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Applied and Environmental Microbiology, October 2000, p. 4595-4597, Vol. 66, No. 10
Laboratoire de Génie Protéique et
Cellulaire, Pôle Sciences, Université de La Rochelle,
La Rochelle, France
Received 23 February 2000/Accepted 7 July 2000
The inhibitory activities of known microcins were evaluated against
some diarrheagenic Escherichia coli strains. Some
antibacterial properties of microcin J25, the most active one, were
studied. A rapid two-step purification was performed. The
MIC and the minimum bactericidal concentration of J25 against E. coli O157:H7 were 1 and 100 µg ml Diarrheagenic Escherichia
coli (DEC) strains are currently associated with food-related
illnesses (13). The notorious DEC serotype O157:H7 has been
the cause of several large food-related epidemics in Europe, North
America, and Japan, mostly after consumption of meat or dairy
products (9). Physicochemical (3, 4, 7) and
biological (2, 8) methods for the control of DEC have been
described, mainly with E. coli O157:H7 as the target strain.
Microcins and colicins are classical bacteriocins produced
by Enterobacteriaceae that inhibit E. coli
and closely related strains (12). Unlike most
colicins, microcins are secreted peptides with low molecular masses
(<10 kDa). Their synthesis is nonlethal for the producing strains and
not mediated by conditions inducing the SOS system (12).
Because of their small size, they are resistant to some proteases and
fairly thermostable. So far, six microcins have been described, namely,
B17 (19), C7 (6), D93 (10), E492
(18), H47 (5), and J25 (1); colicin V
(Col V) can also be considered a microcin (12).
The objective of the present study was to evaluate the inhibitory
activity of microcins, particularly J25, against DEC strains. Our
results showed that all the tested strains were inhibited by at least
two microcins. The antagonistic activity of purified microcin J25, the
most active one under our experimental conditions, was evaluated. This
microcin is a cyclic peptide of 21 unmodified amino acid residues
(1) which apparently block cell division (15).
The operon coding for production, export, and immunity was recently
described (16, 17).
Bacterial strains, preservation, and growth conditions.
Microcin producers were recombinant E. coli strains (MC 4100 for B17, C7, and Col V; VCS 257 for E492; RYC 1000 for H47; and KI 3110 for J25), with each strain harboring the genes required for synthesis,
export, and immunity for a single microcin. Producers of B17, C7, E492,
J25, and Col V were supplied by F. Moreno (Unidad de Genetica
Molecular, Hospital Ramon y Cajal, Madrid, Spain), and the producer of
H47 was supplied by M. Laviña (Instituto de Investigaciones
Biologicas Clemente Estable, Ministerio de Educacion y Cultura,
Montevideo, Uruguay). DEC strains were six collection strains
purchased from Institut Pasteur (Paris, France), named CIP
52.168, CIP 52.170, CIP 52.172, CIP 62.23, CIP 62.24, and CIP 103.571 (respective serotypes: O111:H12, O55:H6, O26:H11, O119, O125, and
O157:H7) and nine clinical isolates obtained from different patients
with acute diarrhea, which were serotypes O26, O55 (three strains),
O86, and O111 and three undetermined serotypes (X1,
X2, and X3).
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Antibacterial Activity Evaluation of Microcin J25
against Diarrheagenic Escherichia coli
ABSTRACT
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1, respectively.
A 104-CFU ml1 contamination by this strain
was destroyed in milk and meat extract by 6.25 µg of J25
ml1 and in half-diluted egg yolk by 50 µg of J25
ml1.
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1. Unless otherwise stated, bacterial cultures were
propagated in BHI at 37°C. Working cultures were maintained at 4°C
on BHI agar slants. Stock cultures were stored at 
80°C.
Inhibition assays.
M63 agar (10 ml) was overlaid with 5 ml of
M63 soft agar seeded with 107 CFU of the target strain
ml1. Sterile glass rings (4-mm inside diameter [i.d.])
were placed on the soft agar and filled with 20 µl of
filter-sterilized samples to be tested. The plates were incubated for
24 h at 37°C and clear inhibition zones were measured.
Inhibition assays were done in triplicate. The results of the
inhibition of DEC strains by culture supernatants of microcin producer
strains are given in Table 1. No strain
was resistant to all the microcins, but the strains showed various
sensitivities to the microcins.
|
Purification of microcin J25 and analysis of its antagonistic
properties against DEC.
A culture (18 h, 37°C) of the J25
producer in M63 medium was centrifuged (13,000 × g, 30 min, 10°C) and heated (10 min, 120°C). Crude extract was prepared
from heated supernatant by ammonium sulfate precipitation (95%
saturation). The pellet was resuspended in water (1/80 of the initial
culture volume) and dialyzed (with a membrane cutoff of 1,000) against
water (24 h, 10°C). The crude microcin was then purified by
reversed-phase high-pressure liquid chromatography (RP-HPLC). Filtered
samples (2 ml) were subjected to semipreparative RP-HPLC (Delta Pak
C18 column, 300 by 19 mm [i.d.]). The mobile phase (12 ml
min1) was 10 mM ammonium acetate buffer (pH 6.0) (eluent
A) and acetonitrile (eluent B). The gradient was 0 to 20% eluent B in
4 min, 20 to 56% eluent B in 36 min, and 56 to 100% eluent B in 9 min. Optical density was measured at 215 nm. The peak fraction of J25
(retention time, 24 min) was collected and freeze-dried. Purified J25
(J25p) was subjected to an additional RP-HPLC analysis
(Delta Pak C18 column, 300 by 3.9 mm [i.d.]) to check its
purity. By peak area integration, J25p had a purity of
96.7%. All the J25p fractions obtained by semipreparative
RP-HPLC were pooled, freeze-dried, and stored at 4°C. Before
utilization, J25p was dissolved in a water-acetonitrile
mixture (60:40, vol/vol) in order to obtain an initial solution with a
concentration of 4 mg ml1.
1 (results
not shown). With the same target strain, the MIC and the MBC of
J25p were 1 and 100 µg ml1, respectively.
|
Ability of microcin J25 to inhibit E. coli O157:H7 in
biological products.
The inhibition activity of J25p
against E. coli O157:H7 was tested in three products:
sterile skim milk, diluted egg yolk (1:1 mixture of sterile egg yolk
and sterile water), and meat extract (50 g of sterile water was added
to 100 g of mincemeat and mixed for 5 min in a stomacher at
maximum speed; the supernatant was harvested by centrifugation and then
filter sterilized before use). Product samples of 500 µl mixed with
20 µl of an appropriate dilution of the initial J25p
solution in sterile saline water (8.5 g liter1) were
inoculated with 5 µl of an E. coli O157:H7 culture in M63 medium (12 h, 37°C) diluted with sterile M63 medium in order to obtain the desired quantity of cells. After 24 h of incubation (37°C), the presence of viable cells in 100-µl samples was checked using a spiral plating method on BHI agar with incubation (37°C, 24 h). Assays were done three times.
1 and an E. coli O157:H7 concentration of
103 CFU ml1, no viable bacteria were detected
after 24 h (0 CFU in three 100-µl product samples). With an
O157:H7 concentration of 104 CFU ml1, we
obtained the same results (i.e., no bacteria were detected) in milk and
meat extract, but complete inhibition required 50 µg of
J25p ml1 in egg yolk, due to the richness of
the medium.
The data presented here show the possibility of using microcins to
control DEC strains, including serotype O157:H7.
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ACKNOWLEDGMENTS |
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We are indebted to F. Moreno and M. Laviña for providing the microcin-producing strains.
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FOOTNOTES |
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* Corresponding author. Mailing address: LGPC, Pôle Sciences, Université de La Rochelle, Avenue Marillac, 17042 La Rochelle, France. Phone: 33-546-458-246. Fax: 33-546-458-247. E-mail: gilles.cottenceau{at}univ-lr.fr.
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Applied and Environmental Microbiology, October 2000, p. 4595-4597, Vol. 66, No. 10
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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