Direct observation of substrate induction of resistance mechanism in Pseudomonas aeruginosa using single live cell imaging.
The resistance mechanism in three strains of Pseudomonas aeruginosa, Delta ABM (devoid of MexAB-OprM), WT, and nalB-1 (overexpression of MexAB-OprM), was investigated using real-time single live cell imaging and fluorescence spectroscopy. Time courses of fluorescence intensity of these three strains in ethidium bromide (EtBr) showed that accumulation kinetics and extrusion machinery were highly dependent upon pump substrate (EtBr) concentration. At high substrate concentration (100 microM), the accumulation kinetic profiles in the cells at earlier incubation times were similar to those observed in low concentration. As EtBr accumulated in the cells reached a critical concentration, the fluorescence intensity of Delta ABM decreased below the fluorescence intensity of EtBr in buffer solution. This result suggested an inductive mechanism in the development of substrate resistance in P. aeruginosa. Substrates appeared to trigger the degradation of EtBr in Delta ABM. Unlike bulk measurements, single live cell imaging overcame the ensemble measurement of bulk analysis and showed that efflux machinery and resistance mechanism in individual cells were not synchronized.
Xu
Xiao-Hong Nancy
XH
Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, USA. xhxu@odu.edu
Wan
Qian
Q
Kyriacou
Sophia V
SV
Brownlow
William J
WJ
Nowak
Michelle E
ME
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001692:Biological Transport; D002455:Cell Division; D024901:Drug Resistance, Multiple, Bacterial; D004996:Ethidium / Q000032:analysis / Q000633:toxicity; D005456:Fluorescent Dyes / Q000032:analysis / Q000633:toxicity; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011550:Pseudomonas aeruginosa / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D001425:Bacterial Outer Membrane Proteins; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D004996:Ethidium