Healing through Music...with a Lil\u27 Bit o\u27 Southern Drawl

OBJECTIVE:Since 1987 all fecal samples referred to the clinical microbiology laboratory of the UZ Brussel were screened for the presence of Shiga toxin-producing E. coli (STEC). In this study all STEC strains isolated over a period of 27 years (1987-2014) were reexamined to achieve deeper insight in the STEC infections in our patient population. METHODS:A total of 606 STEC strains from 604 patients were subjected to molecular methods for shiga toxin (stx) subtyping, detection of additional virulence genes, typing of the O-serogroups, and phylogenetic relatedness assessment of STEC O157:H7/H-. RESULTS:Since the introduction of PCR in 1991 the annual positivity rates varied between 1.1% and 2.7%. The isolation rate of STEC O157:H7/H- remained stable over the years while the isolation rate of non-O157 serotypes increased, mainly since 2011. The majority of the patients were children. Uncomplicated- and bloody diarrhea were the most prevalent gastrointestinal manifestations (respectively 51.9% and 13.6%), 4.3% of the strains were related to the hemolytic uremic syndrome (HUS), and 30.2% of the patients showed none of these symptoms. The strains were very diverse; they belonged to 72 different O-serovars and all stx subtypes except stx1d and stx2g were identified. Out of the 23 stx2f-positives one was associated with HUS and one belonged to the E. albertii species. As seen in other studies, the frequency of strains of the O157:H7/H- serotype and strains carrying stx2a, eaeA and ehxA was higher in patients with HUS. CONCLUSIONS:The characteristics and trends of STEC infection seen in our patient population are similar to those noted in other countries. STEC infections in our hospital are mainly sporadic, and a substantial portion of the patients were asymptomatic carriers. Human STEC Stx2f infection was less rare than previously assumed and we report the first Belgian STEC stx2f HUS case and stx2f positive E. albertii infection

O157:H7 and O104:H4 Vero/Shiga toxin-producing Escherichia coli outbreaks: respective role of cattle and humans

An enteroaggregative Verotoxin (Vtx)-producing Escherichia coli strain of serotype O104:H4 has recently been associated with an outbreak of haemolytic-uremic syndrome and bloody diarrhoea in humans mainly in Germany, but also in 14 other European countries, USA and Canada. This O104:H4 E. coli strain has often been described as an enterohaemorrhagic E. coli (EHEC), i.e. a Vtx-producing E. coli with attaching and effacing properties. Although both EHEC and the German O104:H4 E. coli strains indeed produce Vtx, they nevertheless differ in several other virulence traits, as well as in epidemiological characteristics. For instance, the primary sources and vehicles of typical EHEC infections in humans are ruminants, whereas no animal reservoir has been identified for enteroaggregative E. coli (EAggEC). The present article is introduced by a brief overview of the main characteristics of Vtx-producing E. coli and EAggEC. Thereafter, the O104:H4 E. coli outbreak is compared to typical EHEC outbreaks and the virulence factors and host specificity of EHEC and EAggEC are discussed. Finally, a renewed nomenclature of Vtx-producing E. coli is proposed to avoid more confusion in communication during future outbreaks and to replace the acronym EHEC that only refers to a clinical condition

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

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 Escherichia coli (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

A practical method to implement strain-level metagenomics-based foodborne outbreak investigation and source tracking in routine

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 Escherichia coli (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 E. coli 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

Staphylococcus jettensis sp nov., a coagulase-negative staphylococcal species isolated from human clinical specimens

Eight coagulase-negative, novobiocin-susceptible staphylococcal strains were isolated from human clinical specimens at two different Belgian medical facilities. All strains were non-motile, Gram-stain-positive, catalase-positive cocci. DNA G+C content, peptidoglycan type, menaquinone pattern, the presence of teichoic acid and cellular fatty acid composition were in agreement with the characteristics of species of the genus Staphylococcus. Sequencing of the 16S rRNA gene and four housekeeping genes (dnaJ, tuf, gap and rpoB) demonstrated that these strains constitute a separate taxon within the genus Staphylococcus. Less than 41 % DNA-DNA hybridization with the most closely related species of the genus Staphylococcus (Staphylococcus haemolyticus, Staphylococcus hominis and Staphlococcus lugdunensis) was observed. Key biochemical characteristics that allowed these bacteria to be distinguished from their nearest phylogenetic neighbours are arginine dihydrolase positivity, ornithine decarboxylase negativity and inability to produce acid aerobically from D-mannose, a-lactose and turanose. Acid is produced aerobically from trehalose. Based on these results, a novel species of the genus Staphylococcus is described and named Staphylococcus jettensis sp. nov. The type strain is SE0110(T) (=LMG 26879(T)=CCUG 62657(T)=DSM 26618(T))

Factors associated with the continuum of care of HIV infected patients in Belgium

BREACHinfo:eu-repo/semantics/nonPublishe

Enteropathogenic Escherichia coli

peer reviewedSerogroup O80 was detected in 40% of 104 enteropathogenic Escherichia coli isolates from calves with diarrhea from 42 farms in Belgium during 2008‒2015. These isolates harbored the eae-ξ and fliCH2 genes, similar to the O80 attaching-effacing Shigatoxigenic E. coli isolates found in humans in France. This strain might be emerging

The benefits of whole genome sequencing for foodborne outbreak investigation from the perspective of a national reference laboratory in a smaller country

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 Escherichia coli (STEC) O157:H7 (stx1+, stx2+, eae+) outbreak (2012) and a STEC O157:H7 (stx2+, eae+) 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 (stx1+, stx2+, eae+) and STEC O157:H7 (stx2+, eae+) 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

Validation strategy of a bioinformatics whole genome sequencing workflow for Shiga toxin-producing Escherichia coli using a reference collection extensively characterized with conventional methods

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