Exposure to bile influences biofilm formation by Listeria monocytogenes

Abstract

In the present study we demonstrate that the initial attachment of Listeria monocytogenes cells to plastic surfaces was significantly increased by growth in the presence of bile. Improved biofilm formation was confirmed by crystal violet staining, microscopy and bioluminescence detection of a luciferase-tagged strain. Enhanced biofilm formation in response to bile may influence the ability of L. monocytogenes to form biofilms in vivo during infection and may contribute to survival of this important pathogen in the human gastrointestinal tract and gallbladder.

Figure 1

Biofilm assays. (A) Biofilm assays in 96 well microtitre plates were carried out as described in the text. Briefly, cells that were grown in BHI alone (- bile) or BHI containing 0.3% oxgall (+ bile) were washed and inoculated at equal cell numbers into fresh BHI broth and 200 μl was added to individual wells of a 96 well plate. After 24 hrs incubation at 37°C biofilms were stained with crystal violet and de-stained using ethanol and the optical density at 595 nm of the alcoholic crystal violet solutions was determined. Data is presented as averages +/- standard deviations for three biological repeats in one experiment. This result is representative of three independent experiments. (B) Representative images obtained from microscopic observations of biofilm on Petri dishes. Bacteria stained with crystal violet were observed under a 40× objective.

Figure 2

Bioluminescence imaging of biofilm. L. monocytogenes EGDe lux cells that were grown in BHI (- bile) or BHI containing 0.3% oxgall (+ bile) were washed in Ringer's solution and inoculated at equal cell numbers into fresh BHI broth. 3 ml was added to individual wells of a 6-well plate. After (A) 4 hours and (B) 24 hours the contents of wells were removed and loosely attached bacteria were removed by washing with distilled water. 1 ml of fresh BHI broth was added and luminescence was measured in a Xenogen IVIS100. The color bar indicates bioluminescence signal intensity (in photons s^-1 cm^-2). Results shown are those obtained for three biological repeats in one particular experiment and are representative of three independent experiments.

Figure 3

Microscopy of cells cultured in bile. L. monocytogenes cells were prepared exactly as for biofilm assays (i.e. grown in BHI alone (- bile) and BHI supplemented with 0.3% oxgall (+ bile) and subsequently washed in 1/4 strength Ringers solution). 10 μl of crystal violet was added to 100 μl washed cells in a microcentrifuge tube and mixed. 10 μl was spotted onto slides and viewed with a Leica DMLS microscope containing a digital eyepiece. Three random fields were viewed and representative images were captured.