The urinary antibiotic 5-nitro-8-hydroxyquinoline (Nitroxoline) reduces the formation and induces the dispersal of Pseudomonas aeruginosa biofilms by chelation of iron and zinc

Abstract

Since cations have been reported as essential regulators of biofilm, we investigated the potential of the broad-spectrum antimicrobial and cation-chelator nitroxoline as an antibiofilm agent. Biofilm mass synthesis was reduced by up to 80% at sub-MIC nitroxoline concentrations in Pseudomonas aeruginosa, and structures formed were reticulate rather than compact. In preformed biofilms, viable cell counts were reduced by 4 logs at therapeutic concentrations. Complexation of iron and zinc was demonstrated to underlie nitroxoline's potent antibiofilm activity.

Fig 1

Dosage-dependent effects of sub-MICs of nitroxoline on biofilm mass (A) and planktonic cell density (B) in various P. aeruginosa isolates. Bacteria were grown for 18 h in tryptic soy broth (TSB) medium with or without indicated nitroxoline concentrations. (A) Biofilm formed was stained with crystal violet, measured at 600 nm, and depicted as a percentage of control in medium alone. (B) Density of the planktonic population was determined in parallel by turbidity measurement of the supernatant at 620 nm. Data shown represent respective means ± standard deviations (SD) of three independent experiments, with three replicate tubes per experiment.

Fig 2

Dispersal of mature biofilms by sub-MICs of nitroxoline (NIT). (A) Dosage dependence. Biofilms of strain BK6695-10 were grown in polystyrol tubes (1-ml bathing volume exposed to approximately 5-cm² tube surface, CELLSTAR polystyrene tubes; Greiner Bio-One GmbH, Kremsmünster, Austria) washed and subsequently exposed to the indicated agents or phosphate-buffered saline (PBS) alone (control) for 1.5 h. Viable cell numbers in the supernatant and, after dislodgment by sonication (17), in the biofilm layer were determined by serial dilution and plating. Data represent means ± SD of five independent experiments, and significant differences to controls are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) Kinetics of dispersal. Biofilms were exposed over a period of 12 h and subsequently stained with crystal violet. Results are representative of three independent experiments, with three replicate tubes per experiment.

Fig 3

Effect of sub-MIC nitroxoline on biofilm morphology. Biofilms were grown under static conditions on tissue culture flasks for 18 h in the absence (A, C, E, and G) or presence of 8 μg/ml nitroxoline (B, D, F, and H). (A and B) CLSM images at 100-fold original magnification of acridine orange-stained biofilm. Horizontal optical sections from the midpoint of the biofilms are shown on the left and vertical optical sections on the right. SEM images at 500-fold (C and D), 2,000-fold (E and F) and 10,000-fold (G and H) original magnifications.

Fig 4

Effect of sub-MIC nitroxoline on preexisting biofilms. CLSM images of acridine-stained biofilm that was treated with 8 μg/ml nitroxoline for 4 h (B) and 12 h (D) in comparison to PBS with 2% TSB alone (A and C).

Fig 5

Effect of nitroxoline on twitching motility. Strains were stab inoculated through a thin agar layer containing the indicated nitroxoline concentration onto plastic petri dishes, and the hazy zone forming at the interface between agar and polystyrene surface was measured. Results are presented as the mean percentage change ± SD in twitching zone diameter compared to LB agar alone (control, twitching zone taken as 100% ± SD).

Fig 6

Relative growth of P. aeruginosa PAO1 biofilms in dependence of defined cation concentrations. M9 medium fully supplemented with the respective cations at the concentrations indicated was incubated overnight with or without 200 μg/ml nitroxoline. Pretreated and control media were subsequently chloroform extracted (6 by 50 ml) to remove nitroxoline and its chelates. Biofilm growth of PAO1 in control medium and in nitroxoline-pretreated medium and in pretreated medium resupplemented with the indicated cations was examined. Biofilm growth as determined by crystal violet staining was related to planktonic growth (turbidity measurement at 620 nm), with the growth ratio in the control medium taken as 1. Data represent means ± SD of three independent experiments, and significant differences to controls are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig 7

Effect of urinary concentrations of nitroxoline on the survival of biofilm cells. Biofilms were grown anaerobically in 1% KNO₃ supplemented TSB (PAO1) as previously described (19) or aerobically in TSB (BK6695-10) for 20 h, washed, and subsequently exposed to the indicated agents or PBS with 1% TSB alone (control) for 6 h. Viable cell numbers in the biofilm layer were determined by serial dilution and plating after dislodgment by sonication (17). Data represent means ± SD of three independent experiments, and significant differences to controls are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Colonized tube surface corresponding to approximately 15 cm².

Fig 8

Effect of physiological concentrations of nitroxoline, colistin, and ciprofloxacin on preexisting biofilm. Biofilms of strains PAO1 (A to D) and BK6695-10 (E to H) were grown in x-well tissue culture coverglass chambers (Sarstedt) for 18 h, treated with the compounds indicated for 6 h, and dyed by LIVE/DEAD BacLight bacterial viability kit (Invitrogen). CLSM was performed as z-stacks of 70 μm, with 28 layers each. Images are shown as superposition in the x-z plane (left) and as sections of approximately 30 m² (basal layers; 6 μm above the glass surface) (right). Living cells are colored green (SYTO 9), and dead cells are colored red (propidium iodide).