Effect of biosurfactants on Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a BioFlux channel

Abstract

Recent studies have indicated that biosurfactants play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. A combination of caprylic acid (0.01 % v/v) together with rhamnolipids (0.04 % v/v) was applied to biofilms of Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 9144 and a mixed culture under BioFlux flowthrough conditions and caused disruption of the biofilms. The biofilms were also treated with a combination of rhamnolipids (0.04 % v/v) and sophorolipids (0.01 %). Control treatments with PBS 1× had no apparent effect on biofilm disruption. The Gram-positive bacterium (S. aureus ATCC 9144) was more sensitive than P. aeruginosa ATCC 15442 in terms of disruption and viability as shown by Live/Dead staining. Disruption of biofilms of P. aeruginosa ATCC 15442 was minimal. Oxygen consumption by biofilms, after different treatments with biosurfactants, confirms that sophorolipid on its own is unable to kill/inhibit cells of P. aeruginosa ATCC 15442, and even when used in combination with rhamnolipids, under static conditions, no decrease in the cell viability was observed. Cells in biofilms exposed to mono-rhamnolipids (0.04 % v/v) showed behaviour typical of exposure to bacteriostatic compounds, but when exposed to di-rhamnolipids (0.04 % v/v), they displayed a pattern characteristic of bactericidal compounds.

Keywords: BioFlux; Biofilms; Biosurfactants; Rhamnolipids.

Fig. 1

Biofilm formation and disruption in a BioFlux channel. The images are phase-contrast images and show fully formed biofilms after 48 h of incubation at 30 °C, and the images were recorded with a microscope Evon (10×) (17 % light). a P. aeruginosa ATCC 15442 biofilm before treatment. b P. aeruginosa ATCC 15442 after treatment with rhamnolipid (0.04 %) and caprylic acid (0.01 %). c S. aureus ATCC 9144 before treatment. d S. aureus ATCC 9144 after treatment with rhamnolipid (0.04 %) and caprylic acid (0.01 %). e Mixed culture (P. aeruginosa ATCC 15442/S. aureus ATCC 9144). f Mixed culture after treatment with rhamnolipid (0.04 %) and caprylic acid (0.01 %). g P. aeruginosa ATCC 15442 before treatment. h P. aeruginosa ATCC 15442 after treatment with PBS 1×

Fig. 2

Effect of rhamnolipids and sophorolipids on Biofilm disruption. Biofilms were grown for 48 h at 30 °C and then stained with Live/Dead BacLight and the images were recorded with a microscope Evon (×10) (17 % light). a P. aeruginosa ATCC 15442 biofilm before treatment. b P. aeruginosa ATCC 15442 after treatment with rhamnolipid (0.04 %) and sophorolipid (0.01 %). c S. aureus ATCC 9144 before treatment. d S. aureus ATCC 9144 after treatment with rhamnolipid (0.04 %) and sophorolipid (0.01 %). e Mixed culture (P. aeruginosa ATCC 15442/S. aureus ATCC 9144). f Mixed culture after treatment with rhamnolipid (0.04 %) and sophorolipid (0.01 %)

Fig. 3

Oxygen consumption of P. aeruginosa ATCC 15442 biofilms after 30-min treatment with a combinations of biosurfactants and b mono- and di-rhamnolipids. Shown in a plot of the relative concentration of dissolved oxygen in percentage of saturation concentration versus time after addition of the different substances. Treatment concentrations are indicated