TEM micrograph of UA-CS NPs prepared from 1.2% chitosan and 0.5% UA in the presence of 0.5% TPP.

Figure 5

MIC determination of UA-CS NPs against gram-positive and gram-negative bacteria. (A) Bactericidal effect of UA-CS NPs against E. coli, (B) Bactericidal effect of UA-CS NPs against P. aeruginosa, (C) Bactericidal effect of UA-CS NPs against S. aureus and (D) Bactericidal effect of UA-CS NPs against L. monocytogenes. The MIC was determined by microbroth dilution in a 96-well microtiter plate at 37 ℃ for 24 h under shaking conditions. The cell growth was determined by measuring optical density (OD) at 600 nm. The positive cell growth was considered when the OD₆₀₀ value was found to be > 0.1, since the OD₆₀₀ of broth was found to be ~ 0.1. Each bar is represented as the means of three replicates ± standard deviation.

Figure 6

Killing of stationary phase cells by different concentrations of UA-CS NPs. (A) Log CFU (colony-forming unit) graph of E. coli, (B) E. coli colonies on TSA (tryptic soy agar) plate, (C) Log CFU graph of P. aeruginosa, (D) P. aeruginosa colonies on TSA plate, (E) Log CFU graph of S. aureus, (F) S. aureus colonies on TSA plate, (G) Log CFU graph of L. monocytogenes, and (H) L. monocytogenes colonies on TSA plate. After incubation, the UA-CS NPs treated cell culture were diluted up to 10⁻⁶ dilution and 100 µL mixture spread was plated on the TSA plate. Each plate shows a representative image of bacterial colonies. The arrow in the graphs indicate the complete killing of persister cells. Each bar is represented as the means of three replicates ± standard deviation.

Figure 7

Killing of persister cells obtained from 5 days mature biofilm by different concentrations of UA-CS NPs. (A) Log CFU graph of E. coli, (B) E. coli colonies on TSA plate, (C) Log CFU graph of P. aeruginosa, (D) P. aeruginosa colonies on TSA plate, (E) Log CFU graph of S. aureus, (F) S. aureus colonies on TSA plate, (G) Log CFU graph of L. monocytogenes, and (H) L. monocytogenes colonies on TSA plate. After incubation, the UA-CS NPs treated cell culture were diluted up to 10^-6 dilution and 100 µL mixture spread was plated on the TSA plate. Each plate shows a representative image of bacterial colonies. The arrow in the graphs indicate the complete killing of persister cells. Each bar is represented as the means of three replicates ± standard deviation. ** p < 0.01

Figure 8

Killing of antibiotic-induced persister cells by different concentrations of UA-CS NPs. (A) Log CFU graph of E. coli, (B) E. coli colonies on TSA plate, (C) Log CFU graph of P. aeruginosa, (D) P. aeruginosa colonies on TSA plate, (E) Log CFU graph of S. aureus, (F) S. aureus colonies on TSA plate, (G) Log CFU graph of L. monocytogenes, and (H) L. monocytogenes colonies on TSA plate. After incubation, the UA-CS NPs treated cell culture were diluted up to 10^-6 dilution and 100 µL mixture spread was plated on the TSA plate. Each plate shows a representative image of bacterial colonies. The arrow in the graphs indicate the complete killing of persister cells. Each bar is represented as the means of three replicates ± standard deviation. **, p < 0.01

Figure 9

Microscopic analysis of cell morphology treated with sub-inhibitory concentrations of UA-CS NPs. Arrow indicated the change in cell morphology. (A) Cell morphology of the E. coli cell in the presence of sub-MIC UA-CS NPs, (B) cell morphology of the L. monocytogenes cell in the presence of sub-MIC UA-CS NPs, (C) cell morphology of the P. aeruginosa cell in the presence of sub-MIC UA-CS NPs, and (D) cell morphology of the S. aureus cell in the presence of sub-MIC UA-CS NPs. Deep-sky blue arrows indicate the dead cells, red color arrow-folding of the cell membrane, pink color arrow-elongated/filamented cells, dark orange color arrow-group of microcolonies.