Comparative analysis of virulence factors produced by Pseudomonas aeruginosa strains isolated from chronic wounds or bloodstream infections

Abstract

Pseudomonas aeruginosa is an important pathogen associated with both chronic wounds and bloodstream infections. Virulence factors required for the establishment of acute and chronic infections differ substantially. Since bacteremia can be a severe outcome of wound colonization, we performed a comparative analysis of virulence between P. aeruginosa strains isolated from the bloodstream and chronic wounds. Our results show that, in general, P. aeruginosa strains isolated from bloodstream infections are more virulent than strains that colonize chronic wounds.

Figure 1. Comparison of virulence phenotypes between P. aeruginosa strains isolated from bloodstream infections or chronic wounds.

We analyzed the phenotypic expression of virulence factors of 74 P. aeruginosa strains previously isolated from bloodstream infections or chronic wounds. (A) Overnight biofilms were stained with 0.1% crystal violet and the optical density (OD) of each well was measured at 570 nm (OD ₅₇₀ ) to determine the biofilm formation capability of each strain. Based on the OD ₅₇₀ measurements, strains were also classified into four categories: non-adherent, weakly adherent, moderately adherent, and strongly adherent. Overall, strains isolated from bloodstream infections formed stronger biofilms than strains from chronic wounds. (B) Swimming motility was evaluated by measuring the diameter of colonies grown in LB plates with 0.3% agar for 24 hours. (C) Swarming motility was evaluated by measuring the area of colonies grown in LB plates with 0.6% agar for 24 hours. (D) Twitching motility was evaluated by stabbing P. aeruginosa strains into the agar of LB plates with 1% agar and, after 24 hours, staining the bottom of plates with 0.1% crystal violet. The diameter of the stained area represented the twitching distance of each strain. (E) Proteolytic activity was analyzed by measuring the clearance zone diameter around colonies grown for 24 hours in LB agar with 2% skimmed milk. (F) Pyocyanin production was measured by chloroform extraction from 48-hour cultures and quantification in a spectrophotometer at an OD of 690 nm (OD ₆₉₀ ). The box in each graph extends from the 25th to 75th percentiles and the line in the middle is plotted at the median. Each experiment was performed in triplicate and symbols represent the mean values of the replicates for each P. aeruginosa strain. Whiskers are drawn from the smallest to the largest value. Significance between groups was analyzed using the Mann-Whitney test. ns: p> 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001.