Initial Applications, The Pseudomonas Aeruginosa Outer Membrane Permeability And Drug Resistance Relationships In Drug Screening

Pseudomonas aeruginosa, an organism ubiquitous in nature, has assumed an increasingly prominent role as the aetiological agent in serious infections of hospitalized patients. Because of the drug - resistance of Pseudomonas aeruginosa, the therapy has been difficult. In this paper, we identified the properties of porins in the outer membrane of Pseudomonas aeruginosa, showed the role of membrane permeability in determining antibiotic resistance in Pseudomonas aeruginosa, and studied the mechanism of multi - resistance to some β- lactam antibiotics of clinically isolated Pseudomonas. In order to find new anti - Pseudomonas aeruginosa agents, a primary screening method has been set with the outer membrane as a target, and there was no such studies has been reported so far.To identify the properties of porins in the outer membrane of Pseudomonas aeruginosa, the strain Pseudomonas aeruginosa PAO1 was used in this study. Isolation of outer membrane protein was accomplished by treating the cell envelope with EDTA and lysozyme, followed by centrifugation. The molecular weight of the major outer membrane protein was analyzed by electrophoresis which contained SDS - PAGE. The purification of the porins in Pseudomonas aeruginosa was achieved by DEAE ion - exchange HPLC. The purified outer membrane proteins were reconstituted with phosphatidylcholine and dicetylphosphate into membrane vesicles, and were tested by the liposomes swelling method for the diffusion of antibiotics. The permeability assay using these reconstituted liposomes showed that OprC (Mr,70,000), OprD₂ ( Mr, 46,000) and OprE (Mr, 43,000) are the channel -forming proteins. But only OprD₂ was thought to be the likely route of imipenem (carbapenem) diffusion.To understand the mechanism of multi - resistance to some β- lactam antibiotics such as imipenem of clinically isolated Pseudomonas aeruginosa, four isolates of P. aeruginosa with multi - resistance to the above agents were analyzed by SDS - PAGE outer membrane protein profile and assay of β - lactamase was high. These data favors the conclusion that the mechanism of multi - resistance to the above anti - P. aeruginosa β - lactam might involve both the loss of OprD₂ and a lot of β - lactamase. The main cause of imipenem resistance might be due to the loss of OprD₂.Carbapenems are regarded as potential antibiotics in clinical use. In this paper, the primary screening method which has been set with the outer membrane as a target will help us to discover carbapenems, and will be used in the study of mechanism of anti - bacteria drugs and the evaluation of prospect of new drugs.During the studying of the primary screening method, a OprD₂ - deficient strain P. aeruginosa 7208 was chosen first. The screening method, by using...