Biofilm Formation of Pseudomonas aeruginosa PA14 required lasI and Stimulated by the Pseudomonas Quinolone Signal although Salicylic Acid Inhibition is Independent of the pqs Pathway

Abstract

Pseudomonas aeruginosa is a ubiquitous, opportunistic pathogen responsible for many nosocomial infections, and is often difficult to treat due to its ability to produce biofilm. Biofilms can increase resistance to antimicrobial agents by up to 1000-fold and is controlled by quorum sensing. Salicylic acid has been shown to inhibit biofilm formation in P. aeruginosa by acting as a competitive analogue to the LasR regulator ligand. The las quorum sensing system can control the activation of many different downstream systems. One such pathway las can control is the pqs pathway, where las can indirectly downregulate the formation of the Pseudomonas quinolone signal molecule (PQS), but also directly upregulate the expression of the regulator, pqsR. The PQS molecule facilitates biofilm formation and can be up-regulated through a positive feedback loop. Here, we attempted to elucidate pathways downstream of the las system that are affected by the inhibitory effects of salicylic acid, and whether induction of these pathways would overcome of the effect of SA. Specifically, we focused on the pqs system. To do this, four different strains of P. aeruginosa ( PA14 wildtype, lasI, pqsA, and pqsH mutants) were used. The biofilm densities produced by these four strains were assessed after treating with increasing concentrations of SA and PQS combinations. Quantification of the biofilm densities was made possible with the crystal violet assay. The biofilm densities of the lasI mutant were reduced to approximately 10 – 20% relative to the wild type. The pqsA and pqsH mutants also had less biofilm formation compared to wildtype. An inhibitory effect of salicylic acid on biofilm formation was not observed in any strains. Addition of exogenous PQS increased biofilm density in all strains with the exception of the lasI mutant. In conclusion, the pqs pathway is not essential in P. aeruginosa for biofilm formation, while the las system is required for exogenous PQS to stimulate biofilm formation.