The evolution of virulence in Pseudomonas aeruginosa during chronic wound infection

Abstract

Opportunistic pathogens are associated with a number of chronic human infections, yet the evolution of virulence in these organisms during chronic infection remains poorly understood. Here, we tested the evolution of virulence in the human opportunistic pathogen Pseudomonas aeruginosa in a murine chronic wound model using a two-part serial passage and sepsis experiment, and found that virulence evolved in different directions in each line of evolution. We also assessed P. aeruginosa adaptation to a chronic wound after 42 days of evolution and found that morphological diversity in our evolved populations was limited compared with that previously described in cystic fibrosis (CF) infections. Using whole-genome sequencing, we found that genes previously implicated in P. aeruginosa pathogenesis (lasR, pilR, fleQ, rpoN and pvcA) contained mutations during the course of evolution in wounds, with selection occurring in parallel across all lines of evolution. Our findings highlight that: (i) P. aeruginosa heterogeneity may be less extensive in chronic wounds than in CF lungs; (ii) genes involved in P. aeruginosa pathogenesis acquire mutations during chronic wound infection; (iii) similar genetic adaptations are employed by P. aeruginosa across multiple infection environments; and (iv) current models of virulence may not adequately explain the diverging evolutionary trajectories observed in an opportunistic pathogen during chronic wound infection.

Keywords: Pseudomonas aeruginosa; chronic wounds; evolution of virulence; virulence.

Figure 1.

P. aeruginosa population densities in wound bed and spleen tissues during serial passage experiment are positively correlated. (a) Wound bed CFUs for mice at time of death for each evolutionary round were relatively stable, aside from the 8th mouse in line A. (b) Spleen CFUs for mice at time of death for each evolutionary round were highly variable throughout the experiment, with many values falling below our limit of detection (10² cells). Each CFU count represents one technical replicate. Wound bed and spleen CFUs during the serial passage experiment were positively correlated (Pearson's r₂₈ = 0.44, p = 0.015).

Figure 2.

Changes in morphology, protease production and swimming and swarming motilities. (a) There were five distinct types of colony morphology on CRA at the final round of selection across all three lines of evolution, with line A having two distinct morphology types, and lines B and C each having three distinct colony morphology types (with some colony morphology types being present in multiple lines). (b) Isolates A92, B16, C38 and C62 displayed protease activity comparable with that of the ancestral PA14, while isolates A88, B31, B42 and C31 showed decreased protease activity. (c) Isolates B42, C38 and C62 lost the ability to swim. (d) Isolates A88, B31, B42, C31, C38 and C62 lost the ability to swarm.

Figure 3.

Changes in production of pyoverdine, pyochelin and pyocyanin. (a) Pyoverdine production in the final evolved representative isolates and ancestral PA14. A88 was the only evolved isolate with pyoverdine production significantly different than the ancestor strain (Kruskal–Wallis, Dunn's post hoc test, Benjamini–Hochberg correction, p = 0.018). Error bars indicate SEM. (b) Pyochelin production in the final evolved representative isolates and ancestral PA14. A88 was the only evolved isolate with pyochelin production significantly different than the ancestor strain (Kruskal–Wallis, Dunn's post hoc test, Benjamini–Hochberg correction, p = 0.0082). (c) Pyocyanin production in the final evolved representative isolates and ancestral PA14. C38 and C62 both displayed pyocyanin production significantly different than the ancestor strain (Kruskal–Wallis, Dunn's post hoc test, Benjamini–Hochberg correction, p = 0.01975 and p = 0.01915, respectively).

Figure 4.

Virulence can evolve in diverging directions in a chronic wound. (a) Mice infected by the final evolved population of line B in the sepsis experiment had the highest mortality rate (100%), with no surviving mice at the end of 80 h, while mice infected by line C had the lowest mortality (40%), with three of five mice surviving. (b) Mice infected by the final evolved population of line B in the sepsis experiment had significantly higher mean spleen CFUs at time of death as compared with mice infected by line C, indicating more severe septicaemia (Kruskal–Wallis, Dunn's post hoc test, Holm–Bonferroni correction, p = 0.023). Error bars indicate SEM.