ON-rep-seq as a rapid and cost-effective alternative to whole-genome sequencing for species-level identification and strain-level discrimination of Listeria monocytogenes contamination in a salmon processing plant.

Identification, source tracking, and surveillance of food pathogens are crucial factors for the food-producing industry. Over the last decade, the techniques used for this have moved from conventional enrichment methods, through species-specific detection by PCR to sequencing-based methods, whole-genome sequencing (WGS) being the ultimate method. However, using WGS requires the right infrastructure, high computational power, and bioinformatics expertise. Therefore, there is a need for faster, more cost-effective, and more user-friendly methods. A newly developed method, ON-rep-seq, combines the classical rep-PCR method with nanopore sequencing, resulting in a highly discriminating set of sequences that can be used for species identification and also strain discrimination. This study is essentially a real industry case from a salmon processing plant. Twenty Listeria monocytogenes isolates were analyzed both by ON-rep-seq and WGS to identify and differentiate putative L. monocytogenes from a routine sampling of processing equipment and products, and finally, compare the strain-level discriminatory power of ON-rep-seq to different analyzing levels delivered from the WGS data. The analyses revealed that among the isolates tested there were three different strains. The isolates of the most frequently detected strain (n = 15) were all detected in the problematic area in the processing plant. The strain level discrimination done by ON-rep-seq was in full accordance with the interpretation of WGS data. Our findings also demonstrate that ON-rep-seq may serve as a primary screening method alternative to WGS for identification and strain-level differentiation for surveillance of potential pathogens in a food-producing environment.

Assessment of the bioprotective potential of lactic acid bacteria against Listeria monocytogenes on vacuum-packed cold-smoked salmon stored at 8 °C.

Smoked salmon is a highly appreciated delicatessen product. Nevertheless, this ready-to-eat (RTE) product is considered at risk for Listeria monocytogenes, due to both the prevalence and growth potential of this bacteria on the product. Biopreservation may be considered a mild and natural effective strategy for minimizing this risk. In this study, we evaluated the following three potential bioprotective lactic acid bacterial strains against L. monocytogenes in three smoked salmon types with different physicochemical characteristics, primarily fat, moisture, phenol and acid acetic content: two bacteriocin-like producers that were isolated from smoked salmon and identified as Lactobacillus curvatus and Carnobacterium maltaromaticum and a recognized bioprotective bacteriocin producer from meat origin, Lactobacillus sakei CTC494. L. sakei CTC494 inhibited the growth of L. monocytogenes after 21 days of storage at 8 °C in all the products tested, whereas L. curvatus CTC1742 only limited the growth of the pathogen (<2 log increase). The effectiveness of C. maltaromaticum CTC1741 was dependent on the product type; this strain limited the growth of the pathogen in only one smoked salmon type. These results suggest that the meat-borne starter culture, L. sakei CTC494, may potentially be used as a bioprotective culture to improve the food safety of cold-smoked salmon.

Listeria monocytogenes risk assessment on cold smoked and salt-cured fishery products in Finland - A repeated exposure model.

Listeria monocytogenes causes severe consequences especially for persons belonging to risk groups. Finland is among the countries with highest number of listeriosis cases in the European Union. Although most reported cases appear to be sporadic and the maximum bacterial concentration of 100 cfu/g is not usually exceeded at retail, cold smoked and salt-cured fish products have been noted as those products with great risk especially for the elderly. In order to investigate the listeriosis risk more carefully, an exposure assessment was developed, and laboratory results for cold smoked and salt-cured salmon products were exploited. L. monocytogenes exposure was modeled for consumers in two age groups, the elderly population as a risk group and the working-age population as a reference. Incidence was assessed by estimating bacterial growth in the food products at three temperatures. Bayesian estimation of the risk was based on bacterial occurrence and product consumption data and epidemiological population data. The model builds on a two-state Markov chain describing repeated consumption on consecutive days. The cumulative exposure is probabilistically governed by the daily decreasing likelihood of continued consumption and the increasing bacterial concentrations due to growth. The population risk was then predicted with a Poisson distribution accounting for the daily probabilities of purchasing a contaminated product and the cumulative total probability of infection from its use. According to the model presented in this article, elderly Finns are at a greater risk of acquiring listeriosis than healthy adults. The risk for the elderly does not fully diminish even if the products have been stored at the recommended temperature (between 0 and 3 °C). It can be concluded that the stage after retail, i.e. food handling and storage by consumer or professional kitchens, is essential to protection against listeriosis. The estimation model provides means for assessing the joint impacts of these effects.

Optimization of combinations of bactericidal and bacteriostatic treatments to control Listeria monocytogenes on cold-smoked salmon.

Contamination of cold-smoked salmon by Listeria monocytogenes is a major concern for the seafood industry. The objectives of this study were to (i) determine the most effective bactericidal treatment for L. monocytogenes on salmon and (ii) optimize bactericidal and bacteriostatic treatment combinations to identify cost-effective treatments against L. monocytogenes on salmon. L. monocytogenes challenge trials were conducted in brain heart infusion (BHI) and on salmon disks that were supplemented with bactericidal compounds nisin (NIS), lauric arginate (LAE), ε-polylysine (EPL), and chitosan (CHIT). Subsequently, the most effective bactericidal compound was further tested by concurrent application of a blend of organic acid salts containing potassium lactate and sodium diacetate (PLSDA). L. monocytogenes populations were measured at 7 °C over 60 days, and initial cell density (N0), maximum initial log reduction (Nr), lag phase (λ), maximum growth rate (µmax), and maximum cell density (Nmax) over 60 days storage were estimated. Time to recover to initial cell density (Tinitial) was also compared for combinations of bactericidal and bacteriostatic treatments. Varying degrees of antimicrobial effects were observed with bactericidal compounds in BHI. However, when tested on salmon, only NIS significantly decreased initial L. monocytogenes populations by approximately 2 log CFU/g, and reduced Nmax by approximately 1.5 logCFU/g compared to untreated control (CTRL). Nr achieved by the combined treatment of NIS and PLSDA was approximately 2 log CFU/g regardless of the presence of PLSDA, and a dose-dependent increase in Nr was observed with increasing NIS concentrations. PLSDA alone or in combination with 20 ppm NIS was most effective at delaying growth of L. monocytogenes. The greatest reduction in Nmax was observed with the combination of 20 ppm NIS and PLSDA; Nmax was 3.1 log CFU/g lower compared to CTRL. Comparison of Tinitial indicated that PLSDA with NIS can effectively retard growth of L. monocytogenes to its initial level (following initial reduction) and offers a cost benefit over using high concentrations of NIS alone. In summary, the combined application of NIS (for a bactericidal effect) and PLSDA (for a bacteriostatic effect) proved to be an effective treatment option to reduce initial levels as well as minimize subsequent growth of L. monocytogenes throughout the expected shelf-life of cold-smoked salmon.

The combination of lactate and diacetate synergistically reduces cold growth in brain heart infusion broth across Listeria monocytogenes lineages.

Combinations of organic acids are often used in ready-to-eat foods to control the growth of Listeria monocytogenes during refrigerated storage. The purpose of this study was to quantitatively assess synergy between two organic acid growth inhibitors under conditions similar to those present in cold-smoked salmon, and to assess the effect of evolutionary lineage on response to those growth inhibitors. Thirteen strains of L. monocytogenes, representing lineages I and II, were grown at 7 degrees C in broth at pH 6.1 and 4.65% water-phase NaCl, which was supplemented with 2% potassium lactate, 0.14% sodium diacetate, or the combination of both at the same levels. Our data suggest that lineages adapt similarly to these inhibitors, as the only significant growth parameter difference between lineages was a minor effect (+/- 0.16 day, P = 0.0499) on lag phase (lambda). For all strains, lactate significantly extended lambda, from 2.6 +/- 0.4 to 3.8 +/- 0.5 days (P < 0.001), and lowered the maximum growth rate (mu(max)) from 0.54 +/- 0.06 to 0.49 +/- 0.04 log(CFU/ml)/day (P < 0.001), compared with the control. Diacetate was ineffective alone, but in combination with lactate, synergistically increased lambda to 6.6 +/- 1.6 days (P < 0.001) and decreased mu(max) to 0.34 +/- 0.05 log(CFU/ml)/day (P < 0.001). Monte Carlo simulations provided further evidence for synergy between diacetate and lactate by predicting signficantly slower growth to nominal endpoints for the combination of inhibitors. This study shows potassium lactate and sodium diacetate have significant synergistic effects on both lambda and mu(max) of L. monocytogenes at refrigeration temperature in broth, and justifies combining these inhibitors, at effective levels, in food product formulations.

Global sensitivity analysis applied to a contamination assessment model of listeria monocytogenes in cold smoked salmon at consumption.

In this study, a variance-based global sensitivity analysis method was first applied to a contamination assessment model of Listeria monocytogenes in cold smoked vacuum packed salmon at consumption. The impact of the choice of the modeling approach (populational or cellular) of the primary and secondary models as well as the effect of their associated input factors on the final contamination level was investigated. Results provided a subset of important factors, including the food water activity, its storage temperature, and duration in the domestic refrigerator. A refined sensitivity analysis was then performed to rank the important factors, tested over narrower ranges of variation corresponding to their current distributions, using three techniques: ANOVA, Spearman correlation coefficient, and partial least squares regression. Finally, the refined sensitivity analysis was used to rank the important factors.

Assessment of the microbial contribution to the processing of salted salmon roe (Sujiko).

As the microbial contributions to the processing of salted foods have been little investigated, there remains a possibility that excess sterilization of raw materials for salted foods leads to deterioration in food quality and safety. At a salmon roe (sujiko) processing company, we investigated salted sujiko made identically to commercial products, but that had been processed with or without antibiotics. The antibiotics caused no significant difference in the content of free amino acids, lactic acid or acetic acid. These results show that general aerobic bacteria have no impact on the formation of these flavor compounds.

Quantitative risk assessment of Listeria monocytogenes in French cold-smoked salmon: I. Quantitative exposure assessment.

A quantitative assessment of the exposure to Listeria monocytogenes from cold-smoked salmon (CSS) consumption in France is developed. The general framework is a second-order (or two-dimensional) Monte Carlo simulation, which characterizes the uncertainty and variability of the exposure estimate. The model takes into account the competitive bacterial growth between L. monocytogenes and the background competitive flora from the end of the production line to the consumer phase. An original algorithm is proposed to integrate this growth in conditions of varying temperature. As part of a more general project led by the French Food Safety Agency (Afssa), specific data were acquired and modeled for this quantitative exposure assessment model, particularly time-temperature profiles, prevalence data, and contamination-level data. The sensitivity analysis points out the main influence of the mean temperature in household refrigerators and the prevalence of contaminated CSS on the exposure level. The outputs of this model can be used as inputs for further risk assessment.

Use of Bayesian modelling in risk assessment: application to growth of Listeria monocytogenes and food flora in cold-smoked salmon.

An attempt to use a Bayesian approach to model variability and uncertainty separately in microbial growth in a risk assessment is presented. It was conducted within the framework of a French project aiming at assessing the exposure to Listeria monocytogenes in cold-smoked salmon. The chosen model describes the effect of time and temperature on bacterial growth. A Bayesian approach close to the one proposed by Pouillot et al. [Int. J. Food Microbiol. 81 (2003) 87] is used to estimate the variability and uncertainty of growth parameters from both literature data and data experimentally acquired during the project. Variability between strains and between products is taken into account. The growth of the food flora of cold-smoked salmon is also modelled by the same method. The results obtained for both models are used to predict the simultaneous growth of L. monocytogenes and food flora in cold-smoked salmon with a competitive model, expressing variability and uncertainty through a second-order Monte Carlo simulation.

Quantitative risk assessment for Listeria monocytogenes in smoked or gravad salmon and rainbow trout in Sweden.

The objective of the present work was to develop a quantitative risk assessment model in which the exposure and risk of acquiring listeriosis from consumption of packaged smoked or gravad salmon and rainbow trout were estimated. An Excel spreadsheet model was constructed in which variables were represented by distributions based on surveys of L. monocytogenes in these food products, and on demographic and consumption data. Growth or inactivation was not included in the model. The model was run through Monte Carlo simulations using the @Risk software (Palisade Corporation). The probability of illness per serving was calculated using two dose-response models from the literature. The first was an exponential model in which the species specific constant R, that helps define the dose-response curve, previously has been estimated to be 1.18 x 10(-10) based on German data (GR). In this study, R was estimated to 5.6 x 10(-10) based on Swedish data. The second model was a flexible Weibull-Gamma model (WG), with different coefficients for high- and low-risk groups. The exponential model (GR), although conservative and generally overestimating the risk, still predicted a lower probability of illness than the WG-model. The estimated mean risk per serving was 2.8 x 10(-5) (GR, high-risk group), 2.0 x 10(-3) (WG, low-risk group) and 0.016 (WG, high-risk group), respectively. The average number of reported listeriosis cases in Sweden is 37 per year. In comparison, the mean number of annual cases predicted by the risk assessment model was 168 (range 47 to 2800, GR, high-risk group), and 95 000 (range 34 000 to 1.6 x 10(6), WG high-risk group), respectively. If 1 to 10% (uniform distribution) of strains, instead of all, were considered virulent, the mean number of predicted cases would decrease to nine (GR) and 5200 (WG), respectively. The mean annual cumulative individual risk in the high-risk group based on a monthly exposure was estimated to be 4.0 x 10(-4) (range 8.0 x 10(-8) to 5.4 x 10(-3), GR). This risk increased to 1.5 x 10(-3) (range 1.7 x 10(-5) to 9.2 x 10(-3), GR) based on a weekly exposure. The risk assessment model was most sensitive to the input distribution describing the level of contamination and to a lesser degree on the prevalence of L. monocytogenes, the proportion of virulent strains, and serving sizes. A lack of data on the prevalence and concentration of L. monocytogenes in these products, dose-response data and quantitative information on the proportion of virulent strains were identified.

Import risk assessment for salmon meat.

The authors discuss the risk assessment currently being conducted by the Australian Quarantine and Inspection Service (AQIS) on the importation of salmon products. AQIS conducted a public consultation on the proposal, in line with Australian Government policy on transparency and accountability in the quarantine decision-making process. The authors examine the factors which should be taken into account in the assessment of the risk associated with the importation of such products, and note the difficulties encountered with the epidemiology of fish diseases.

Assessment of the antimicrobial sensitivity of Aeromonas salmonicida isolates from farmed Atlantic salmon in Scotland.

Sixty-five isolates of Aeromonas salmonicida were assessed for antimicrobial sensitivity by disc diffusion tests and the results compared with the minimum inhibitory concentration for each isolate. The results demonstrated that disc diffusion, using a standard technique, may be used to categorize isolates as sensitive or resistant provided that the corresponding minimum inhibitory concentrations are known.