It is a broad-spectrum antibiotic with greater activity against gram- negative than gram-positive bacteria (4). The antibacterial activity of polymyxin B results from ...
Vol. 30, No. 2
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1986, p. 340-341 0066-4804/86/080340-02$02.00/0 Copyright X 1986, American Society for Microbiology
In Vitro Cytotoxicity and Antibiotic Activity of Polymyxin B Nonapeptide AXEL K. DUWE', C. ANTHONY RUPAR,2* GREG B. HORSMAN,1 AND STEPHEN I. VAS' Department of Microbiology, Toronto Western Hospital and University of Toronto, Toronto, Ontario MST 2S8,1 and Department of Biochemistry, Children's Psychiatric Research Institute and University of Western Ontario, London, Ontario N6A 4G6,2 Canada Received 27 November 1985/Accepted 15 May 1986
Polymyxin B nonapeptide, prepared by enzymic removal of the fatty acyl diaminobutyric acid side chain from polymyxin B, was about 100-fold less toxic to K562 cells than polymyxin B. MICs of polymyxin B nonapeptide against a test panel of bacteria were 2- to 64-fold lower than those of polymyxin B.
5"Cr release assay with 20,000 5'Cr-labeled K562 cells grown
Polymyxin B is a peptide antibiotic containing a heptapeptide ring and a fatty acylated tripeptide side chain. It is a broad-spectrum antibiotic with greater activity against gramnegative than gram-positive bacteria (4). The antibacterial activity of polymyxin B results from its ability to bind and disorganize the outer membrane of gram-negative bacteria, resulting in changes to the membrane permeability barrier (4). Immobilized polymyxin B can disrupt the Escherichia coli outer membrane permeability barrier, indicating that penetration of the outer membrane is not required for activity (3). The inhibition of polymyxin B binding to outer membranes by divalent cations and the polycationic nature of polymyxin B suggest that antibiotic-outer membrane interactions are electrostatic (4). Polymyxin B nonapeptide (PMBN), a derivative of polymyxin B which lacks the side chain terminal fatty acyl diaminobutyrate, binds with relatively high affinity to the outer membrane (6), disrupts the outer membrane (5), and sensitizes gram-negative bacteria to several hydrophobic antibiotics (8) and serum complement (7). Alterations in the fatty acyl side chain of the related antibiotic, colistin, influence both antimicrobial activity and acute toxicity toward mice (1). In the present study, the eucaryotic cytotoxicity against K562 cells and antimicrobial activities of polymyxin B and PMBN against a panel of gram-positive and gram-negative bacteria were compared. The procedure for the preparation of PMBN from polymyxin B sulfate (Sigma Chemical Co., St. Louis, Mo.) with ficin (Sigma) was similar to procedures previously described for the degradation of colistin (2), with the following modification. The aqueous phase remaining after butanol extractions to remove N-acyl diaminobutyric acid and undegraded polymyxin B was concentrated and dialyzed with a UM-2 membrane (Amicon, Oakville, Ontario, Canada). The purity of the PMBN was checked by thin-layer chromatography on cellulose plates (Merck, Toronto, Ontario, Canada) with solvent butanol, pyridine, acetic acid, and water (15:10:3:12). The amount of polymyxin B contaminant in PMBN preparations was determined to be 128
P. aeruginosa ATCC 27853
0.125
4
1/32
4 ml of Trypticase soy broth (BBL) and incubated for 3 h at 37°C. This was adjusted to a McFarland standard (0.5) giving approximately 108 CFU/ml and further diluted in sterile distilled water with 0.02% Tween 80 to 107 CFU. Using a Dynatech 96-prong semiautomated inoculator, 0.0015 ml was inoculated into each well containing 0.1 ml of MuellerHinton broth. This gave a final concentration of 105 CFU/ml. The plates were incubated at 35°C for 18 h. Each test was done in triplicate, and the endpoint was taken as the lowest antibiotic concentration at which there was no visible growth. PMBN was a less effective antibiotic than polymyxin B (Table 2). The MICs of PMBN were 2- to 8-fold higher for gram-positive bacteria and 32- to 64-fold higher for the susceptible gram-negative bacteria tested. S. marcescens was resistant to polymyxin B and was not affected by a twofold higher concentration of PMBN. A test tube assay for MIC and MBC in which E. coli ATCC 25922 was grown in 2 ml of broth for 2 h was within one tube dilution of the microtiter plate data, and the MBCs were >25 ,ug/ml for both PMBN and polymyxin B.
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The toxicity of PMBN toward K562 cells was nearly 100-fold less than that of polymyxin B, whereas the antibiotic activity was decreased only 2- to 64-fold against a panel of both gram-negative and gram-positive bacteria. The results suggest that the removal of the fatty acyl diaminobutyric acid side chain terminal from polymyxin B had a greater effect on eucaryotic cytotoxicity than on antimicrobial activity. It may therefore be useful to screen the nonapeptides of other polymyxins for both antibiotic activity and eucaryotic toxicity to determine whether some of these nonapeptides may be useful against bacteria resistant to more conventional antibiotics. Although PMBN was less toxic to eucaryotic cells in vitro, caution should be exercised in extrapolating these results to in vivo toxicity. Further experiments are required to determine how PMBN is metabolized in vivo. Our results indicate that in vivo studies are appropriate and suggest a useful methodology for a preliminary screening of polymyxin peptides. LITERATURE CITED 1. Chihara, S., A. Ito, M. Yahata, T. Tobita, and Y. Koyama. 1974. Chemical synthesis, isolation and characterization of a-Nfattyacyl colistin nonapeptide with special reference to the correlation between antimicrobial activity and carbon number of fattyacyl moiety. Agric. Biol. Chem. 38:521-529. 2. Chihara, S., T. Tobita, M. Yahata, A. Ito, and Y. Koyama. 1973. Enzymatic degradation of colistin: isolation and identification of a-N-acyl a,-y-diaminobutyric acid and colistin nonapeptide. Agric. Biol. Chem. 37:2455-2463. 3. Rosenthal, K. S., and D. R. Storm. 1977. Disruption of the Escherichia coli outer membrane permeability barrier by immobilized polymyxin B. J. Antibiot. 30:1087-1092. 4. Storm, D. R., K. S. Rosenthal, and P. E. Swanson. 1977. Polymyxin and related peptide antibiotics. Annu. Rev. Biochem. 46:723-763. 5. Vaara, M., and T. Vaara. 1983. Polycations as outer membranedisorganizing agents. Antimicrob. Agents Chemother. 24: 114-122. 6. Vaara, M., and P. ViUanen. 1985. Binding of polymyxin B nonapeptide to gram-negative bacteria. Antimicrob. Agents Chemother. 27:548-554. 7. Vaara, M., P. ViUanen, T. Vaara, and P. H. Makela. 1984. An outer membrane-disorganizing peptide PMBN sensitizes E. coli strains to serum bactericidal action. J. Immunol. 132:2582-2589. 8. ViUanen, P., and M. Vaara. 1984. Susceptibility of gram-negative bacteria to polymyxin B nonapeptide. Antimicrob. Agents Chemother. 25:701-705.
