Characterization of the dispirotripiperazine derivative PDSTP as antibiotic adjuvant and antivirulence compound against Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a major human pathogen, able to establish difficult-to-treat infections in immunocompromised and people with cystic fibrosis (CF). The high rate of antibiotic treatment failure is due to its notorious drug resistance, often mediated by the formation of persistent biofilms. Alternative strategies, capable of overcoming P. aeruginosa resistance, include antivirulence compounds which impair bacterial pathogenesis without exerting a strong selective pressure, and the use of antimicrobial adjuvants that can resensitize drug-resistant bacteria to specific antibiotics. In this work, the dispirotripiperazine derivative PDSTP, already studied as antiviral, was characterized for its activity against P. aeruginosa adhesion to epithelial cells, its antibiotic adjuvant ability and its biofilm inhibitory potential. PDSTP was effective in impairing the adhesion of P. aeruginosa to various immortalized cell lines. Moreover, the combination of clinically relevant antibiotics with the compound led to a remarkable enhancement of the antibiotic efficacy towards multidrug-resistant CF clinical strains. PDSTP-ceftazidime combination maintained its efficacy in vivo in a Galleria mellonella infection model. Finally, the compound showed a promising biofilm inhibitory activity at low concentrations when tested both in vitro and using an ex vivo pig lung model. Altogether, these results validate PDSTP as a promising compound, combining the ability to decrease P. aeruginosa virulence by impairing its adhesion and biofilm formation, with the capability to increase antibiotic efficacy against antibiotic resistant strains.

Keywords: Pseudomonas aeruginosa; antiadhesion; antibiofilm; antibiotic adjuvant; antivirulence; combination therapy; drug resistance.

Figure 1

Chemical structure of the compound PDSTP.

Figure 2

Differential adhesion abilities to immortalized human cell lines of Pseudomonas aeruginosa strains treated with PDSTP. Adhesion of Pseudomonas aeruginosa PA01 on A549 cell monolayers in the presence of increasing concentrations of PDSTP (from 1 to 400 μg/mL) (A). Adhesion of Pseudomonas aeruginosa PA01 on 16HBE14o- and CFBE41o- cell monolayers in the presence of 50 μg/mL of PDSTP (B). Adhesion of Pseudomonas aeruginosa BT2 and BT72 on A549 cell monolayers in the presence of 50 and 200 μg/mL of PDSTP (C). Adhesion of Pseudomonas aeruginosa PA01 on A549 cell monolayers in the presence of 50 μg/mL of PDSTP, 50 μg/mL heparin and a mixture of PDSTP and heparin (at the previously used concentrations) (D). Each experiment was performed three times, each time in duplicate. Statistically significant differences are indicated (One-way ANOVA test for panel (A,C,D); ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Student’s t test for panel (B); *, p < 0.05).

Figure 3

ImageStream flow cytometry analysis of PDSTP anti-adhesive effect against Pseudomonas aeruginosa PA01. Adhesion of GFP-expressing Pseudomonas aeruginosa PA01 on A549 cell monolayers in the presence of 50 μg/mL of PDSTP (A). Events showing the number of adhered GFP-expressing Pseudomonas aeruginosa PA01 per A549 cell on 10.000 acquisitions of the control (black column) and samples treated with 50 μg/mL of PDSTP (green column) (B). Number of events normalized on the total number of cells with adhered bacteria, expressed in percentage, of the control (black column) and cells treated with 50 μg/mL of the compound (green column) (C). The experiment was performed twice, each time in duplicate. Statistically significant differences are indicated (Student’s t test; ns, not significant; *, p < 0.05).

Figure 4

Pseudomonas aeruginosa PA01 time-killing assays of tobramycin (A), ciprofloxacin (B), ceftazidime (C) and colistin (D) combined with either 50 or 200 μg/mL of PDSTP, represented as the difference in log₁₀(CFU/mL) of each time point against the log₁₀ (CFU/mL) at time 0. Black line, untreated sample; orange line, treatment with 50 μg/mL of PDSTP; red line, treatment with 200 μg/mL of PDSTP; blue line, treatment with a concentration equal to ½ MIC of the antibiotic; light green line, combination of 50 μg/mL of PDSTP with the antibiotic; dark green line, combination of 200 μg/mL of PDSTP with the antimicrobial.

Figure 5

Kaplan–Meier survival curve of Galleria mellonella larvae infected with Pseudomonas aeruginosa PA01 and treated with physiological saline (black), PDSTP (red), ceftazidime (blue) or the combination of PDSTP and ceftazidime (green). The experiment was performed three times. Statistically significant differences are indicated (Log-rank test; *, p < 0.05).

Figure 6

Effect of PDSTP against biofilm formation in Pseudomonas aeruginosa PA01, NN2 and SG2 strains using crystal violet assay (A). CLSM images (400x magnification) of Pseudomonas aeruginosa biofilms formed with or without PDSTP (B). 2D images acquired at equal distances along the Z-axis were stacked to reconstruct the 3D biofilm images. Analysis of biofilm properties by COMSTAT2 (C–E). Experiments were performed three times. Statistically significant differences are indicated (One-way ANOVA test; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 7

Effect of PDSTP on Pseudomonas aeruginosa PA01, NN2 and SG2 strains biofilm formation in an ex vivo pig lung model (EVPL), represented as the number of CFU/mL recovered from treated and untreated tissues (A). CLSM images (400x magnification) of Pseudomonas aeruginosa PA01 biofilms formed on EVPL with or without PDSTP (B). 2D images acquired at equal distances along the Z-axis were stacked to reconstruct the 3D biofilm images. Analysis of biofilm properties by COMSTAT2 (C). Experiments were performed three times. Statistically significant differences are indicated (One-way ANOVA test; *, p < 0.05; **, p < 0.01).