Comparative analysis of the activity of MDR pumps in Salmonella enterica using different indicatory compounds and methods of assay

Abstract

Multidrug resistance (MDR) caused by efflux pumps is one ofthe most common reasons of bacterial resistance to antibiotics. Itis important to assay the efficiency of efflux pumps in bacteriaand to identify the selectivity of these pumps. In our study weused well-known efflux pump substrates – ethidium (Et+), tetraphenylphosphonium (TPP+) and Nile red (NR) as the indicatorycompounds. The main distinctness of Et+is the binding thisindicatory ion to DNA leading to the increase of the fluorescenceof studied samples. TPP+does not bind to intracellular structures and leaks out of deenergized cells. These two lipophiliccations and lipophilic stain NR were simultaneously used asagents competing for the interaction with efflux pumps. BesideEt+fluorescence measurements we applied potentiometric analysis using selective electrodes to determine Et+and TPP+uptakeby the cells. Phenylalanyl-arginyl-b-naphtylamide (PAbN) wasused to evaluate the input of RND-family pumps in total effluxactivity of the cells. Antibiotic polymyxin B (PMB), EDTA andheat treatment were used to permeabilize and deenergize the cells.Our experiments demonstrated the important role of the assayconditions on the efflux pump activity. pH, ionic strength, temperature of the incubation medium considerably affected the efficiency and kinetics of the efflux. The level of aeration of S.enterica cell suspension also affected the efficiency of the pumps.