5-Fluorouracil Inhibits Bacterial Growth and Reduces Biofilm in Addition to Having Synergetic Effects with Gentamicin Against Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a multidrug-resistant pathogen known for chronic infections, mainly due to biofilm formation. This study aimed to explore the potential repurposing of 5-fluorouracil (5-FU), an anticancer drug, to treat P. aeruginosa infections. Firstly, we investigated the inhibitory effects of 5-FU on bacterial growth using the microdilution method. Secondly, the impact of 5-FU on biofilm formation and disassembly was assessed via biofilm biomass measurements with the crystal violet staining method and confocal microscopy analyses. Lastly, the potential synergy between 5-FU and the antibiotics gentamicin and meropenem was evaluated using a checkerboard assay. Results revealed that 5-FU inhibited bacterial growth in a dose-dependent manner, with 100% inhibition observed at concentrations of 25 µg/mL and higher. Also, 70% and 100% reductions in biofilm biomass were demonstrated at concentrations of 12 and 100 µg/mL, respectively. Controversy, these higher concentrations unexpectedly increased biofilm biomass in pre-formed biofilms. Synergistic interactions were observed between 5-FU and gentamicin in both growth inhibition (FICI 0.31) and biofilm inhibition (ZIP 14.1), while no synergy was found with meropenem. These findings highlight the potential of 5-FU as an adjunctive therapy for P. aeruginosa infections, especially in combination with gentamicin. However, further research is required to address 5-FU limitations against mature biofilms.

Keywords: 5-fluorouracil; Pseudomonas aeruginosa; biofilm; growth inhibition; synergism.

Figure 1

Efficacy of different concentrations of 5-fluorouracil on the growth of PAO1. (A) Average OD₆₀₀ reading and % inhibition of 5-fluorouracil concentrations ranging from 0 to 500 µg/mL; (B) % inhibition of bacterial growth at different concentrations of 5-fluorouracil. The potency of the drug decreased significantly at concentrations below 0.5 µg/mL, while it remained above 95% at concentrations of 12 µg/mL and above. Data points represent the mean values of at least 3 replicates. For all concentrations, there was a significant decrease in growth with a p value of 0.0044 (p < 0.05) for a concentration of 0.1 µg/mL and p < 0.0001 for concentrations of 0.5 to 500 µg/mL.

Figure 1

Efficacy of different concentrations of 5-fluorouracil on the growth of PAO1. (A) Average OD₆₀₀ reading and % inhibition of 5-fluorouracil concentrations ranging from 0 to 500 µg/mL; (B) % inhibition of bacterial growth at different concentrations of 5-fluorouracil. The potency of the drug decreased significantly at concentrations below 0.5 µg/mL, while it remained above 95% at concentrations of 12 µg/mL and above. Data points represent the mean values of at least 3 replicates. For all concentrations, there was a significant decrease in growth with a p value of 0.0044 (p < 0.05) for a concentration of 0.1 µg/mL and p < 0.0001 for concentrations of 0.5 to 500 µg/mL.

Figure 2

Heatmap displaying the OD₆₀₀ readings from a checkerboard assay in a 96-well plate (horizontal axis, µg/mL) and gentamicin (vertical axis, 0–8 µg/mL). A darker color represents a higher increase in absorbance, which corresponds to increased bacterial growth. The FICI was calculated from each drug with the MIC (the lowest concentration of the drug showing 80% inhibition). (A) Combined treatment of gentamicin and 5-fluorouracil indicated synergy with an IFCI value of 0.31. (B) Combined treatment of meropenem and 5-fluorouracil indicated an additive effect with an FICI value of 0.56.

Figure 2

Heatmap displaying the OD₆₀₀ readings from a checkerboard assay in a 96-well plate (horizontal axis, µg/mL) and gentamicin (vertical axis, 0–8 µg/mL). A darker color represents a higher increase in absorbance, which corresponds to increased bacterial growth. The FICI was calculated from each drug with the MIC (the lowest concentration of the drug showing 80% inhibition). (A) Combined treatment of gentamicin and 5-fluorouracil indicated synergy with an IFCI value of 0.31. (B) Combined treatment of meropenem and 5-fluorouracil indicated an additive effect with an FICI value of 0.56.

Figure 3

Absorbance measurements at 490 nm showing biofilm biomass at different concentrations of 5-fluorouracil. Values are adjusted for background absorbance. 5-fluorouracil significantly reduced biofilm biomass compared to the untreated control group with a maximum reduction observed at concentrations of 12 and 100 µg/mL. * p < 0.01; ** p < 0.001.

Figure 4

Absorbance measurements at 490 nm showing biofilm dispersion after the addition of different concentrations of 5-fluorouracil. Values are adjusted for background absorbance. Established biofilms were treated with increasing concentrations of 5-fluorouracil (0.1, 0.5, 12, and 100 µg/mL) for 24 h. 5-fluorouracil resulted in a dose-dependent increase in biofilm biomass rather than disassembly. ** p < 0.01; *** p < 0.001.

Figure 5

Dose-dependent effect of 5-fluorouracil on P. aeruginosa biofilms. Representative CLSM images of PAO1 biofilm 48 h after treatment with 5-fluorouracil (100 µm scale). Biofilms obtained with (A) untreated control and treatments with (B) 0.1, (C) 0.5, (D) 12, and (E) 100 µg/mL 5-fluorouracil. (F) Biofilm thickness measurement expressed in µm. (G) Cell viability graph showing the percentage of live cells (green bars) and dead cells (red bars). Data are expressed as the mean. * p < 0.05; ** p < 0.01; *** p < 0.001; ****p < 0.0001.

Figure 6

The average synergy score for a drug combination is determined based on all the dose combination measurements. The 2D and 3D synergy maps use red and green colors to indicate synergistic and antagonistic dose regions, respectively. (A) Gentamicin and 5-fluorouracil ZIP synergy score of 14.2 (>10), likely indicating synergy. (B) Meropenem and 5-fluorouracil ZIP synergy score of 0.97 (<10), indicating no synergy.

Figure 7

Overview of the key findings of this study.