Ethidium Binding to Salmonella enterica ser. Typhimurium Cells and Salmon Sperm DNA.
Bacterial resistance to antibiotics due to increased efficiency of the efflux is a serious problem in clinics of infectious diseases. Knowledge of the factors affecting the activity of efflux pumps would help to find the solution. For this, fast and trustful methods for efflux analysis are needed. Here, we analyzed how the assay conditions affect the accumulation of efflux indicators ethidium (Et+) and tetraphenylphosphonium in Salmonella enterica ser. Typhimurium cells. An inhibitor phenylalanyl-arginyl-β-naphtylamide was applied to evaluate the input of RND family pumps into the total efflux. In parallel to spectrofluorimetric analysis, we used an electrochemical assessment of Et+ concentration. The results of our experiments indicated that Et+ fluorescence increases immediately after the penetration of this indicator into the cells. However, when cells bind a high amount of Et+, the intensity of the fluorescence reaches the saturation level and stops reacting to the accumulated amount of this indicator. For this reason, electrochemical measurements provide more trustful information about the efficiency of efflux when cells accumulate high amounts of Et+. Measurements of Et+ interaction with the purified DNA demonstrated that the affinity of this lipophilic cation to DNA depends on the medium composition. The capacity of DNA to bind Et+ considerably decreases in the presence of Mg2+, Polymyxin B or when DNA is incubated in high ionic strength media.
['Sakalauskaitė|Sandra|S|', 'Mikalayeva|Valeryia|V|0000-0003-2848-816X', 'Daugelavičius|Rimantas|R|0000-0001-9721-6200']
D000818:Animals D001426:Bacterial Proteins D004247:DNA D024901:Drug Resistance, Multiple, Bacterial D004996:Ethidium D008297:Male D009861:Onium Compounds D009943:Organophosphorus Compounds D012474:Salmon D012486:Salmonella typhimurium D013050:Spectrometry, Fluorescence D013094:Spermatozoa
ethidium tetraphenylphosphonium phenylalanyl-arginyl-β-naphtylamide Polymyxin B efflux inhibitor multidrug resistance outer membrane permeability