In vitro potentiation of tetracyclines in Pseudomonas aeruginosa by RW01, a new cyclic peptide

Abstract

The pipeline for new drugs against multidrug-resistant Pseudomonas aeruginosa remains limited, highlighting the urgent need for innovative treatments. New strategies, such as membrane-targeting molecules acting as adjuvants, aim to enhance antibiotic effectiveness and combat resistance. RW01, a cyclic peptide with low antimicrobial activity, was selected as an adjuvant to enhance drug efficacy through membrane permeabilization. RW01's activity was evaluated via antimicrobial susceptibility testing in combination with existing antibiotics on 10 P. aeruginosa strains and analog synthesis. Synergy was assessed using checkerboard assays, and one-step mutants were generated to identify altered pathways through whole-genome sequencing and variant analysis. Permeabilizing activity was studied using flow cytometry and real-time fluorescence measurement. In vivo toxicity was assessed in female C57BL/6J mice, and possible interaction with mouse serum was also evaluated. Susceptibility testing revealed specific synergy with tetracyclines, with up to a 16-fold reduction in minimum inhibitory concentrations. Sequencing revealed that resistance to the RW01-minocycline combination involved mutations in the pmrB gene, affecting outer membrane lipopolysaccharide composition. This was further confirmed by the identification of cross-resistance to colistin in these mutants. RW01 reduced the mutant prevention concentration of minocycline from 64 to 8 mg/L. RW01 was demonstrated to enhance membrane permeabilization and therefore minocycline uptake with statistical significance. Synthetic derivatives of RW01 showed a complete loss of activity, highlighting the importance of RW01's D-proline(NH2) residue. No acute or cumulative in vivo toxicity was observed in mice. These findings suggest that RW01 could revitalize obsolete antimicrobials and potentially expand therapeutic options against multidrug-resistant P. aeruginosa.

Keywords: Pseudomonas aeruginosa; adjuvants; antimicrobial resistance; cyclic peptides; membrane disruption.

Fig 1

Chemical structure of RW01 cyclic peptide and its chemical analogs.

Fig 2

Minimum inhibitory concentration of each antibiotic with and without 100 mg/L of RW01. In green, ≥4-fold MIC reduction; in orange, 1- to 4-fold MIC reduction; in red, ≤1-fold MIC reduction.

Fig 3

MIC distribution of antibiotic vs combined treatment with RW01 among the P. aeruginosa strains used in this study. The x axis represents the concentration of the antibiotic used in milligram per liter. The y axis represents all the antibiotics tested. In blue, MIC distribution of antibiotic itself; in orange, MIC distribution of the combination. ATB, antibiotic.

Fig 4

Results of checkerboard assays performed with PAO1 (a and b) and PAOUW (c and d). Color intensity indicates the frequency of observed growth: 0 (white) represents no growth, while 1 (dark blue/green) indicates growth observed in all replicates. Light blue/green denotes growth observed in one replicate; medium blue/green denotes growth observed in two replicates. On the x axis, minocycline and tigecycline concentration in milligram per liter. On the y axis, RW01 concentration in milligram per liter. N, negative control column.

Fig 5

Population analysis through CFU per milliliter recounts of PAO1 in increasing concentrations of minocycline alone or in combination with RW01 at 100 µg/mL. MPC, mutant prevention concentration.

Fig 6

PI⁺ percentage (%) obtained from flow cytometry recounts of wild-type PAO1, at different concentrations of RW01 (0.2× MIC, 100 mg/L; 0.5× MIC, 250 mg/L; 1× MIC, 500 mg/L; 5× MIC, 2.5 g/L; and 10× MIC, 5 g/L) and colistin (0.2× MIC, 0.2 mg/L; 0.5× MIC, 0.5 mg/L; 1× MIC, 1 mg/L; 5× MIC, 5 mg/L; and 10× MIC – 10 mg/L) and at different times (4 and 24 h). *Difference between 4 h RW01 treatment and 4 h colistin treatment was statistically significant.

Fig 7

Visualization of RW01’s permeabilization activity in PAO1 at increasing concentrations under confocal microscopy (0.2× MIC, 100 mg/L; 0.5× MIC, 250 mg/L; 1× MIC, 500 mg/L; 5× MIC, 2.5 g/L; and 10× MIC, 5 g/L). VC, viability control, no treatment.

Fig 8

Fluorescence peak measurements of minocycline that relate to intracellular accumulation. Dotted lines represent error bars of the three replicates for each treatment.