%0 Journal Article %J J Clin Microbiol %D 2024 %T Closing the gap: Oxford Nanopore Technologies R10 sequencing allows comparable results to Illumina sequencing for SNP-based outbreak investigation of bacterial pathogens. %A Bert Bogaerts %A An Van den Bossche %A Bavo Verhaegen %A Laurence Delbrassinne %A Wesley Mattheus %A Stéphanie Nouws %A Maxime Godfroid %A Stefan Hoffman %A Nancy Roosens %A Sigrid C.J. De Keersmaecker %A Kevin Vanneste %X

Whole-genome sequencing has become the method of choice for bacterial outbreak investigation, with most clinical and public health laboratories currently routinely using short-read Illumina sequencing. Recently, long-read Oxford Nanopore Technologies (ONT) sequencing has gained prominence and may offer advantages over short-read sequencing, particularly with the recent introduction of the R10 chemistry, which promises much lower error rates than the R9 chemistry. However, limited information is available on its performance for bacterial single-nucleotide polymorphism (SNP)-based outbreak investigation. We present an open-source workflow, Prokaryotic Awesome variant Calling Utility (PACU) (https://github.com/BioinformaticsPlatformWIV-ISP/PACU), for constructing SNP phylogenies using Illumina and/or ONT R9/R10 sequencing data. The workflow was evaluated using outbreak data sets of Shiga toxin-producing and by comparing ONT R9 and R10 with Illumina data. The performance of each sequencing technology was evaluated not only separately but also by integrating samples sequenced by different technologies/chemistries into the same phylogenomic analysis. Additionally, the minimum sequencing time required to obtain accurate phylogenetic results using nanopore sequencing was evaluated. PACU allowed accurate identification of outbreak clusters for both species using all technologies/chemistries, but ONT R9 results deviated slightly more from the Illumina results. ONT R10 results showed trends very similar to Illumina, and we found that integrating data sets sequenced by either Illumina or ONT R10 for different isolates into the same analysis produced stable and highly accurate phylogenomic results. The resulting phylogenies for these two outbreaks stabilized after ~20 hours of sequencing for ONT R9 and ~8 hours for ONT R10. This study provides a proof of concept for using ONT R10, either in isolation or in combination with Illumina, for rapid and accurate bacterial SNP-based outbreak investigation.

%B J Clin Microbiol %8 2024 Mar 05 %G eng %R 10.1128/jcm.01576-23 %0 Journal Article %J Water Environment Research %D 2024 %T Development of a reverse transcriptase digital droplet polymerase chain reaction‐based approach for SARS‐CoV‐2 variant surveillance in wastewater %A Laura Van Poelvoorde %A Andrea Gobbo %A S. Nauwelaerts %A Bavo Verhaegen %A Marie Lesenfants %A Raphael Janssens %A Veronik Hutse %A Marie-Alice Fraiture %A Sigrid C.J. De Keersmaecker %A Philippe Herman %A Koenraad Van Hoorde %A Nancy Roosens %B Water Environment Research %V 96 %8 Jan-03-2024 %G eng %N 3 %R 10.1002/wer.10999 %0 Report %D 2024 %T Wastewater-based epidemiological surveillance - Methodological appendix %A Raphael Janssens %A Hadrien Maloux %A Sven Hanoteaux %A Laura Van Poelvoorde %A Nancy Roosens %A Bavo Verhaegen %A Julie Linussio %A Koenraad Van Hoorde %A Steven Van Gucht %A Karin De Ridder %A Koen Blot %A Veronik Hutse %A Marie Lesenfants %K applied genomics %K environment %K epidemiology %K Surveillance %K wastewater %K wastewater surveillance %K wastewater-based epidemiology %I Sciensano %C Brussels, Belgium %P 17 %8 feb 2024 %G eng %U https://www.sciensano.be/en/biblio/wastewater-based-epidemiological-surveillance-methodological-appendix %M D/2024.14.440/27 %0 Report %D 2024 %T Wastewater-based epidemiological surveillance - Weekly report %A Raphael Janssens %A Hadrien Maloux %A Sven Hanoteaux %A Laura Van Poelvoorde %A Nancy Roosens %A Bavo Verhaegen %A Julie Linussio %A Koenraad Van Hoorde %A Steven Van Gucht %A Karin De Ridder %A Koen Blot %A Veronik Hutse %A Marie Lesenfants %K applied genomics %K environment %K epidemiology %K Surveillance %K wastewater %K wastewater surveillance %K wastewater-based epidemiology %I Sciensano %C Brussels, Belgium %P 26 %8 2024 %G eng %U https://www.sciensano.be/en/biblio/wastewater-based-epidemiological-surveillance-weekly-report %M D/2024.14.440/28 %0 Generic %D 2023 %T BSFM 2023 - L. monocytogenes in Plant-Based Foods : insights from of a recent foodborne outbreak linked to vegan alternative to cheese %A Laurence Delbrassinne %A An Van den Bossche %A Marie Polet %A Wesley Mattheus %A Anneline Christiaens %A Bavo Verhaegen %A Koenraad Van Hoorde %K foodborne outbreak %K foodborne outbreak investigation %K Listeria monocytogenes %X

Introduction

Listeriosis outbreaks have been associated with a wide range of animal-derived foods, including unpasteurized milk, dairy products, raw or undercooked meat and raw or smoked fish products.

A survey conducted by Smartproteinproject (funded by the EU Horizon 2020) on European consumer attitudes towards plant-based foods has indicated an increasing consumption of plant-based food products in Europe over the years. In Belgium, there was an 8 % increase in sales volume between 2019-2020, with plant-based plain milk (primarily made from soy, almond and oat) showing the highest sales volume. Recently, a foodborne outbreak caused by L. monocytogenes was traced back to the consumption of almond-milk & cashew alternative to cheese. This report presents the first occurrence of such a matrix in listeriosis outbreaks in Belgium.

Materials and Methods

Food samples were collected by the Belgian Federal Agency for the Safety of the Food Chain (FASFC) and analysed at the National Reference Laboratory (NRL) for Foodborne outbreaks following ISO 11290 – 1 and – 2. A human strain was collected at the NRC Listeria. All isolated strains underwent serotyping for further characterisation at the NRC Listeria, and Whole Genome Sequencing (WGS) was performed for clustering analysis.

Discussion

Due to the growing demand for novel and alternative food products among the population, concerns are raised about the potential risks associated with plant-based food production. For example, challenge tests performed on bovine milk and plant-based milk demonstrated similar growth of L. monocytogenes. The outbreak described here provides strong evidence of the contamination of plant-based alternative to cheese with L. monocytogenes: the strain isolated from the affected individual (case) matched the strain found in the consumed vegan cheese-like product, indicating it as the source of contamination. A third isolate was obtained by analysing a different product of vegan alternative to cheese from the same brand, suggesting a potential contamination in the processing environment. The company has voluntarily implemented withdrawal and recall measures on all its cheese-like vegetable specialties (RASFF 2023.0500). Additional cases were also reported in Germany, France and The Netherlands (EpiPulse 2022-FWD-00102).

In conclusion, this report underscores the occurrence of L. monocytogenes in plant-based foods. It emphasizes the need for further investigation into the prevalence of this pathogen in plant-based foods and its survival throughout the plant-based food supply chain.  

%B BSFM %I Belgian Society for Food Microbiology (BSFM) %C Brussels, Belgium %8 10/2023 %G eng %0 Journal Article %J Microorganisms %D 2023 %T Development of a Droplet Digital PCR to Monitor SARS-CoV-2 Omicron Variant BA.2 in Wastewater Samples %A Laura Van Poelvoorde %A Corinne Picalausa %A Andrea Gobbo %A Bavo Verhaegen %A Marie Lesenfants %A Philippe Herman %A Koenraad Van Hoorde %A Nancy Roosens %K SARS-CoV-2; omicron; VOC; mutation; RT-ddPCR; variant detection; wastewater surveillance %X

Wastewater-based surveillance can be used as a complementary method to other SARS-CoV-2 surveillance systems. It allows the emergence and spread of infections and SARS-CoV-2 variants to be monitored in time and place. This study presents an RT-ddPCR method that targets the T19I amino acid mutation in the spike protein of the SARS-CoV-2 genomes, which is specific to the BA.2 variant (omicron). The T19I assay was evaluated both in silico and in vitro for its inclusivity, sensitivity, and specificity. Moreover, wastewater samples were used as a proof of concept to monitor and quantify the emergence of the BA.2 variant from January until May 2022 in the Brussels-Capital Region which covers a population of more than 1.2 million inhabitants. The in silico analysis showed that more than 99% of the BA.2 genomes could be characterized using the T19I assay. Subsequently, the sensitivity and specificity of the T19I assay were successfully experimentally evaluated. Thanks to our specific method design, the positive signal from the mutant probe and wild-type probe of the T19I assay was measured and the proportion of genomes with the T19I mutation, characteristic of the BA.2 mutant, compared to the entire SARS-CoV-2 population was calculated. The applicability of the proposed RT-ddPCR method was evaluated to monitor and quantify the emergence of the BA.2 variant over time. To validate this assay as a proof of concept, the measurement of the proportion of a specific circulating variant with genomes containing the T19I mutation in comparison to the total viral population was carried out in wastewater samples from wastewater treatment plants in the Brussels-Capital Region in the winter and spring of 2022. This emergence and proportional increase in BA.2 genomes correspond to what was observed in the surveillance using respiratory samples; however, the emergence was observed slightly earlier, which suggests that wastewater sampling could be an early warning system and could be an interesting alternative to extensive human testing.

%B Microorganisms %V 11 %8 12/03/2023 %G eng %N 3 %& 729 %R https://doi.org/10.3390/microorganisms11030729 %0 Journal Article %J International Journal of Food Microbiology %D 2023 %T Hepatitis E virus in pork meat products and exposure assessment in Belgium %A Tatjana Locus %A Lambrecht, Ellen %A Michael Peeters %A Vanessa Suin %A Bavo Verhaegen %A Koenraad Van Hoorde %A Lamoral, Sophie %A Thomas Vanwolleghem %A Steven Van Gucht %B International Journal of Food Microbiology %V 397 %8 Jan-07-2023 %G eng %R 10.1016/j.ijfoodmicro.2023.110198 %0 Journal Article %J Int J Food Microbiol %D 2023 %T Hepatitis E virus in pork meat products and exposure assessment in Belgium. %A Tatjana Locus %A Lambrecht, Ellen %A Michael Peeters %A Vanessa Suin %A Bavo Verhaegen %A Koenraad Van Hoorde %A Lamoral, Sophie %A Thomas Vanwolleghem %A Steven Van Gucht %X

Zoonotic hepatitis E virus (HEV) genotype 3 infections are the predominant cause of acute viral hepatitis in Europe, mostly associated with the consumption of HEV contaminated pork meat. In this study we looked at the HEV RNA positivity rate of pork meat products readily available from Belgian supermarkets and evaluated the overall HEV consumer exposure in a Belgian context. Two basic assessments were performed in a 'worst-case' scenario setting: one solely focusing on the contamination level of the product itself (ingredients and processing parameters) and another estimating the overall consumer exposure, taking into account consumption habits in Belgium. Non-thermal-processed ready-to-eat (i.e. ready for consumption without additional cooking step by consumer) pork meat products (e.g. raw dried sausages), had a high estimated HEV contamination level, while thermal-processed ready-to-eat pork meat products (e.g. pork liver pâté) had the highest overall consumer exposure estimates. Following these assessments, pork liver pâtés, raw dried hams and raw dried sausages (n = 54) were purchased from Belgian supermarkets (n = 3) and analyzed for HEV RNA by RT-PCR. In total, 31 % (n = 17) products tested positive. HEV RNA was found in 65 % of the pork liver pâtés, 15 % of raw dried hams and 0 % of raw dried sausages. Phylogenetic analysis of four isolates (all gt3c) from pork liver pâté samples showed similarities with human clinical cases from Germany and Belgium.

%B Int J Food Microbiol %V 397 %8 2023 Apr 05 %G eng %R 10.1016/j.ijfoodmicro.2023.110198 %0 Journal Article %J Animals %D 2023 %T SARS-CoV-2 Infection in Captive Hippos (Hippopotamus amphibius), Belgium %A Francis Vercammen %A Ann Brigitte Cay %A Sophie Gryseels %A Nadège Balmelle %A Léa Joffrin %A Koenraad Van Hoorde %A Bavo Verhaegen %A Elisabeth Mathijs %A Rianne Van Vredendaal %A Tanmay Dharmadhikari %A Koen Chiers %A Tim Van Olmen %A Gianfilippo Agliani %A Judith Van den Brand %A Herwig Leirs %B Animals %V 13 %8 Jan-01-2023 %G eng %N 2 %R 10.3390/ani13020316 %0 Generic %D 2023 %T Strain-level characterization without culture enrichment? Easing and accelerating outbreak investigation using shotgun metagenomics facilitated with nanopore adaptive sampling %A Florence E Buytaers %A Bavo Verhaegen %A Tom Van Nieuwenhuysen %A Nancy Roosens %A Kevin Vanneste %A Kathleen Marchal %A Sigrid C.J. De Keersmaecker %K adaptive sampling %K nanopore %K outbreak investigation %K shotgun metagenomics %X

Introduction

Traditionally, the investigation of suspect food samples during an outbreak investigation requires the samples to be enriched in order to reach detectable levels. However, a bias can occur during the enrichment of the food due to competition between the various living organisms or the cultivation parameters. Moreover, the current methods require a subsequent but not always successful isolation. In part due to these two drawbacks, not all outbreaks can be resolved. Shotgun metagenomics is a sequencing method involving the analysis of the genetic material directly extracted from a sample, without the need for an isolation of the bacteria. By sequencing and analysing the DNA of the sample, it can identify the presence of various pathogens to the strain level. Moreover, adaptive sampling is a new tool proposed during nanopore sequencing to target certain DNA strands to be sequenced preferably. The combination of metagenomics and adaptive sampling could allow to circumvent the isolation but also the enrichment of the food sample.

Materials and Methods

As a proof-of-concept, we explored the use of adaptive sampling using various databases (depletion of the food matrix i.e. potato, enrichment of a selection of foodborne pathogens, enrichment of a database of Staphylococcus aureus) compared to shotgun metagenomics without adaptive sampling on DNA of mashed potatoes spiked with DNA of S. aureus at a level of 0.5%. The strain was previously associated with a foodborne outbreak. Additionally, the living S. aureus strain was spiked into mashed potatoes at a level of 105 CFU per 25 grams and three DNA extraction kits were tested, in combination with two databases for adaptive sampling, following whole genome amplification to increase the amount of genetic material to meet sequencing standards. The strain-level data analysis was performed as previously described (Buytaers et al. 2021).

Discussion

Our results showed that the combination of whole genome amplification, metagenomics and adaptive sampling using a database of S. aureus genomes outperformed shotgun metagenomics and adaptive sampling using other databases. It allowed strain-level analysis of foodborne outbreaks without the need for culture enrichment, thereby enabling faster investigations and facilitating precise pathogen characterization, contributing to improved food safety and public health.

%B BSFM %I BSFM %C Brussels, Belgium %8 2023-10-13 %G eng %0 Journal Article %J Science of The Total Environment %D 2023 %T Time series modelling for wastewater-based epidemiology of COVID-19: A nationwide study in 40 wastewater treatment plants of Belgium, February 2021 to June 2022 %A Xander Bertels %A Sven Hanoteaux %A Raphael Janssens %A Hadrien Maloux %A Bavo Verhaegen %A Peter Delputte %A Tim Boogaerts %A Alexander L.N. van Nuijs %A Delphine Brogna %A Catherine Linard %A Marescaux, Jonathan %A Christian Didy %A Rosalie Pype %A Nancy Roosens %A Koenraad Van Hoorde %A Marie Lesenfants %A Lies Lahousse %K ARIMA %K COVID-19 %K Flow rate %K PMMoV %K wastewater surveillance %X

Background: Wastewater-based epidemiology (WBE) has been implemented to monitor surges of COVID-19. Yet,
multiple factors impede the usefulness of WBE and quantitative adjustment may be required.
Aim: We aimed to model the relationship between WBE data and incident COVID-19 cases, while adjusting for
confounders and autocorrelation.
Methods: This nationwide WBE study includes data from 40 wastewater treatment plants (WWTPs) in Belgium
(02/2021–06/2022). We applied ARIMA-based modelling to assess the effect of daily flow rate, pepper mild
mottle virus (PMMoV) concentration, a measure of human faeces in wastewater, and variants (alpha, delta, and
omicron strains) on SARS-CoV-2 RNA levels in wastewater. Secondly, adjusted WBE metrics at different lag times
were used to predict incident COVID-19 cases. Model selection was based on AICc minimization.
Results: In 33/40 WWTPs, RNA levels were best explained by incident cases, flow rate, and PMMoV. Flow rate
and PMMoV were associated with -13.0 % (95 % prediction interval: -26.1 to +0.2 %) and +13.0 % (95 %
prediction interval: +5.1 to +21.0 %) change in RNA levels per SD increase, respectively. In 38/40 WWTPs,
variants did not explain variability in RNA levels independent of cases. Furthermore, our study shows that RNA
levels can lead incident cases by at least one week in 15/40 WWTPs. The median population size of leading
WWTPs was 85.1 % larger than that of non‑leading WWTPs. In 17/40 WWTPs, however, RNA levels did not lead
or explain incident cases in addition to autocorrelation.
Conclusion: This study provides quantitative insights into key determinants of WBE, including the effects of
wastewater flow rate, PMMoV, and variants. Substantial inter-WWTP variability was observed in terms of
explaining incident cases. These findings are of practical importance to WBE practitioners and show that the
early-warning potential of WBE is WWTP-specific and needs validation.

%B Science of The Total Environment %V 899 %8 19-07-2023 %G eng %R 10.1016/j.scitotenv.2023.165603 %0 Journal Article %J Science of The Total Environment %D 2023 %T Time series modelling for wastewater-based epidemiology of COVID-19: A nationwide study in 40 wastewater treatment plants of Belgium, February 2021 to June 2022 %A Xander Bertels %A Sven Hanoteaux %A Raphael Janssens %A Hadrien Maloux %A Bavo Verhaegen %A Peter Delputte %A Tim Boogaerts %A Alexander L.N. van Nuijs %A Delphine Brogna %A Catherine Linard %A Marescaux, Jonathan %A Christian Didy %A Rosalie Pype %A Nancy Roosens %A Koenraad Van Hoorde %A Marie Lesenfants %A Lies Lahousse %B Science of The Total Environment %V 899 %8 Jan-11-2023 %G eng %R 10.1016/j.scitotenv.2023.165603 %0 Report %D 2023 %T Toxi-infections alimentaires en Belgique - Rapport annuel 2022 %A Laurence Delbrassinne %A Bavo Verhaegen %A Inge Van Damme %A Koenraad Van Hoorde %K Campylobacter %K foodborne outbreak %K Norovirus %K Salmonella %K toxi-infection alimentaire %I Sciensano %C Brussels, Belgium %P 47 %8 12/2023 %G eng %M D/2023.14.440/65 %0 Journal Article %J Front Microbiol %D 2023 %T Transforming Shiga toxin-producing surveillance through whole genome sequencing in food safety practices. %A Stéphanie Nouws %A Bavo Verhaegen %A Sarah Denayer %A Florence Crombé %A Denis Piérard %A Bert Bogaerts %A Kevin Vanneste %A Kathleen Marchal %A Nancy Roosens %A Sigrid C.J. De Keersmaecker %K Food Safety %K implementation %K Shiga toxin-producing Escherichia coli %K Surveillance %K whole genome sequencing %X

INTRODUCTION: Shiga toxin-producing (STEC) is a gastrointestinal pathogen causing foodborne outbreaks. Whole Genome Sequencing (WGS) in STEC surveillance holds promise in outbreak prevention and confinement, in broadening STEC epidemiology and in contributing to risk assessment and source attribution. However, despite international recommendations, WGS is often restricted to assist outbreak investigation and is not yet fully implemented in food safety surveillance across all European countries, in contrast to for example in the United States.

METHODS: In this study, WGS was retrospectively applied to isolates collected within the context of Belgian food safety surveillance and combined with data from clinical isolates to evaluate its benefits. A cross-sector WGS-based collection of 754 strains from 1998 to 2020 was analyzed.

RESULTS: We confirmed that WGS in food safety surveillance allows accurate detection of genomic relationships between human cases and strains isolated from food samples, including those dispersed over time and geographical locations. Identifying these links can reveal new insights into outbreaks and direct epidemiological investigations to facilitate outbreak management. Complete WGS-based isolate characterization enabled expanding epidemiological insights related to circulating serotypes, virulence genes and antimicrobial resistance across different reservoirs. Moreover, associations between virulence genes and severe disease were determined by incorporating human metadata into the data analysis. Gaps in the surveillance system were identified and suggestions for optimization related to sample centralization, harmonizing isolation methods, and expanding sampling strategies were formulated.

DISCUSSION: This study contributes to developing a representative WGS-based collection of circulating STEC strains and by illustrating its benefits, it aims to incite policymakers to support WGS uptake in food safety surveillance.

%B Front Microbiol %V 14 %8 2023 %G eng %R 10.3389/fmicb.2023.1204630 %0 Report %D 2023 %T Voedselvergiftigingen in België en Vlaanderen - Jaaroverzicht 2022 %A Laurence Delbrassinne %A Bavo Verhaegen %A Inge Van Damme %A Koenraad Van Hoorde %K Campylobacter %K foodborne outbreak %K Norovirus %K Salmonella Food Poisoning %I Sciensano %C Brussels, Belgium %P 49 %8 12/2023 %G eng %M D/2023.14.440/64 %0 Report %D 2023 %T Voedselvergiftigingen in België - Jaaroverzicht 2022 %A Laurence Delbrassinne %A Bavo Verhaegen %A Inge Van Damme %A Koenraad Van Hoorde %K Campylobacter %K foodborne outbreak %K Norovirus %K Salmonella %K toxi-infection alimentaire %I Sciensano %C Brussels, Belgium %P 48 %8 12/2023 %G eng %M D/2023.14.440/66 %0 Generic %D 2022 %T BSFM 2022 - International outbreak of Salmonella Typhimurium linked to a chocolate factory in 2022: Belgian findings %A Laurence Delbrassinne %E Dieter Van Cauteren %E Bavo Verhaegen %E Valeska Laisnez %E Wesley Mattheus %Y Vera Cantaert %Y Géraldine de Muylder %Y Tiffany Dierinck %Y Naïma Hammami %Y Veronica Jaramillo %Y Inne Nauwelaers %Y Koenraad Van Hoorde %K foodborne outbreak %K foodborne outbreak investigation %K Salmonella %X

Introduction

A multi-country outbreak caused by monophasic Salmonella Typhimurium is presented. A link with chocolate products from a Belgian factory was established by epidemiological, microbiological and traceback investigations. Whole genome sequencing (WGS) analysis of human and food isolates revealed the presence of two clusters:  HC5_296366 (cluster 1) and HC5_298160 (cluster 2).  The investigation of the outbreak as well as  measures taken are presented.

Materials and Methods

Probable and confirmed cases were identified using ECDC case definitions. Raw materials and finished food products collected at the factory were analysed for Salmonella spp. using ISO 6579-1:2017 and real-time PCR. WGS analysis of selected food and human isolates was performed.

Results & Discussion

In Belgium 64 confirmed and 2 probable cases were identified (89% children aged 1-9 years old, 43% hospitalizations), with illness onset from mid-January until April and a peak in cases mid-February 2022. WGS data revealed two distinct clusters at 62 allelic differences from each other. Seven of 229 food products tested positive for Salmonella Typhimurium. WGS analysis of the food isolates indicated matches with both clusters. Moreover, in December 2021, Salmonella isolates matching with the later identified clusters had also been found in samples during an own-check in the factory. Eleven types of products were recalled worldwide and food safety authorities shut down the factory 8 April 2022. A strong collaboration and information sharing between different stakeholders resulted in comprehensive measures to stop the spread of this international outbreak.

References

Joint ECDC-EFSA Rapid Outbreak Assessment  https://www.ecdc.europa.eu/sites/default/files/documents/1st-update-ROA_monophasic-S-Typhimurium-ST34_2022-00014_UK-amended-8-June.pdf

RASFF 2022.1799 ; RASFF 2022.2201; RASFF 2022.2452

%B BSFM %I Sciensano %C Brussels, Belgium %P 1 %8 2022/10 %G eng %0 Generic %D 2022 %T ESCAIDE 2022 Oral presentation: International outbreak of Salmonella Typhimurium linked to a chocolate factory in 2022: Belgian findings %A Valeska Laisnez %A Vera Cantaert %A Laurence Delbrassinne %A Géraldine de Muylder %A Tiffany Dierinck %A Hammami, Naïma %A Veronica Jaramillo %A Wesley Mattheus %A Inne Nauwelaers %A Bavo Verhaegen %A Dieter Van Cauteren %X

Background

Mid-February 2022, the United Kingdom reported via EpiPulse a cluster of monophasic Salmonella Typhimurium infections. Epidemiological and traceback investigations revealed a multi-country outbreak linked to chocolate products from a Belgian factory of an international brand. Microbiological investigations indicated two clusters:  HC5_296366 (cluster 1) and HC5_298160 (cluster 2).  We assessed the extent of the outbreak in Belgium and took measures to limit further spread.

Methods

Probable and confirmed cases were identified using ECDC case definitions. Case-interviews focused on exposure to chocolate products of the concerned brand. Raw materials and finished food products collected at the factory were analysed for Salmonella spp. using real-time PCR. Whole genome sequence (WGS) analysis of isolates of probable cases and positive food samples is still ongoing.

Results

We identified 62 probable cases (39 cluster 1, 23 cluster 2), with illness onset from mid-January until April and a peak in cases mid-February 2022. Of these 62 cases, 54 were aged 1-9 years old. Among the 44 interviewed cases, 19 have been hospitalized and 41 reported consumption of products of the factory among whom 35 reported consumption of Kinder Surprise. Seven of 229 food products tested positive for Salmonella; WGS analysis indicated matches with both clusters. In December 2021, Salmonella was found in samples during a self-check in the factory, these isolates matched with the later identified clusters. Eleven types of products were recalled worldwide and food safety authorities shut down the factory 8 April 2022.

Conclusions

Epidemiological and microbiological investigations confirmed the link between Salmonella cases and products from a Belgian chocolate factory. A strong collaboration and information sharing between different stakeholders resulted in comprehensive measures to stop the spread of this international outbreak.

%B ESCAIDE %8 2022-11-25 %G eng %0 Generic %D 2022 %T Introduction of WGS in food microbiology: advantages and challenges %A Bavo Verhaegen %A N Botteldoorn %A Vera Cantaert %A de Zutter, Lieven %A Véronique Delcenserie %A Annemie Geeraerd %A Mahillon, Jacques %A Marcella Mori %A Pochet, Brigitte %A Nancy Roosens %A Koenraad Van Hoorde %A Kim Feys %A Herman, Lieve %K antimicrobial resistance (AMR) %K Food Safety %K foodborne outbreak investigation %K Monitoring %K SciCom %K Whole genome sequencing (WGS) %X

In October 2021 the Scientific Committee of the Federal Agency for the Safety of the Food Chain (FASFC) published the opinion 18-2021 regarding whole genome sequencing (WGS) for the detection of foodborne outbreaks and bacterial risk assessment. This opinion was prepared using a self-tasking mandate and published after public consultation.

The added value of WGS for the detection of foodborne outbreaks and bacterial risk assessment has been demonstrated on many occasions in the last couple of years. Therefore, the implementation of WGS in the Belgian context needs some serious reflection. In the future, WGS will largely replace the various older typing methods in the field of bacterial food safety investigation, due to its high discriminatory power and the general use at the international level. Therefore, the Scientific Committee advises the FASFC to gradually make the transition to WGS for the analysis of food isolates.

While the advantages of the WGS method are considerable, some challenges still need to be taken into account for routine and uniform implementation. As with the implementation of any method the validations of the WGS methodology requires a significant effort. Furthermore, the interpretation of the results should be done with care. Therefore, in outbreak investigations it is recommended that WGS-based results be interpreted by a multidisciplinary team (microbiologists, molecular biologists, bioinformaticians, epidemiologists) with sufficient expertise. Only by putting the WGS-based results, the epidemiological evidence and the strains metadata together can a hypothesis be formulated. Further care should be given to the communication of these interpretations or hypotheses to the different actors (competent authority, food business operator, consumer). Finally, effort should be made to facilitate data sharing in an appropriate way.

%B 26th Conference on Food Microbiology %I Belgian Society for Food Microbiology %C Brussels, Belgium %8 10/2022 %G eng %0 Generic %D 2022 %T The key role of WGS-based surveillance of Listeria monocytogenes in both human and food isolates in outbreak investigations %A An Van den Bossche %A Sigrid C.J. De Keersmaecker %A Wesley Mattheus %A Nancy Roosens %A Bert Bogaerts %A Kevin Vanneste %A Koenraad Van Hoorde %A Bavo Verhaegen %K cgMLST %K Listeria monocytogenes %K NGS %K outbreak %B 26th Conference on Food Microbiology %I BSFM %C Brussels, Belgium %8 13-14/10/2022 %G eng %N BSFM %0 Journal Article %J Foods %D 2022 %T Metagenomics to Detect and Characterize Viruses in Food Samples at Genome Level? Lessons Learnt from a Norovirus Study %A Florence E Buytaers %A Bavo Verhaegen %A Mathieu Gand %A Jolien D'aes %A Kevin Vanneste %A Nancy Roosens %A Kathleen Marchal %A Sarah Denayer %A Sigrid C.J. De Keersmaecker %B Foods %V 11 %8 Jan-11-2022 %G eng %N 21 %R 10.3390/foods11213348 %0 Journal Article %J Viruses %D 2022 %T SARS-CoV-2 Surveillance in Belgian Wastewaters %A Raphael Janssens %A Sven Hanoteaux %A Hadrien Maloux %A Sofieke Klamer %A Valeska Laisnez %A Bavo Verhaegen %A Catherine Linard %A Lies Lahousse %A Peter Delputte %A Matthieu Terwagne %A Jonathan Marescaux %A Rosalie Pype %A Christian Didy %A Katelijne Dierick %A Koenraad Van Hoorde %A Marie Lesenfants %K alerting indicator %K Correlation %K public health authority %K SARS-CoV-2 %K Surveillance %K viral load per capita %K viral to faecal ratio %K wastewater-based epidemiology %X

Wastewater-based surveillance was conducted by the national public health authority to monitor SARS-CoV-2 circulation in the Belgian population. Over 5 million inhabitants representing 45% of the Belgian population were monitored throughout 42 wastewater treatment plants for 15 months comprising three major virus waves. During the entire period, a high correlation was observed between the daily new COVID-19 cases and the SARS-CoV-2 concentration in wastewater corrected for rain impact and covered population size. Three alerting indicators were included in the weekly epidemiological assessment: High Circulation, Fast Increase, and Increasing Trend. These indicators were computed on normalized concentrations per individual treatment plant to allow for a comparison with a reference period as well as between analyses performed by distinct laboratories. When the indicators were not corrected for rain impact, rainy events caused an underestimation of the indicators. Despite this negative impact, the indicators permitted us to effectively monitor the evolution of the fourth virus wave and were considered complementary and valuable information to conventional epidemiological indicators in the weekly wastewater reports communicated to the National Risk Assessment Group.

%B Viruses %V 14 %8 02-09-2022 %G eng %N 9 %R 10.3390/v14091950 %0 Generic %D 2022 %T Shotgun metagenomics as a ONE Health tool for better protecting human health %A Mathieu Gand %A Florence E Buytaers %A Bram Bloemen %A Assia Saltykova %A Sarah Denayer %A Bavo Verhaegen %A Wesley Mattheus %A Denis Piérard %A Bert Bogaerts %A Kathleen Marchal %A The FARMED consortium %A Nancy Roosens %A Kevin Vanneste %A Sigrid C.J. De Keersmaecker %X

Introduction: Open approaches to identify all biological material in a sample without a priori knowing what to look for, could transform the way specific key public health questions can be addressed, in a ONE Health context. Metagenomics is defined as the study of all genetic material within a sample, using sequencing technologies. Shotgun metagenomics is capable of detecting a wide range of microorganisms in a sample, as well as their composing genes such as virulence or antimicrobial resistance (AMR) genes, without prior knowledge, nor the need to isolate. Such approaches could offer efficient and fast solutions to limitations faced by conventional methods in e.g. the field of pathogen detection/characterization along the food chain, starting from the food production environment (including detection of AMR genes) until foodborne outbreak investigation and clinical/veterinary diagnostics. This could also allow a better understanding of infectious diseases and accurate risk (safety) assessment in food safety issues. However, before it can be used in these settings, this approach should be appropriately developed and validated from sampling and DNA extraction to sequencing and data analysis.

Methodology: Third generation sequencing technologies have had a breakthrough with the launch of nanopore sequencing, which through the production of long sequencing reads, portability and real-time data generation, represents a disruptive innovation for microbiology. Compared to short read sequencing (Illumina), this technology allows to unambiguously detect and scaffold microbial genes to their host chromosomes, even for complex metagenomics samples, allowing taxonomic classification up to an unprecedented sensitivity, including the identification of linked specific genes, such as AMR or virulence genes. We are developing and optimizing the protocols (from DNA extraction to data analysis) for sensitive metagenomics, by analyzing various materials collected in food-producing environments, and spiked with defined mock communities, composed of different bacterial species in various concentrations and carrying AMR and/or virulence genes, taking the specific need of each application into account. Moreover, the short read and long read approaches are being compared to each other, and also to the results obtained with conventional methods.

Results: A commercial kit was selected and optimized for the extraction of sufficient high-molecular weight DNA, to match the needs for long-read sequencing. The analysis of animal and environmental samples collected at a production farm, subsequently spiked with bacterial species present at various concentrations and carrying AMR genes, allowed to identify the spiked species and to make the link with AMR genes detected in the same sample when found on the same reads used for taxonomic identification, except for species present at low concentrations. Comparable results were obtained using short-read sequencing, without being able to link the identified species with the detected AMR genes. Our approach was also successful on artificially contaminated food samples. It allowed the same characterization of the foodborne pathogen (serotyping, virulence genes, relatedness to other cases) as the conventional methods used in reference laboratories, however without isolation and in a shorter time-frame. This could be achieved to the strain level even in samples with several E. coli strains. The method was also used to solve a Salmonella food-borne outbreak that occurred in Belgium in 2019.

Discussion: By applying the shotgun metagenomics approach to specific case studies, we delivered proof-of-concepts and demonstrated its added value compared to the conventional methods. We proved that it can characterize a bacterial pathogen to the strain level in spiked food as well as in the context of a real outbreak, and resolve the investigation in a faster time frame. EFSA called for such proof-of-concepts in a recent opinion about the use of metagenomics for food safety.  Additionally, we showed that the use of long-read sequencing can help to achieve a higher level of resolution by identifying specific bacteria and the AMR genes present in their genomes, with only one analysis. Future studies will explore at the technical level how this new technology can be transferred for fast, easy and direct use on-site, in a food-producing environment. This will open up ample opportunities for future research in the different fields, and even beyond, where the metagenomics approach would then be a well-established tool to be used to address specific public health questions at a more detailed and extended/comprehensive level, contributing to better protection of human health.

%B EFSA One %8 21/06/2022 %G eng %N EFSA %0 Report %D 2022 %T Toxi-infections alimentaires en Belgique - Rapport annuel 2021 %A Laurence Delbrassinne %A Bavo Verhaegen %A Inge Van Damme %A Koenraad Van Hoorde %K Campylobacter %K foodborne outbreak %K Norovirus %K Salmonella %K toxi-infection alimentaire %I Sciensano %C Brussels, Belgium %P 41 %8 12/2022 %G eng %M D/2022.14.440/63 %0 Report %D 2022 %T Voedselvergiftigingen in België en Vlaanderen - Jaaroverzicht 2021, %A Laurence Delbrassinne %E Bavo Verhaegen %E Inge Van Damme %E Koenraad Van Hoorde %K voedsel-toxi infectie %I Sciensano %C Belgium %P 42 %8 2022 %G eng %M D/2023.14.440/49 %0 Report %D 2022 %T Voedselvergiftigingen in België - Jaaroverzicht 2021 %A Laurence Delbrassinne %A Bavo Verhaegen %A Inge Van Damme %A Koenraad Van Hoorde %K Campylobacter %K foodborne outbreaks %K Norovirus %K Salmonella %K voedsel-toxi infectie %I Sciensano %C Brussels, Belgium %P 41 %8 12/2022 %G eng %M D/2022.14.440/64 %0 Journal Article %J Microbial Genomics %D 2021 %T Application of a strain-level shotgun metagenomics approach on food samples: resolution of the source of a Salmonella food-borne outbreak %A Florence E Buytaers %A Assia Saltykova %A Wesley Mattheus %A Bavo Verhaegen %A Nancy Roosens %A Kevin Vanneste %A Valeska Laisnez %A Hammami, Naïma %A Pochet, Brigitte %A Vera Cantaert %A Marchal, Kathleen %A Sarah Denayer %A Sigrid C.J. De Keersmaecker %K food surveillance %K Metagenomics %K outbreak %K Salmonella %K SNP analysis %K strain-level %X

Food-borne outbreak investigation currently relies on the time-consuming and challenging bacterial isolation from food, to be able to link food-derived strains to more easily obtained isolates from infected people. When no food isolate can be obtained, the source of the outbreak cannot be unambiguously determined. Shotgun metagenomics approaches applied to the food samples could circumvent this need for isolation from the suspected source, but require downstream strain-level data analysis to be able to accurately link to the human isolate. Until now, this approach has not yet been applied outside research settings to analyse real food-borne outbreak samples. In September 2019, a Salmonella outbreak occurred in a hotel school in Bruges, Belgium, affecting over 200 students and teachers. Following standard procedures, the Belgian National Reference Center for human salmonellosis and the National Reference Laboratory for Salmonella in food and feed used conventional analysis based on isolation, serotyping and MLVA (multilocus variable number tandem repeat analysis) comparison, followed by whole-genome sequencing, to confirm the source of the contamination over 2 weeks after receipt of the sample, which was freshly prepared tartar sauce in a meal cooked at the school. Our team used this outbreak as a case study to deliver a proof of concept for a short-read strain-level shotgun metagenomics approach for source tracking. We received two suspect food samples: the full meal and some freshly made tartar sauce served with this meal, requiring the use of raw eggs. After analysis, we could prove, without isolation, that Salmonella was present in both samples, and we obtained an inferred genome of a Salmonella enterica subsp. enterica serovar Enteritidis that could be linked back to the human isolates of the outbreak in a phylogenetic tree. These metagenomics-derived outbreak strains were separated from sporadic cases as well as from another outbreak circulating in Europe at the same time period. This is, to our knowledge, the first Salmonella food-borne outbreak investigation uniquely linking the food source using a metagenomics approach and this in a fast time frame.

%B Microbial Genomics %V 7 %8 Jul-04-2021 %G eng %N 4 %R 10.1099/mgen.0.000547 %0 Journal Article %J Curr Issues Mol Biol %D 2021 %T Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution. %A Laura Van Poelvoorde %A Mathieu Gand %A Marie-Alice Fraiture %A Sigrid C.J. De Keersmaecker %A Bavo Verhaegen %A Koenraad Van Hoorde %A Ann Brigitte Cay %A Nadège Balmelle %A Philippe Herman %A Nancy Roosens %K COVID-19 %K COVID-19 Nucleic Acid Testing %K Diagnostic Tests, Routine %K Evolution, Molecular %K Humans %K RNA, Viral %K Roc Curve %K SARS-CoV-2 %X

The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples.

%B Curr Issues Mol Biol %V 43 %8 2021 Nov 06 %G eng %N 3 %R 10.3390/cimb43030134 %0 Journal Article %J Frontiers in Microbiology %D 2021 %T Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies %A Florence E Buytaers %A Assia Saltykova %A Sarah Denayer %A Bavo Verhaegen %A Kevin Vanneste %A Nancy Roosens %A Piérard, Denis %A Marchal, Kathleen %A Sigrid C.J. De Keersmaecker %K Flongle %K food surveillance %K Metagenomics %K nanopore %K outbreak %K SNP analysis %K STEC %K strain-level %X

The current routine laboratory practices to investigate food samples in case of foodborne outbreaks still rely on attempts to isolate the pathogen in order to characterize it. We present in this study a proof of concept using Shiga toxin-producing Escherichia coli spiked food samples for a strain-level metagenomics foodborne outbreak investigation method using the MinION and Flongle flow cells from Oxford Nanopore Technologies, and we compared this to Illumina short-read-based metagenomics. After 12 h of MinION sequencing, strain-level characterization could be achieved, linking the food containing a pathogen to the related human isolate of the affected patient, by means of a single-nucleotide polymorphism (SNP)-based phylogeny. The inferred strain harbored the same virulence genes as the spiked isolate and could be serotyped. This was achieved by applying a bioinformatics method on the long reads using reference-based classification. The same result could be obtained after 24-h sequencing on the more recent lower output Flongle flow cell, on an extract treated with eukaryotic host DNA removal. Moreover, an alternative approach based on in silico DNA walking allowed to obtain rapid confirmation of the presence of a putative pathogen in the food sample. The DNA fragment harboring characteristic virulence genes could be matched to the E. coli genus after sequencing only 1 h with the MinION, 1 h with the Flongle if using a host DNA removal extraction, or 5 h with the Flongle with a classical DNA extraction. This paves the way towards the use of metagenomics as a rapid, simple, one-step method for foodborne pathogen detection and for fast outbreak investigation that can be implemented in routine laboratories on samples prepared with the current standard practices.

%B Frontiers in Microbiology %V 12 %8 May-11-2021 %G eng %R 10.3389/fmicb.2021.738284 %0 Report %D 2021 %T Toxi-infections alimentaires en Belgique - Rapport annuel 2020 %A Laurence Delbrassinne %A Bavo Verhaegen %A Sarah Denayer %A Koenraad Van Hoorde %K Campylobacter %K foodborne outbreak %K Norovirus %K Salmonella %K toxi-infection alimentaire %I Sciensano %C Brussels, Belgium %P 41 %8 12/2021 %G eng %M D/2021/14.440/71 %0 Journal Article %J Microbial Genomics %D 2021 %T Validation strategy of a bioinformatics whole genome sequencing workflow for Shiga toxin-producing Escherichia coli using a reference collection extensively characterized with conventional methods %A Bert Bogaerts %A Stéphanie Nouws %A Bavo Verhaegen %A Sarah Denayer %A Julien Van Braekel %A Raf Winand %A Qiang Fu %A Florence Crombé %A Denis Piérard %A Kathleen Marchal %A Nancy Roosens %A Sigrid C.J. De Keersmaecker %A Kevin Vanneste %X

Whole genome sequencing (WGS) enables complete characterization of bacterial pathogenic isolates at single nucleotide resolution, making it the ultimate tool for routine surveillance and outbreak investigation. The lack of standardization, and the variation regarding bioinformatics workflows and parameters, however, complicates interoperability among (inter)national laboratories. We present a validation strategy applied to a bioinformatics workflow for Illumina data that performs complete characterization of Shiga toxin-producing Escherichia coli (STEC) isolates including antimicrobial resistance prediction, virulence gene detection, serotype prediction, plasmid replicon detection and sequence typing. The workflow supports three commonly used bioinformatics approaches for the detection of genes and alleles: alignment with blast+, kmer-based read mapping with KMA, and direct read mapping with SRST2. A collection of 131 STEC isolates collected from food and human sources, extensively characterized with conventional molecular methods, was used as a validation dataset. Using a validation strategy specifically adopted to WGS, we demonstrated high performance with repeatability, reproducibility, accuracy, precision, sensitivity and specificity above 95 % for the majority of all assays. The WGS workflow is publicly available as a ‘push-button’ pipeline at https://galaxy.sciensano.be. Our validation strategy and accompanying reference dataset consisting of both conventional and WGS data can be used for characterizing the performance of various bioinformatics workflows and assays, facilitating interoperability between laboratories with different WGS and bioinformatics set-ups.

%B Microbial Genomics %V 7 %8 Jan-03-2021 %G eng %N 3 %R 10.1099/mgen.0.000531 %0 Report %D 2021 %T Voedselvergiftigingen in België - Jaaroverzicht 2020 %A Laurence Delbrassinne %A Bavo Verhaegen %A Sarah Denayer %A Koenraad Van Hoorde %K Campylobacter %K foodborne outbreak %K Norovirus %K Salmonella %K voedselvergiftiging; voedseltoxi-infectie %I Sciensano %C Brussels, Belgium %P 41 %8 12/2021 %G eng %M D/2021/14.440/70 %0 Journal Article %J Frontiers in Microbiology %D 2021 %T Whole Genome Sequencing Provides an Added Value to the Investigation of Staphylococcal Food Poisoning Outbreaks %A Stéphanie Nouws %A Bert Bogaerts %A Bavo Verhaegen %A Sarah Denayer %A Lasse Laeremans %A Kathleen Marchal %A Nancy Roosens %A Kevin Vanneste %A Sigrid C.J. De Keersmaecker %K DNA extraction kits %K enterotoxin gene profiling %K outbreak investigation %K relatedness determination %K Staphylococcus aureus, staphylococcal food poisoning (SFP) %K whole genome sequencing %X

Through staphylococcal enterotoxin (SE) production, Staphylococcus aureus is a common cause of food poisoning. Detection of staphylococcal food poisoning (SFP) is mostly performed using immunoassays, which, however, only detect five of 27 SEs described to date. Polymerase chain reactions are, therefore, frequently used in complement to identify a bigger arsenal of SE at the gene level (se) but are labor-intensive. Complete se profiling of isolates from different sources, i.e., food and human cases, is, however, important to provide an indication of their potential link within foodborne outbreak investigation. In addition to complete se gene profiling, relatedness between isolates is determined with more certainty using pulsed-field gel electrophoresis, Staphylococcus protein A gene typing and other methods, but these are shown to lack resolution. We evaluated how whole genome sequencing (WGS) can offer a solution to these shortcomings. By WGS analysis of a selection of S. aureus isolates, including some belonging to a confirmed foodborne outbreak, its added value as the ultimate multiplexing method was demonstrated. In contrast to PCR-based se gene detection for which primers are sometimes shown to be non-specific, WGS enabled complete se gene profiling with high performance, provided that a database containing reference sequences for all se genes was constructed and employed. The custom compiled database and applied parameters were made publicly available in an online user-friendly interface. As an all-in-one approach with high resolution, WGS additionally allowed inferring correct isolate relationships. The different DNA extraction kits that were tested affected neither se gene profiling nor relatedness determination, which is interesting for data sharing during SFP outbreak investigation. Although confirming the production of enterotoxins remains important for SFP investigation, we delivered a proof-of-concept that WGS is a valid alternative and/or complementary tool for outbreak investigation.

%B Frontiers in Microbiology %V 12 %8 Feb-11-2021 %G eng %R 10.3389/fmicb.2021.750278 %0 Journal Article %J Foods %D 2020 %T The Benefits of Whole Genome Sequencing for Foodborne Outbreak Investigation from the Perspective of a National Reference Laboratory in a Smaller Country. %A Stéphanie Nouws %A Bert Bogaerts %A Bavo Verhaegen %A Sarah Denayer %A Florence Crombé %A Klara De Rauw %A Denis Piérard %A Kathleen Marchal %A Kevin Vanneste %A Nancy Roosens %A Sigrid C.J. De Keersmaecker %K Food Safety %K foodborne outbreak investigation %K Shiga toxin-producing Escherichia coli %K STEC %K Surveillance %K WGS %K whole genome sequencing %X

Gradually, conventional methods for foodborne pathogen typing are replaced by whole genome sequencing (WGS). Despite studies describing the overall benefits, National Reference Laboratories of smaller countries often show slower uptake of WGS, mainly because of significant investments required to generate and analyze data of a limited amount of samples. To facilitate this process and incite policy makers to support its implementation, a Shiga toxin-producing (STEC) O157:H7 (+, +, +) outbreak (2012) and a STEC O157:H7 (+, +) outbreak (2013) were retrospectively analyzed using WGS and compared with their conventional investigations. The corresponding results were obtained, with WGS delivering even more information, e.g., on virulence and antimicrobial resistance genotypes. Besides a universal, all-in-one workflow with less hands-on-time (five versus seven actual working days for WGS versus conventional), WGS-based cgMLST-typing demonstrated increased resolution. This enabled an accurate cluster definition, which remained unsolved for the 2013 outbreak, partly due to scarce epidemiological linking with the suspect source. Moreover, it allowed detecting two and one earlier circulating STEC O157:H7 (+, +, +) and STEC O157:H7 (+, +) strains as closely related to the 2012 and 2013 outbreaks, respectively, which might have further directed epidemiological investigation initially. Although some bottlenecks concerning centralized data-sharing, sampling strategies, and perceived costs should be considered, we delivered a proof-of-concept that even in smaller countries, WGS offers benefits for outbreak investigation, if a sufficient budget is available to ensure its implementation in surveillance. Indeed, applying a database with background isolates is critical in interpreting isolate relationships to outbreaks, and leveraging the true benefit of WGS in outbreak investigation and/or prevention.

%B Foods %V 9 %8 2020 Aug 01 %G eng %N 8 %R 10.3390/foods9081030 %0 Journal Article %J International Journal of Food Microbiology %D 2020 %T First detection of a plasmid located carbapenem resistant blaVIM-1 gene in E. coli isolated from meat products at retail in Belgium in 2015 %A Cristina Garcia-Graells %A Bas Berbers %A Bavo Verhaegen %A Kevin Vanneste %A Marchal, Kathleen %A Nancy Roosens %A N Botteldoorn %A Sigrid C.J. De Keersmaecker %B International Journal of Food Microbiology %V 324 %8 Jan-07-2020 %G eng %R 10.1016/j.ijfoodmicro.2020.108624 %0 Journal Article %J Sci Rep %D 2020 %T Impact of DNA extraction on whole genome sequencing analysis for characterization and relatedness of Shiga toxin-producing Escherichia coli isolates. %A Stéphanie Nouws %A Bert Bogaerts %A Bavo Verhaegen %A Sarah Denayer %A Piérard, Denis %A Marchal, Kathleen %A Nancy Roosens %A Kevin Vanneste %A Sigrid C.J. De Keersmaecker %K NGS %K pathogenic E. coli %K STEC %K WGS %X

Whole genome sequencing (WGS) has proven to be the ultimate tool for bacterial isolate characterization and relatedness determination. However, standardized and harmonized workflows, e.g. for DNA extraction, are required to ensure robust and exchangeable WGS data. Data sharing between (inter)national laboratories is essential to support foodborne pathogen control, including outbreak investigation. This study evaluated eight commercial DNA preparation kits for their potential influence on: (i) DNA quality for Nextera XT library preparation; (ii) MiSeq sequencing (data quality, read mapping against plasmid and chromosome references); and (iii) WGS data analysis, i.e. isolate characterization (serotyping, virulence and antimicrobial resistance genotyping) and phylogenetic relatedness (core genome multilocus sequence typing and single nucleotide polymorphism analysis). Shiga toxin-producing Escherichia coli (STEC) was selected as a case study. Overall, data quality and inferred phylogenetic relationships between isolates were not affected by the DNA extraction kit choice, irrespective of the presence of confounding factors such as EDTA in DNA solution buffers. Nevertheless, completeness of STEC characterization was, although not substantially, influenced by the plasmid extraction performance of the kits, especially when using Nextera XT library preparation. This study contributes to addressing the WGS challenges of standardizing protocols to support data portability and to enable full exploitation of its potential.

%B Sci Rep %V 10 %8 2020 09 04 %G eng %N 1 %R 10.1038/s41598-020-71207-3 %0 Journal Article %J Scientific Reports %D 2020 %T Impact of DNA extraction on whole genome sequencing analysis for characterization and relatedness of Shiga toxin-producing Escherichia coli isolates %A Stéphanie Nouws %A Bert Bogaerts %A Bavo Verhaegen %A Sarah Denayer %A Piérard, Denis %A Marchal, Kathleen %A Nancy Roosens %A Kevin Vanneste %A Sigrid C.J. De Keersmaecker %K DNA extraction kits %K outbreak investigation %K Shiga toxin producing Escherichia coli (STEC) %K Whole genome sequencing (WGS) %X

Whole genome sequencing (WGS) has proven to be the ultimate tool for bacterial isolate characterization and relatedness determination. However, standardized and harmonized workflows, e.g. for DNA extraction, are required to ensure robust and exchangeable WGS data. Data sharing between (inter)national laboratories is essential to support foodborne pathogen control, including outbreak investigation. This study evaluated eight commercial DNA preparation kits for their potential influence on: (i) DNA quality for Nextera XT library preparation; (ii) MiSeq sequencing (data quality, read mapping against plasmid and chromosome references); and (iii) WGS data analysis, i.e. isolate characterization (serotyping, virulence and antimicrobial resistance genotyping) and phylogenetic relatedness (core genome multilocus sequence typing and single nucleotide polymorphism analysis). Shiga toxin-producing Escherichia coli (STEC) was selected as a case study. Overall, data quality and inferred phylogenetic relationships between isolates were not affected by the DNA extraction kit choice, irrespective of the presence of confounding factors such as EDTA in DNA solution buffers. Nevertheless, completeness of STEC characterization was, although not substantially, influenced by the plasmid extraction performance of the kits, especially when using Nextera XT library preparation. This study contributes to addressing the WGS challenges of standardizing protocols to support data portability and to enable full exploitation of its potential.

%B Scientific Reports %V 10 %8 Jan-12-2020 %G eng %N 1 %R 10.1038/s41598-020-71207-3 %0 Journal Article %J Microorganisms %D 2020 %T A Practical Method to Implement Strain-Level Metagenomics-Based Foodborne Outbreak Investigation and Source Tracking in Routine. %A Florence E Buytaers %A Assia Saltykova %A Sarah Denayer %A Bavo Verhaegen %A Kevin Vanneste %A Nancy Roosens %A Piérard, Denis %A Marchal, Kathleen %A Sigrid C.J. De Keersmaecker %K food surveillance %K Metagenomics %K outbreak %K SNP analysis %K STEC %K whole genome %X

The management of a foodborne outbreak depends on the rapid and accurate identification of the responsible food source. Conventional methods based on isolation of the pathogen from the food matrix and target-specific real-time polymerase chain reactions (qPCRs) are used in routine. In recent years, the use of whole genome sequencing (WGS) of bacterial isolates has proven its value to collect relevant information for strain characterization as well as tracing the origin of the contamination by linking the food isolate with the patient's isolate with high resolution. However, the isolation of a bacterial pathogen from food matrices is often time-consuming and not always successful. Therefore, we aimed to improve outbreak investigation by developing a method that can be implemented in reference laboratories to characterize the pathogen in the food vehicle without its prior isolation and link it back to human cases. We tested and validated a shotgun metagenomics approach by spiking food pathogens in specific food matrices using the Shiga toxin-producing (STEC) as a case study. Different DNA extraction kits and enrichment procedures were investigated to obtain the most practical workflow. We demonstrated the feasibility of shotgun metagenomics to obtain the same information as in ISO/TS 13136:2012 and WGS of the isolate in parallel by inferring the genome of the contaminant and characterizing it in a shorter timeframe. This was achieved in food samples containing different strains, including a combination of different STEC strains. For the first time, we also managed to link individual strains from a food product to isolates from human cases, demonstrating the power of shotgun metagenomics for rapid outbreak investigation and source tracking.

%B Microorganisms %V 8 %8 2020 Aug 05 %G eng %N 8 %R 10.3390/microorganisms8081191 %0 Journal Article %J Int J Mol Sci %D 2020 %T Strain-Level Metagenomic Data Analysis of Enriched In Vitro and In Silico Spiked Food Samples: Paving the Way towards a Culture-Free Foodborne Outbreak Investigation Using STEC as a Case Study. %A Assia Saltykova %A Florence E Buytaers %A Sarah Denayer %A Bavo Verhaegen %A Piérard, Denis %A Nancy Roosens %A Marchal, Kathleen %A Sigrid C.J. De Keersmaecker %K foodborne outbreak investigation %K public health %K strain-level metagenomics %X

Culture-independent diagnostics, such as metagenomic shotgun sequencing of food samples, could not only reduce the turnaround time of samples in an outbreak investigation, but also allow the detection of multi-species and multi-strain outbreaks. For successful foodborne outbreak investigation using a metagenomic approach, it is, however, necessary to bioinformatically separate the genomes of individual strains, including strains belonging to the same species, present in a microbial community, which has up until now not been demonstrated for this application. The current work shows the feasibility of strain-level metagenomics of enriched food matrix samples making use of data analysis tools that classify reads against a sequence database. It includes a brief comparison of two database-based read classification tools, Sigma and Sparse, using a mock community obtained by in vitro spiking minced meat with a Shiga toxin-producing (STEC) isolate originating from a described outbreak. The more optimal tool Sigma was further evaluated using in silico simulated metagenomic data to explore the possibilities and limitations of this data analysis approach. The performed analysis allowed us to link the pathogenic strains from food samples to human isolates previously collected during the same outbreak, demonstrating that the metagenomic approach could be applied for the rapid source tracking of foodborne outbreaks. To our knowledge, this is the first study demonstrating a data analysis approach for detailed characterization and phylogenetic placement of multiple bacterial strains of one species from shotgun metagenomic WGS data of an enriched food sample.

%B Int J Mol Sci %V 21 %8 2020 Aug 08 %G eng %N 16 %R 10.3390/ijms21165688 %0 Report %D 2020 %T Toxi-infections alimentaires en Belgique - Rapport annuel 2019 %A Sarah Denayer %A Bavo Verhaegen %A Koenraad Van Hoorde %X

En 2019, 571 toxi-infections alimentaires collectives ont été enregistrées en Belgique par le LNR TIA. Il s’agit du nombre le plus élevé depuis le début des enregistrements en Belgique (1999). Au total, au moins 2 457 personnes sont tombées malades et 28 personnes ont été hospitalisées.

Salmonella était l’agent le plus souvent rapporté comme étant à l’origine d’un foyer de toxi-infection alimentaire en 2019 et a rendu au moins 216 personnes malades. Norovirus était deuxième agent le plus souvent identifié comme étant la cause d’infections alimentaires et a rendu au moins 41 personnes malades. Clostridium perfringens, Listeria monocytogenes, Campylobacter, Bacillus cereus et les souches pathogènes d’Escherichia coli O157 font parties des autres germes identifiés. Des amines biogènes telles que l’histamine ont déclenché un foyer d’origine alimentaire.

Les sources d’infection sont très diverses, mais ce sont surtout des repas composés (67,3%) qui ont été envoyés au laboratoire pour analyse. Pour 70,4% des foyers, c’est dans un restaurant qu’a eu lieu l’exposition à une denrée alimentaire contaminée.

%P 43 %8 2020 %G eng %& 1 %0 Report %D 2020 %T Voedselvergiftigingen in Belgie en Vlaanderen - Jaaroverzicht 2019 %A Sarah Denayer %A Bavo Verhaegen %A Koenraad Van Hoorde %K Bacillus cereus %K foodborne outbreak %K Salmonella %X

In 2019 werden in België 571 collectieve voedseltoxi-infecties geregistreerd door het NRL-VTI, waarvan 272 in Vlaanderen. Dit is het hoogste cijfer sinds de registraties in België (1999). In totaal werden minstens 2457 personen ziek en werden 28 personen gehospitaliseerd.

Salmonella was het meest gerapporteerde agens als oorzaak van een uitbraak in 2019 en veroorzaakte minstens 216 zieken. Norovirus was het tweede meest geïdentificeerde agens als oorzaak van voedselinfecties en veroorzaakte daarbij minstens 41 zieken. Andere geïdentificeerde kiemen zijn Clostridium perfringens, Listeria monocytogenes, Campylobacter, Bacillus cereus en pathogene Escherichia coli O157. Biogene aminen zoals histamine veroorzaakte één voedselgebonden uitbraak.

De bron van de infectie is heel divers en vooral samengestelde maaltijden (67.3%) werden voor onderzoek doorgestuurd naar het laboratorium.

Restaurants waren in 70.4% van de uitbraken de plaats van blootstelling aan een besmet voedingsmiddel.

%I Sciensano %C Brussels, Belgium %P 42 %8 04/2020 %G eng %M D/2020/14.440/43 %) Sciensano %& 1 %0 Report %D 2020 %T Voedselvergiftigingen in België - jaaroverzicht 2019 %A Sarah Denayer %A Bavo Verhaegen %A Koenraad Van Hoorde %K Bacillus cereus %K coagulase positive staphylococci %K foodborne outbreak %K Salmonella %X

In 2019 werden in België 571 collectieve voedseltoxi-infecties geregistreerd door het NRL-VTI. Dit is het hoogste cijfer sinds de registraties in België (1999). In totaal werden minstens 2457 personen ziek en werden 28 personen gehospitaliseerd.

Salmonella was het meest gerapporteerde agens als oorzaak van een uitbraak in 2019 en veroorzaakte minstens 216 zieken. Norovirus was het tweede meest geïdentificeerde agens als oorzaak van voedselinfecties en veroorzaakte daarbij minstens 41 zieken. Andere geïdentificeerde kiemen zijn Clostridium perfringens, Listeria monocytogenes, Campylobacter, Bacillus cereus en pathogene Escherichia coli O157. Biogene aminen zoals histamine veroorzaakte één voedselgebonden uitbraak.

De bron van de infectie is heel divers en vooral samengestelde maaltijden (67.3%) werden voor onderzoek doorgestuurd naar het laboratorium.  Restaurants waren in 70.4% van de uitbraken de plaats van blootstelling aan een besmet voedingsmiddel.

%I Sciensano %C Brussels, Belgium %P 42 %8 2020 %G eng %M D/2020/14.440/41 %& 1 %0 Journal Article %J New Microbes New Infect %D 2020 %T Whole-genome sequencing of serotype 4b isolated from ready-to-eat lentil salad in Algiers, Algeria. %A R Drali %A A Deriet %A Bavo Verhaegen %A Sigrid C.J. De Keersmaecker %A N Botteldoorn %A Kevin Vanneste %A Nancy Roosens %A F Mouffok %X

is a Gram-positive food-borne pathogen causing a serious threat for public health. Here we announce the whole genome sequence (3 011 693 bp) of serotype 4b, isolated from ready-to-eat lentil salad in Algiers and belonging to sequence type 2, lineage I and clonal complex 2.

%B New Microbes New Infect %V 33 %8 2020 Jan %G eng %R 10.1016/j.nmni.2019.100628 %0 Journal Article %J Frontiers in Immunology %D 2019 %T QuilA-Adjuvanted T. gondii Lysate Antigens Trigger Robust Antibody and IFNγ+ T Cell Responses in Pigs Leading to Reduction in Parasite DNA in Tissues Upon Challenge InfectionImage %A Mizanur Rahman %A Devriendt, Bert %A I. Gisbert Algaba %A Bavo Verhaegen %A Dorny, Pierre %A Katelijne Dierick %A Cox, Eric %K magnetic capture-qPCR %K Pigs %K QuilA %K TLA vaccine %K Toxoplasma gondii %X

Toxoplasma gondii is an intracellular parasite of all mammals and birds, responsible for toxoplasmosis. In healthy individuals T. gondii infections mostly remain asymptomatic, however this parasite causes severe morbidity and mortality in immunocompromised patients and congenital toxoplasmosis in pregnant women. The consumption of raw or undercooked pork is considered as an important risk factor to develop toxoplasmosis in humans. Since effective therapeutic interventions to treat toxoplasmosis are scarce, vaccination of meat producing animals may prevent T. gondii transmission to humans. Here, we evaluated the elicited immune responses and the efficacy of a potential vaccine candidate, generated by size fractionation of T. gondii lysate proteins, to reduce the parasite burden in tissues from experimentally T. gondii infected pigs as compared to vaccination with total lysate antigens (TLA). Our results show that both the vaccine candidate and the TLA immunization elicited strong serum IgG responses and elevated percentages of CD4+CD8+IFNγ+ T cells in T. gondii infected pigs. However, the TLA vaccine induced the strongest immune response and reduced the parasite DNA load below the detection limit in brain and skeletal muscle tissue in most animals. These findings might inform the development of novel vaccines to prevent T. gondii infections in livestock species and humans.

%B Frontiers in Immunology %V 10 %8 Aug-09-2020 %G eng %R 10.3389/fimmu.2019.0222310.3389/fimmu.2019.02223.s00110.3389/fimmu.2019.02223.s00210.3389/fimmu.2019.02223.s00310.3389/fimmu.2019.02223.s00410.3389/fimmu.2019.02223.s00510.3389/fimmu.2019.02223.s006 %0 Government Document %D 2019 %T Toxi-infections alimentaires en Belgique - Rapport annuel 2018 %A Sarah Denayer %E Bavo Verhaegen %Y Koenraad Van Hoorde %K Salmonella %K toxi-infection alimentaire %K toxin %X

En 2018, 397 toxi-infections alimentaires collectives ont été enregistrées en Belgique par le LNR TIA. Il s’agit du nombre le plus élevé depuis le début des enregistrements en Belgique (1999). Au total, au moins 2 216 personnes sont tombées malades et 23 personnes ont été hospitalisées.  Bacillus cereus a été l'agent le plus souvent rapporté comme étant à l'origine d'un foyer de toxi-infection alimentaire en 2018.

Norovirus et Salmonella ont été les deuxièmes agents les plus souvent identifiés comme cause d'infections alimentaires. Salmonella a provoqué un foyer dans plusieurs écoles et au moins 546 enfants sont tombés malades. Campylobacter et E. coli O157 pathogène font partie des autres germes identifiés. Des amines biogènes telles que l'histamine et la tyramine ont chacune déclenché un foyer d’origine alimentaire. Pour la tyramine, il s’agit de la première notification en Belgique.

Les sources d'infection sont très diverses, mais ce sont surtout des repas composés (60,7%) qui ont été envoyés au laboratoire pour analyse. Pour 55,2% des foyers, c’est dans un restaurant que l'exposition à une denrée alimentaire contaminée a eu lieu.

%C Brussels, Belgium %P 39 %8 oct 2019 %G eng %M D/2019/14.440/46 %& 1 %0 Report %D 2019 %T Voedselvergiftigingen in België en in Vlaanderen 2018 %A Sarah Denayer %A Bavo Verhaegen %A Koenraad Van Hoorde %A Katelijne Dierick %K voedselvergiftiging; voedseltoxi-infectie %X

In 2018 werden in België 397 collectieve voedseltoxi-infecties geregistreerd door het NRL-VTI. Dit is het hoogste cijfer sinds de registraties in België (1999).

In totaal werden minstens 2216 personen ziek en werden 23 personen gehospitaliseerd.

Bacillus cereus was het meest gerapporteerde agens als oorzaak van een uitbraak in 2018.

Norovirus en Salmonella waren het tweede meest geïdentificeerde agens als oorzaak van voedselinfecties. Salmonella was verantwoordelijk voor een uitbraak in verschillende scholen en veroorzaakte daarbij minstens 546 zieke kinderen.

Andere geïdentificeerde kiemen zijn Campylobacter en pathogene E. coli O157.

Biogene aminen zoals histamine en tyraminen veroorzaakten elk één voedselgebonden uitbraak. Voor tyramine is dit de eerste melding in België.

De bron van de infectie is heel divers en vooral samengestelde maaltijden (60.7%) werden voor onderzoek doorgestuurd naar het laboratorium.

Restaurants waren in 55.2% van de uitbraken de plaats van blootstelling aan een besmet voedingsmiddel.

%C Brussels, Belgium %P 39 %8 10/2019 %G eng %M D/2019/14.440/48 %0 Report %D 2019 %T Voedselvergiftigingen in Belgie - Jaaroverzicht 2018 %A Sarah Denayer %A Bavo Verhaegen %A Koenraad Van Hoorde %A Katelijne Dierick %K foodborne outbreak %K Salmonella %K toxin %X

In 2018 werden in België 397 collectieve voedseltoxi-infecties geregistreerd door het NRL-VTI. Dit is het hoogste cijfer sinds de registraties in België (1999).

In totaal werden minstens 2216 personen ziek en werden 23 personen gehospitaliseerd.

Bacillus cereus was het meest gerapporteerde agens als oorzaak van een uitbraak in 2018.

Norovirus en Salmonella waren het tweede meest geïdentificeerde agens als oorzaak van voedselinfecties. Salmonella was verantwoordelijk voor een uitbraak in verschillende scholen en veroorzaakte daarbij minstens 546 zieke kinderen.

Andere geïdentificeerde kiemen zijn Campylobacter en pathogene E. coli O157.

Biogene aminen zoals histamine en tyraminen veroorzaakten elk één voedselgebonden uitbraak. Voor tyramine is dit de eerste melding in België.

De bron van de infectie is heel divers en vooral samengestelde maaltijden (60.7%) werden voor onderzoek doorgestuurd naar het laboratorium.

Restaurants waren in 55.2% van de uitbraken de plaats van blootstelling aan een besmet voedingsmiddel.

%C Brussels, Belgium %P 39 %8 10/2019 %G eng %M D/2019/14.440/46 %0 Journal Article %J Microbiol Resour Announc %D 2019 %T Whole-Genome Sequencing of Multidrug-Resistant Escherichia coli Strains Harboring the Gene, Isolated from Seawater of the Algiers Coast in Algeria. %A M Berrazeg %A A Deriet %A Sigrid C.J. De Keersmaecker %A Bavo Verhaegen %A Kevin Vanneste %A N Botteldoorn %A Nancy Roosens %A F Mouffok %A R Drali %X

Colistin resistance has emerged worldwide and is threatening the treatment efficacy of multiresistant strains in humans and animals. Here, we communicate the whole-genome sequencing (WGS) of two colistin-resistant strains, M49 and M78, with genomes sizes of 4,947,168 and 5,178,716 bp, respectively, isolated from seawaters of the Algiers coast.

%B Microbiol Resour Announc %V 8 %8 2019 Aug 22 %G eng %N 34 %R 10.1128/MRA.00638-19 %0 Journal Article %J Microbiol Resour Announc %D 2019 %T Whole-Genome Sequencing of Six Strains of Salmonella enterica Isolated from Imported Meat in Algeria. %A A Deriet %A M Berrazeg %A Sigrid C.J. De Keersmaecker %A N Botteldoorn %A Kevin Vanneste %A Bavo Verhaegen %A Nancy Roosens %A F Mouffok %A R Drali %X

Nontyphoidal (NTS) is one of the main causes of foodborne disease worldwide. In this report, we announce the first whole-genome sequencing of six strains of isolated from imported meat in Algeria. The genome sizes ranged from 4,601,209 to 4,958,962 bp. Antimicrobial resistance (AMR) genes, plasmids, and virulence factors were detected.

%B Microbiol Resour Announc %V 8 %8 2019 Aug 29 %G eng %N 35 %R 10.1128/MRA.00615-19 %0 Journal Article %J Int J Parasitol %D 2018 %T Pork as a source of transmission of Toxoplasma gondii to humans: a parasite burden study in pig tissues after infection with different strains of Toxoplasma gondii as a function of time and different parasite stages. %A I. Gisbert Algaba %A Bavo Verhaegen %A Jennes, Malgorzata %A Mizanur Rahman %A Wim Coucke %A Cox, Eric %A Dorny, Pierre %A Katelijne Dierick %A Stéphane De Craeye %X

Toxoplasma gondii is an ubiquitous apicomplexan parasite which can infect any warm-blooded animal including humans. Humans and carnivores/omnivores can also become infected by consumption of raw or undercooked infected meat containing muscle cysts. This route of transmission is considered to account for at least 30% of human toxoplasmosis cases. To better assess the role of pork as a source of infection for humans, the parasite burden resulting from experimental infection with different parasite stages and different strains of T. gondii during the acute and chronic phases was studied. The parasite burden in different tissues was measured with a ISO 17025 validated Magnetic Capture-quantitative PCR. A high burden of infection was found in heart and lungs during the acute phase of infection and heart and brain were identified as the most parasitised tissues during the chronic phase of infection, independent of the parasite stage and the strain used. Remarkably, a higher parasite burden was measured in different tissues following infection with oocysts of a type II strain compared with a tissue cyst infection with three strains of either type II or a type I/II. However, these results could have been affected by the use of different strains and euthanasia time points. The parasite burden resulting from a tissue cyst infection was not significantly different between the two strains.

%B Int J Parasitol %V 48 %8 2018 Jun %G eng %N 7 %R 10.1016/j.ijpara.2017.12.009 %0 Report %D 2018 %T Toxi-infections alimentaires en Belgique - Rapport annuel 2017 %A Sarah Denayer %A Laurence Delbrassinne %A Bavo Verhaegen %A N Botteldoorn %K toxi-infection alimentaire %X

En 2017, 304 toxi-infections alimentaires collectives ont été enregistrées en Belgique par le LNR TIA.

Au total, au moins 1409 personnes sont tombées malades et 49 personnes ont été hospitalisées.

Campylobacter était l'agent le plus souvent rapporté comme étant à l'origine d'un foyer de toxi-infection alimentaire en 2017.

Le norovirus et l’histamine étaient les deuxièmes agents les plus identifiés comme cause d'infections alimentaires. Les poissons gras tels que le thon ou les préparations à base de thon étaient la source présentant le plus de risques lors des foyers d’histamine.

Salmonella, Bacillus cereus, Clostridium perfringens et E. coli O157 pathogène faisaient partie des autres germes identifiés.

Les sources d'infection sont très diverses mais ce sont surtout des repas composés (43.4%) qui ont été transmis au laboratoire en vue d’une analyse.

Pour 52.6% des foyers, c’est dans un restaurant que l'exposition à une denrée alimentaire contaminée s'est produite.

%I Sciensano %C Brussels, Belgium %P 36 %8 2018 %G eng %M D/2018/14.440/50 %& 1 %0 Report %D 2018 %T Voedselvergiftigingen in België - Jaaroverzicht 2017 %A Sarah Denayer %A Laurence Delbrassinne %A Bavo Verhaegen %A N Botteldoorn %K voedselvergiftiging; voedseltoxi-infectie %X

In 2017 werden in België 304 collectieve voedseltoxi-infecties geregistreerd door het NRL-VTI.

In totaal werden minstens 1409 personen ziek en werden 49 personen gehospitaliseerd.

Campylobacter was het meest gerapporteerde agens als oorzaak van een uitbraak in 2017.

Norovirus en Histamine waren het tweede meest geïdentificeerde agens als oorzaak van voedselinfecties. Vetrijke vis zoals tonijn of tonijnbereidingen waren de meest risicovolle bron voor de histamine uitbraken.

Andere geïdentificeerde kiemen zijn Salmonella, Bacillus cereus, Clostridium perfringens en pathogene E. coli O157.

De bron van de infectie is heel divers en vooral samengestelde maaltijden (43.4%) werden voor onderzoek doorgestuurd naar het laboratorium.

Restaurants waren in 52.6% van de uitbraken de plaats van blootstelling aan een besmet voedingsmiddel.

%I Sciensano %C Brussels, Belgium %P 37 %8 2018 %G eng %M D/2018/14.440/49 %& 1 %0 Report %D 2017 %T Intoxications alimentaires en Belgique en 2016 %A Sarah Denayer %A Laurence Delbrassinne %A Bavo Verhaegen %A N Botteldoorn %K foyer %K intoxication %K toxi-infection alimentaire %I WIV-ISP %C Bruxelles, Belgique %P 37 %8 07/2017 %G eng %M D/2017/2505/14 %0 Conference Proceedings %B Labinfo - FAVV %D 2017 %T Opsporen van STEC-uitscheidende runderen aan de hand van Droplet Digital PCR: Een detectie met duizenden druppels. %A Bavo Verhaegen %A E. Van Coillie %A K. De Reu %X

De darmbacterie Escherichia coli is een over het algemeen onschadelijke bacterie die deel uitmaakt van de gezonde darmflora bij mens en dier. Toch kunnen enkele types E. coli, zoals de Shiga-toxine producerende E. coli of STEC, milde tot zeer ernstige ziektes veroorzaken bij de mens. Het vermogen van deze bacterie om Shiga-toxines als specifieke gifstoffen te produceren kan leiden tot diarree, bloedstollingstoornissen en nierfalen met soms de dood tot gevolg, vooral bij jonge kinderen en ouderen (1). Herkauwers en vooral runderen worden beschouwd als het belangrijkste reservoir van STEC. Terwijl deze dieren zelf zelden of nooit ziektetekens vertonen, liggen ze vaak aan de basis van STEC uitbraken bij de mens door direct contact met de besmette dieren of door voeding gecontamineerd met STEC. Hoe STEC zich weet te onderhouden binnen de runderpopulatie is echter nog niet volledig duidelijk. Geïnfecteerde dieren kunnen opgespoord worden door STEC-aanwezigheid te bepalen in mest. Sommige runderen scheiden opmerkelijk hoge aantallen STEC uit. Deze worden “super-shedders” genoemd en spelen waarschijnlijk een cruciale rol in het onderhouden van STEC infecties in de runderpopulatie (2). Het bepalen van de uitgescheiden hoeveelheden STEC in mest kan veel duidelijkheid scheppen omtrent de overdracht van STEC, de contaminatiebronnen en de effectiviteit van bepaalde behandelingen op hoeveniveau.

%B Labinfo - FAVV %V 16 %& 32 %0 Report %D 2017 %T Voedselvergiftigingen in België en Vlaanderen in 2016 %A Sarah Denayer %A Laurence Delbrassinne %A Bavo Verhaegen %A N Botteldoorn %K uitbraak %K voedseltoxi-infecties %K Voedselvergiftigingen %I WIV-ISP %C Brussel, België %P 37 %8 07/2017 %G eng %M D/2017/2505/15 %0 Conference Proceedings %B VMT Voedingsmiddelen, management en technologie %D 2016 %T Analyseren van duizenden druppels – Voedselveiligheidsrisico’s detecteren met droplet PCR %A Bavo Verhaegen %A E. Van Coillie %A M. De Loose %A I. Taverniers %A K. De Reu %X

Het ILVO ontwikkelt de nieuwe droplet digital PCR. Deze heeft ten opzichte van de kwantitatieve PCR als belangrijk voordeel dat een standaardcurve op basis van referentiemateriaal achterwege kan blijven. Zeker nu monsters gemakkelijker kunnen worden verdeeld in zeer kleine deeltjes of druppeltjes neemt de populariteit van deze methode toe bij het detecteren van pathogenen en virussen, maar ook voor ggo’s en allergenen.

%B VMT Voedingsmiddelen, management en technologie %7 10 %& 20 %0 Journal Article %J Toxins (Basel) %D 2016 %T Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces. %A Bavo Verhaegen %A De Reu, Koen %A de Zutter, Lieven %A Verstraete, Karen %A Heyndrickx, Marc %A Van Coillie, Els %K Animals %K Cattle %K Feces %K Genes, Bacterial %K polymerase chain reaction %K Shiga-Toxigenic Escherichia coli %K virulence %X

Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan(®) Environmental Master Mix 2.0; UMM: TaqMan(®) Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material.

%B Toxins (Basel) %V 8 %8 2016 05 18 %G eng %N 5 %R 10.3390/toxins8050157 %0 Journal Article %J Int J Food Microbiol %D 2016 %T Evaluation of detection methods for non-O157 Shiga toxin-producing Escherichia coli from food. %A Bavo Verhaegen %A Inge Van Damme %A Heyndrickx, Marc %A N Botteldoorn %A Mohamed Elhadidy %A Verstraete, Karen %A Katelijne Dierick %A Sarah Denayer %A de Zutter, Lieven %A De Reu, Koen %K Animals %K Cattle %K Food Contamination %K Food Microbiology %K Humans %K Lettuce %K Meat %K Real-Time Polymerase Chain Reaction %K Shiga-Toxigenic Escherichia coli %K Soybeans %X

Shiga toxin-producing Escherichia coli (STEC) remains a major foodborne pathogen of concern across the globe. Rapid detection and isolation of this pathogen is of great importance for public health reasons. In this study the detection and isolation of four non-O157 STEC strains (O26, O103, O111, O145) from different artificially contaminated matrices, namely ground (minced) beef, cattle carcass swab, lettuce mix and sprouted soy beans, were evaluated. Low amounts of STEC were used (0.25-1.40 cfu/g) to spike the samples. All samples were enriched in parallel in Buffered Peptone Water (BPW) and Brila broth. After enrichment, detection was performed using real-time PCR (qPCR), and isolation using two chromogenic agar media, CHROMagar™ STEC and ChromID™ EHEC. Inoculation on the agar media was performed either directly after enrichment or after the use of an acid treatment procedure. Furthermore, the use of this procedure was also tested on naturally contaminated food products, using 150 stx-positive samples. Although the qPCR Cycle Threshold (Ct) values were lower after enrichment in Brila broth, no significant differences in recovery were observed between both enrichment broths. Both agar media were equally suitable for the isolation of STEC, although a significantly higher recovery was obtained when using both agar media in parallel. For samples with a Ct value above 25, an acid treatment step prior to isolation ensured a significant improvement in the recovery of STEC due to the reduction in background microbiota. This acid treatment procedure proved especially useful for the isolation of STEC from sprouted soy bean samples.

%B Int J Food Microbiol %V 219 %P 64-70 %8 2016 Feb 16 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26736066?dopt=Abstract %& 64 %R 10.1016/j.ijfoodmicro.2015.12.006 %0 Generic %D 2016 %T Genetic relatedness and virulence profile of Shiga toxin-producing Escherichia coli isolated from food in Belgium %A Bavo Verhaegen %A E. Barbau-Piednoir %A L. De Zutter %A Inge Van Damme %A Sarah Denayer %A Sigrid C.J. De Keersmaecker %A Heyndrickx,M. %K Belgium %K conference %K Escherichia coli %K food %K Food Microbiology %K Genetic %K Luminex %K microbiology %K ON %K profile %K virulence %K VTEC/STEC %B Twenty-first Conference on Food Microbiology %I NA %C NA %8 0/0/2016 %G eng %N BSFM %1 5283 %2 09/15-16/2016 %0 Report %D 2016 %T Voedselvergiftigingen in België 2016 %A Sarah Denayer %A Laurence Delbrassinne %A Bavo Verhaegen %A N Botteldoorn %K uitbraak %K voedsel-toxi infectie %K voedselvergiftiging %I WIV-ISP %C Brussels, Belgium %P 37 %8 07/2017 %G eng %M D/2017/2505/13 %0 Journal Article %J Int J Environ Res Public Health %D 2015 %T Comparison of Six Chromogenic Agar Media for the Isolation of a Broad Variety of Non-O157 Shigatoxin-Producing Escherichia coli (STEC) Serogroups. %A Bavo Verhaegen %A De Reu, Koen %A Heyndrickx, Marc %A de Zutter, Lieven %K agar %K Culture Media %K Escherichia coli Infections %K Humans %K Serogroup %K Shiga-Toxigenic Escherichia coli %X

The isolation of non-O157 STEC from food samples has proved to be challenging. The selection of a suitable selective isolation agar remains problematic. The purpose of this study was to qualitatively and quantitatively evaluate six chromogenic agar media for the isolation of STEC: Tryptone Bile X-glucuronide agar (TBX), Rainbow® Agar O157 (RB), Rapid E. coli O157:H7 (RE), Modified MacConkey Agar (mMac), CHROMagarTM STEC (Chr ST) and chromIDTM EHEC (Chr ID). During this study, 45 E. coli strains were used, including 39 STEC strains belonging to 16 different O serogroups and 6 non-STEC E. coli. All E. coli strains were able to grow on TBX and RB, whereas one STEC strain was unable to grow on Chr ID and a number of other STEC strains did not grow on mMac, CHROMagar STEC and Rapid E. coli O157:H7. However, only the latter three agars were selective enough to completely inhibit the growth of the non-STEC E. coli. Our conclusion was that paired use of a more selective agar such as CHROMagar STEC together with a less selective agar like TBX or Chr ID might be the best solution for isolating non-O157 STEC from food.

%B Int J Environ Res Public Health %V 12 %8 2015 Jun 17 %G eng %N 6 %R 10.3390/ijerph120606965 %0 Journal Article %J Microbiology %D 2015 %T Genetic diversity of Shiga toxin-producing Escherichia coli O157 : H7 recovered from human and food sources. %A Mohamed Elhadidy %A Elkhatib, Walid F %A Eman A Abo Elfadl %A Verstraete, Karen %A Sarah Denayer %A Barbau-Piednoir, Elodie %A de Zutter, Lieven %A Bavo Verhaegen %A De Rauw, Klara %A Piérard, Denis %A De Reu, Koen %A Heyndrickx, Marc %K Escherichia coli Infections %K Escherichia coli O157 %K Food Microbiology %K Genes, Bacterial %K Genetic Variation %K Genotype %K Humans %K Multilocus Sequence Typing %K Polymorphism, Genetic %K Shiga Toxin %X

The aim of this study was to identify an epidemiological association between Shiga toxin-producing Escherichia coli O157 : H7 strains associated with human infection and with food sources. Frequency distributions of different genetic markers of E. coli O157 : H7 strains recovered from human and food sources were compared using molecular assays to identify E. coli O157 : H7 genotypes associated with variation in pathogenic potential and host specificity. Genotypic characterization included: lineage-specific polymorphism assay (LSPA-6), clade typing, tir (A255T) polymorphism, Shiga toxin-encoding bacteriophage insertion site analysis and variant analysis of Shiga toxin 2 gene (stx2a and stx2c) and antiterminator Q genes (Q933 and Q21). The intermediate lineage (LI/II) dominated among both food and human strains. Compared to other clades, clades 7 and 8 were more frequent among food and human strains, respectively. The tir (255T) polymorphism occurred more frequently among human strains than food strains. Q21 and Q933 + Q21 were found at significantly higher frequencies among food and human strains, respectively. Moreover, stx2a and stx2a+c were detected at significantly higher frequencies among human strains compared to food strains. Bivariate analysis revealed significant concordance (P<0.05) between the LSPA-6 assay and the other typing methods. Multivariable regression analysis suggested that tir (255T) was the most distinctive genotype that can be used to detect bacterial clones with potential risk for human illness from food sources. This study supported previous reports of the existence of diversity in genetic markers among different isolation sources by including E. coli O157 : H7 strains from both food and human sources. This might enable tracking genotypes with potential risk for human illness from food sources.

%B Microbiology %V 161 %P 112-9 %8 2015 Jan %G eng %N Pt 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25411313?dopt=Abstract %R 10.1099/mic.0.083063-0 %0 Journal Article %J J Food Prot %D 2015 %T Growth of Stressed Strains of Four Non-O157 Shiga Toxin-Producing Escherichia coli Serogroups in Five Enrichment Broths. %A Bavo Verhaegen %A De Reu, Koen %A Heyndrickx, Marc %A Inge Van Damme %A de Zutter, Lieven %K Culture Media %K Serogroup %K Shiga-Toxigenic Escherichia coli %X

The purpose of this study was to evaluate (i) the behavior of several strains of non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O103, O111, and O145) exposed to different stress conditions and (ii) the growth dynamics of stressed and nonstressed non-O157 STEC cells in five enrichment media. STEC strains were exposed to acid, cold, and freeze stresses. Lethal and sublethal injuries were determined by plating in parallel on selective and nonselective agar media. Freeze stress (8 days, 20°C) caused the most lethal (95.3% ± 2.5%) injury, as well as the most sublethal (89.1% ± 8.8%) injury in the surviving population. Growth of stressed and nonstressed pure cultures of non-O157 STEC on modified tryptic soy broth, buffered peptone water (BPW), BPW with sodium pyruvate, Brila, and STEC enrichment broth (SEB) was determined using total viable counts. To compare growth capacities, growth after 7 and 24 h of enrichment was measured; lag phases and maximum growth rates were also calculated. In general, growth on BPW resulted in a short lag phase followed by a high maximum growth rate during the enrichment of all tested strains when using all three stress types. Furthermore, BPW ensured the highest STEC count after 7 h of growth. Supplementing the medium with sodium pyruvate did not improve the growth dynamics. The two selective media, Brila and SEB, were less efficient than BPW, but Brila's enrichment performance was remarkably better than that of SEB. This study shows that irrespective of the effect of background flora, BPW is still recommended for resuscitation of non-O157 STEC.

%B J Food Prot %V 78 %8 2015 Nov %G eng %N 11 %R 10.4315/0362-028X.JFP-15-019 %0 Journal Article %J J Food Prot %D 2014 %T Evaluation of a new chromogenic medium for direct enumeration of Campylobacter in poultry meat samples. %A Seliwiorstow, Tomasz %A Julie Baré %A Bavo Verhaegen %A Uyttendaele, Mieke %A de Zutter, Lieven %K agar %K Animals %K Campylobacter %K Colony Count, Microbial %K Culture Media %K Food Contamination %K Food Microbiology %K Meat %K Poultry %X

The present study was conducted to compare Campylobacter counts obtained by three selective media: modified charcoal cefoperazonedeoxycholate agar (mCCDA), Campy Food agar (CFA), and a novel agar RAPID' Campylobacter agar. Analysis of 12 artificially and 36 naturally contaminated samples indicated no significant differences in Campylobacter counts obtained with all three selective media. Lin's concordance correlation coefficient (CCC) and the Bland-Altman plot revealed a high level of agreement between Campylobacter counts when evaluating RAPID versus mCCDA and CFA plates. RAPID agar was the only medium tested that could effectively suppress the growth of the background microflora with naturally contaminated samples. Results of this study clearly indicated that RAPID agar is highly selective without loss of sensitivity for recovering Campylobacter. Results obtained are in agreement with those for other commonly used media; therefore, RAPID medium is suitable for Campylobacter enumeration in poultry meat samples.

%B J Food Prot %V 77 %P 2111-4 %8 2014 Dec %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/25474058?dopt=Abstract %R 10.4315/0362-028X.JFP-14-237 %0 Generic %D 2014 %T Genetic diversity of shiga-toxin producing Escherichia coli O157:H7 recovered from human and food sources in Belgium %A Mohamed Elhadidy %A W. Elkhatib %A Elfadi,E.A.A %A Verstraeten,K. %A Sarah Denayer %A E. Barbau-Piednoir %A L. De Zutter %A Bavo Verhaegen %A K. De Rauw %A Denis Piérard %A K De Reu %A Heyndrickx,M. %K Belgium %K conference %K diversity %K Escherichia coli %K Escherichia coli O157:H7 %K food %K Food Microbiology %K food sources %K Genetic %K Human %K microbiology %K ON %K Shiga Toxin %K STEC %B Nineteenth Conference on Food Microbiology %I NA %C NA %8 0/0/2014 %G eng %N =Belgian Society for Food Microbiology vzw/aslb (BSFM %1 38709 %2 18-19/09/2014 %0 Generic %D 2013 %T Enrichment efficiency of cold and freeze stressed O26 and O111 STEC serotypes %A Bavo Verhaegen %A Heyndrickx,M. %A N Botteldoorn %A L. De Zutter %K conference %K Efficiency %K food %K Food Microbiology %K microbiology %K STEC %B 18 th conference of food microbiology %I NA %C NA %8 12/9/2013 %G eng %N BSFM %1 340 %2 12-13 september 2013 %0 Journal Article %J Vlaams Diergeneeskundig Tijdschrift %D 2012 %T Knelpunten in aquacultuur: enkele risicovolle aspecten van de vroege levensstadia van de vis. %A Bavo Verhaegen %A A. Rekecki %A A. Decostere %A W. Van den Broeck %X

Aquacultuur is een zeer snel groeiende sector, maar er zijn nog talrijke knelpunten bij het opkweken van larven (larvicultuur) en van adulte vissen. Veel van deze knelpunten zijn van nutritionele aard. Het voedingsregime is nog vaak onvoldoende afgesteld op de behoeften van specifieke vissoorten waardoor uithongering een grote invloed blijft hebben. Deze problemen kunnen opgelost worden door de ontwikkeling van het gastro-intestinale stelsel per vissoort in kaart te brengen. Parameters, zoals de enterocytenhoogte, de aanwezigheid van supranucleaire vacuoles in de enterocyten en ‘the point of no return’ kunnen als maatstaf gebruikt worden om uithongering te monitoren en te voorkomen. Aan de hand van deze gegevens kan een optimaal voedingsregime met rotiferen (radardiertjes), Artemia  (pekelkreeftjes) en Copepoda (eenoogkreeftjes) uitgewerkt worden.

Aquaculture is a fast growing sector. However, during larviculture and fish husbandry, many bottlenecks still occur. Many problems concern nutritional factors and can be avoided by a clear understanding of the development of the gastrointestinal system of the various fish species. Parameters, such as enterocyte height, the presence of enterocytic supranuclear vacuoles and 'the point of no return', can be used to monitor and prevent food deprivation. This information can also be used to compose a feeding regime with rotifers, Artemia en copepods.

%B Vlaams Diergeneeskundig Tijdschrift %V 81 %G eng %& 63 %0 Generic %D 0 %T Molecular characterization of human pathogenic norovirus circulating in Belgium: 10 years of systematic molecular surveillance data %A Bavo Verhaegen %A Alexandra Vodolazkaia %A Marina Mukovnikova %A Katelijne Dierick %A Koenraad Van Hoorde %G eng %0 Journal Article %D 0 %T Whole genome sequencing en voedselveiligheid in België %A K. Feys %A N Botteldoorn %A V. Cantaert %A L. De Zutter %A V. Delcenserie %A A.G. Ameryckx %A J. Mahillon %A Marcella Mori %A B. Pochet %A Nancy Roosens %A Koenraad Van Hoorde %A Bavo Verhaegen %A L. Herman %G eng