Biological characterization of Pseudomonas aeruginosa endotoxin released by antibiotic treatment in vitro

Abstract

The supernatants taken from Pseudomonas aeruginosa cultures in human sera or chemically defined M9 medium in the presence of ceftazidime (CAZ) contained high levels of endotoxin, while those of imipenem (IPM) yielded relatively lower levels of endotoxin. The membrane-filtered supernatants were used as a source of endotoxin and the biological activities of the endotoxin were examined in comparison with those of hot-phenol water-extracted LPS. The CAZ-released endotoxin preparation contained large amounts of protein. The protein, however, appeared to lack significant endotoxic activity through the endotoxin-protein component, since the endotoxin did not show any toxic effect in D(+)-galactosamine (GaIN)-sensitized C3H/HeJ mice in vivo or macrophage activation in vitro. The activities of CAZ- and IPM-released endotoxins as assessed by chromogenic Limulus amebocyte coagulation test were fundamentally identical to those of purified P. aeruginosa LPS and Escherichia coli LPS, since their regression lines were parallel. The biological effects of CAZ-released endotoxin, such as lethal toxicity in GaIN-sensitized mice, in vitro induction of TNF and NO production by peritoneal macrophages, and MAP-kinase activation in macrophages of LPS-responsive C3H/He and LPS-low responsive C3H/HeJ mice, were similar to those of the LPS. Macrophage activation by CAZ-released endotoxin as well as LPS was mainly dependent on serum factors and CD14 antigen. Polymyxin B blocked the activity. These findings indicate that the endotoxic activity of CAZ-released endotoxin is due primarily to LPS (lipid A), although such preparations contain a high level of protein released from or produced by the organisms. Finally, the possibility that the treatment of P. aeruginosa infection with some kind of antibiotics may induce endotoxic shock was suggested in a mouse model.

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