In Vitro Cytotoxicity and Antibiotic Activity of Polymyxin B Nonapeptide

In vitro cytotoxicity and antibiotic activity of
polymyxin B nonapeptide.
A K Duwe, C A Rupar, G B Horsman and S I Vas
Antimicrob. Agents Chemother. 1986, 30(2):340. DOI:
10.1128/AAC.30.2.340.
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1986, p. 340-341
0066-4804/86/080340-02$02.00/0
Copyright X 1986, American Society for Microbiology
Vol. 30, No. 2
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.
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

VOL. 30, 1986
TABLE 2. MICs of polymyxin B and PMBN against
gram-negative and gram-positive bacteria
MIC (,ug/ml)
MIC ratio
Strain Polymyxin B PMBN (polymyxin B/PMBN)
S. aureus
ATCC 25923 32 128 1/4
ATCC 29213 16 64 1/4
S. epidermidis
ATCC 27626 64 128 1/2
ATCC 14990 16 128 1/8
E. coli ATCC 0.5 32 1/64
25922
K. pneumoniae 2 128 1/64
ATCC 13883
S. marcescens 64 >128
ATCC 13880
P. aeruginosa 0.125 4 1/32
ATCC 27853
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 Mueller-
Hinton 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.
NOTES 341
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.
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