Sarah Denayer

Sudden death of a young adult associated with Bacillus cereus food poisoning.

ABSTRACT A lethal intoxication case, which occurred in Brussels, Belgium, is described. A 20-year-old man died following the ingestion of pasta contaminated with Bacillus cereus. Emetic strains of B. cereus were isolated, and high levels of cereulide (14.8 μg/g) were found in the spaghetti meal.

Novel norovirus recombinants and GII.4 sub-lineages associated with outbreaks between 2006 and 2010 in Belgium

BackgroundNoroviruses (NoVs) are an important cause of acute gastroenteritis in humans worldwide. To gain insight into the epidemiologic patterns of NoV outbreaks and to determine the genetic variation of NoVs strains circulating in Belgium, stool samples originating from patients infected with NoVs in foodborne outbreak investigations were analysed between December 2006 and December 2010.ResultsNoVs were found responsible of 11.8% of all suspected foodborne outbreaks reported in the last 4 years and the number of NoV outbreaks reported increased along the years representing more than 30% of all foodborne outbreaks in 2010. Genogroup II outbreaks largely predominated and represented more than 90% of all outbreaks. Phylogenetic analyses were performed with 63 NoV-positive samples for the partial polymerase (N = 45) and/or capsid gene (N = 35) sequences. For 12 samples, sequences covering the ORF1-ORF2 junction were obtained. A variety of genotypes was found among genogroups I and II; GII.4 was predominant followed in order of importance by GII.2, GII.7, GII.13, GI.4 and GI.7. In the study period, GII.4 NoVs variants 2006a, 2006b, 2007, 2008 and 2010 were identified. Moreover, phylogenetic analyses identified different recombinant NoV strains that were further characterised as intergenotype (GII.e/GII.4 2007, GII.e/GII.3 and GII.g/GII.1) and intersub-genotype (GII.4 2006b/GII.4 2007 and GII.4 2010/GII.4 2010b) recombinants.ConclusionsNoVs circulating in the last 4 years in Belgium showed remarkable genetic diversity either by small-scale mutations or genetic recombination. In this period, GII.4 2006b was successfully displaced by the GII.4 2010 subtype, and previously reported epidemic GII.b recombinants seemed to have been superseded by GII.e recombinants in 2009 and GII.g recombinants in 2010. This study showed that the emergence of novel GII.4 variants together with novel GII recombinants could lead to an explosion in NoV outbreaks, likewise to what was observed in 2008 and 2010. Among recombinants detected in this study, two hitherto unreported strains GII.e/GII.3 and GII.g/GII.1 were characterised. Surveillance will remain important to monitor contemporaneously circulating strains in order to adapt preventive and curative strategies.

A Review of Known and Hypothetical Transmission Routes for Noroviruses

Human noroviruses (NoVs) are considered a worldwide leading cause of acute non-bacterial gastroenteritis. Due to a combination of prolonged shedding of high virus levels in feces, virus particle shedding during asymptomatic infections, and a high environmental persistence, NoVs are easily transmitted pathogens. Norovirus (NoV) outbreaks have often been reported and tend to affect a lot of people. NoV is spread via feces and vomit, but this NoV spread can occur through several transmission routes. While person-to-person transmission is without a doubt the dominant transmission route, human infective NoV outbreaks are often initiated by contaminated food or water. Zoonotic transmission of NoV has been investigated, but has thus far not been demonstrated. The presented review aims to give an overview of these NoV transmission routes. Regarding NoV person-to-person transmission, the NoV GII.4 genotype is discussed in the current review as it has been very successful for several decades but reasons for its success have only recently been suggested. Both pre-harvest and post-harvest contamination of food products can lead to NoV food borne illness. Pre-harvest contamination of food products mainly occurs via contact with polluted irrigation water in case of fresh produce or with contaminated harvesting water in case of bivalve molluscan shellfish. On the other hand, an infected food handler is considered as a major cause of post-harvest contamination of food products. Both transmission routes are reviewed by a summary of described NoV food borne outbreaks between 2000 and 2010. A third NoV transmission route occurs via water and the spread of NoV via river water, ground water, and surface water is reviewed. Finally, although zoonotic transmission remains hypothetical, a summary on the bovine and porcine NoV presence observed in animals is given and the presence of human infective NoV in animals is discussed.

Characterization of Escherichia coli from raw poultry in Belgium and impact on the detection of Campylobacter jejuni using Bolton broth.

A comparative study examining Bolton broth and Preston broth for enrichment and reliable detection of Campylobacter jejuni (both healthy and freeze stressed cells) was performed. Tested as pure cultures, Bolton broth enabled faster resuscitation and growth of C. jejuni compared to Preston broth. When C. jejuni was co-incubated with extended-spectrum-beta-lactamase (ESBL) producing Escherichia coli isolated from Belgian poultry meat preparations, the latter dominated in the Bolton enrichment broth and crowded the mCCDA plates. This resulted in the inability to recover C. jejuni by ISO 10272-1:2006 standard method. Preston broth did not support the growth of the ESBL E. coli isolates, but showed longer detection time of C. jejuni compared to Bolton broth. The use of the same antibiotic (sodium cefoperazone) in Bolton broth and in mCCDA plates may explain the problems encountered for detection of C. jejuni, as high numbers of ESBL E. coli present after enrichment in Bolton broth, also caused overgrowth and masked the few C. jejuni colonies present on the mCCDA plates. The use of Campylobacter spp. specific real-time PCR circumvented these problems and enabled rapid detection of the pathogen after 24h enrichment in both Bolton and Preston broth, for both healthy and freeze stressed cells.

Molecular Detection and Genotyping of Noroviruses

Noroviruses (NoVs) are a major cause of gastroenteritis worldwide in humans and animals and are known as very infectious viral agents. They are spread through feces and vomit via several transmission routes involving person-to-person contact, food, and water. Investigation of these transmission routes requires sensitive methods for detection of NoVs. As NoVs cannot be cultivated to date, detection of these viruses relies on the use of molecular methods such as (real-time) reverse transcriptase polymerase chain reaction (RT-PCR). Regardless of the matrix, detection of NoVs generally requires three subsequent steps: a virus extraction step, RNA purification, and molecular detection of the purified RNA, occasionally followed by molecular genotyping. The current review mainly focused on the molecular detection and genotyping of NoVs. The most conserved region in the genome of human infective NoVs is the ORF1/ORF2 junction and has been used as a preferred target region for molecular detection of NoVs by methods such as (real-time) RT-PCR, NASBA, and LAMP. In case of animal NoVs, broad range molecular assays have most frequently been applied for molecular detection. Regarding genotyping of NoVs, five regions situated in the polymerase and capsid genes have been used for conventional RT-PCR amplification and sequencing. As the expected levels of NoVs on food and in water are very low and inhibition of molecular methods can occur in these matrices, quality control including adequate positive and negative controls is an essential part of NoV detection. Although the development of molecular methods for NoV detection has certainly aided in the understanding of NoV transmission, it has also led to new problems such as the question whether low levels of human NoV detected on fresh produce and shellfish could pose a threat to public health.

Prevalence and levels of Bacillus cereus emetic toxin in rice dishes randomly collected from restaurants and comparison with the levels measured in a recent foodborne outbreak.

Whereas the prevalence of Bacillus cereus emetic strains in the environment has been shown to be very low, there is a lack of information on the prevalence of its toxin, cereulide, in food. Yet, the rice leftovers of a family outbreak which occurred after the consumption of dishes taken away from an Asian restaurant revealed significant amounts of cereulide, reaching up to 13,200 ng/g of food. The occurrence of cereulide in rice dishes collected from various restaurants was therefore evaluated using the liquid chromatography coupled with tandem mass spectrometry method, which allows for the direct quantification of the toxin in food. The cereulide prevalence was found to be 7.4% when samples were analyzed at the day of sampling, but reached 12.9% when exposed to temperature abuse conditions (25°C). The cereulide concentrations observed in cooked rice dishes were low (approximately 4 ng/g of food). However, since little is known yet about the potential chronic toxicity of cereulide, one needs to be very careful and vigilant.

Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test

The detection and identification of botulinum neurotoxins (BoNT) is complex due to the existence of seven serotypes, derived mosaic toxins and more than 40 subtypes. Expert laboratories currently use different technical approaches to detect, identify and quantify BoNT, but due to the lack of (certified) reference materials, analytical results can hardly be compared. In this study, the six BoNT/A1–F1 prototypes were successfully produced by recombinant techniques, facilitating handling, as well as improving purity, yield, reproducibility and biosafety. All six BoNTs were quantitatively nicked into active di-chain toxins linked by a disulfide bridge. The materials were thoroughly characterized with respect to purity, identity, protein concentration, catalytic and biological activities. For BoNT/A1, B1 and E1, serotypes pathogenic to humans, the catalytic activity and the precise protein concentration were determined by Endopep-mass spectrometry and validated amino acid analysis, respectively. In addition, BoNT/A1, B1, E1 and F1 were successfully detected by immunological assays, unambiguously identified by mass spectrometric-based methods, and their specific activities were assigned by the mouse LD50 bioassay. The potencies of all six BoNT/A1–F1 were quantified by the ex vivo mouse phrenic nerve hemidiaphragm assay, allowing a direct comparison. In conclusion, highly pure recombinant BoNT reference materials were produced, thoroughly characterized and employed as spiking material in a worldwide BoNT proficiency test organized by the EQuATox consortium.

Whole genome sequence analysis of Salmonella Enteritidis PT4 outbreaks from a national reference laboratory’s viewpoint

Introduction: In April and May 2014, two suspected egg-related outbreaks of Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) were investigated by the Belgian National Reference Laboratory of Foodborne Outbreaks. Both the suspected food and human isolates being available, and this for both outbreaks, made these the ideal case study for a retrospective whole genome sequencing (WGS) analysis with the goal to investigate the feasibility of this technology for outbreak investigation by a National Reference Laboratory or National Reference Centre without thorough bioinformatics expertise. Methods: The two outbreaks were originally investigated epidemiologically with a standard questionnaire and with serotyping, phage typing, multiple-locus variable-number of tandem repeats analysis (MLVA) and antimicrobial susceptibility testing as classical microbiological methods. Retrospectively, WGS of six outbreak isolates was done on an Illumina HiSeq. Analysis of the WGS data was performed with currently available, user-friendly software and tools, namely CLC Genomics Workbench, the tools available on the server of the Center for Genomic Epidemiology and BLAST Ring Image Generator (BRIG). Results: To all collected human and food outbreak isolates, classical microbiological investigation assigned phage type PT4 (variant phage type PT4a for one human isolate) and MLVA profile 3-10-5-4-1, both of which are common for human isolates in Belgium. The WGS analysis confirmed the link between food and human isolates for each of the outbreaks and clearly discriminated between the two outbreaks occurring in a same time period, thereby suggesting a non-common source of contamination. Also, an additional plasmid carrying an antibiotic resistance gene was discovered in the human isolate with the variant phage type PT4a. Discussion: For the two investigated outbreaks occurring at geographically separated locations, the gold standard classical microbiological subtyping methods were not sufficiently discriminative to distinguish between or assign a common origin of contamination for the two outbreaks, while WGS analysis could do so. This case study demonstrated the added value of WGS for outbreak investigations by confirming and/or discriminating food and human isolates between and within outbreaks. It also proved the feasibility of WGS as complementary or even future replacing (sub)typing method for the average routine laboratory.

Evaluation of detection methods for non-O157 Shiga toxin-producing Escherichia coli from food.

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.

Food-Borne Outbreak Investigation and Molecular Typing: High Diversity of Staphylococcus aureus Strains and Importance of Toxin Detection

Staphylococcus aureus is an important aetiological agent of food intoxications in the European Union as it can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Reported enterotoxin dose levels causing food-borne illness are scarce and varying. Three food poisoning outbreaks due to enterotoxin-producing S. aureus strains which occurred in 2013 in Belgium are described. The outbreaks occurred in an elderly home, at a barbecue event and in a kindergarten and involved 28, 18, and six cases, respectively. Various food leftovers contained coagulase positive staphylococci (CPS). Low levels of staphylococcal enterotoxins ranging between 0.015 ng/g and 0.019 ng/g for enterotoxin A (SEA), and corresponding to 0.132 ng/g for SEC were quantified in the food leftovers for two of the reported outbreaks. Molecular typing of human and food isolates using pulsed-field gel electrophoresis (PFGE) and enterotoxin gene typing, confirmed the link between patients and the suspected foodstuffs. This also demonstrated the high diversity of CPS isolates both in the cases and in healthy persons carrying enterotoxin genes encoding emetic SEs for which no detection methods currently exist. For one outbreak, the investigation pointed out to the food handler who transmitted the outbreak strain to the food. Tools to improve staphylococcal food poisoning (SFP) investigations are presented.