Epinephrine affects motility, and increases adhesion, biofilm and virulence of Pseudomonas aeruginosa H103

Abstract

Microbial endocrinology has demonstrated for more than two decades, that eukaryotic substances (hormones, neurotransmitters, molecules of the immune system) can modulate the physiological behavior of bacteria. Among them, the hormones/neurotransmitters, epinephrine (Epi) and norepinephrine (NE), released in case of stress, physical effort or used in medical treatment, were shown to be able to modify biofilm formation in various bacterial species. In the present study, we have evaluated the effect of Epi on motility, adhesion, biofilm formation and virulence of Pseudomonas aeruginosa, a bacterium linked to many hospital-acquired infections, and responsible for chronic infection in immunocompromised patients including persons suffering from cystic fibrosis. The results showed that Epi increased adhesion and biofilm formation of P. aeruginosa, as well as its virulence towards the Galleria mellonella larvae in vivo model. Deciphering the sensor of this molecule in P. aeruginosa and the molecular mechanisms involved may help to find new strategies of treatment to fight against this bacterium.

Figure 1

Effect of Epi on P. aeruginosa H103 motility. (a) Covered distance by swarming motility on LB 0.6% agar, after 24 h, (b) Covered distance by twitching motility on LB 1% agar, (c) Macroscopic and microscopic observations of twitching. Micrographs were taken with an Axio Vert A.1 inverted microscope, scale bar: 0.1 mm. The error bars indicate the standard error of the mean (SEM). Two-tailed paired t-test was used, (n = 6 and n = 3 for swarming and twitching assays respectively), *p = 0.0125, ns: not significant.

Figure 2

Effect of Epi on phage PO4 sensitivity. Phage PO4 sensitivity towards P. aeruginosa H103 was tested in presence of 1 and 10 µM of Epi after 24 h. Plaque lysis were enumerated and expressed in PFU/mL, fold change is expressed for each condition. The error bars indicate the standard error of the mean (SEM). Two-tailed paired t-test was used (n = 4), *p = 0.025.

Figure 3

Effect of Epi on adhesion of P. aeruginosa H103. (a) Top view of P. aeruginosa H103-GFP adhesion observed by CLSM with two different concentrations of Epi (1 and 10 µM), after 2 h. (b) Image analysis by COMSTAT. Data were normalized according to the untreated control set at 100%. (c) Adhesion assay on Caco-2/TC7 cells, *p = 0.0418. The error bars indicate the standard error of the mean (SEM). Two-tailed paired t-test was used (n = 5), **p = 0.001222, ***p = 0.000413, ns: not significant.

Figure 4

Effect of Epi on P. aeruginosa H103 biofilm formation. (a) Three dimensional (3D) and side views of P. aeruginosa H103-GFP 24 h biofilm observed by CLSM, in presence of 1 or 10 µM of Epi. (b) Images analysis by COMSTAT. Data were normalized according to the untreated control set at 100%. The error bars indicate the standard error of the mean (SEM) of at least three independent experiments. Two-tailed paired t-test was used (n = 4), *p < 0.05, ***p < 0.001, ns: not significant.

Figure 5

Virulence assay on G. mellonella. The virulence of P. aeruginosa H103 was tested towards the caterpillar G. mellonella larvae, during 17 h. Log-rank (Mantel-Cox) test was used, **p = 0.0086.