Pseudomonas aeruginosa increases formation of multidrug-tolerant persister cells in response to quorum-sensing signaling molecules

Abstract

Bacterial persister cells constitute a small portion of a culture which is tolerant to killing by lethal doses of bactericidal antibiotics. These phenotypic variants are formed in numerous bacterial species, including those with clinical relevance like the opportunistic pathogen Pseudomonas aeruginosa. Although persisters are believed to contribute to difficulties in the treatment of many infectious diseases, the underlying mechanisms affecting persister formation are not well understood. Here we show that even though P. aeruginosa cultures have a significantly smaller fraction of multidrug-tolerant persister cells than cultures of Escherichia coli or Staphylococcus aureus, they can increase persister numbers in response to quorum-sensing-related signaling molecules. The phenazine pyocyanin (and the closely related molecule paraquat) and the acyl-homoserine lactone 3-OC12-HSL significantly increased the persister numbers in logarithmic P. aeruginosa PAO1 or PA14 cultures but not in E. coli or S. aureus cultures.

FIG. 1.

Comparison of the persister fractions of cultures of P. aeruginosa, E. coli, or S. aureus. P. aeruginosa PAO1 (diamonds), E. coli K-12 (circles), or S. aureus RN4220 (triangles) cultures were grown to mid-logarithmic (A) or stationary phase (B) at 37°C with aeration and challenged with carbenicillin or ciprofloxacin, respectively, for 3.5 h. Samples were plated on LB agar plates for CFU counting. Nonchallenged control samples (0 mg/ml) were taken directly prior to antibiotic treatment. Cell numbers are plotted either as CFU/ml (left) or as the percentage of nonchallenged cell numbers (right). The values are averages of three independent experiments, and the error bars indicate the standard error of the mean. These experiments were reproduced for P. aeruginosa PA14 with similar results (data not shown).

FIG. 2.

Effect of the phenazine PYO on persister formation in logarithmic P. aeruginosa cultures. (A) P. aeruginosa PAO1 cultures were treated with different concentrations of PYO at early logarithmic phase and subsequently challenged with carbenicillin (1 mg/ml) in mid-logarithmic phase for 3.5 h (circles) or cultivated without antibiotic (diamonds). The values are averages of three independent experiments, and the error bars indicate the standard error of the mean. (B) P. aeruginosa PAO1 cultures were treated with 2 mM PYO (circles) or cultivated without PYO (diamonds) at early logarithmic phase and subsequently challenged with increasing concentrations of carbenicillin. Samples were plated on LB agar plates for CFU counting. The experiment was repeated three times using different carbenicillin concentrations with similar results. The effect of PYO was reproduced for P. aeruginosa PA14 with similar results (data not shown).

FIG. 3.

Effect of the phenazine PYO on persister formation in stationary P. aeruginosa cultures. P. aeruginosa PAO1 cultures were grown to stationary phase at 37°C with aeration in presence or absence of 2 mM PYO, which was added at early logarithmic phase. (A) Bacterial growth was followed by measuring of the optical density (OD₆₀₀). (B) After 18 h of growth, cells were challenged with ciprofloxacin (15 μg/ml) for 3.5 h. Samples were plated on LB agar plates for CFU counting. Nonchallenged control samples (0 μg/ml) were taken directly prior to antibiotic treatment. The values are averages of three independent experiments, and the error bars indicate the standard error of the mean.

FIG. 4.

Effect of the phenazine PYO on persister formation in logarithmic cultures of E. coli and S. aureus. S. aureus RN4220 (A) or E. coli K-12 (B) cultures were treated with different concentrations of PYO at early logarithmic phase and subsequently challenged with ampicillin (1 mg/ml) in mid-logarithmic phase for 3.5 h (circles) or cultivated without antibiotic (diamonds). Samples were plated on LB agar plates for CFU counting. The values are averages of three independent experiments, and the error bars indicate the standard error of the mean.

FIG. 5.

Effect of paraquat or 3-OC12-HSL on P. aeruginosa persister formation. P. aeruginosa PAO1 cultures were treated with different concentrations of paraquat (A) or 3-OC12-HSL (B) at early log phase and subsequently challenged with carbenicillin (1 mg/ml) in mid-log phase (circles) for 3.5 h or cultivated without antibiotic (diamonds). Samples were plated on LB agar plates for CFU counting. The effect of paraquat was reproduced for P. aeruginosa PA14 with similar results (data not shown). The values are averages of three independent experiments, and the error bars indicate the standard error of the mean.

FIG. 6.

Synergistic positive effects of PYO and 3-OC12-HSL on P. aeruginosa PAO1 persister formation. P. aeruginosa PAO1 cultures were treated with different concentrations of 3-OC12-HSL and 0.2 mM PYO (circles) or without PYO (diamonds) at early log phase and subsequently challenged with carbenicillin (5 mg/ml, filled symbols) in mid-log phase or cultivated without antibiotic (open symbols). Samples were plated on LB agar plates for CFU counting. Cell numbers are either plotted as CFU/ml (left) or as the percentage of nonchallenged cell numbers (right). The values are averages of three independent experiments, and the error bars indicate the standard error of the mean.

FIG. 7.

Effect of cell-free supernatants of stationary cultures of the PA14 WT and the PA14 Δphz mutant on persister formation. P. aeruginosa PA14 cultures were grown to early log phase, collected, and resuspended in LB (−) or cell-free supernatants of stationary PA14 WT or PA14 Δphz mutant cultures (supplemented with LB powder). After another 1 h 15 min of cultivation, the cultures were challenged with carbenicillin or piperacillin (1 mg/ml) for 3.5 h (solid bars). Samples were plated on LB agar plates for CFU counting. Nonchallenged control samples (shaded bars) were taken directly prior to antibiotic treatment. The values are averages of three independent experiments, and the error bars indicate the standard error of the mean.