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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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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 (J25_p) 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, J25_p had a purity of 96.7%. All the J25_p fractions obtained by semipreparative RP-HPLC were pooled, freeze-dried, and stored at 4°C. Before utilization, J25_p 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.

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Ability of microcin J25 to inhibit E. coli O157:H7 in biological products. The inhibition activity of J25_p 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 J25_p 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.

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