Tolerance of dormant and active cells in Pseudomonas aeruginosa PA01 biofilm to antimicrobial agents

Abstract

Objectives: The aim of the study was to determine the susceptibility of active and dormant cell populations from Pseudomonas aeruginosa biofilms to non-antibiotic antimicrobial agents such as chlorine, hydrogen peroxide and silver ions in comparison with antibiotics. Methods: Active cells in colony biofilm were differentially labelled by induction of a green fluorescent protein (GFP). Active and dormant cells were sorted in phosphate buffered solution by flow cytometry. Reductions in viability were determined with plate counts. Results: The spatial pattern of metabolic activity in colony biofilm was verified, and the active and dormant cells were successfully sorted according to the GFP intensity. Active cells had bigger cell size and higher intracellular density than dormant cells. While dormant cells were more tolerant to tobramycin and silver ions, active cells were more tolerant to chlorine. Metabolically active cells contain denser intracellular components that can react with highly reactive oxidants such as chlorine, thereby reducing the available concentrations of chlorine. In contrast, the concentrations of silver ions and hydrogen peroxide were constant during treatment. Aerobically grown stationary cells were significantly more tolerant to chlorine unlike other antimicrobial agents. Conclusions: Chlorine was more effective in inactivation of metabolically inactive dormant cells and also more effective under anaerobic conditions. The high oxidative reactivity and rapid decay of chlorine might influence the different antimicrobial actions of chlorine compared with antibiotics. This study contributes to understanding the effects of dormancy and the presence of oxygen on the susceptibility of P. aeruginosa biofilm to a wide range of antimicrobial agents.