Evolution of Listeria monocytogenes in a Food Processing Plant Involves Limited Single-Nucleotide Substitutions but Considerable Diversification by Gain and Loss of Prophages.

Whole-genome sequencing (WGS) is becoming the standard method for subtyping Listeria monocytogenes Interpretation of WGS data for isolates from foods and associated environments is, however, challenging due to a lack of detailed data on Listeria evolution in processing facilities. Here, we used previously collected WGS data for 40 L. monocytogenes isolates obtained from a cold-smoked salmon processing facility between 1998 and 2015 to probe the L. monocytogenes molecular evolution in this facility, combined with phenotypic assessment of selected isolates. Isolates represented three clusters (1, 2, and 3); cluster 3 isolates (n = 32) were obtained over 18 years. The average mutation rate for cluster 3 was estimated as 1.15 × 10-7 changes per nucleotide per year (∼0.35 changes per genome per year); the most recent common ancestors (MRCAs) of subclusters 3a and 3b were estimated to have occurred around 1958 and 1974, respectively, within the age of the facility, suggesting long-term persistence in this facility. Extensive prophage diversity was observed within subclusters 3a and 3b, which have one shared and six unique prophage profiles for each subcluster (with 16 prophage profiles found among all 40 isolates). The plasmid-borne sanitizer tolerance operon bcrABC was found in all cluster 2 and 3 isolates, while the transposon-borne sanitizer tolerance gene qacH was found in one cluster 1 isolate; presence of these genes was correlated with the ability to survive increased concentrations of sanitizers. Selected isolates showed significant variation in the ability to attach to surfaces, with persistent isolates attaching better than transient isolates at 21°C.IMPORTANCE Knowledge about the genetic evolution of L. monocytogenes in food processing facilities over multiple years is generally lacking. This information is critical to interpret WGS findings involving food or food-associated isolates. This study suggests that L. monocytogenes that persists in processing facilities may evolve with a low single-nucleotide mutation rate mostly driven by negative (i.e., purifying) selection but with rapid diversification of prophages. Hence, isolation of L. monocytogenes with few single-nucleotide polymorphism (SNP) differences in different locations (e.g., supplier plants and receiving plants) is possible, highlighting the importance of epidemiological and detailed isolate metadata for interpreting WGS data in traceback investigation. Our study also shows how advanced WGS data analyses can be used to support root cause analysis efforts and may, for example, pinpoint the time when a persistence event started (which then potentially could be linked to facility changes, introduction of new equipment, etc.).

Implications of Mobile Genetic Elements for Salmonella enterica Single-Nucleotide Polymorphism Subtyping and Source Tracking Investigations.

Single-nucleotide polymorphisms (SNPs) are widely used for whole-genome sequencing (WGS)-based subtyping of foodborne pathogens in outbreak and source tracking investigations. Mobile genetic elements (MGEs) are commonly present in bacterial genomes and may affect SNP subtyping results if their evolutionary history and dynamics differ from that of the bacterial chromosomes. Using Salmonella enterica as a model organism, we surveyed major categories of MGEs, including plasmids, phages, insertion sequences, integrons, and integrative and conjugative elements (ICEs), in 990 genomes representing 21 major serotypes of S. enterica We evaluated whether plasmids and chromosomal MGEs affect SNP subtyping with 9 outbreak clusters of different serotypes found in the United States in 2018. The median total length of chromosomal MGEs accounted for 2.5% of a typical S. enterica chromosome. Of the 990 analyzed S. enterica isolates, 68.9% contained at least one assembled plasmid sequence. The median total length of assembled plasmids in these isolates was 93,671 bp. Plasmids that carry high densities of SNPs were found to substantially affect both SNP phylogenies and SNP distances among closely related isolates if they were present in the reference genome for SNP subtyping. In comparison, chromosomal MGEs were found to have limited impact on SNP subtyping. We recommend the identification of plasmid sequences in the reference genome and the exclusion of plasmid-borne SNPs from SNP subtyping analysis.IMPORTANCE Despite increasingly routine use of WGS and SNP subtyping in outbreak and source tracking investigations, whether and how MGEs affect SNP subtyping has not been thoroughly investigated. Besides chromosomal MGEs, plasmids are frequently entangled in draft genome assemblies and yet to be assessed for their impact on SNP subtyping. This study provides evidence-based guidance on the treatment of MGEs in SNP analysis for Salmonella to infer phylogenetic relationship and SNP distance between isolates.

Broad-Spectrum Supramolecularly Reloadable Antimicrobial Coatings.

Antimicrobial surfaces limit the spread of infectious diseases. To date, there is no antimicrobial coating that has widespread use because of short-lived and limited spectrum efficacy, poor resistance to organic material, and/or cost. Here, we present a paint based on waterborne latex particles that is supramolecularly associated with quaternary ammonium compounds (QACs). The optimal supramolecular pairing was first determined by immobilizing selected ions on self-assembled monolayers exposing different groups. The QAC surface loading density was then increased by using polymer brushes. These concepts were adopted to develop inexpensive paints to be applied on many different surfaces. The paint could be employed for healthcare and food production applications. Its slow release of QAC allows for long-lasting antimicrobial action, even in the presence of organic material. Its efficacy lasts for more than 90 washes, and importantly, once lost, it can readily be restored by spraying an aqueous solution of the QAC. We mainly tested cetyltrimethylammonium as QAC as it is already used in consumer care products. Our antimicrobial paint is broad spectrum as it showed excellent antimicrobial efficiency against four bacteria and four viruses.

Inactivation of food-borne viruses using natural biochemical substances.

Food-borne viruses such as human Noroviruses (NoVs), hepatitis A virus (HAV), Rotaviruses (RoVs) are a public health concern worldwide. Biochemical substances, which occur naturally in plants, animals or microorganisms, might possess considerable antimicrobial properties. In this study, the reported effects of biochemical substances on food-borne viruses are reviewed. The biochemical substances are grouped into several categories including (i) polyphenols and proanthocyanins, (ii) saponin, (iii) polysaccharides, (iv) organic acids, (v) proteins and polypeptides, (vi) essential oils. Although not fully understood, the mechanism of action for the antiviral activity of the natural compounds is presented. Generally, it is thought to be the prevention of the viral attachment to host cells, either by causing damage on the viral capsids or change of the receptors on the cell membranes. It is recommended that further studies are undertaken not only on the wide-range screening for novel antiviral substances, but also on the mechanism in-depth as well as the exploration for their potential application in controlling virus contamination in foods or food processing.

Effect of grape seed extract on human norovirus GII.4 and murine norovirus 1 in viral suspensions, on stainless steel discs, and in lettuce wash water.

The anti-norovirus (anti-NoV) effect of grape seed extract (GSE) was examined by plaque assay for murine norovirus 1 (MNV-1), cell-binding reverse transcription-PCR for human NoV GII.4, and saliva-binding enzyme-linked immunosorbent assay for human NoV GII.4 P particles, with or without the presence of interfering substances (dried milk and lettuce extract). GSE at 0.2 and 2 mg/ml was shown to reduce the infectivity of MNV-1 (>3-log PFU/ml) and the specific binding ability of NoV GII.4 to Caco-2 cells (>1-log genomic copies/ml), as well as of its P particles to salivary human histo-blood group antigen receptors (optical density at 450 nm of >0.8). These effects were decreased as increasing concentrations of dried milk (0.02 and 0.2%) or lettuce extract were added. Under an electron microscope, human NoV GII.4 virus-like particles showed inflation and deformation after treatment with GSE. Under conditions that simulated applications in the food industry, the anti-NoV effect of GSE using MNV-1 as a target organism was shown to be limited in surface disinfection (<1-log PFU/ml, analyzed in accordance with EN 13697:2001). However, a 1.5- to 2-log PFU/ml reduction in MNV-1 infectivity was noted when 2 mg of GSE/ml was used to sanitize water in the washing bath of fresh-cut lettuce, and this occurred regardless of the chemical oxygen demand (0 to 1,500 mg/ml) of the processing water.

Inactivation of murine norovirus 1, coliphage phiX174, and Bacteroides [corrected] fragilis phage B40-8 on surfaces and fresh-cut iceberg lettuce by hydrogen peroxide and UV light.

In this study, the inactivating properties of liquid hydrogen peroxide (L-H(2)O(2)), vaporized hydrogen peroxide (V-H(2)O(2)), UV light, and a combination of V-H(2)O(2) and UV light were tested on murine norovirus 1 (MNV-1) and bacteriophages (φX174 and B40-8) as models for human noroviruses. Disinfection of surfaces was examined on stainless steel discs based on European Standard EN 13697 (2001). For fresh-produce decontamination, a mixture of the viruses was inoculated onto shredded iceberg lettuce and treated after overnight incubation at 2°C. According to our results, L-H(2)O(2) (2.1%) was able to inactivate MNV-1 and φX174 on stainless steel discs by approximately 4 log(10) units within 10 min of exposure, whereas for B40-8, 15% of L-H(2)O(2) was needed to obtain a similar reduction in 10 min. Only a marginal reduction (≤1 log(10) unit after 5 min of exposure) by V-H(2)O(2) (2.52%) was achieved for the tested model viruses, although in combination with UV light, a 4-log(10)-unit decrease within 5 min of treatment was observed on stainless steel discs. Similar trends were observed for the decontamination of shredded iceberg lettuce, but the viral decline was reduced. These results demonstrated that both L-H(2)O(2) and a combination of V-H(2)O(2) and UV light can be used for norovirus inactivation on surfaces; V-H(2)O(2) (2.52%) in combination with UV light is promising for decontamination of fresh produce with much less consumption of water and disinfectant.

Evaluation of a norovirus detection methodology for soft red fruits.

In the present study, a proposed methodology for detection of GI and GII noroviruses (NoV) in soft red fruits was evaluated. The murine norovirus-1 (MNV-1), a recently described cultivable NoV surrogate was integrated in the detection methodology as full process control, reverse transcription control and real-time PCR internal amplification control. Both the performance and robustness of the proposed methodology were analyzed. Firstly, the performance of the method was examined by analysis of the recovery of MNV-1, GI and/or GII NoV inoculated on frozen raspberry crum samples. Results showed that the recovery of MNV-1 was not significantly influenced by the inoculum incubation time (30 min or overnight incubation) or the inoculum level (10(6) or 10(8) genomic MNV-1 copies/10 g of frozen raspberry crum sample). In contrast, a significant influence of the GI and GII NoV inoculum level (10(4) or 10(6) genomic MNV-1 copies/10 g of frozen raspberry crum sample) was noticed on the recovery of respectively GI and GII NoV from frozen raspberry crum samples. Secondly, the robustness of the methodology was evaluated by subjecting three types of artificially MNV-1, GI and/or GII NoV contaminated soft red fruit products (deepfrozen forest fruit mix, fresh raspberries and fresh strawberry puree) to the method. Results showed a significant influence of the soft red fruit product type on the recovery efficiency of GI NoV and MNV-1, while no significant differences could be shown for GII NoV. In general, the recovery of GI and GII NoV in strawberry puree was more efficient from the strawberry puree compared to the two other soft red fruit types. In conclusion, results show that this methodology can be used for detection of NoV in different soft red fruits, although NoV recovery efficiencies can be influenced by (1) the NoV concentration on the soft red fruit type and (2) the tested soft red fruit type.

Genetic changes are introduced by repeated exposure of Salmonella spiked in low water activity and high fat matrix to heat.

WGS is used to define if isolates are "in" or "out" of an outbreak and/or microbial root cause investigation. No threshold of genetic differences is fixed and the conclusions on similarity between isolates are mainly based on the knowledge generated from previous outbreak investigations and reported mutation rates. Mutation rates in Salmonella when exposed to food processing conditions are lacking. Thus, in this study, the ability of heat and dry stress to cause genetic changes in two Salmonella serotypes frequently isolated from low moisture foods was investigated. S. enterica serovars S. Agona ATCC 51,957 and S. Mbandaka NCTC 7892 (ATCC 51,958) were repeatedly exposed to heat (90 °C for 5 min) in a low water activity and high fat matrix. No increased fitness of the strains was observed after 10 repeated heat treatments. However, genetic changes were introduced and the number of genetic differences increased with every heat treatment cycle. The genetic changes appeared randomly in the genome and were responsible for a population of diverse isolates with 0 to 28 allelic differences (0 to 38 SNPs) between them. This knowledge is key to interpret WGS results for source tracking investigations as part of a root cause analysis in a contamination event as isolates are exposed to stress conditions.

Identification of Closely Related Listeria monocytogenes Isolates with No Apparent Evidence for a Common Source or Location: A Retrospective Whole Genome Sequencing Analysis.

ABSTRACT: Public health and regulatory agencies worldwide sequence all Listeria monocytogenes isolates obtained as part of routine surveillance and outbreak investigations. Many of these entities submit the sequences to the National Center for Biotechnology Information Pathogen Detection (NCBI PD) database, which groups the L. monocytogenes isolates into single nucleotide polymorphism (SNP) clusters based on a pairwise SNP difference threshold of 50 SNPs. Our goal was to assess whether isolates with metadata that suggest different sources or locations could show evidence for close genetic relatedness indicating a recent common ancestor and a possible unknown common source. We compared the whole genome sequencing (WGS) data of 249 L. monocytogenes isolates sequenced here, which have detailed metadata, with WGS data of nonclinical isolates on NCBI PD. The 249 L. monocytogenes isolates originated from natural environments (n = 91) as well as from smoked fish (n = 62), dairy (n = 56), and deli meat (n = 40) operations in the United States. Using a combination of subtyping by core genome multilocus sequence typing and high-quality SNP, we observed five SNP clusters in which study isolates and SNP cluster isolates seemed to be closely related and either (i) shared the same geolocation but showed different source types (one SNP cluster); (ii) shared the same source type but showed different geolocations (two SNP clusters); or (iii) shared neither source type nor geolocation (two SNP clusters). For one of the two clusters under (iii), there was, however, no strong bootstrap support for a common ancestor shared between the study isolates and SNP cluster isolates, indicating the value of in-depth evolutionary analyses when WGS data are used for traceback and epidemiological investigations. Overall, our results demonstrate that some L. monocytogenes subtypes may be associated with specific locations or commodities; these associations can help in investigations involving multi-ingredient foods such as sandwiches. However, at least some L. monocytogenes subtypes can be widespread geographically and can be associated with different sources, which may present a challenge to traceback investigations involving these subtypes.

The effect of pH on the growth rate of Bacillus cereus sensu lato: Quantifying strain variability and modelling the combined effects of temperature and pH.

In this study, the effect of pH, alone or in combination with temperature, on the maximum growth rate (µmax) of B. cereus sensu lato was investigated. In phase 1, the effect of pH at 30 °C was studied for 16 mesophilic strains and 2 psychrotrophic strains of Bacillus cereus sensu lato. The µmax vs. pH relationship was found to show a similar pattern for all the strains. Several pH models from literature were evaluated and the best performing 'growth rate vs. pH' model selected. A stochastic model was then developed to predict the maximum specific growth rate of mesophilic B. cereus at 30 °C as a function of pH, the intra-species variability being incorporated via considering the model parameters (e.g. pHmin) randomly distributed. The predicted maximum specific growth rates were acceptably close to independent published data. In phase 2, the combined effects of temperature and pH were studied. Growth rates were also generated at 15, 20 and 40 °C for a selection of strains and the pH model was fitted at each temperature. Interestingly, the results showed that the estimates for the pHmin parameter for mesophilic strains were lower at 20-30 °C than near the optimum temperature (40 °C), suggesting that experiments for the determination of this parameter should be conducted at lower-than-optimum temperatures. New equations were proposed for the relationship between temperature and the minimum pH-values, which were also consistent with the experimental growth boundaries. The parameters defining this equation quantify the minimum temperature for growth observed experimentally, the temperature of maximum enzyme stability and the maximum temperature for growth. Deviations from the Gamma hypothesis (multiplicative effects of environmental factors on the maximum specific growth rate) were observed near the growth limits, especially at 40 °C. To improve model performance, two approaches, one based on a minimum pH-term (doi: https://doi.org/10.3389/fmicb.2019.01510) and one based on an interaction term (doi: http://dx.doi.org/10.1016/S0168-1605(01)00640-7) were evaluated.

Inactivation of murine norovirus 1 and Bacteroides fragilis phage B40-8 by mesophilic and thermophilic anaerobic digestion of pig slurry.

Mesophilic (37 degrees C) and thermophilic (52 degrees C) anaerobic digestion of pig slurry induced at least a 4-log decrease in murine norovirus 1, used as a surrogate virus for porcine norovirus, after 13 and 7 days, respectively. Bacteroides fragilis phage B40-8, employed as a universal viral model, was lowered by 2.5 log after 7 days. The viral titer declined due to temperature and matrix effects.

Biogenic silver for disinfection of water contaminated with viruses.

The presence of enteric viruses in drinking water is a potential health risk. Growing interest has arisen in nanometals for water disinfection, in particular the use of silver-based nanotechnology. In this study, Lactobacillus fermentum served as a reducing agent and bacterial carrier matrix for zerovalent silver nanoparticles, referred to as biogenic Ag(0). The antiviral action of biogenic Ag(0) was examined in water spiked with an Enterobacter aerogenes-infecting bacteriophage (UZ1). Addition of 5.4 mg liter(-1) biogenic Ag(0) caused a 4.0-log decrease of the phage after 1 h, whereas the use of chemically produced silver nanoparticles (nAg(0)) showed no inactivation within the same time frame. A control experiment with 5.4 mg liter(-1) ionic Ag+ resulted in a similar inactivation after 5 h only. The antiviral properties of biogenic Ag(0) were also demonstrated on the murine norovirus 1 (MNV-1), a model organism for human noroviruses. Biogenic Ag(0) was applied to an electropositive cartridge filter (NanoCeram) to evaluate its capacity for continuous disinfection. Addition of 31.25 mg biogenic Ag(0) m(-2) on the filter (135 mg biogenic Ag(0) kg(-1) filter medium) caused a 3.8-log decline of the virus. In contrast, only a 1.5-log decrease could be obtained with the original filter. This is the first report to demonstrate the antiviral efficacy of extracellular biogenic Ag(0) and its promising opportunities for continuous water disinfection.

Comparison of enrichment conditions for rapid detection of low numbers of sublethally injured Escherichia coli O157 in food.

A comparative study of lag phases and growth rates of healthy, stressed, and sublethally injured Escherichia coli O157 cells in 10 enrichment broths was performed. The evaluation of enrichment protocols was validated by different end point detection methods (two PCR and two combined capture-plate methods). Tryptic soy broth b [TSB (b)] provided the fastest growth (max = 1.00 1 0.06 h- ) but failed to recover oxidative-stressed E. coli O157. TSB (a), TSB-yeast extract medium, TSB supplemented with 8 mg/liter novobiocin plus 16 mg/liter vancomycin (TSB+), buffered peptone water (BPW), and BPW supplemented with 8 mg/liter vancomycin (BPW+V) enabled resuscitation of E. coli O157 cells independent from precultural conditions. Modified TSB plus 10 mg/liter novobiocin (mTSB+N), EC medium, EC reduced bile salts medium (ECred), TSB (b), and TSB supplemented with 8 mg/liter novobiocin plus 16 mg/liter vancomycin plus 2 mg/liter rifampin plus 1 mg/liter K-Telluriet plus 1.5 g/liter bile salts no. 3 (TSB++) all failed to recover E. coli O157 cells for at least one type of stress. The use of TSB (a), TSB+, BPW, and BPW+V was compared with that of mTSB+N (International Organization for Standardization reference broth) for reliable detection of low numbers of healthy, stressed, and sublethally injured E. coli O157 (approximately 10 CFU/10 g) from foods (sprouted seeds, fermented sausage, raw milk, and raw ground beef). When low numbers of healthy cells were inoculated, BPW, BPW+V, TSB, TSB+, and mTSB+N enabled growth until detectable numbers within 6 h of enrichment at 41.5 degrees C. Results showed that mTSB+N failed to recover to detectable numbers E. coli O157 cells sublethally injured by freeze and food stresses, in contrast to what was obtained with BPW and BPW+V. This study highlights that using mTSB+N for recovery of E. coli O157 from foods may yield false-negative results.

Efficacy of sodium hypochlorite and peroxyacetic acid to reduce murine norovirus 1, B40-8, Listeria monocytogenes, and Escherichia coli O157:H7 on shredded iceberg lettuce and in residual wash water.

The efficiency of sodium hypochlorite (NaOCl) and peroxyacetic acid (PAA) to reduce murine norovirus 1 (MNV-1), a surrogate for human norovirus, and Bacteroides fragilis HSP40-infecting phage B40-8 on shredded iceberg lettuce was investigated. The levels of removal of viruses MNV-1 and B40-8 were compared with the reductions observed for bacterial pathogens Listeria monocytogenes and Escherichia coli O157:H7. Two inoculation levels, one with a high organic load and the other containing a 10-fold lower number of pathogens and organic matter, showed that the effectiveness of NaOCl was greatly influenced by the presence of organic material, which was not observed for PAA. Moreover, the present study showed that 200 mg/liter NaOCl or 250 mg/liter PAA is needed to obtain an additional reduction of 1 log (compared with tap water) of MNV-1 on shredded iceberg lettuce, whereas only 250 mg/liter PAA achieved this for bacterial pathogens. None of the treatments resulted in a supplementary 1-log PFU/g reduction of B40-8 compared with tap water. B40-8 could therefore be useful as an indicator of decontamination processes of shredded iceberg lettuce based on NaOCl or PAA. Neither MNV-1, B40-8, nor bacterial pathogens could be detected in residual wash water after shredded iceberg lettuce was treated with NaOCl and PAA, whereas considerable numbers of all these microorganisms were found in residual wash water consisting solely of tap water. This study illustrates the usefulness of PAA and NaOCl in preventing cross-contamination during the washing process rather than in causing a reduction of the number of pathogens present on lettuce.

Comparative Analysis of Tools and Approaches for Source Tracking Listeria monocytogenes in a Food Facility Using Whole-Genome Sequence Data.

As WGS is increasingly used by food industry to characterize pathogen isolates, users are challenged by the variety of analysis approaches available, ranging from methods that require extensive bioinformatics expertise to commercial software packages. This study aimed to assess the impact of analysis pipelines (i.e., different hqSNP pipelines, a cg/wgMLST pipeline) and the reference genome selection on analysis results (i.e., hqSNP and allelic differences as well as tree topologies) and conclusion drawn. For these comparisons, whole genome sequences were obtained for 40 Listeria monocytogenes isolates collected over 18 years from a cold-smoked salmon facility and 2 other isolates obtained from different facilities as part of academic research activities; WGS data were analyzed with three hqSNP pipelines and two MLST pipelines. After initial clustering using a k-mer based approach, hqSNP pipelines were run using two types of reference genomes: (i) closely related closed genomes ("closed references") and (ii) high-quality de novo assemblies of the dataset isolates ("draft references"). All hqSNP pipelines identified similar hqSNP difference ranges among isolates in a given cluster; use of different reference genomes showed minimal impacts on hqSNP differences identified between isolate pairs. Allelic differences obtained by wgMLST showed similar ranges as hqSNP differences among isolates in a given cluster; cgMLST consistently showed fewer differences than wgMLST. However, phylogenetic trees and dendrograms, obtained based on hqSNP and cg/wgMLST data, did show some incongruences, typically linked to clades supported by low bootstrap values in the trees. When a hqSNP cutoff was used to classify isolates as "related" or "unrelated," use of different pipelines yielded a considerable number of discordances; this finding supports that cut-off values are valuable to provide a starting point for an investigation, but supporting and epidemiological evidence should be used to interpret WGS data. Overall, our data suggest that cgMLST-based data analyses provide for appropriate subtype differentiation and can be used without the need for preliminary data analyses (e.g., k-mer based clustering) or external closed reference genomes, simplifying data analyses needs. hqSNP or wgMLST analyses can be performed on the isolate clusters identified by cgMLST to increase the precision on determining the genomic similarity between isolates.

Whole genome sequencing used in an industrial context reveals a Salmonella laboratory cross-contamination.

In 2013, during a routine laboratory analysis performed on food samples, one finished product from a European factory was tested positive for Salmonella Hadar. At the same period, one environmental isolate in the same laboratory was serotyped Salmonella Hadar. Prior to this event, the laboratory performed a proficiency testing involving a sample spiked with NCTC 9877 Salmonella Hadar. The concomitance of Salmonella Hadar detection led to the suspicion of a laboratory cross-contamination between the Salmonella Hadar isolate used in the laboratory proficiency testing and the Salmonella Hadar isolate found on the finished product by the same laboratory. Since the classical phenotypic serotyping method is able to attribute a serotype to Salmonella isolates with a common antigenic formula, but cannot differentiate strains of the same serotype within the subspecies, whole genome sequencing was used to test the laboratory cross-contamination hypothesis. Additionally, 12 Salmonella Hadar from public databases, available until the time of the event, were included in the whole genome sequencing analysis to better understand the genomic diversity of this serotype in Europe. The outcome of the analysis showed a maximum of ten single nucleotide polymorphisms (SNPs) between the isolates coming from the laboratory and the finished product, and thus confirmed the laboratory cross-contamination. These results combined with all additional investigations done at the factory, allowed to release finished product batches produced and thus circumvented unnecessary food waste and economic losses for the factory.

Detection of murine norovirus 1 by using plaque assay, transfection assay, and real-time reverse transcription-PCR before and after heat exposure.

The correlation between the detection of murine norovirus 1 RNA by real-time reverse transcription-PCR and the infectivity by plaque assay before and after heat exposure (80 degrees C) was examined. No correlation was found in the current study. Moreover, heat inactivation had a much stronger detrimental effect on virus infectivity than on the integrity of the viral genome.

Evaluation of viral extraction methods on a broad range of Ready-To-Eat foods with conventional and real-time RT-PCR for Norovirus GII detection.

Noroviruses (NoV) are a common cause of foodborne outbreaks. In spite of that, no standard viral detection method is available for food products. Therefore, three viral elution-concentration methods and one direct RNA isolation method were evaluated on a broad range of Ready-To-Eat (RTE) food products (mixed lettuce, fruit salad, raspberries and two RTE dishes) artificially seeded with a diluted stool sample contaminated with NoV genogroup II. These seeding experiments revealed two categories of RTE products, fruits and vegetables grouped together and RTE dishes (penne and tagliatelle salads) which are rich in proteins and fat formed another category. The RNA extracts were amplified and detected with two conventional RT-PCR systems (Booster and Semi-nested GII) and one real-time RT-PCR (Real-time GII) assay. A fast direct RNA isolation method detected 10(2) RT-PCRU on 10 g penne and tagliatelle salads with the conventional RT-PCR assays. However real-time RT-PCR was less sensitive for penne salad. A viral elution-concentration method, including a buffer solution for the elution step and one polyethylene glycol (PEG) precipitation step, was able to detect 10(2) RT-PCRU on 50 g frozen raspberries with conventional and real-time RT-PCR assays. Moreover the latter extraction method used no environmental hazardous chemical reagents and was easy to perform.

Survival and transfer of murine norovirus 1, a surrogate for human noroviruses, during the production process of deep-frozen onions and spinach.

The reduction of murine norovirus 1 (MNV-1) on onions and spinach by washing was investigated as was the risk of contamination during the washing procedure. To decontaminate wash water, the industrial sanitizer peracetic acid (PAA) was added to the water, and the survival of MNV-1 was determined. In contrast to onions, spinach undergoes a heat treatment before freezing. Therefore, the resistance of MNV-1 to blanching of spinach was examined. MNV-1 genomic copies were detected with a real-time reverse transcription PCR assay in PAA-treated water and blanched spinach, and PFUs (representing infectious MNV-1 units) were determined with a plaque assay. A < or = 1-log reduction in MNV-1 PFUs was achieved by washing onion bulbs and spinach leaves. More than 3 log PFU of MNV-1 was transmitted to onion bulbs and spinach leaves when these vegetables were washed in water containing approximately 5 log PFU/ml. No decline of MNV-1 occurred in used industrial spinach wash water after 6 days at room temperature. A concentration of 20 ppm of PAA in demineralized water (pH 4.13) and in potable water (pH 7.70) resulted in reductions of 2.88 +/- 0.25 and 2.41 +/- 0.18 log PFU, respectively, after 5 min of exposure, but no decrease in number of genomic copies was observed. No reduction of MNV-1 PFUs was observed on frozen onions or spinach during storage for 6 months. Blanching spinach (80 degrees C for 1 min) resulted in at least 2.44-log reductions of infectious MNV-1, but many genomic copies were still present.

Optimization of subculture and DNA extraction steps within the whole genome sequencing workflow for source tracking of Salmonella enterica and Listeria monocytogenes.

This work shows that an incubation time reduced to 4-5 h to prepare a culture for DNA extraction followed by an automated DNA extraction can shorten the hands-on time, the turnaround time by 30% and increase the throughput while maintaining the WGS quality assessed by high quality Single Nucleotide Polymorphism analysis.