A major virulence factor of bacteria that cause generalized infections is their resistance to the lytic action of the complement cascade, an important defence mechanism of the host. Invasive Gram-negative enteric bacteria, which cause about one-third of all bacteraemic infections, are completely resistant to lysis by complement, even in the presence of hyperimmune serum. The same bacteria are also resistant to many antibiotics that are effective therapeutic agents against other bacteria, as the outermost surface layer (the outer membrane) of the bacteria functions as a permeability barrier. Here we show that it is possible to sensitize such bacteria to both complement and antibiotics by using an agent that binds to the outer membrane. This agent is a nontoxic derivative of polymyxin which by itself has no bactericidal action.

Polymyxin B nonapeptide, a polymyxin B derivative which lacks the fatty acyl part and the bactericidal activity of polymyxin, was shown to sensitize smooth encapsulated Escherichia coli (018:K1) and smooth Salmonella typhimurium to hydrophobic antibiotics (novobiocin, fusidic acid, erythromycin, clindamycin, nafcillin, and cloxacillin). The polymyxin B nonapeptide-treated bacteria were as sensitive to these antibiotics as are deep rough mutahts. A lysine polymer with 20 lysine residues (lysine2o) had a largely similar effect. Larger lysine polymers and the protamine salmine were bactericidal but, at sublethal concentrations, sensitized the strains to the antibiotics mentioned above, whereas lysine4, streptomycin, cytochrome c, lysozyme, and the polyamines cadaverine, spermidine, and spermine had neither bactericidal nor sensitizing activity.Gram-negative bacteria have an exceptionally efficient barrier against many external influences: the outer membrane (OM), a unique structure located outside the peptidoglycan. This membrane makes gram-negative bacteria resistant to host defense factors, such as lysozyme, which are toxic to gram-positive bacteria. In gram-negative bacteria that live in the gut, the OM is an eflctive barrier, also giving protection from bile and digestive enzymes (17, Z1). At the same time, their OM excludes many antibiotics (e.g., erythromycin, lincomycin, clindamycin, novobiocin, fusidic acid, nafcillin, and cloxacillini) (10, 21) and reduces the penetration of several others (especially penicillin, ampicillin, carbenicillin, and most cephalosporins) (21,24,29,39). The well-known difficulty of treating gram-negative infections is partially due to the permeability barrier properties of the OM, suggesting the possibility of making the bacteria sensitive to many more antibiotics by disorganizing their OM.But is this disorganizing possible? Polymyxin, a polycationic amphipathic antibiotic, is bactericidal to gram-negative bacteria by virtue of a ddal mechanism of action: it disorganizes and penetrates the OM to reach its final bactericidal target, the cytoplasmic membrane (28,31

The outer membrane-disorganizing effect of a short (10-ain) treatment with polycationic agents was studied with smooth Salmonella typhimurium used as a test organism. The polycationic agents were the protamine salmine, a lysine polymer with 20 lysine residues (lysine2g), and the deacylated polymyxin B derivative polymyxin B nonapeptide. Two different types of outer membranedis&$anizing action were foupd. Protamine and lysine20 released 20 to 30%o of the lipopohlys&charide from the outer membrane and sensitized the bacteria to the anioniq detergent sodium dodecyl sulfate but did not (under these conditions) make the bacteria permeable to the hydrophobic probes fusidic acid and actinomycin'D. In contrast, polymyxin B nonapeptide did not release lipopolysaccharide'or 'sensitize the bacteria to sodium dodecyl sulfate but made the outer membrane permeable to the hydrophobic probes. None of the agents was bactericidal under the conditions used or caused any leakage of periplasmic ,Blactamase. Polymyxin B was used as a reference and showe4 characteristic outer membrane-disorganizing action. In thin-section electron microscopy, polymyxin B nQnapeptide caused the appearance of long, narrow, finger-like projections on the outer membrane. Protamine and lysine20 caused a distinctly wrinkled appearance of the outer membrane but no projections.In the accompanying paper (30), we showed that growth in the presence of subinhibitory concentrations of a number of polycationic agents sensitizes smooth enteric bacteria to the hydrophobic antibiotics to which these bacteria are very resistant by nature. The active polycationic agents included protamine, a lysine polymer with 20 residues (lysine2O), and a polymyxin derivative, polymyxin B nonapeptide (PMBN), which lacks the fatty acid tail and also the antibacterial activity of polymyxin. The mechanism by which the polycations sensitize the bacteria to antibiotics was not studied. However, the polycations were shown to make the bacteria as sensitive as are the deep rough (Re) mutants that have a very defective lipopolysaccharide (LPS) in their outer membrane (OM) and, as a consequence, a profoundly disorganized OM (18). Therefore, the polycationic agents could disorganize the OM, as do also polymyxin (26) and EDTA (7).In the present paper, we characterize the action of the polycations on various parameters of OM disorganization. It should be emphasized that this paper deals with the effect of short treatment with polycations, in contrast to the long, continuous treatment described in the accompanying paper (30). We demonstrate herein a number of qualitative differences between the action of protamine and lysine20 on the one hand and PMBN and polymyxin on the other. MATERIALS AND METHODSBacterial strains. The test organism used was smooth Salmonella typhimurium SL6% (metA22 trpB2 H-i-b H2-e,n,xflaA66 rpsLI20) (34) also used in the accompanying paper. Strain SH7794 is strain SL696 carrying R plasmid R471a coding for TEM-type p-lactamase, transferred from SH9178 (29)

The lack of novel antibiotics against gram-negative bacteria has reinstated polymyxins as the drugs of last resort to treat serious infections caused by extremely multiresistant gram-negative organisms. However, polymyxins are nephrotoxic, and this feature may complicate therapy or even require its discontinuation. Like that of aminoglycosides, the nephrotoxicity of polymyxins might be related to the highly cationic nature of the molecule. Colistin and polymyxin B carry five positive charges. Here we show that novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. NAB739 has a cyclic peptide portion identical to that of polymyxin B, but in the linear portion of the peptide, it carries the threonyl-D-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). The MICs of NAB739 for 17 strains of Escherichia coli were identical, or very close, to those of polymyxin B. Furthermore, NAB739 was effective against other polymyxin-susceptible strains of Enterobacteriaceae and against Acinetobacter baumannii. At subinhibitory concentrations, it dramatically sensitized A. baumannii to low concentrations of antibiotics such as rifampin, clarithromycin, vancomycin, fusidic acid, and meropenem. NAB739 methanesulfonate was a prodrug analogous to colistin methanesulfonate. NAB740 was the most active derivative against Pseudomonas aeruginosa. NAB7061 (linear portion of the peptide, threonyl-aminobutyryl) lacked direct antibacterial activity but sensitized the targets to hydrophobic antibiotics by factors up to 2,000. The affinities of the NAB compounds for isolated rat kidney brush border membrane were significantly lower than that of polymyxin B.