Polymyxins and Circulin

Upon exposure to 6,000 U of polymyxin B sulfate per ml, cells of the polymyxinsensitive PAO 1 strain of Pseudomonas aeruginosa displayed in thin sections long projections arising from the outer membrane of the cell wall and extensive cytoplasmic degradation with accumulation of cytoplasmic membrane infoldings. Polymyxin-resistant isolates derived from the PAO 1 strain, however, grew well in the presence of 6,000 U of polymyxin per ml and exhibited none of these effects, having instead the appearance of a typically healthy cell. Freeze-etching of cells of the sensitive strain grown in basal medium without polymyxin revealed a concave cell wall layer studded with numerous particles. Freeze-etching of cells of the resistant isolates grown in basal medium containing 6,000 U of polymyxin per ml revealed a concave cell wall layer (i.e., the outer half of the outer membrane) in which most of these particles were absent. Thus, acquisition of resistance to polymyxin was correlated with an alteration in the architecture of the outer membrane. When the resistant isolates were grown in the basal medium lacking polymyxin and then freeze-etched, the particle distribution in the concave cell wall layer resembled that of the sensitive parent strain. The cells had regained sensitivity to polymyxin upon suspension in medium containing 6,000 U/ml as determined by their failure to grow and by internal damages seen in thin sections. These cells also had acquired increased sensitivity to ethylenediaminetetraacetate, whereas the polymyxin-resistant cells grown in the presence of polymyxin were resistant to lysis by ethylenediaminetetraacetate. The polymyxin-resistant isolates were not stable mutants but instead represented an adaptive response to the presence of polymyxin in the medium.There has been considerable interest about both how polymyxin exerts its antimicrobial action on Pseudomonas aeruginosa and how resistance to polymyxin is acquired. Polymyxin is thought to interact with phospholipids in the cell envelope (30), causing a disorganization of the cytoplasmic membrane that results in disruption of the osmotic equilibrium of the cell and leakage of the cell contents (21,24,30). Koike et al. (15) have shown that treatment of P. aeruginosa with polymyxin caused projections to appear on the cell wall and damaged the cytoplasmic membrane with part of the cytoplasmic contents extruding through cracks in the membrane.Studies of polymyxin-resistant cells have been mainly chemical in nature and without reference to an ultrastructural basis for resistance. An increase in cell wall lipid content appears to be a consistent finding, in resistance 'Present address:

Section: Resultsmentioning

confidence: 98%

mentioning

confidence: 99%

Ultrastructural study of polymyxin-resistant isolates of Pseudomonas aeruginosa

Gilleland¹,

Murray²

1976

“…The spikes of the tail plate attached closely to the dense line which was visible in the profile of the LPS strand. Table 4 shows the dis-DISCUSSION PLB and CL act by damaging the cell envelope (20). We have shown that treatment of gram-negative bacteria with polymyxin results in the formation of projections from the outer layer of the cell envelope and the liberation of cytoplasm through cracks in the membrane (1 1).…”

Section: Resultsmentioning

confidence: 99%

Effect of Polymyxin on the Bacteriophage Receptors of the Cell Walls of Gram-Negative Bacteria

Koike

Iida

1971

Treatment of gram-negative bacteria with lethal doses of polymyxin B and colistin resulted in the formation of projections of the outer layer of the cell wall. Phages T3, T4, and T7, which use wall lipopolysaccharide as receptors, were specifically prevented from adsorbing to Escherichia coli B cells treated with polymyxin, whereas phages T1, T2, T5, and T6 were not. In the systems of phage P22C-Salmonella typhimurium LT2 and phage C21-S. typhimurium variant SL1069, the phage were prevented from adsorbing to the host cell treated with the antibiotics. Electron microscopic observations show that phage T2 adsorbed irreversibly to the normal smooth surface between the projections on the outer layer caused by the drug treatment. These results indicate that lipopolysaccharide is affected by polymyxin functionally and morphologically, but lipoprotein is not. The purified lipopolysaccharide showed a ribbon-like structure when viewed face on and showed trilamellar structure when viewed edge on. The lipopolysaccharide from E. coli B was irreversibly adsorbed by phages T3, T4, and T7, but not phage T2. Often, phage T4 adsorbed to both sides of the lipopolysaccharide strand at comparable distances. Phage P22C adsorbed through the spikes of the tail-plates to the lipopolysaccharide from S. typhimurium LT2. Lipopolysaccharide which was treated with low doses of the drug (2.5 to 6.25 gg of polymyxin B per ml to 100 ug of lipopolysaccharide per ml) turned into the coiled form and was partially broken down into short segments with coiled form. The loosely coiled lipopolysaccharide retains both its function as the receptor and its trilamellar structure. Treatment with high doses of the drug (12.5 to 25 ug of polymyxin B per ml to 100l g of lipopolysaccharide per ml) caused the collapse of the trilamellar structure of the strand. These collapsed lipopolysaccharides became flat and fused with each other, making an amorphous mass, and finally they were broken into small collapsed fragments.

“…Neomycin, streptomycin, and kanamycin are similar in structure and, although normally considered inhibitors of protein synthesis, may act as surface-active agents (14). Polymyxin is a cationic surface-active agent (20,21,26). Other known surface-active agents, sodium deoxycholate, sodium lauryl sulfate, and Roccal, a quaternary ammonium compound, also decreased recovery of UHTtreated spores at concentrations which were only slightly inhibitory to non-UHT-treated spores.…”

Section: Fig 3 Effect Of Uht Alkali Treatment On Survivalmentioning

confidence: 99%

Spore membrane(s) as the site of damage within heated Clostridium perfringens spores

Flowers¹,

Adams²

1976

Clostridium perfringens spores were injured by ultrahigh-temperatuie treatment at 105 C for 5 min. Injury was manifested as an increased sensitivity to polymyxin and neomycin. Since many of the survivors could not germinate normally the ultrahigh-temperature-treated spores were sensitized to and germinated by lysozyme. Polymyxin reportedly acts upon the cell membrane. Neomycin may inhibit protein synthesis and has surface-active properties. Injured spores were increasingly sensitive to known surface-active agents, sodium lauryl sulfate, sodium deoxycholate, and Roccal, a quaternary ammonium compound. Injured spores sensitive to polymyxin and neomycin also were osmotically fragile and died during outgrowth in a liquid medium unless the medium was supplemented with 20% sucrose, 10% dextran, or 10% polyvinylpyrrolidone. The results suggested that a spore structure destined to become cell membrane or cell wall was the site of injury. Repair of injury during outgrowth in the presence of protein, deoxyribonucleic acid, ribonucleic acid and cell wall synthesis inhibitors was corsistent with this hypothesis.