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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Detection of food-borne viruses such as noroviruses, rotaviruses and hepatitis A virus in food products differs from detection of most food-borne bacteria, as most of these viruses cannot be ...cultivated in cell culture to date. Therefore, detection of food-borne viruses in food products requires multiple steps: first, virus extraction; second, purification of the viral genomic material (RNA for the majority of food-borne viruses); and last, molecular detection. This review is focused on the first step, the virus extraction. All of the numerous published protocols for virus extraction from food samples are based on 3 main approaches: 1) (acid adsorption–) elution–concentration; 2) direct RNA extraction; and 3) proteinase K treatment. This review summarizes these virus extraction approaches and the results obtained from published protocols. The use of process controls is also briefly described.
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► This paper gives an overview of methods for extraction of viruses from foods. ► 3 food categories: fat/protein foods, water/carbohydrate foods, shellfish. ► Different approaches for extraction of viruses from these food categories. ► Quality control: process controls.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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
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
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.
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°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.
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 Ag⁰. The antiviral action of biogenic Ag⁰ was examined in water spiked with an Enterobacter aerogenes-infecting bacteriophage (UZ1). Addition of 5.4 mg liter⁻¹ biogenic Ag⁰ caused a 4.0-log decrease of the phage after 1 h, whereas the use of chemically produced silver nanoparticles (nAg⁰) 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 Ag⁰ were also demonstrated on the murine norovirus 1 (MNV-1), a model organism for human noroviruses. Biogenic Ag⁰ was applied to an electropositive cartridge filter (NanoCeram) to evaluate its capacity for continuous disinfection. Addition of 31.25 mg biogenic Ag⁰ m⁻² on the filter (135 mg biogenic Ag⁰ 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 Ag⁰ and its promising opportunities for continuous water disinfection.
Whole genome sequencing (WGS), using high throughput sequencing technology, reveals the complete sequence of the bacterial genome in a few days. WGS is increasingly being used for source tracking, ...pathogen surveillance and outbreak investigation due to its high discriminatory power. In the food industry, WGS used for source tracking is beneficial to support contamination investigations. Despite its increased use, no standards or guidelines are available today for the use of WGS in outbreak and/or trace-back investigations. Here we present a validation of our complete (end-to-end) WGS workflow for
and
including: subculture of isolates, DNA extraction, sequencing and bioinformatics analysis. This end-to-end WGS workflow was evaluated according to the following performance criteria: stability, repeatability, reproducibility, discriminatory power, and epidemiological concordance. The current study showed that few single nucleotide polymorphism (SNPs) were observed for
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when comparing genome sequences from five independent colonies from the first subculture and five independent colonies after the tenth subculture. Consequently, the stability of the WGS workflow for
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was demonstrated despite the few genomic variations that can occur during subculturing steps. Repeatability and reproducibility were also demonstrated. The WGS workflow was shown to have a high discriminatory power and has the ability to show genetic relatedness. Additionally, the WGS workflow was able to reproduce published outbreak investigation results, illustrating its capability of showing epidemiological concordance. The current study proposes a validation approach comprising all steps of a WGS workflow and demonstrates that the workflow can be applied to
or
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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.
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CEKLJ, GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Human noroviruses (NoVs) are considered a worldwide leading cause of acute non-bacterial gastroenteritis. Due to a combination of prolonged shedding of high virus levels in feces, virus particle ...shedding during asymptomatic infections, and a high environmental persistence, NoVs are easily transmitted pathogens. Norovirus (NoV) outbreaks have often been reported and tend to affect a lot of people. NoV is spread via feces and vomit, but this NoV spread can occur through several transmission routes. While person-to-person transmission is without a doubt the dominant transmission route, human infective NoV outbreaks are often initiated by contaminated food or water. Zoonotic transmission of NoV has been investigated, but has thus far not been demonstrated. The presented review aims to give an overview of these NoV transmission routes. Regarding NoV person-to-person transmission, the NoV GII.4 genotype is discussed in the current review as it has been very successful for several decades but reasons for its success have only recently been suggested. Both pre-harvest and post-harvest contamination of food products can lead to NoV food borne illness. Pre-harvest contamination of food products mainly occurs via contact with polluted irrigation water in case of fresh produce or with contaminated harvesting water in case of bivalve molluscan shellfish. On the other hand, an infected food handler is considered as a major cause of post-harvest contamination of food products. Both transmission routes are reviewed by a summary of described NoV food borne outbreaks between 2000 and 2010. A third NoV transmission route occurs via water and the spread of NoV via river water, ground water, and surface water is reviewed. Finally, although zoonotic transmission remains hypothetical, a summary on the bovine and porcine NoV presence observed in animals is given and the presence of human infective NoV in animals is discussed.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
► We compared four viral concentration methods for their recovery in water. ► MNV-1 and MS2 phages were used as surrogate viruses during this study. ► All methods were evaluated in processing water ...and four types of irrigation water. ► The best method used a negatively charged membrane with the Tr alk elution buffer. ► The best method was tested for its recovery of HAV, GI and GII NoV and RV.
Four viral concentration methods were evaluated for their efficiency in recovering murine norovirus-1 (MNV-1) (surrogate for human noroviruses (NoV)) and MS2 bacteriophages from processing water (1L) and four different types of irrigation water (bore hole water, rain water, open well and river water) (2–5L). Three methods were based on the viral adsorption and elution principle, two methods using an electronegative HA-membrane (Katayama et al., 2002), one method using an electropositive Zetapor membrane according to CEN/TC275/WG6/TAG4 and the fourth method was based on size exclusion using a tangential flow filtration system. Detection of MNV-1 was achieved by real-time RT-PCR and detection of MS2 by double-layer plaque assay.
For the recovery of MNV-1, the method using an electronegative HA-filter in combination with an elution buffer earlier optimized by Hamza et al. (2009) (Method 1) performed best for all types of water (recovery: 5.8–21.9%). In case of MS2 detection, the best method depended upon the type of water although Method 1 provided the most consistent recovery.
To complete this evaluation, the Method 1 was evaluated further for the concentration of human enteric viruses (GI and GII NoV, hepatitis A virus (HAV) and rotaviruses) in the same five types of water. Although detection of rotaviruses (RV) was somewhat less efficient, Method 1 proved reliable for the detection of NoV and HAV in all water types. Mean recovery efficiencies ranging from 4.8% for detection of GI NoV in open well water to 32.1% for detection of HAV in bore hole water, depending on the water type and the viral pathogen analyzed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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