Organic Acids and Their Salts Potentiate the Activity of Selected Antibiotics against Pseudomonas aeruginosa Biofilms Grown in a Synthetic Cystic Fibrosis Sputum Medium

Abstract

The failure of antibiotic therapy in respiratory tract infections in cystic fibrosis is partly due to the high tolerance observed in Pseudomonas aeruginosa biofilms. This tolerance is mediated by changes in bacterial metabolism linked to growth in biofilms, opening up potential avenues for novel treatment approaches based on modulating metabolism. The goal of the present study was to identify carbon sources that increase the inhibiting and/or eradicating activity of tobramycin, ciprofloxacin, and ceftazidime against P. aeruginosa PAO1 biofilms grown in a synthetic cystic fibrosis sputum medium (SCFM2) and to elucidate their mode of action. After screening 69 carbon sources, several combinations of antibiotics + carbon sources that showed markedly higher anti-biofilm activity than antibiotics alone were identified. d,l-malic acid and sodium acetate could potentiate both biofilm inhibiting and eradicating activity of ciprofloxacin and ceftazidime, respectively, while citric acid could only potentiate biofilm inhibitory activity of tobramycin. The mechanisms underlying the increased biofilm eradicating activity of combinations ciprofloxacin/d,l-malic acid and ceftazidime/sodium acetate are similar but not identical. Potentiation of ceftazidime activity by sodium acetate was linked to increased metabolic activity, a functional TCA cycle, increased ROS production, and high intracellular pH, whereas the latter was not required for d,l-malic acid potentiation of ciprofloxacin. Finally, our results indicate that the potentiation of antibiotic activity by carbon sources is strain dependent.

Keywords: Pseudomonas aeruginosa; biofilms; cystic fibrosis.

FIG 1

Effect of the selected carbon sources on P. aeruginosa PAO1 biofilm inhibition in SCFM2 by 2.5 μg/mL tobramycin, 0.3 μg/mL ciprofloxacin and 1.56 μg/mL ceftazidime. Values shown are the mean (n ≥ 3). Error bars indicate standard error. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

Effect of different combinations of (A) ciprofloxacin/d,l-malic acid and (B) ceftazidime/sodium acetate on eradication of P. aeruginosa PAO1 biofilms. Values shown are the mean (n ≥ 3). Error bars indicate standard error. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Maximum metabolic rate of treated and untreated (control) P. aeruginosa PAO1 biofilms. (A) MA: 60 mM d,l-malic acid; CIP: 1.2 μg/ml ciprofloxacin; MA-CIP: 60 mM d,l-malic acid combined with 1.2 μg/ml ciprofloxacin. (B) SA: 120 mM sodium acetate; CAZ: 12.48 μg/ml ceftazidime; SA-CAZ: combination of 120 mM sodium acetate and 12.48 μg/ml ceftazidime. Values shown are the mean (n ≥ 3). Error bars indicate standard error, **, P < 0.01; ***, P < 0.001.

FIG 4

Evaluation of the role of SDH, PMF and intracellular pH on the potentiating effect of additional carbon source on antibiotic during eradication of P. aeruginosa PAO1 biofilms. 24-h-old-biofilm growth treated by antibiotic (or not) in SCFM2 supplemented with or without additional carbon source in the presence or absence of (A) the SDH inhibitor TTFA, (B) the proton motive force disruptor CCCP, (C) the K⁺ ionophore nigericin and excess levels of K⁺. MA: 60 mM d,l-malic acid; CIP: 1.2 μg/ml ciprofloxacin; MA-CIP: 60 mM d,l-malic acid combined with 1.2 μg/ml ciprofloxacin; SA: 120 mM sodium acetate; CAZ: 12.48 μg/ml ceftazidime; SA-CAZ: combination of 120 mM sodium acetate and 12.48 μg/ml ceftazidime. Values shown are the mean (n ≥ 3). Error bars indicate standard error, **, P < 0.01; ***, P ≤ 0.001; ****, P < 0.0001.

FIG 5

ROS-induced fluorescence in P. aeruginosa PAO1 biofilms. (A) Fluorescence generated over time. (B) Fluorescence at 24 h and 45 h. CIP: 1.2 μg/ml ciprofloxacin; MA-CIP: 60 mM d,l-malic acid combine with 1.2 μg/ml ciprofloxacin; CAZ: 12.48 μg/ml ceftazidime; SA-CAZ: combination of 120 mM sodium acetate and 12.48 μg/ml ceftazidime. Values shown are the mean (n = 3). Error bars indicate standard error, **, P < 0.01.

FIG 6

Eradication of biofilms formed by P. aeruginosa strains LES B58, AA2, AA44, and DK2. Control: no carbon source, no antibiotic; MA: 60 mM d,l-malic acid; CIP: 1.2 μg/ml ciprofloxacin; MA-CIP: 60 mM d,l-malic acid + 1.2 μg/ml ciprofloxacin; SA: 120 mM sodium acetate; CAZ: 12.48 μg/ml ceftazidime; SA-CAZ: 120 mM sodium acetate + 12.48 μg/ml ceftazidime. Values shown are the mean (n = 3). Error bars indicate standard error.