Antibacterial synergism of polymyxin B nonapeptide and hydrophobic antibiotics in experimental gram-negative infections in mice

Abstract

Polymyxin B nonapeptide, derived by cleavage of the fatty acyl diaminobutyric acid from polymyxin B, is considerably less toxic, lacks bactericidal activity, and retains its ability to render gram-negative bacteria susceptible to several antibiotics by permeabilizing their outer membranes. The peptide rendered all 53 polymyxin-susceptible strains tested more susceptible to novobiocin, lowering the MIC of novobiocin eightfold or more. The combination of polymyxin B nonapeptide with novobiocin or with erythromycin administered intraperitoneally in multiple doses synergistically protected mice infected with gram-negative bacteria. This combination may be clinically useful because of the apparent rarity of the acquisition of resistance.Resistance to hydrophobic antibiotics in gram-negative bacteria may be either due to a decrease in the penetration of the antibiotic through the outer membrane or due to specific mechanisms, such as gene mutation or acquisition of resistance genes. The distinction between the two types of resistance is important, because it allows the search for antibiotics to which the development of specific mechanisms of resistance are rare but to which the bacterial membrane is impermeable. Such active but nonpenetrating antibiotics may gain clinical use when combined with membrane-active agents that increase their penetration. Polycations at sublethal concentrations increase the permeability of the outer membranes of gramnegative bacteria and render these microorganisms susceptible to many hydrophobic antibiotics (15)(16)(17)(18). Among the various polycations tested, polymyxin B nonapeptide (PMBN), derived by cleaving the fatty acid moiety from polymyxin B, has been studied the most. PMBN lacks antibacterial activity but renders most polymyxin-susceptible gram-negative bacteria susceptible to hydrophobic antibiotics by disorganizing the bacterial outer membranes to enhance antibiotic penetration (17)(18)(19). In the present study, we sought to further determine the incidence of strains that exhibit permeability-associated resistance in assays with PMBN and to examine the therapeutic efficacy of hydrophobic antibiotics in combination with PMBN in experimental gram-negative infections in mice.All clinical isolates were obtained from various body sites at regional outpatient clinics (see Table 2). The bacteria were grown on agar slants (nutrient agar; Difco Laboratories, Detroit, Mich.) and kept at 4°C for up to 2 weeks. The MICs of each antibiotic were determined as described elsewhere (7). 0.5 ml of phosphate-buffered saline (0.1 M NaCl, 0.02 M P04; pH 7.2) containing either 5 x 105 CFU of Klebsiella pneumoniae serotype K2 (1) harvested from the logarithmic phase of growth in nutrient broth (Difco) at 37°C or 1 x 108 CFU of Pseudomonas aeruginosa 33347 (kindly provided by Nehama Garber, Bar-Ilan University, Ramat-Gan, Israel) harvested from agar slants after overnight growth at 37°C (8).The efficacy of PMBN either alone or in combination with other antibiotics was evaluated following intraperiton...

Antimicrobial Peptides Activate the Rcs Regulon through the Outer Membrane Lipoprotein RcsF

“…PMBN resulted in Rcs activation at 2.5 g/ml, which is substantially below its MIC of Ն20 g/ml in MHB. This concentration of PMBN leads to a substantial increase in OM sensitivity to lipophilic compounds without a release in periplasmic proteins (35,49). This same effect is seen with PMB exposure but is masked by its bactericidal effects at low concentrations (46,48,49).…”

“…The Rcs response to PMBN is particularly interesting because its predominate activity at subinhibitory concentrations is selective permeabilization of the outer membrane to hydrophobic compounds (11,35,49). PMBN resulted in Rcs activation at 2.5 g/ml, which is substantially below its MIC of Ն20 g/ml in MHB.…”

Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa

“…Interestingly, tetracain demonstrated strong synergism with novobiocin, a property already noted for some other local anesthetics (22,37). These results confirm that efficient permeabilizers may produce sufficient damage to the outer membrane bilayer to increase strongly the penetration of lipophilic antibiotics via the LPS uptake route (36,53). By contrast, polymyxin B and DAPB failed to sensitize strain PAO1 to the hydrophilic antibiotics ceftazidime and ciprofloxacin (data not shown).…”

Development of Des-Fatty Acyl-Polymyxin B Decapeptide Analogs with Pseudomonas aeruginosa-Specific Antimicrobial Activity

“…7 ]-PMB (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) activity of 10 and 11 was not reflected in their bactericidal potency against E. coli, which was moderate, with MIC values of 8 and 16 nmol/ml, respectively (Table 3, Fig. 3B).…”

“…1) It is well-known that the enzymatic removal of the fatty acyl-Dab 1 of PMB with ficin yields PMB nonapeptide (PMBN), 3,4) which retains considerable lipopolysaccharide (LPS)-binding activity 5) and has reduced toxicity. 6) However, PMBN has low bactericidal activity, with a minimum inhibitory concentration (MIC) value of Ͼ256 nmol/ml towards Escherichia coli (E. coli).…”