Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

A real-time PCR assay was designed to detect a 162-bp fragment of the ssrA gene in Listeria monocytogenes. The specificity of the assay for L. monocytogenes was confirmed against a panel of 6 Listeria species and 26 other bacterial species. A detection limit of 1-10 genome equivalents was determined for the assay. Application of the assay in natural and artificially contaminated culture enriched foods, including soft cheese, meat, milk, vegetables and fish, enabled detection of 1-5 CFU L. monocytogenes per 25g/ml of food sample in 30h. The performance of the assay was compared with the Roche Diagnostics 'LightCycler foodproof Listeria monocytogenes Detection Kit'. Both methods detected L. monocytogenes in all artificially contaminated retail samples (n=27) and L. monocytogenes was not detected by either system in 27 natural retail food samples. The method developed in this study has the potential to enable the specific detection of L. monocytogenes in a variety of food types in a time-frame considerably faster than current standard methods. The potential of the ssrA gene as a nucleic acid diagnostic (NAD) target has been demonstrated in L. monocytogenes. We are currently developing NAD tests based on the ssrA gene for a range of common foodborne and clinically relevant bacterial pathogens.     

Anaerobic oxidation of methane: mechanisms, bioenergetics, and the ecology of associated microorganisms

Microbially mediated anaerobic oxidation of methane (AOM) moderates the input of methane, an important greenhouse gas, to the atmosphere by consuming methane produced in various marine, terrestrial, and subsurface environments. AOM coupled to sulfate reduction has been most extensively studied because of the abundance of sulfate in marine systems, but electron acceptors otherthan sulfate are more energetically favorable. Phylogenetic trees based on 16S rRNA gene clone libraries derived from microbial communities where AOM occurs show evidence of diverse, methanotrophic archaea (ANME) closely associated with sulfate-reducing bacteria, but these organisms have not yet been isolated as pure cultures. Several biochemical pathways for AOM have been proposed, including reverse methanogenesis, acetogenesis, and methylogenesis, and both culture-dependent and independent techniques have provided some clues to howthese communities function. Still, questions remain regarding the diversity, physiology, and metabolic restrictions of AOM-related organisms.     

Factors influencing the national distribution of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in British soils

The polycyclic aromatic hydrocarbons (PAHs) naphthalene, acenaphthylene, acenaphthene, fluorene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, ideno[1,2,3,-cd]-pyrene, dibenz[a,h]anthracene, benzo[g,h,i]perylene and the polychlorinated biphenyls (PCBs) 8, 18, 28, 29, 31, 52, 77, 101, 105, 114, 118, 123, 126, 128, 138, 141, 149, 153, 156, 157, 163, 169, 170, 171, 180, 183, 187, 189, 194, 199, 201, 206, and 209 were measured in -200 rural soils across Great Britain (GB). Dominance of soil PAH profiles by heavier compounds (4-6 rings) provided initial evidence for the importance of source in governing soil PAH concentrations. No relationship was found between soil organic matter (SOM) and sum concentration of total and "heavy" PAHs, although there was a weak positive relationship with lighter compounds. A spatial statistical technique showed that highest soil PAH concentrations were usually found close to industrial/urban centers where presumably source intensity is highest. PCBs clustered into seven groups, five of which contained a single "dioxin like" PCB, one contained lighter congeners (2-4 chlorines), and one contained heavy congeners (5-10 chlorines). Linear regressions with SOM explained up to 24.3% of variation for the sum concentration of penta- to deca- congeners, but <1% for the lighter congener groups. No significant relationships were found with latitude. Spatial statistical techniques showed clusters of high soil PCB concentrations predominantly in west and south east GB, either associated with urbanized areas or on the West coast.     

Effect of addition of sucrose and aspartame on the compression resistance of hydrocolloids gels

The effect of adding sucrose (5–25% w/w) and aspartame (0.04–0.16% w/w) on the compression resistance of three hydrocolloid gelled systems: κ‐carrageenan, gellan gum and κ‐carrageenan/locust bean gum at three different concentrations (0.3, 0.75 and 1.2% w/w) was studied. Sucrose addition increased true rupture stress in the three‐gelled systems, this effect being stronger in gellan gels. The deformability modulus increased with sucrose concentration in gellan gels, but not in the other systems. Rupture stress and deformability modulus increased with the addition of sucrose only in the harder gels (0.75 and 1.2% w/w). The effect of sucrose addition on the true rupture strain was significant but, in general, not important, mainly for lower gum concentrations. Aspartame addition did not affect the compression parameters.     

Ovarian activity in Fleckvieh, Brown Swiss and two strains of Holstein-Friesian cows in pasture-based, seasonal calving dairy systems

The objectives of the study were to compare the ovarian activity of Holstein-Friesian (CH HF), Fleckvieh (CH FV) and Brown Swiss (CH BS) dairy cows of Swiss origin with that of Holstein-Friesian (NZ HF) dairy cows of New Zealand origin, the latter being used as a reference for reproductive performance in pasture-based seasonal calving systems. Fifty, second-lactation NZ HF cows were each paired with a second-lactation Swiss cow (17, 15 and 18 CH HF, CH FV and CH BS respectively) in 13 pasture-based, seasonal-calving commercial dairy farms in Switzerland. Ovarian activity was monitored by progesterone profiling from calving to first breeding service. CH BS cows produced less energy-corrected milk (mean 22·8 kg/d) than the other breeds (26·0-26·5 kg/d) during the first 100 d of lactation. CH HF cows had the lowest body condition score (BCS) at calving and the greatest BCS loss from calving to 30 d post partum. Commencement of luteal activity (CLA) was later for NZ HF than for CH FV (51·5 v. 29·2 d; P <0·01), with CH HF and CH BS intermediate (43 d). On average, NZ HF and CH HF cows had one oestrous cycle before the onset of the seasonal breeding period; this was less (P<0·01) than either CH FV (1·7) or CH BS (1·6). There was a low prevalence of luteal persistency (3%) among the studied cows. First and second oestrous cycle inter-ovulatory intervals did not differ between breeds (20·5-22·6 d). The luteal phase length of CH BS during the second cycle was shorter (10·6 d) than that of the other breeds (13·8-16·0 d), but the inter-luteal interval was longer (9·8 d v. 7·0-8·0 d). The results suggest that the Swiss breeds investigated have a shorter interval from calving to CLA than NZ HF cows.     

Effect of acid deposition on quantity and quality of dissolved organic matter in soil-water

The aim of this study was to explore how acid deposition may affect the concentration and quality of dissolved organic matter (DOM) in soil-water. This was done by a small-scale acidification experiment during two years where 0.5 × 0.5 m(2) plots were artificially irrigated with water with different sulfuric acid content, and soil-water was sampled using zero-tension lysimeters under the O-horizon. The DOM was characterized using absorbance, fluorescence, and size exclusion chromatography analyses. Our results showed lower mobility of DOM in the high acid treatment. At the same time, there was a significant change in the DOM quality. Soil-water in the high acid treatment exhibited DOM that was less colored, less hydrophobic, less aromatic, and of lower molecular weight, compared to the low acid treatment. This supports the hypothesis that reduction in sulfur deposition is an important driver behind the ongoing brownification of surface waters in many regions.     

A novel method for determination of size resolved, submicrometer particle traffic emission factors

A novel approach to determine size-segregated particle number emission factors for traffic is presented. It was proven that using limited data sets (800-2000 samples) statistically significant emission factors from road traffic can be extracted. In this study data from four sites were used for calculating emission factors (rural and urban roadside, urban rooftop, and urban background). The measurements were performed using SMPS/DMPS (scanning or differential particle sizers) from TSI and commercial gas analyzers. Describing the particle concentration as a ratio to an exhaust trace gas, e.g. NOx, the dilution effect will be minimized. This ratio is easily compared among different studies. By knowledge of the emission factor of the chosen trace gas the emission ratio can be converted to an emission factor for particle numbers of defined particle sizes. For the presented method only one measurement site is needed, where the difference between high and low (background) traffic exposure is used. To define high and low traffic exposure, the best result was obtained using high ratio of [NO] to [NO2] and low [NOx], respectively. Emission ratios for 10-100-nm particles at two road sites, one high-speed 90-kmph rural case and one urban, slower, and more congested situation, were determined to (35 +/- 15) x 10(14) and (24 +/- 8) x 10(14) particles per mole NOx, respectively.     

Occurrence of foodborne pathogens in Irish farmhouse cheese

Food safety is a critical factor in the production of farmhouse cheese. In Ireland the varieties of farmhouse cheese produced reflect a much broader range than those produced commercially and some of these cheese varieties are associated with greater microbiological risk. These include cheese produced from unpasteurised milk and soft ripened cheese such as mould or smear-ripened cheeses which have high pH and relatively short ripening times. The aim of this study was to determine the microbiological quality of farmhouse cheeses in Ireland. Three hundred and fifty one cheese samples, from 15 cheese producers, were analysed for microbiological quality on a monthly basis throughout the year. The analyses included enumeration of Escherichia coli, Staphylococcus aureus and Listeria monocytogenes (using the relevant agars) and enrichment for L. monocytogenes. The cheeses selected were produced from ovine, caprine and bovine milk. Both unpasteurised and pasteurised milk cheeses were sampled and these included hard, semi-hard and soft cheeses, internal/external mould-ripened and smear-ripened cheeses and the cheeses represented different geographic regions. Of the cheeses tested, 94% were free of L. monocytogenes, all were within the EU limits for E. coli and only one cheese variety had S. aureus levels above the recommended numbers for the first 6 months of the year. Due to a modified production process the numbers were within the guidelines for the second six months. The results indicate that Irish farmhouse cheeses are of a high microbiological quality.     

Isoflavone determination in spontaneous legumes identified by DNA barcodes

Isoflavones have been associated with several health protective effects. In this work spontaneous legume plants were screened as putative sources of dietary isoflavones. A molecular identification of the collected species was performed throughout DNA barcoding using ITS, rbcL, rpoC1 and matK sequences. The use of a multi-locus barcoding system complemented with basic morphological information allowed the unequivocal identification at the species level of 90% of the samples. The determination of isoflavone content was performed by high-performance liquid chromatography with diode-array detection. Total average contents in the studied species were significantly different, Ononis natrix and Cytisus scoparius possessing the highest total isoflavones content (396 and 273 mg kg⁻¹, respectively) and Lotus creticus, the lowest (20 mg kg⁻¹). The correlation of total isoflavone content with the phylogeny of this set of plants as determined by the rpoC1 sequences was evaluated for the first time.     

Cold atmospheric pressure plasma treatment of ready-to-eat meat: inactivation of Listeria innocua and changes in product quality

The application of cold atmospheric pressure plasma for decontamination of a sliced ready-to-eat (RTE) meat product (bresaola) inoculated with Listeria innocua was investigated. Inoculated samples were treated at 15.5, 31, and 62 W for 2–60 s inside sealed linear-low-density-polyethylene bags containing 30% oxygen and 70% argon. Treatments resulted in a reduction of L. innocua ranging from 0.8 ± 0.4 to 1.6 ± 0.5 log cfu/g with no significant effects of time and intensity while multiple treatments at 15.5 and 62 W of 20 s with a 10 min interval increased reduction of L. innocua with increasing number of treatments. Concentrations of thiobarbituric acid reactive substances (TBARS) increased with power, treatments and storage time and were significantly higher than those of control samples after 1 and 14 days of storage at 5 °C. However, the levels were low (from 0.1 to 0.4 mg/kg) and beneath the sensory threshold level. Surface colour changes included loss of redness of ∼40% and 70% after 1 and 14 days of storage, respectively, regardless of plasma treatment. The results indicate that plasma may be applicable in surface decontamination of pre-packed RTE food products. However, oxidation may constitute an issue in some products.