Characterization of an E2-type colicin and its application to treat alfalfa seeds to reduce Escherichia coli O157:H7.

Several outbreaks of Escherichia coli O157:H7 infections have been associated with contaminated alfalfa seeds. A recently isolated E. coli strain Hu194 was capable of inhibiting 22 strains of E. coli O157:H7 and this inhibition was mediated by the production of a colicin named Hu194. The objectives of this study were to test the efficacy of treating alfalfa seeds with colicin Hu194 against E. coli O157:H7 strains, and to characterize this antimicrobial protein. Significant reductions (approximately 5 log CFU ml-1) in the viable cell counts of strains 43890 and 43895 were observed after 1-day incubation with semi-crude colicin, and after 2 days for strain 3081. Strain 43890 was successfully eliminated (5 log CFU g-1) from inoculated alfalfa seeds after soaking in a colicin suspension at a concentration of 10,000 AU/g. Treatment of alfalfa seeds inoculated with strains 43895 and 3081 required 20-fold higher concentrations of colicin Hu194 to achieve as much as 3 log CFU g-1 reductions. The genes encoding the colicin Hu194 operon were located on a 6 kb plasmid, and the sequence analysis revealed that this colicin was an E-type DNAse. From the sequence data, the estimated molecular masses of colicin Hu194, its immunity protein and lysis protein were 61.3, 10.0 and 4.8 kDa, respectively. Based on DNA and protein sequence comparisons with other E-type colicin, colicin Hu194 belonged to the type E2-colicin cluster. However, cross-immunity tests between E-group colicins suggested that Hu194 colicin was divergent from the previously characterized E2 colicins.

Inhibition of Listeria monocytogenes , Escherichia coli O157:H7, and Micrococcus luteus by Linear Furanocoumarins in a Model Food System.

Lime peel, parsnip, lemon peel, dried parsley flakes, cold pressed lime oil, and distilled lime oil samples were analyzed for the presence and concentration of the linear furanocoumarins (LFs) psoralen, 5-methoxypsoralen (5- MOP), and 8-methoxypsoralen (8-MOP) by thin layer chromatography and gas chromatography mass spectrometry. Cold-pressed lime oil had the highest LF content (psoralen, 67 ± 29 µg/ml, 5-MOP, 1,634 ± 62 µg/ml, and 8-MOP, 44 ± 2 µg/ml). The antimicrobial effectiveness of LFs against Listeria monocytogenes , Escherichia coli O157:H7, and Micrococcus luteus was tested in a model food system consisting of a slurry of 25% commercial "garden vegetables" baby food in 0.1% peptone water. Inhibition required UV activation after the addition of the LFs to the model system. Lime peel extract, cold-pressed lime oil, and a 5-MOP standard inhibited the growth of L. monocytogenes , but not E. coli O157:H7. M. luteus was inhibited only by the cold-pressed lime oil. The minimum LF concentration that caused inhibition of the growth of L. monocytogenes was 32 µg/g and the minimum bactericidal concentration was 43 µg/g. Cold-pressed lime oil inhibited L. monocytogenes even at the lowest concentration added to the model system (10 µg/g), while the corresponding LF standard did not. This suggested the presence of other antimicrobial agents in the oil.

Inhibition of Listeria monocytogenes , Escherichia coli O157:H7, and Micrococcus luteus by Linear Furanocoumarins in Culture Media.

The linear furanocoumarins psoralen, 5-methoxypsoralen (5-MOP), and 8-methoxypsoralen (8-MOP) were tested as antimicrobial agents against Listeria monocytogenes , Escherichia coli O157:H7 and Micrococcus luteus . The linear furanocoumarins were activated with UV light at 365 nm for 60 min. MICs of these furanocoumarins in tryptic soy broth supplemented with 0.6% yeast extract were determined at room temperature for 48 h. Psoralen was the most effective antimicrobial agent of the three studied. This compound inhibited the three test microorganisms at concentrations of 5 µg/ml or lower. Only L. monocytogenes was inhibited by 5-MOP.Concentrations of at least 10µg of 8-MOP per ml were required to inhibit the test microorganisms. Mixtures containing at least 7.5 µg of psoralen per ml inhibited the growth of the three microorganisms. In the absence of psoralen, mixtures of 5- MOP and 8-MOP were ineffective as growth inhibitors of M. luteus and E. coli O157:H7.