Investigating the Role of the Antimicrobial Peptide TTO-53 in the Disruption of Pseudomonas aeruginosa Biofilm

Abstract

Pseudomonas aeruginosa infections are commonly acquired in hospital settings and are oftentimes severe and deadly. P. aeruginosa is known for its resistance to a wide range of antibiotics due to the low permeability of its outer membrane, possession of various efflux pumps and modifying enzymes, and its ability to form biofilms. Biofilms are aggregates of bacterial cells enclosed in an extracellular matrix. This extracellular matrix is composed of an assortment of extracellular polymeric substances (EPS’s) that can interact with and influence the rate of transport of antimicrobial agents. Previous research using a synthetic antimicrobial peptide made of unnatural amino acids named TTO-53 has shown its ability to disrupt pre-established P. aeruginosa biofilm. The goal of this study is to investigate how this disruption is taking place and the influence of this peptide on the efficacy of antibiotics in killing P. aeruginosa. Due to the role of quorum sensing, a communication system between cells in biofilm, in the maintenance of biofilms, we hypothesized that exposing P. aeruginosa to TTO-53 causes disruption of quorum sensing resulting in dispersion of pre-established biofilm. To test this hypothesis, we used quantitative real-time PCR (qRT-PCR) to determine if there was differential expression in genes within various quorum sensing pathways. Results from qRT-PCR showed that exposing overnight cultures to TTO-53 did not cause differential expression in genes within the LasR, PQS, and RhlI/RhlR quorum sensing pathways. To investigate the influence of TTO-53 on the efficacy of various antibiotics, we exposed cultures of PAO1 to varying concentrations of each antibiotic and compared the colony-forming units per milliliter (cfu/mL) of those cultures to the cfu/mL of cultures treated with those same concentrations of each antibiotic and TTO-53. These results indicated TTO-53 increases the efficacy of Chloramphenicol and Tazobactam/Piperacillin, while decreasing the efficacy of Tobramycin. This study revealed that an hour-long exposure to TTO-53 does not impact P. aeruginosa quorum sensing pathways and the combination treatment of TTO-53 has a synergistic effect with some antibiotics while inhibiting others.