Potential uptake of Escherichia coli O157:H7 from organic manure into crisphead lettuce.

To investigate the potential transfer of Escherichia coli O157:H7 from contaminated manure to fresh produce, lettuce seedlings were transplanted into soil fertilized with bovine manure which had been inoculated with approximately 10(4) CFU g(-1) E. coli O157:H7. The lettuce was grown for approximately 50 days in beds in climate-controlled rooms in a greenhouse. As the bacterium was not detected in the edible parts of the lettuce, the outer leaves of the lettuce, or the lettuce roots at harvest it was concluded that transmission of E. coli O157:H7 from contaminated soil to lettuce did not occur. The pathogen persisted in the soil for at least 8 weeks after fertilizing but was not detected after 12 weeks. Indigenous E. coli was detected only sporadically on the lettuce at harvest, and enterococci were not detected at all. The numbers of enterococci declined more rapidly than those of E. coli in the soil. Pseudomonas fluorescens, which inhibited growth of E. coli O157:H7 in vitro, was isolated from the rhizosphere.

Development and application of new nucleic acid-based technologies for microbial community analyses in foods.

Several challenges still persist in the analysis of microorganisms in foods, particularly in studies of complex communities. Nucleic acid-based methods are promising tools in addressing new questions concerning microbial communities. We have developed several new methods in the field of nucleic acid-based microbial community analyses. These methods cover both sample preparation and detection approaches. The sample preparation method involves simplified DNA purification using paramagnetic beads. As an extension of this method, the same paramagnetic beads are used for both cell separation and DNA purification. This enables full automation. The separate detection of viable and dead bacteria is a major issue in nucleic acid-based diagnostics. We have applied a living/dead dye that binds covalently to DNA and inhibits the PCR from dead cells. In addition, a DNA array-based detection assay has been developed. The assay combines the specificity obtained by enzymatic labeling of DNA probes with the possibility of detecting several targets simultaneously by DNA array hybridization. In combination with 16S rDNA amplification, this is a promising tool for community analyses. Also, we have developed a novel approach for multiplex quantitative PCR. The multiplex PCR has been combined with our DNA array-based detection method. Finally, we are now in the process of adapting a system for monitoring microbial growth and death in real-time through the tagging of bacteria with green fluorescent protein (GFP) combined with fluorescence detection using a high-resolution confocal laser scanner.

Characterization of Listeria monocytogenes isolates from the meat, poultry and seafood industries by automated ribotyping.

A total of 564 Listeria monocytogenes isolates were characterized by automated ribotyping. The samples were taken from equipment, personnel and the environment after cleaning procedures and during food processing, as well as from raw materials and products from six meat, two poultry and five seafood processing plants located in the Faroe Islands, Finland, Iceland, Norway and Sweden. Altogether, 25 different ribotypes (RTs) were generated. Two RTs occurred in the samples from all three food sectors--meat, poultry and seafood. Four RTs occurred in meat and poultry plant samples and other four RTs occurred in meat and seafood plant samples. Five RTs occurred only in meat plant samples, five only in poultry plant samples and five only in seafood plant samples. Eight of the thirteen plants had their own in-house L. monocytogenes ribotype. There was geographical differences between the RTs, but no correlation between RTs and food sectors was detected. The discrimination power of automated ribotyping was satisfactory to trace the contamination sources in the food processing plants clearly indicating the sites at which improved cleaning procedures were necessary. In addition, it was possible to screen a large number of isolates with two instruments located at different institutes and to make a reliable combination of the results.