Activity of the carbapenem panipenem and role of the OprD (D2) protein in its diffusion through the Pseudomonas aeruginosa outer membrane

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Abstract
Evidence of permeation of panipenem through the OprD (D2) channel of Pseudomonas aeruginosa outer membrane was shown by using OprD protein-producing and -nonproducing strains which contained plasmid pHN4, which codes for L-1 beta-lactamase of Xanthomonas maltophilia. Permeation by panipenem was determined by measuring hydrolysis of the carbapenem by beta-lactamase in the periplasmic space. Permeation by panipenem was also determined by counting uptake of [14C]panipenem into P. aeruginosa PAO1 and its OprD protein-deficient mutant, and this permeation of PAO1 was inhibited by L-lysine. These results indicate that panipenem, as well as imipenem, uses the OprD channel, which functions as a specific channel for diffusion of basic amino acids. Panipenem and imipenem showed stronger activities against PAO1 and clinical isolates in human serum than in Mueller-Hinton broth, which contains more amino acids than human serum does. The activities of the carbapenems were reduced by addition of L-lysine to human serum. Similar results were obtained with mouse serum and ascitic fluid. In contrast, such a change in the activities of carbapenems was not observed with an OprD protein-deficient mutant, suggesting that the main reason for the strong activities of carbapenems in biological fluids is a decrease in competition between the antibiotics and basic amino acids for permeation through the OprD channel. Panipenem and imipenem showed much stronger therapeutic efficacies against experimental infections with P. aeruginosa in mice than did the reference antibiotics. Their in vivo activities were more consistent with their MICs in biological fluids than with those in Mueller-Hinton broth.