Fingolimod Inhibits Exopolysaccharide Production and Regulates Relevant Genes to Eliminate the Biofilm of K. pneumoniae

Abstract

Klebsiella pneumoniae (K. pneumoniae) exhibits the ability to form biofilms as a means of adapting to its adverse surroundings. K. pneumoniae in this biofilm state demonstrates remarkable resistance, evades immune system attacks, and poses challenges for complete eradication, thereby complicating clinical anti-infection efforts. Moreover, the precise mechanisms governing biofilm formation and disruption remain elusive. Recent studies have discovered that fingolimod (FLD) exhibits biofilm properties against Gram-positive bacteria. Therefore, the antibiofilm properties of FLD were evaluated against multidrug-resistant (MDR) K. pneumoniae in this study. The antibiofilm activity of FLD against K. pneumoniae was assessed utilizing the Alamar Blue assay along with confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and crystal violet (CV) staining. The results showed that FLD effectively reduced biofilm formation, exopolysaccharide (EPS), motility, and bacterial abundance within K. pneumoniae biofilms without impeding its growth and metabolic activity. Furthermore, the inhibitory impact of FLD on the production of autoinducer-2 (AI-2) signaling molecules was identified, thereby demonstrating its notable anti-quorum sensing (QS) properties. The results of qRT-PCR analysis demonstrated that FLD significantly decreased the expression of genes associated with the efflux pump gene (AcrB, kexD, ketM, kdeA, and kpnE), outer membrane (OM) porin proteins (OmpK35, OmpK36), the quorum-sensing (QS) system (luxS), lipopolysaccharide (LPS) production (wzm), and EPS production (pgaA). Simultaneously, FLD exhibited evident antibacterial synergism, leading to an increased survival rate of G. mellonella infected with MDR K. pneumoniae. These findings suggested that FLD has substantial antibiofilm properties and synergistic antibacterial potential for colistin in treating K. pneumoniae infections.

Keywords: K. pneumoniae; antibacterial; biofilm; fingolimod (FLD); synergistic.

Figure 1

(A) Structure of FLD. (B) Influence of FLD on growth and metabolic activity of K. pneumonia. The line graphs and bar graph show the growth and metabolic activity of FLD, respectively. n = 3; results from all experiments are presented as the mean ± SD of three replicates. (C) Bar graph represents dose-dependent inhibition of K. pneumoniae biofilms upon treatment with FLD. Biofilm formation inhibition at various concentrations of FLD (0.25, 0.5, 1, 2, 4, 8, 16, and 32 μg/mL) for 24 h. **** = p < 0.0001, *** = p < 0.001. Results from all experiments are presented as the mean ± SD of three replicates. (D) Biofilm destruction at various concentrations of FLD (0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg/mL). **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01. Results from all experiments are presented as the mean ± SD of three replicates.

Figure 2

CLSM images of viable bacteria in K. pneumoniae biofilms ((A) without FLD, (B) treated by 4 µg/mL FLD, (C) treated by 8 µg/mL FLD, (D) treated by 16 µg/mL FLD, (E) treated by 32 µg/mL FLD, and (F) treated by 64 µg/mL FLD).

Figure 3

SEM images (×3000, 10,000, and 20,000) of K. pneumoniae morphology effected by FLD at 32 μg/mL.

Figure 4

(A) Results of EPS inhibition (%) at various concentrations of FLD (0, 4, 8, 16, 32, 64, and 128 µg/mL) for 24 h. (B) Motility inhibition of K. pneumoniae with FLD. (a) Quantitative estimation of motility based on the diameter of the halo zone. (b) Images of motility following incubation with K. pneumonia at various concentrations of FLD (4, 8, 16, and 32 µg/mL). (C) Number of viable bacteria in biofilms under different concentrations of FLD treatment. (D) The inhibition of AI-2 activity was assessed by treating it with various concentrations of FLD (4, 8, 16, 32, and 64 µg/mL). n = 3. Results from all experiments are presented as the mean ± SD of three replicates. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Figure 5

The contents of lipids (A), polysaccharide slime (B), total DNA (C), and eDNA (D) in the biofilm matrix upon FLD treatment. Each bar represents the mean ± SD of three independent experiments, **** indicates p < 0.0001 when compared with control group.

Figure 6

(A) The impact of FLD (32 µg/mL) on the transcription of biofilm-regulated genes. The qRT-PCR analysis demonstrated a noteworthy disparity in eleven genes in comparison to the control. ns indicates not significant **** indicates p < 0.001 when compared with control group. (B) The survival rate of G. mellonella in the three groups with different treatments for 120 h. (C) The survival rate of G. mellonella. The control group exhibited no mortality within 4 days. Conversely, the model, colistin, and FLD groups all succumbed to death within 36 h following bacterial infection. Notably, the survival rate of subjects receiving a combination treatment of FLD and colistin reached 80%.