Kristine Naterstad

Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

ABSTRACT 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 monocytogenesbased 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 otherListeria 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.

Interactions of the bacteriocins sakacin P and nisin with food constituents

Bacteriocins are amphiphilic peptides susceptible to adsorption to food macromolecules and proteolytic degradation. These properties may limit their use as preservation agents. The aim of the present work has been to elucidate the fate of the bacteriocin sakacin P in food. Nisin was used in a few experiments for comparison. Recovery of bacteriocins was studied in homogenates of cold-smoked salmon, chicken cold cuts and raw chicken, with verification of the results in the corresponding food products. More than 80% of the added sakacin P and nisin were quickly adsorbed to proteins in the food matrix. In foods that had not been heat-treated, proteolytic activity caused a rapid degradation of the bacteriocins, with less than 1% of the total activity left after 1 week in cold-smoked salmon, and even less in raw chicken. In heat-treated foods, the bacteriocin activity was stable for more than 4 weeks. The high fat content in salmon compared to chicken had no adverse effect on bacteriocin recovery or activity. However, mixing of triglyceride oils and bacteriocin solutions caused a considerable loss of activity. No principal differences were observed between sakacin P and nisin, but less nisin was adsorbed to muscle proteins at low pH, and the negative effect of oils was less pronounced for nisin. Growth of Listeria monocytogenes was completely inhibited for at least 3 weeks in both chicken cold cuts and cold-smoked salmon by addition of sakacin P (3.5 microg/g), despite the proteolytic degradation in the salmon.

Detection of viable and dead Listeria monocytogenes on gouda-like cheeses by real-time PCR

Aims:  Surface contamination by Listeria monocytogenes of gouda‐like cheeses during processing represents a potential public health problem. The aim of this work was to develop novel real‐time PCR diagnostics to detect the presence of viable, dead or viable but not culturable (VBNC) cells on gouda‐like cheeses.

In vitro testing of commercial and potential probiotic lactic acid bacteria.

Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host. The objective of this study was to investigate the diversity of selected commercial and potential probiotic lactic acid bacteria using common in vitro screening assays such as transit tolerance in the upper human gastrointestinal tract, adhesion capacity to human intestinal cell lines and effect on epithelial barrier function. The selected bacteria include strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus farciminis, Lactobacillus sakei, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri and Pediococcus pentosaceus. Viable counts after simulated gastric transit tolerance showed that L. reuteri strains and P. pentosaceus tolerate gastric juice well, with no reduction of viability, whereas L. pentosus, L. farciminis and L. sakei strains lost viability over 180min. All strains tested tolerate the simulated small intestinal juice well. The bacterial adhesion capacity to human intestinal cells revealed major species and strain differences. Overall, L. plantarum MF1298 and three L. reuteri strains had a significant higher adhesion capacity compared to the other strains tested. All strains, both living and UV-inactivated, had little effect on the epithelial barrier function. However, living L. reuteri strains revealed a tendency to increase the transepithelial electrical resistance (TER) from 6 to 24h. This work demonstrates the diversity of 18 potential probiotic bacteria, with major species and strain specific effects in the in vitro screening assays applied. Overall, L. reuteri strains reveal some interesting characteristics compared to the other strains investigated.

Construction of vectors for inducible gene expression in Lactobacillus sakei and L. plantarum

We have constructed vectors for inducible expression of genes in Lactobacillus sakei and Lactobacillus plantarum. The key elements of these vectors are a regulatable promoter involved in the production of the bacteriocins sakacin A and sakacin P and the genes encoding the cognate histidine protein kinase and response regulator that are necessary to activate this promoter upon induction by a peptide pheromone. The vectors are built up of cassettes that permit easy exchange of all parts through restriction enzyme digestion and ligation. Using beta-glucuronidase as a reporter enzyme, variants of these vectors were compared with each other, and with a corresponding system based on genes involved in the production of nisin. Several of the new vectors permitted tightly controlled and efficient expression of beta-glucuronidase in both L. sakei and L. plantarum.

Fourier Transform Infrared and Raman Spectroscopy for Characterization of Listeria monocytogenes Strains

ABSTRACT The purpose of this study was to characterize the variation in biochemical composition of 89 strains of Listeria monocytogenes with different susceptibilities towards sakacin P, using Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy. The strains were also analyzed using amplified fragment length polymorphism (AFLP) analysis. Based on their susceptibilities to sakacin P, the 89 strains have previously been divided into two groups. Using the FTIR spectra and AFLP data, the strains were basically differentiated into the same two groups. Analyses of the FTIR and Raman spectra revealed that the strains in the two groups contained differences in the compositions of carbohydrates and fatty acids. The relevance of the variation in the composition of carbohydrates with respect to the variation in the susceptibility towards sakacin P for the L. monocytogenes strains is discussed.

Inhibition of Listeria monocytogenes in chicken cold cuts by addition of sakacin P and sakacin P-producing Lactobacillus sakei

Aims: To evaluate the potential of sakacin P and sakacin P‐producing Lactobacillus sakei for the inhibition of growth of Listeria monocytogenes in chicken cold cuts, by answering the following questions. (i) Is sakacin P actually produced in food? (ii) Is sakacin P produced in situ responsible for the inhibiting effect? (iii) How stable is sakacin P in food?

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.

A candidate probiotic with unfavourable effects in subjects with irritable bowel syndrome: a randomised controlled trial

BackgroundSome probiotics have shown efficacy for patients with irritable bowel syndrome (IBS). Lactobacillus (L.) plantarum MF1298 was found to have the best in vitro probiotic properties of 22 strains of lactobacilli. The aim of this study was to investigate the symptomatic effect of L. plantarum MF1298 in subjects with IBS. Primary outcome was treatment preference and secondary outcomes were number of weeks with satisfactory relief of symptoms and IBS sum score.MethodsThe design was a randomised double blind placebo-controlled crossover trial. 16 subjects with IBS underwent two three-week periods of daily intake of one capsule of 1010 CFU L. plantarum MF 1298 or placebo separated by a four-week washout period.ResultsThirteen participants (81%; 95% CI 57% to 93%; P = 0.012) preferred placebo to L. plantarum MF1298 treatment. The mean (SD) number of weeks with satisfactory relief of symptoms in the periods with L. plantarum MF1298 and placebo were 0.50 (0.89) and 1.44 (1.26), respectively (P = 0.006). IBS sum score was 6.44 (1.81) in the period with L. plantarum MF1298 treatment compared with 5.35 (1.77) in the period with placebo (P = 0.010). With a clinically significant difference in the IBS sum score of 2 in disfavour of active treatment, the number needed to harm was 3.7, 95% CI 2.3 to 10.9.ConclusionsThis trial shows for the first time an unfavourable effect on symptoms in subjects with IBS after intake of a potential probiotic.The trial registration numberClinical trials NCT00355810.

Diversity of Lactobacillus sakei strains investigated by phenotypic and genotypic methods

The diversity of 10 strains of Lactobacillus sakei, a commercially important species of lactobacilli, was characterized by studying food isolates. Growth characteristics varied among the strains when examined after growth in a complex medium and a defined medium with either glucose or ribose. A commercial starter culture strain showed the fastest growth rates and high biomass formation on all media, while two of the strains hardly grew on ribose. Based on acidification properties in a meat model, some of the strains had the ability to compete with the indigenous microbiota of the meat batter in addition to being fast acid producers. Carbohydrate-fermentation abilities revealed a relatively wide variation, clustering the strains into two phenotypic groups. The isolates were analyzed using different genetic fingerprinting techniques, demonstrating a distinction between two genetic groups, a grouping consistent with previous studies dealing with L. sakei strains. Comparative genome hybridization (CGH) was introduced for clustering the strains and the same division into two genetic groups was observed. Chromosomal sizes of the strains were estimated by pulsed field gel electrophoresis (PFGE) and were found to vary from 1884 to 2175 kb. The genetic groups did not correlate with the clustering obtained with carbohydrate-fermenting abilities or with chromosomal sizes.