| Abstract [eng] |
Multidrug resistance (MDR) pumps are one of the major causes of resistance in opportunistic pathogens, such as Pseudomonas aeruginosa. Consequently, fast and reliable methods are needed to assay the efficiency of MDR pumps in these bacteria. However, direct efflux pump functions’ real-time assessment methods are missing. Such assays would improve the evaluation of MDR efflux pump activities and the search for potential inhibitors. The assay for monitoring of MDR efflux pumps, employing voltage (Δψ) indicator tetraphenylphosphonium (TPP+) and TPP+-selective electrodes was developed. The assay was applied to evaluate the activity of tetracycline specific efflux pump TetA(B) in E. coli. Competitive inhibition of TetA(B) activity was demonstrated. Moreover, it was shown that tetracycline-specific efflux pump TetA(B) has higher selectivity for TPP+ than tetracycline. The designed method can be used for real-time monitoring of MDR efflux pumps in P. aeruginosa. The main factors influencing the activities of MDR efflux pumps were addressed: outer membrane permeability, Δψ, aeration, temperature and cell growth stage. It was also shown that TPP+ in combination with the pump inhibitor phenylalanyl-arginyl-β-naphtylamide (PAβN) can be used to monitor the activity of RND-type efflux pumps in P. aeruginosa. By controlling the outer membrane permeability and Δψ, the real-time measurements of the RND pump activity were performed. It was shown that PAβN depolarizes plasma membrane of the cell in the medium without any nutrients. However, under optimal growth conditions P. aeruginosa cells are able to recreate the outer membrane barrier covering the activities of MDR efflux pumps. Furthermore, it was shown that the activities of P. aeruginosa MDR efflux pumps diminish in lower than room temperature. Altogether, it was demonstrated that the activity of RND-type efflux pumps in P. aeruginosa is highly sensitive to incubation conditions such as the presence of nutrients, incubation medium, the level of aeration and temperature. Thus, it is of great importance to standardize the experimental conditions to obtain quantitative and comparative results. |
