rpoS-mutation variants are selected in Pseudomonas aeruginosa biofilms under imipenem pressure

Abstract

Background: Pseudomonas aeruginosa is a notorious opportunistic pathogen causing various types of biofilm-related infections. Biofilm formation is a unique microbial strategy that allows P. aeruginosa to survive adverse conditions such as antibiotic treatment and human immune clearance.

Results: In this study, we experimentally evolved P. aeruginosa PAO1 biofilms for cyclic treatment in the presence of high dose of imipenem, and enriched hyperbiofilm mutants within six cycles in two independent lineages. The competition assay showed that the evolved hyperbiofilm mutants can outcompete the ancestral strain within biofilms but not in planktonic cultures. Whole-genome sequencing analysis revealed the hyperbiofilm phenotype is caused by point mutations in rpoS gene in all independently evolved mutants and the same mutation was found in P. aeruginosa clinical isolates. We further showed that mutation in rpoS gene increased the intracellular c-di-GMP level by turning on the expression of the diguanylate cyclases. Mutation in rpoS increased pyocyanin production and virulence in hyperbiofilm variants.

Conclusion: Here, our study revealed that antibiotic treatment of biofilm-related P. aeruginosa infections might induce a hyperbiofilm phenotype via rpoS mutation, which might partially explain antimicrobial treatment failure of many P. aeruginosa biofilm-related infections.

Keywords: Biofilms; Cyclic-di-GMP; Experimental biofilm evolution; Pseudomonas aeruginosa; Sigma factor RpoS; Virulence.

Fig. 1

Experimental biofilm evolution of P. aeruginosa under antibiotic stress. A The setup of experimental biofilm evolution of P. aeruginosa. The biofilm of six independent lineages of the P. aeruginosa PAO1 were grown on 5 mm glass bead. After 24 h cultivation, one bead was vortex and sonicated for CFU counts, another bead was transferred to a 24 well microplate and treated with 160 μg/mL imipenem. After 24 h treatment, the surviving cells were grown overnight in fresh medium and start another cycle. B Evolution of biofilm bacteria exposed to imipenem resulted in a rapid increase in biofilm bacteria CFU on bead. C Crystal violet (CV) staining of biofilms formed by ancestral and hyperbiofilm variant strains on PVC plate. Data are presented as the mean ± s.d. of five biological replicates. Significance was determined using a Student’s t test: *P < 0.05, **P < 0.01 and ***P < 0.001. D and E The time frame of emergence of hyperbiofilm variants in linage W1 (D) and W6 (E). The biofilm formation by the different colonies was displayed with CFU of biofilm cells on 5 mm glass bead

Fig. 2

Hyperbiofilm phenotype is caused by rpoS mutation. rpoS point mutation, full deletion (A) and regions deletion strains (B) were increased the biofilm formation. The biofilm formation by the indicated strains was displayed with CFU of biofilm cells on 5 mm glass bead. Data are presented as the mean ± s.d. of four biological replicates. Significance was determined using a Student’s t test: ***P < 0.001. EV represents the empty vector pHERD20T in this assay

Fig. 3

Hyperbiofilm variants prevail in biofilm competitions. A–F The competition of ancestor and hyperbiofilm variants in planktonic cultures and biofilms when inoculated at the same ratio (A–C) or ancestral: hyperbiofilm mutant = 5:1 (D–F). G Disc diffusion antibiotic sensitivity testing, H growth curves measurement, and I biofilm growth curve was measured. Data are presented as the mean ± s.d. of four biological replicates. Significance was determined using a Student’s t test: n.s indicates no significant difference (P ≥ 0.05); ***P < 0.001

Fig. 4

Expression of P_cdrA-gfp, P_rsmY-gfp and P_rsmZ-gfp reporter fusions in rpoS mutants and PAO1 wild-type strain. Relative fluorescence intensity (reflected as GFP/OD₆₀₀) was measured in representative strains containing the P_cdrA-gfp (A), rsmY (B) and rsmZ (C) reporter fusions. Data are presented as the mean ± s.d. of five biological replicates

Fig. 5

rpoS mutations were existed in P. aeruginosa clinical isolates. The distribution of non-synonymous mutation A and the top 5 mutations B on RpoS of P. aeruginosa. 4000 sequences of rpoS were download form pseudomonas genome database, and the non-synonymous mutations were analyzed by CLC Genomics Workbench. C Biofilm formation ability assessment of clinical isolates. A total of 288 clinical isolates from the first affiliated hospital of Guangxi Medical University (shown in black) were analyzed for rpoS mutations and biofilm assays on 96 well PVC plates

Fig. 6

Pyocyanin production and virulence are increased in rpoS mutants. The production of pyocyanin (A) and cytotoxicity effect against macrophage cells (B) of P. aeruginosa PAO1 wild-type, RpoS^P251L, RpoS^Q266stop and ΔrpoS. Data are presented as the mean ± s.d. of four biological replicates. Significance was determined using a Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001