PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenes based on the 5-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hlyA) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all other Listeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies were evaluated, since these systems are simple and relatively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approximately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h. In conclusion, a complete quantitative method for L. monocytogenes in water and in skimmed and raw milk was developed.

D . L IL L EH AU G . 1997. This report summarizes the results from an effort to optimize the double-agar plate assay for visualization of the plaques made by six temperate bacteriophages induced from industrial strains of Lactococcus lactis. Among the several parameters found to influence the plaque assay, the effect of incorporating glycine into the growth medium was most striking, resulting in extensive increase in the plaque size of all of the 13 phage-host pairs tested. Notable effects on the plaque size of other factors such as the procedure for sterilization of the agar medium, the volume and softness of the top and bottom layers, and the number and growth stage of the bacterial cells added to the lawn, were also observed. By exploiting these findings in an optimized procedure for plaque assaying, several indicator strains were identified which were unable to support the development of plaques on standard double-agar plates. Since bacterial hosts usually are identified by their ability to support the development of plaques, this observation suggests that the severe difficulty experienced in identifying lactococcal starter strains that are sensitive towards a temperate phage, partly is a problem of methodology.

The genetic elements required for the integration of the temperate lactococcal bacteriophage 4LC3 into the chromosome of its bacterial host, Lactococcus lactis subsp. cremoris, were identified and characterized. The 4LC3 phage attachment site, attP, was mapped and sequenced. DNA sequence analysis of attP and of the bacterial attachment site, attB, as well as the two phage-host junctions, attR and attL, in the chromosome of a +LC3 lysogen, identified a 9-bp common core region, 5'-TTCTTCATG'-3, within which the strand exchange reaction takes place during integration. Most temperate bacteriophages characterized thus far insert their DNA into the chromosome of their bacterial host through site-specific recombination between specific phage (attP) and bacterial (attB) attachment sites, in accordance with the model of phage X integration (10, 15). This integrative recombination is catalyzed by site-specific recombinases (integrases) identified in several temperate coliphages (for a review, see reference 37) but also in temperate phages from several other bacterial genera (14,35,70,71). The integrase (Int) family of site-specific recombinases also includes the recombinases of several other genetic elements, such as certain transposons and plasmids (18,20,27,41,48,51), and regulatory elements of fimbria synthesis in Escherichia coli (26). Although the members of the Int family constitute a highly divergent group of enzymes, they all share limited sequence similarity, especially in a C-termal region believed to make up part of the catalytic site of these recombinases (4, 56).The integrative recombination system of bacteriophage X has been extensively studied (46,47,65,67) and has provided a model for the characterization of the components as well as for the elucidation of the regulatory mechanisms involved in this recombination process. Despite numerous reports on temperate bacteriophages from lactococci, there is so far no information on the molecular biology of lysogeny among this group of gram-positive bacteria. Bacteriophage (LC3, a temperate phage isolated from Lactococcus lactis subsp. cremoris after induction with UV light, recently was described (39). In the present study, the site-specific recombination system of phage 4LC3 was analyzed. The 4LC3 attachment sites were mapped and sequenced, and a common core sequence present in both attP and attB and in the prophage-host hybrid regions in lysogens was Preparation of 4LC3 antiserum. 4)LC3 phage particles, purified twice on a CsCl gradient, were dialyzed against 0.9% NaCl and mixed with an equal volume of Freund's complete adjuvant, and the mixture was shaken to yield an emulsion. A 0.5-ml sample containing ca. 1012 PFU was injected subcutaneously into the neck of a rabbit. The rabbit was reinoculated after 4 and 6 weeks, with Freund's incom-1745

Aims: The objective of the study was to evaluate a high-throughput liquid microcultivation protocol and FTIR spectroscopy for the differentiation of food spoilage filamentous fungi. Methods and Results: For this study, fifty-nine food-related fungal strains were analysed. The cultivation of fungi was performed in liquid medium in the Bioscreen C microtitre plate system with a throughput of 200 samples per cultivation run. Mycelium was prepared for FTIR analysis by a simple procedure, including a washing and a homogenization step. Hierarchical cluster analysis was used to study affinity among the different species. Based on the hierarchical cluster analysis, a classification and validation scheme was developed by artificial neural network analysis. The classification network was tested by an independent test set. The results show that 93Á9 and 94Á0% of the spectra were correctly identified at the species and genus level, respectively. Conclusions: The use of high-throughput liquid microcultivation protocol combined with FTIR spectroscopy and artificial neural network analysis allows differentiation of food spoilage fungi on the phylum, genus and species level. Significance and Impact of the Study: The high-throughput liquid microcultivation protocol combined with FTIR spectroscopy can be used for the detection, classification and even identification of food-related filamentous fungi. Advantages of the method are high-throughput characteristics, high sensitivity, low costs and relatively short time of analysis.

Characterization and identification of fungi in food industry is an important issue both for routine analysis and trouble-shooting incidences. Present microbial techniques for fungal characterization suffer from a low throughput and are time consuming. In this study we present a protocol for high-throughput microcultivation and spectral characterization of fungi by Fourier transform infrared spectroscopy. For the study 11 species of in total five different fungal genera (Alternaria, Aspergillus, Mucor, Paecilomyces, and Phoma) were analyzed by FTIR spectroscopy. All the strains were isolated from trouble-shooting incidents in the production of low and high acid beverages. The cultivation was performed in malt extract broth (liquid medium) in a Bioscreen C system, allowing high-throughput cultivation of 200 samples at the same time. Mycelium was subsequently investigated by high-throughput Fourier transform infrared spectroscopy. Four spectral regions, fatty acids + lipid (3200-2800 cm(-1), 1300-1000 cm(-1)), protein-lipid (1800-1200 cm(-1)), carbohydrates (1200-700 cm(-1)) and "finger print" (900-700 cm(-1)) were evaluated for reproducibility and discrimination ability. The results show that all spectral regions evaluated can be used as spectroscopic biomarkers for differentiation of fungi by FTIR. The influence of different growth times on the ability of species discrimination by FTIR spectroscopy was investigated, and an optimal separation of all five genera was observed after five days of growth. This work presents a novel concept for high-throughput cultivation of fungi for FTIR spectroscopy that enables characterization or identification of hundreds of strains per day.