Effects of Pseudomonas aeruginosa PAO1 mutants on the growth of Listeria biofilms

Abstract

Listeria monocytogenes is a Gram-negative, rod-shaped pathogen with the ability to form resistant biofilms in food-processing environments. Substances secreted by Pseudomonas aeruginosa PAO1 have been found to naturally inhibit growth of L. monocytogenes biofilms. Transposon mutants were created to determine genes encoding for these substances. In this study, thirteen transposon mutants of P. aeruginosa PAO1 were evaluated to determine whether their ability to inhibit Listeria biofilms was changed. P. aeruginosa PAO1 wild-type and the thirteen mutants were grown in sterile Tryptic Soy Broth (TSB) for 48 hours at 37 ˚C. Cells were then separated by centrifugation and supernatants were sterilized by filtration through a 0.22 μm filter. These supernatants (PCM) were subsequently used to treat Listeria biofilms, which were grown for 24 hours at 30 ˚C in polystyrene microtitre plates. PCM prepared from Pseudomonas wild type and mutants was added to the biofilms followed by incubation for a further 24 hours at 30 ˚C. Controls included biofilms treated with TSB alone. Finally, biofilms were stained with crystal violet, and quantified in a FLUOStar OPTIMA microtitre plate absorbency reader. Treatment in all controls yielded dense biofilm formation, while the P. aeruginosa wild type PCM effectively reduced the biofilms grown in the polystyrene microtitre plates. Two of the original thirteen mutants exhibited impaired biofilm removal, with four other mutants showing a potential loss of biofilm removal characteristics. This indicates that these mutants posses the transposon inserted in a gene of P. aeruginosa that plays a role in Listeria biofilm removal. Inverse PCR will be used to identify the genomic location of the transposon in these two mutants.