Relevance of porin OprE on adaptive resistance of Pseudomonas aeruginosa biofilms to benzalkonium chloride

Abstract

Biofilm formation is a phenomenon that happens naturally in the environment, representing one of the strategies adopt by microorganisms in attempt to protect themselves from external pressures. In clinical area, the most common practice to eliminate unwanted biofilms is the application of antimicrobial agents. However, current disinfection practices show often inefficacy in the eradication of biofilms, contributing for decreased susceptibility of microorganisms. The high tolerance of Pseudomonas aeruginosa to several antibiotics has been reported and is caused, partially, by reduced permeability through membrane. Concerning biocide resistance, the membrane alterations adopted by microorganisms is still unclear. The main goal of this work was the characterization of the membrane protein profile of sessile P. aeruginosa after induction of adaptive phenotype by exposed to antimicrobial stress with the biocide benzalkonium chloride (BC). Previous to biofilm exposure to BC, biofilms were formed on glass wool for 5 days. Biofilm adaptation was made by serially exposure of P. aeruginosa in increasing concentrations for each 48 hours. After 6 days of adaptation, alterations in outer membrane proteins expressed by sessile bacteria and matrix proteins were examined by SDS-PAGE. From adaptation resulted biofilms able to survive in media with 324 mg/L of BC. Analyzing the protein profiles of adapted and non-adapted biofilms, it was verified the decrease expression of a serie of proteins with 45 kDa. Certainly, the outer membrane porin OprE must be present on this serie. Indeed, this OprE is often associated with phenomena of resistance [1]. The results suggest that, as similar as chromate resistance, OprE can have a role in efflux of BC from periplasm to cell exterior [2]. Regarding protein profiles of the polymeric matrix of biofilms were found differences between adapted and non-adapted biofilms. However, there was no evidence that these changes could be associated with resistance phenomenon. This work revealed that a simple in vitro process of induction of adaptive resistance generated P. aeruginosa more resistant to BC action. This finding may, partially, explain the rapid development of resistance by microorganisms in a real environment, notably in clinical area, where antimicrobials are often used and sometimes at inadequate levels. Knowledge about mechanisms underlying the biocide resistance can be useful to design new antimicrobial agents with specific target as OprE.