FTY720 Reduces the Biomass of Biofilms in Pseudomonas aeruginosa in a Dose-Dependent Manner

Abstract

Pseudomonas aeruginosa, a nosocomial pathogen, has strong biofilm capabilities, representing the main source of infection in the human body. Repurposing existing drugs has been explored as an alternative strategy to combat emerging antibiotic-resistant pathogens. Fingolimod hydrochloride (FTY720), an immunomodulatory drug for multiple sclerosis, has shown promising antimicrobial effects against some ESKAPE pathogens. Therefore, the effects of FTY720 on the biofilm capabilities of Pseudomonas aeruginosa were investigated in this study. It was determined that FTY720 inhibited the growth of P. aeruginosa PAO1 at 100 µM. The significant reduction in PAO1 cell viability was observed to be dose-dependent. Additional cytotoxicity analysis on human cell lines showed that FTY720 significantly reduced viabilities at sub-inhibitory concentrations of 25-50 µM. Microtiter assays and confocal analysis confirmed reductions in biofilm mass and thickness and the cell survivability ratio in the presence of FTY720. Similarly, virulence production and biofilm-related gene expression (rhlA, rhlB, pilA, pilI, fliC, fliD and algR) were determined. The results demonstrate that pigment production was affected and quantitative real-time PCR analysis showed a variable degree of reduced gene expression in response to FTY720 at 12.5-50 µM. These findings suggest that FTY720 could be repurposed as an alternative antibiofilm agent against Pseudomonas aeruginosa.

Keywords: FTY720; Pseudomonas aeruginosa; biofilm biomass; biomass reduction; fingolimod.

Figure 1

Chemical structure of fingolimod hydrochloride.

Figure 2

(A) Resazurin-based MIC determination of FTY720 against P. aeruginosa PAO1. (B) Inhibition percentages on the bacterial growth of P. aeruginosa upon FTY720 exposure. Data are expressed as mean ± SD (n = 9).

Figure 3

Effects on the viability of (A) immortalized human bone marrow mesenchymal stem cells and (B) human gingival fibroblasts exposed to FTY720 treatment. Data are presented as mean ± SD (n = 9); * p < 0.05; ** p < 0.01.

Figure 4

(A) Effects of FTY720 treatment on the growth of P. aeruginosa PAO1. (B) Effects on the bacterial cell viability of P. aeruginosa upon FTY720 exposure. Data are expressed as mean ± SD (n = 6); * p < 0.05; ** p < 0.01.

Figure 5

Effects of the different concentrations of FTY720 on the biofilm formation of P. aeruginosa PAO1 at 24 h. Data are expressed as mean ± SD (n = 6); * p < 0.05; ** p < 0.01.

Figure 6

Disruptive effects of FTY720 on P. aeruginosa biofilms. Representative images of PAO1 biofilms after treatment for 48 h with FTY720 using CLSM. Biofilms of (A) untreated control and treatment with (B) 6.25, (C) 12.5, (D) 25, and (E) 50 µM FTY720. (F) Biofilm thickness measurements. Data are expressed as mean ± SD (n = 6); ** p < 0.01; *** p < 0.001.

Figure 7

Effects on bacterial biofilm inhibition using the LIVE/DEAD assay with confocal laser scanning microscopy after 48 h of treatment with FTY720. Live (green) and dead (red) biofilm cells of PAO1. (A) PAO1 percentages and (B) representative images for control, 6.25, 12.5, 25, and 50 µM FTY720 exposure, respectively. Data are expressed as mean ± SD (n = 6); ** p < 0.01. Scale bar: 50 µm.

Figure 8

(A) Effects on the pyocyanin production of P. aeruginosa after treatment with FTY720 compared to control cultures. (B) Effects on the biosynthesis of pyoverdine siderophores by P. aeruginosa after exposure to FTY720. Data are expressed as mean ± SD (n = 6); * p < 0.05; ** p < 0.01.

Figure 9

Effects on the expression levels of selected genes involved in the motility and biofilm formation of P. aeruginosa after 24 and 48 h exposure to FTY720. Expression of (A) rhlA and (B) rhlB for rhamnolipid production; (C) pilA and (D) pilI for pilin; (E) fliC and (F) fliD for flagellar filament; and (G) algR for alginate biosynthesis. Data are presented as mean fold change ± SD (n = 9); * p < 0.05.