A novel library-independent approach based on high-throughput cultivation in Bioscreen and fingerprinting by FTIR spectroscopy for microbial source tracking in food industry.

Microbiological source tracking (MST) for food industry is a rapid growing area of research and technology development. In this paper, a new library-independent approach for MST is presented. It is based on a high-throughput liquid microcultivation and FTIR spectroscopy. In this approach, FTIR spectra obtained from micro-organisms isolated along the production line and a product are compared to each other. We tested and evaluated the new source tracking approach by simulating a source tracking situation. In this simulation study, a selection of 20 spoilage mould strains from a total of six genera (Alternaria, Aspergillus, Mucor, Paecilomyces, Peyronellaea and Phoma) was used. The simulation of the source tracking situation showed that 80-100% of the sources could be correctly identified with respect to genus/species level. When performing source tracking simulations, the FTIR identification diverged for Phoma glomerata strain in the reference collection. When reidentifying the strain by sequencing, it turned out that the strain was a Peyronellaea arachidicola. The obtained results demonstrated that the proposed approach is a versatile tool for identifying sources of microbial contamination. Thus, it has a high potential for routine control in the food industry due to low costs and analysis time. SIGNIFICANCE AND IMPACT OF THE STUDY: The source tracking of fungal contamination in the food industry is an important aspect of food safety. Currently, all available methods are time consuming and require the use of a reference library that may limit the accuracy of the identification. In this study, we report for the first time, a library-independent FTIR spectroscopic approach for MST of fungal contamination along the food production line. It combines high-throughput microcultivation and FTIR spectroscopy and is specific on the genus and species level. Therefore, such an approach possesses great importance for food safety control in food industry.

Characterization of food spoilage fungi by FTIR spectroscopy.

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 93.9 and 94.0% 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.

Analysis of a regulator involved in the genetic switch between lysis and lysogeny of the temperate Lactococcus lactis phage phi LC3.

Sequencing of a 1.3-kb fragment of DNA from the temperate Lactococceus lactis subsp. cremoris phage phiLC3 revealed a pair of two divergently oriented ORFs, orf63 and orf286. The deduced amino acid sequence of the product of orf286 showed extensive homology to those of repressors of the temperate lactococcal phages rlt, Tuc2009 and BK5-T. A mutant with an amber mutation in orf286 gave rise to a clear plaque phenotype, indicating that this gene is involved in the lytic and lysogenic development of phiLC3. Gel mobility shift assays showed that the partially purified Orf286 protein bound specifically to the 224-bp intergenic region located between orf286 and orf63, and further characterization by DNase I footprinting analysis revealed that Orf286 protects two distinct sites within this region. Sequence analysis of the intergenic region revealed two putative, divergently oriented promoters, P1 and P2; orf286 and orf63 are probably transcribed from P1 and P2, respectively. In vivo analyses of P1 and P2 using beta-galactosidase as a reporter enzyme in L. lactis showed that transcription from P1 was repressed while transcription from P2 was stimulated in the presence of the Orf286 protein. These results suggest a complex role for the Orf286 protein in regulating the genetic switch between lytic and lysogenic growth of phiLC3.

The life cycles of the temperate lactococcal bacteriophage phiLC3 monitored by a quantitative PCR method.

We present here a new and general approach for monitoring the life cycles of temperate bacteriophages which establish lysogeny by inserting their genomes site-specifically into the bacterial host chromosome. The method is based on quantitative amplification of specific DNA sites involved in various cut-and-join events during the life cycles of the phages (i.e. the cos, attP, attB, attL and attR sites) with the use of sequence-specific primers. By comparing the amounts of these specific DNA sites at different intervals, we were able to follow the development of the lytic and lysogenic life cycles of the temperate lactococcal bacteriophage phiLC3 after infection of its bacterial host Lactococcus lactis ssp. cremoris IMN-C18.

Application of 5'-nuclease PCR for quantitative detection of Listeria monocytogenes in pure cultures, water, skim milk, and unpasteurized whole milk.

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.

Detection and quantification of Salmonella in pure cultures using 5'-nuclease polymerase chain reaction.

Advances in detection and quantification assays based on nucleic acids conceivably will revolutionize the ability to quickly and specifically detect and quantify microorganisms in foods. Among these assays, the polymerase chain reaction (PCR) assay and the TaqMan PCR Detection System (Perkin-Elmer) probably are among the most promising. Since a 5'-nuclease PCR renders possible the automated and direct detection and quantification of PCR products (Holland et al., 1991. Proc. Natl. Acad. Sci. USA 88, 7276-7280), microorganisms in foods can be detected and quantified indirectly within a few hours through analysis of the microbial DNA or RNA sequences present. In the present report we have adapted a 5'-nuclease-based kit for the quantification of Salmonella.

An improved plaque assay for poor plaque-producing temperate lactococcal bacteriophages.

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.

A highly efficient and stable system for site-specific integration of genes and plasmids into the phage phiLC3 attachment site (attB) of the Lactococcus lactis chromosome.

An integration vector system based on the site-specific integration apparatus of the temperate lactococcal bacteriophage phiLC3 was developed. A 1.6-kb recombinogenic DNA cassette, containing the phiLC3 integrase gene (int) and the phage attachment site (attP), mediated site-specific integration of a single marker-gene, as well as of a replication-thermosensitive (-ts) plasmid (pINT2), into the phiLC3 attB site of Lactococcus lactis subsp. lactis LM0230 chromosome after introduction of the DNA into the cells by electroporation. Both the marker gene and the pINT2 plasmid were stably inserted as single copies in an orientation-specific and integrase-dependent manner, the pINT2-ts replicon being stably maintained at temperatures both permissive and non-permissive for plasmid-directed replication. Essentially all transformants obtained with the pINT2 plasmid appeared to be integrants, demonstrating the remarkably high efficiency of the system. This high efficiency rendered possible the detection of transformation-plus-integration events using DNA directly obtained from ligase reaction mixtures, thus avoiding initial subcloning in a non-lactococcal strain and subsequent cointegration of foreign replication functions into the chromosome of L. lactis. The above results, the observation that the phiLC3 attB site appear to be conserved in L. lactis, and the fact that the int-attP cassette functions efficiently in a non-phiLC3-host strain, show that the phiLC3 site-specific integration apparatus provides an efficient and 'food grade' tool for stable integration of genetic elements into the chromosome of L. lactis.

Characterization of genetic elements required for site-specific integration of the temperate lactococcal bacteriophage phi LC3 and construction of integration-negative phi LC3 mutants.

The genetic elements required for the integration of the temperate lactococcal bacteriophage phi LC3 into the chromosome of its bacterial host, Lactococcus lactis subsp. cremoris, were identified and characterized. The phi LC3 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 phi LC3 lysogen, identified a 9-bp common core region, 5'-TTCTTCATG'-3, within which the strand exchange reaction takes place during integration. The attB core sequence is located within the C-terminal part of an open reading frame of unknown function. The phi LC3 integrase gene (int), encoding the phi LC3 site-specific recombinase, was identified and is located adjacent to attP. The phi LC3 Int protein, as deduced from the nucleotide sequence, is a basic protein of 374 amino acids that shares significant sequence similarity with other site-specific recombinases of the integrase family. Phage phi LC3 int- and int-attP-defective mutants, conferring an abortive lysogenic phenotype, were constructed.

The plasmid-encoded lactococcal envelope-associated proteinase is encoded by a chromosomal gene in Lactococcus lactis subsp. cremoris BC101.

The plasmid-free strain Lactococcus lactis subsp. cremoris BC101 produced an extracellular proteinase physicochemically similar to the proteinase encoded by the plasmid-linked prtP gene of other lactococcal strains. The absence of detectable plasmids in strain BC101 indicated that the prtP proteinase gene may be chromosomally located. The chromosomal linkage of the prtP proteinase gene in BC101 was confirmed by pulsed-field electrophoresis of chromosomal DNA and hybridization, using as a probe the plasmid-linked prtP gene from L. lactis subsp. cremoris Wg2. The prtM gene necessary for the maturation of the proteinase was also chromosomally located adjacent to prtP in BC101. By using as a hybridization probe the ISS1-like element ISS1W, which is found adjacent to the proteinase genes in both pWV05 and pSK111, specific homology to the chromosomal fragment containing the proteinase gene was found. DNA sequencing of a polymerase chain reaction product of chromosomal DNA upstream from prtM revealed a 123-nucleotide sequence which was 100% identical to the equivalent sequence in the ISS1W-containing plasmid. The terminal inverted repeat (18 nucleotides) of the ISS1W element was found in this sequenced DNA. These findings suggest that the chromosomal proteinase gene is organized in a fashion similar to that of the plasmid-linked proteinase gene.

Characterization of phiLC3, a Lactococcus lactis subsp. cremoris temperature bacteriophage with cohesive single-stranded DNA ends.

The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp. cremoris, has an isometric head and a flexible tail containing 1 major protein and 8 minor proteins. Infection of a permissive L. lactis host strain yields a burst of about 50 phages per infected cell with a latent period of 60 min. A detailed restriction map of the phage chromosome was constructed by using 12 different restriction enzymes. The phage chromosome is a 33-kb linear double-stranded DNA molecule with unique cohesive ends and with a G + C content of 36.5%. Chemical sequencing of the DNA ends revealed 13-base 3' extended complementary single strands with a relatively high percentage of G + C. Pulsed-field gel electrophoretic analysis of DNA from a strain lysogenized with phiLC3 was used to localize the prophage to a 320-kb BamHI restriction endonuclease fragment from the host chromosomal DNA. This result indicates that lysogeny involves integration of the phage into the host chromosome. A spontaneous phiLC3 clear plaque mutant that was unable to give rise to lysogens was isolated.