Table 1.
Sequences and MICs (μM) for the peptides and azithromycin used in this work.
Fig 1.
D-11 synergizes with azithromycin against P. aeruginosa.
(A) Representative heat plots of the microdilution checkerboard assay for the combination of azithromycin and D-11 against PAO1. (B) Time killing curves for D-11 and azithromycin monotherapy and combination therapy against PAO1 during 24 h incubation at the indicated concentrations. Error bars represent mean ± SD for three biological replicates.
Fig 2.
D-11 increases membranes permeability, interacts with LPS and phospholipids and dissipates the membrane potential.
(A) D-11 dosage-related outer membrane permeability increment. Polymyxin B at 0.5mg/L was used as a positive control. (B) D-11 dosage-related inner membrane permeability increment. 0.5 mg/L polymyxin B was used as a positive control. (C) SYTO9 and PI live/dead cell staining of PAO1 treated with D-11. P. aeruginosa cells were treated with indicated concentrations of D-11 for 60 min. Cells were washed with PBS buffer and stained with SYTO9 and PI before being inspected with a microscope. (D) D-11 activity in the presence of BSA, PC, PE, PG, CL or LPS, the activity for the synergistic relation D-11/azithromycin in the presence of LPS (E), CL (F), and PG (G). D-11 lost the synergistic effect in the presence of LPS, CL or PG. The MIC of azithromycin was determined by checkerboard microdilution assays in the presence of LPS, CL or PG (0–256 mg/L) with 4 μM of D-11 or not. (H) DiSC3(5) membrane potential measurement for several D-11 dosages. Polymyxin B at 0.5mg/L was used as a positive control. The DiSC3(5) dye was incubated with PAO1 for 30 min followed by self-quenching and stabilization, then indicated concentrations of D-11 and SDS were added. The fluorescence units were monitored for 60 min. (I) PMF disruption measurement. CCCP was used as a positive control. All the tests were performed in triplicate and all the data are presented as mean ± SD. **, P < 0.01; ***, P < 0.001 by Student’s t-test.
Fig 3.
D-11 impairs ATP and NADH levels, induces the accumulation of ROS, inhibits efflux pumps and promotes intracellular antibiotic accumulation.
(A) Intracellular ATP levels at different D-11 concentrations. 40 mg/L CCCP was used as a positive control. (B) Resazurin to resorufin reduction by NADH at different D-11 doses. (C) Intracellular NADH concentration after different D-11 doses. (D) Total ROS accumulation in PAO1 treated with D-11. Exogenous addition of NAC (6 mM) diminished the accumulation of ROS induced by 16 μM of D-11. (E) D-11 dosage-related EtBr efflux inhibition. PAO1 cells were incubated with EtBr in HEPES for 30 min at 37 °C then the cells were collected and resuspended in MHB medium containing the indicated concentrations of D-11 or 40mg/L CCCP. Then the fluorescence was monitored for 90 min at 37°C. (F) D-11 antagonizes the effect of the efflux pumps inhibitor PAβN. (G) Intracellular D-11 dosage-related increment of azithromycin. (H) β-Galactosidase activity for the combination of azithromycin and D-11. (I) Relative lacZ mRNA expression levels for D-11 and D-11/azithromycin-treated cells. All the tests were performed in triplicate and all the data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant by Student’s t-test.
Fig 4.
Transcriptome analysis and proposed synergy action model.
(A) The effect of D-11 alone, azithromycin alone or the combination over the expression of several genes involved in stress response, oxidative phosphorylation, metabolism and virulence. AZM, 8 μM azithromycin treatment alone; D-11, 4 μM D-11 treatment alone; AZM + D-11, the combination of azithromycin and D-11 treatment. (B) Proposed action mechanism for the synergy of D-11 in combination with antibiotics. D-11 restores the susceptibility of P. aeruginosa to antibiotics through membrane-mediated mechanisms by interacting with LPS, CL and PG. These reactions trigger membrane depolarization and induce membrane dysfunction, further resulting in metabolic perturbations and ROS accumulation. In addition, more antibiotics rapidly accumulate inside bacteria due to the increased membrane permeability and dysfunction of efflux pumps mediated by the dissipation of membrane potential. The accumulation of ROS and antibiotics working together resulted in the death of cells.
Fig 5.
D-11 increase the anti-biofilm ability of azithromycin.
(A) The biofilm was grown at the indicated conditions and stained with crystal violet. The crystal violet was dissolved in ethanol and measured at a wavelength of 595 nm. (B) The biofilm was grown at the indicated conditions and dispersed by ultrasound. The bacterial count inside of biofilm was determined by plating. The established biofilm was treated with indicated conditions for 24 hours. The bacterial count inside the biofilm (C) or in the supernatant (D) was determined by plating. All the tests were performed in triplicate and all the data are presented as mean ± SEM. **, P < 0.01; ***, P < 0.001; ns, not significant by Student’s t-test.
Fig 6.
D-11 enhances the activity of multiple antibiotics.
(A) synergistic effects of D-11 with multiple antibiotics. The FICI values of D-11 combined with multiple antibiotics and the fold change of MICs of those antibiotics in the presence of 4 μM D-11. (B) The heat map of the FIC indices for the synergistic effects of multiple antimicrobial agents combined with 4 μM D-11 against three standard P. aeruginosa strain and nine clinical isolates. Synergy is indicated by an FIC index ≤ 0.5.
Fig 7.
D-11 enhances the activities of multiple antibiotics ex vivo and in vivo.
(A) Ex vivo bacteremia model for antibiotics, D-11, and their combinations antimicrobial activity. 10 μL of serial decimal dilutions of each one of the combinations of antibiotics with/without D-11 (4 μM) tested in blood were dropped. In vivo efficacy of azithromycin (7.5 mg/kg) (B), chloramphenicol (8 mg/kg) (C), doxycycline (2 mg/kg) (D) and D-11 (10 mg/kg) alone or their combination against P. aeruginosa PAO1 (n = 7 for doxycycline and D-11, n = 8 for the other groups). All the three experiments are using the same group of negative control which treatment with saline. All the data are presented as mean ± SD. ***, P < 0.001; ****, P < 0.0001; ns by Student’s t test.