Action of Polymyxin B on Bacterial Membranes: Morphological Changes in the Cytoplasm and in the Outer Membrane of Salmonella typhimurium and Escherichia coli B

Abstract

Though the primary action of the cationic antibiotic polymyxin B is against the membrane of susceptible bacteria, severe morphological changes are detected in the cytoplasm. Using fluorescence microscopy and a mono-N-dansylpolymyxin B derivative, we could demonstrate aggregations of the antibiotic with cellular material, possibly nucleic acids and/or ribosomes. These aggregations were only produced by minimum inhibitory or higher concentrations of the antibiotic as shown with Salmonella and Escherichia strains differing in their polymyxin susceptibility. The outer membrane of Salmonella typhimurium revealed characteristic blebs when treated with polymyxin B. This was investigated by the gentle methods of spray-freezing and freeze-etching. The obtained electron micrographs suggest that the polymyxin-induced blebs are projections of the outer monolayer of the outer membrane. A possible mechanism of penetration of polymyxin B through the cell envelope of gram-negative bacteria is presented.The basic polypeptide polymyxin B (PX) acts specifically on gram-negative bacteria by electrostatic and hydrophobic interactions with anionic cell envelope components like phospholipids (10, 11, 21, 34, 36) and lipopolysaccharides (LPS) (3, 26). These molecules are most likely the primary receptor molecules and responsible for the classification of the polymyxins as membrane-specific antibiotics. A possible penetration of the antibiotic into the cytoplasm, however, has not been directly demonstrated, although Few and Schulmann (7) supposed from binding studies that such a penetration could occur. In the same sense, Handley et al. (9) interpreted the condensation of nuclear material in PX-treated bacteria as revealed by electron maicroscopy as polymyxin-deoxyribonucleic acid complexes. These have been described in vitro (18). By the use of mono-N-dimethylaminonaphthalene-sulfonyl-polymyxin B (dansyl-PX), we were able to prove the intracellular accumulation of PX. A preliminary report has been presented (37).An additional structural change, the formation of blebs in the outer membrane of gram-negative bacteria (9, 13, 31, 40), was reinvestigated by the application of spray-freezing and freeze-etching (1, 2, 24). Freeze-etching has been described for Escherichia coli B to give a relatively close approximation to the living state (5). It could be excluded that PX-induced bleb formation is an artifact of fixation of detergent-weakened membranes. The technique used suggests these blebs to be evaginations of the cell surface monolayer of Salmonella typhimurium. MATERIALS AND METHODSBacterial strains and culture conditions. S. typhimurium SL 1135 (complete 0-antigenic side chain of LPS) and SL 1102 (chemotype Re) were obtained from S. Schlecht, Max-Planck-Institut fur Immunbiologie, Freiburg (Germany). S. typhimurium G 30 (a uridine 5'-diphosphate-galactose-4-epimerase-lacking mutant, chemotype Rc) was provided by M. J. Osbom, University of Connecticut at Farmington. S. typhimurium SL 1102-RE was a polymyxin-resistant mutant isolated in ...

Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action

“…Unlike bulges, which are typically observed as a single protrusion per cell (Fig. 6), blebs almost always occur as multiple protrusions per cell [171,[182][183][184]. Blebs are also typically smaller than bulges (diameters of just 10-100nm) [26,184], though enlargement and peptidoglycan rupture during some treatments allows them to develop into bulges [179].…”

“…Antibacterial agent-induced bleb formation has been reported in numerous Gram-negative species including E. coli [41,143,179,180,182], L. pneumophila [83,171], P. aeruginosa [142,[181][182][183], and Salmonella Typhimurium [184]. Unlike bulges, which are typically observed as a single protrusion per cell (Fig.…”

“…6), blebs almost always occur as multiple protrusions per cell [171,[182][183][184]. Blebs are also typically smaller than bulges (diameters of just 10-100nm) [26,184], though enlargement and peptidoglycan rupture during some treatments allows them to develop into bulges [179]. Bleb formation occurs following treatment with membrane-active agents [24,41,181,184] and inhibitors of RNA [143] and protein synthesis [83,179,181].…”

“…Blebs are also typically smaller than bulges (diameters of just 10-100nm) [26,184], though enlargement and peptidoglycan rupture during some treatments allows them to develop into bulges [179]. Bleb formation occurs following treatment with membrane-active agents [24,41,181,184] and inhibitors of RNA [143] and protein synthesis [83,179,181]. All three types of agent also induce filamentation [39, 142,143], and this can accompany the bleb formation [142,143].…”

“…Disruption of the outer membrane by the antibiotic polymyxin B [41,171,180,184], the biocide chlorhexidine [24,184], and the chelating agent EDTA [181] all induce bleb formation. In the case of polymyxin B, blebbing is induced at concentrations ranging from 1xMIC to ~12.5xMIC [41,171,180], the blebs appearing as early as 3 mins after treatment [184], and remaining observable as long as 5 h [171].…”