Studies on the selectivity of action of colistin, colistin nonapeptide and colistin heptapeptide on the cell envelope of Escherichia coli.

KOIKE e t al. reported that a cationic acylpeptide antibiotic, colistin (polymyxin E) caused the formation of blebs on the outer layer of the Gram-negative bacterial cell envelope". They have also reported that colistin affects the cytoplasmic membrane, leads to leakage of cytoplasmic 260 nm-absorbing materials and has specific bursting-activity on spheroplasts of Gram-negative bacteria'). On the other hand, COOPERSTOCK showed with Limulus assay that endotoxic lipopolysaccharide (LPS), which was located in the cell envelope, was inactivated with colistinmethate derived from colistin sulfate'. Successively, the authors4-' reported that colistin nonapeptide was obtained from the hydrolysate of colistin after treatment with the proteolytic enzyme, ficin, and that colistin was cleaved selectively by colistin-inactivating enzyme, colistinase, to yield colistin heptapeptide. These peptides exhibited no antibiotic activity. However, recently, VAARA and VAARA reported that mixtures of polymyxin B nonapeptide and a hydrophobic antibiotic had a synergistic effect against Escherichia coli°D. SIxL and GALLA demonstrated by fluorescense polarization measurements that colistin nonapeptide bound to phosphatidic acid in the cell membrane and, consequently, led to a rigidification of the cell membrane'. The present study reports the investigation, using colistin nonapeptide and colistin heptapeptide, of the importance of the cyclic peptide moiety and the tripeptide side chain of colistin in the action of colistin on the cell envelope of E. coli NIHJ. E. coli NIHJ, used as the test organism in this study, was inoculated into 10 ml of medium in a test tube and incubated overnight at 37°C. The medium had the following composition per liter: glucose 1.0 g, Casamino Acids 1.0 g, K~HPO, 7.0g, KH_PO, 2.0g, MgSO, • 7H.,O 0.1g, (NH,),SO, 1.0g, sodium citrate 0.5-1 L-tryptophan 20 mg. Erythromycin, rifampicin and novobiocin were obtained from Boehringer Mannhein GmbH, West Germany. Fusidic acid, cloxacillin and ficin (EC 3.4.22.3) were obtained from Signia Chemical Co., U.S.A. Lipopolysaccharide w from E. coli 0111: B4 was obtained from Difco Laboratories, U.S.A. "PYRODICK", which was used for colorimetric determination of endotoxin, was obtained from Seikagaku Ko.-yo Co., Ltd., Tokyo. Colistin nonapeptide (CSNP) was prepared by the method of CHIHARA et al. using ficin41, and colistin heptapeptide (CSHP) using colistinase by the method of the authors". Minimum inhibitory concentration (MIC) was determined by a dilution method using tissue culture plates with 96-wells (Falcon, catalog No. 3072; Becton Dickinson Labware). Culture medium (200 1;.1) containing the indicated amount of each antibiotic and CSNP or CSHP was pipetted into each well of a tissue culture plate. An aliquot (10 pl) containing 2':10 bacterial cells per m! suspended in a fresh medium was added to each well. The plates were covered, mixed on a plate mixer and incubated at 37'C for 18 hours. The lowest antibiotic concentration that completely inhibited growth was defined as the minimum inhibitory concentration (MIC). Fig. I shows chemical structures of colistin (CS), colistin nonapeptide (CSNP) and colistin heptapeptide (CSNP). Minimum inhibitory concentrations of five