Antivirulence Properties of a Low-Molecular-Weight Quaternized Chitosan Derivative against Pseudomonas aeruginosa

Abstract

The co-occurrence of increasing rates of resistance to current antibiotics and the paucity of novel antibiotics pose major challenges for the treatment of bacterial infections. In this scenario, treatments targeting bacterial virulence have gained considerable interest as they are expected to exert a weaker selection for resistance than conventional antibiotics. In a previous study, we demonstrated that a low-molecular-weight quaternized chitosan derivative, named QAL, displays antibiofilm activity against the major pathogen Pseudomonas aeruginosa at subinhibitory concentrations. The aim of this study was to investigate whether QAL was able to inhibit the production of relevant virulence factors of P. aeruginosa. When tested in vitro at subinhibiting concentrations (0.31-0.62 mg/mL), QAL markedly reduced the production of pyocyanin, pyoverdin, proteases, and LasA, as well as inhibited the swarming motility of three out of four P. aeruginosa strains tested. Furthermore, quantitative reverse transcription PCR (qRT-PCR) analyses demonstrated that expression of lasI and rhlI, two QS-related genes, was highly downregulated in a representative P. aeruginosa strain. Confocal scanning laser microscopy analysis suggested that FITC-labelled QAL accumulates intracellularly following incubation with P. aeruginosa. In contrast, the reduced production of virulence factors was not evidenced when QAL was used as the main polymeric component of polyelectrolyte-based nanoparticles. Additionally, combination of sub-MIC concentrations of QAL and tobramycin significantly reduced biofilm formation of P. aeruginosa, likely due to a synergistic activity towards planktonic bacteria. Overall, the results obtained demonstrated an antivirulence activity of QAL, possibly due to polymer intracellular localization and QS-inhibition, and its ability to inhibit P. aeruginosa growth synergizing with tobramycin.

Keywords: LasA; Pseudomonas aeruginosa; biofilm; chitosan; motility; proteases; pyocyanin; pyoverdin; quaternized chitosan; quorum sensing; tobramycin; virulence factors.

Figure 1

Growth curves of P. aeruginosa strains incubated in LB broth alone (CTRL, control) or in the presence of QAL 0.31 or 0.62 mg/mL. Values are the mean of two independent experiments.

Figure 2

Effects of QAL at sub-MIC concentrations on the production of virulence factors by P. aeruginosa strains. Virulence factors were quantified in supernatant cultures after incubation of each bacterial strain in the presence or absence (CTRL) of QAL for 48 h in LB broth in static conditions. Each experiment was carried out at least three times. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Effect of QAL at sub-MIC levels on swarming motility of P. aeruginosa strains. Swarming motility was evaluated by measuring the diameter of the swarming area (mm) on agar soft medium after incubation at 37 °C for 24 h. Each experiment was carried out three times. * p < 0.05; ** p < 0.01; *** p < 0.001. Representative swarm plate images taken 24 h after inoculation are also shown for each strain. Plates without QAL (CTRL, on the left) and plates containing QAL 0.62 mg/mL (on the right) are shown.

Figure 4

Relative expression of the QS-associated genes (rhlI and lasI) evaluated by qRT-PCR in P. aeruginosa cells untreated (CTRL) or incubated with QAL (0.31 and 0.62 mg/mL) for 48 h. The levels of QS gene expression were normalized to those of the house-keeping rpoD gene and then compared with the level of expression of the control. Results are expressed as the mean and standard error of the mean from three independent experiments. Statistically significant differences (** p < 0.01; *** p < 0.001) between the test compound and the corresponding controls are indicated.

Figure 5

Flow cytometric analysis and CLSM images of P. aeruginosa cells incubated for 48 h with QAL-FITC at 0.62 mg/mL; (a) Cytofluorimetric analysis of QAL-FITC-treated P. aeruginosa; (b) CLSM image of P. aeruginosa treated with FITC only (negative control); (c,d) different fields of view of P. aeruginosa treated with QAL-FITC showing typical cytoplasmic granular fluorescent staining; (e) confocal Z-stack images of QAL-FITC-treated bacterial cells. A representative experiment is depicted out of two performed with similar results.

Figure 6

Effect of subinhibiting concentrations of QAL used alone and in combination (comb) with tobramycin (tob) on (a) biofilm formation and (b) planktonic growth of P. aeruginosa strains. CTRL, control. Data represent the average of two technical replicates from three independent experiments and error bars indicate standard error of the mean. Asterisks represent statistical significance of the comparisons between each sample with the corresponding combination. * p < 0.05; ** p < 0.01; *** p < 0.001.