Impact of cell signals and pyocyanin on mixed-biofilms of Pseudomonas aeruginosa PaO1 and Escherichia coli K-12

Abstract

Biofilms occur when microorganisms attach to surfaces, replicate and secrete dense polymers to the extent where the organisms become physically encapsulated. As biofilms often arise where there is a high level of diversity of microbial species, there is a high-likelihood that biofilms will contain more than one species of microorganism. These mixed-species biofilms have been shown to be advantageous to either one or multiple species within these biofilms. Pseudomonas aeruginosa and Escherichia coli are two bacteria of interest that are commonly involved in infections of humans and are known biofilm-producers that have generated dual-species biofilms in vitro. In recent years it has been shown that the formation of mixed-species biofilms of these two bacterial species is beneficial to either one or both bacteria in terms of adherence to surfaces, formation of the biofilm matrix, viable cell concentrations within mixed-species biofilms and resistance to external threats in the form of synthetic antimicrobial agents. The interactions between these two organisms in large part had been attributed to secretion of quorum-sensing signals and others molecules produced as a by-product of metabolic processes. This study investigated how the cell-signalling molecules, indole produced by E. coli and n-acyl homoserine lactones, n-butyryl homoserine lactone (C4-HSL) and n-3-oxo-dodecanoyl (3-oxo-C12-HSL) produced by P. aeruginosa would affect interactions between these organisms within mixed-species biofilms. Previous work had suggested that cell signally molecules would result in cross species effects. The work conducted within this study had found that n-acyl homoserine lactones had no discernible effect on E. coli biofilm formation and that indole did not have a strong influence over biofilm and production of the metabolic by-product and virulence factor pyocyanin, produced by P. aeruginosa PaO1. Pyocyanin however produced within mixed-species biofilms was shown to reduce viable wild-type E. coli K-12 to similar concentrations to that of an indole-deficient strain. The results within this thesis also found that reducing the incubation temperature increased biofilm formation of E. coli. For instance, viable cell retention increased from 2.20x 105 cfu at 37°C to 8.73x107 cfu at 25°C within 72 hours whereas total biofilm production increased from an absorbance reading of 0.22 at 37°C to an absorbance reading of 0.98 at 30°C within 24 hours. Reductions in pyocyanin in P. aeruginosa PaO1 cultures also coincided with reductions in temperature thus reducing the temperature from 37°C to 25°C reduced pyocyanin concentrations of P. aeruginosa PaO1 cultures, grown for 72 hours from 149.34 µM to 23.10 µM. A result of reducing the incubation temperature of mixed-species biofilm cultures, other than noticeable reductions in concentrations of pyocyanin, was an increase in E. coli cell recovery. For instance, within 72 hours E. coli viable cell recovery increased from 1.9x102 cfu to 2.0 x105 cfu when the temperature was reduced from 37°C to 25°C. This indicated that E. coli within mixed-species biofilms alongside P. aeruginosa in an enclosed system was more likely to survive at lower temperatures than higher temperatures ergo are more likely to survive within mixed-species biofilms outside rather than inside of the human body with a core temperature of approximately 37°C. This highlights a potential issue where the bacteria contained within these biofilms that have been shown in some instances to be more resistant to antimicrobials than single-species biofilms, have a higher likelihood of remaining viable for an extended period of time outside of the host reservoir. Increasing the duration of viability of E. coli and P. aeruginosa in mixed-species biofilms in areas that are prone to containing biofilms and are commonly associated with the presence of human beings can potentially increase the chances of contracting these infectious agents.

Date of Award	2014
Original language	English