Repurposing metformin as a quorum sensing inhibitor in Pseudomonas aeruginosa

Abstract

Background: Quorum sensing is a mechanism of intercellular communication that controls the production of virulence factors in Pseudomonas aeruginosa. Inhibition of quorum sensing can disarm the virulence factors without exerting stress on bacterial growth that leads to emergence of antibiotic resistance.

Objectives: Finding a new quorum sensing inhibitor and determining its inhibitory activities against virulence factors of Pseudomonas aeruginosa PAO1 strain.

Methods: Quorum sensing was evaluated by estimation of violacein production by Chromobacterium violaceum CV026. Molecular docking was used to investigate the possible binding of metformin to LasR and rhlR receptors. The inhibition of pyocyanin, hemolysin, protease, elastase in addition to swimming and twitching motilities, biofilm formation and resistance to oxidative stress by metformin was also assessed.

Results: Metformin significantly reduced the production of violacein pigment. Significant inhibition of pyocyanin, hemolysin, protease and elastase was achieved. Metformin markedly decreased biofilm formation, swimming and twitching motilities and increased the sensitivity to oxidative stress. In the molecular docking study, metformin could bind to LasR by hydrogen bonding and electrostatic interaction and to rhlR by hydrogen bonding only.

Conclusion: Metformin can act as a quorum sensing inhibitor and virulence inhibiting agent that may be useful in the treatment of Pseudomonas aeruginosa infection.

Keywords: Metformin; Pseudomonas aeruginosa; quorum sensing; virulence inhibition.

Figure 1

Effect of metformin on growth of PAO1. The growth was measured by measuring OD600 after overnight incubation in the absence and presence of 1/10 MIC of metformin. No statistically significant difference was found between the growth rates of the untreated or treated cultures.

Figure 2

Inhibition of biofilm formation of PAO1 by 1/10 MIC of metformin.*, significant P< 0.05.Microscopic visualization of biofilm under the light microscope (X1000) in the treated culture (left) and untreated (right).

Figure 3

Inhibition of production violacein pigment of ChhromobacteriumviolaceumCV026 by 1/10 MIC of metformin.*, significant P< 0.05.

Figure 4

Inhibition of protease production by 1/10 MIC of metformin by the skim milk agar method. Metformin reduced the diameter of clear zone around the wells made in skim milk agar. *, significant P< 0.05.

Figure 5

Pyocyanin production in PAO1 untreated (left) and metformin treated (right). The supernatants of both treated and untreated cultures were measured at 691nm. Metformin significantly reduced pyocyanin production. *, significant P< 0.05.

Figure 6

Inhibition of elastase activity by 1/10 MIC of metformin. The supernatants of untreated and metformin treated cultures were incubated with elastin congo red. After centrifugation to remove the insoluble dye, the supernatants were measured at 495nm. Significant decrease in elastase activity was found with metformin. *, significant P< 0.05.

Figure 7

Effect of metformin on resistance to oxidative stress in PAO1.The diameter of inhibition zone of hydrogen peroxide in the presence of metformin (left) and in absence of metformin (right).*, significant P< 0.05.

Figure 8

Inhibition of swimming motility of PAO1 by 1/10 MIC of metformin. Metformin decreased swimming (left) as compared to the control (right). *, significant P< 0.05.

Figure 9

Inhibition of twitching motility of PAO1 by 1/10 MIC of metformin. Metformin decreased twitching (left) as compared to the control (right). *, significant P< 0.05.

Figure 10

Inhibition of hemolysin by metformin. Hemoglobin was released by incubation of culture supernatants with 2% erythrocyte suspension in saline for 2h and the absorbance was measured at 540nm. Metformin showed a significant hemolysin inhibiting activity. *, significant P< 0.05.

Figure 11

The Molecular docking of metformin into the active site of LasR enzyme 3D (Left) and 2D schematic view of the binding (Right).

Figure 12

The Molecular docking of natural ligand (Top) and C30 furanone (Bottom) into the active site of LasR enzyme 3D (Left) and 2D schematic view of the binding (Right).

Figure 13

The Molecular docking of C4-HSL (Top) and metformin (Bottom) into the active site of rhlr receptor model 3D (Left) and 2D schematic view of the binding (Right).