Rapid, accurate bacterial identification in biological samples is an important task for microbiology laboratories, which 16S~rRNA gene Sanger sequencing of cultured isolates frequently used. In contrast, next-generation does not require intermediate culturing steps and can be directly applied on communities, but its performance has been extensively evaluated. We present a comparative evaluation second (Illumina) third (Oxford Nanopore Technologies (ONT)) generation technologies 16S targeted...

ABSTRACT 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) gained prominence may offer advantages over sequencing, particularly recent introduction R10 chemistry, which promises much lower error rates than R9 chemistry. However, limited information is available on its performance...

Despite being a well-established research method, the use of whole genome sequencing (WGS) for routine molecular typing and pathogen characterization remains substantial challenge due to required bioinformatics resources and/or expertise. Moreover, many national reference laboratories centers, as well other working under quality system, require extensive validation demonstrate that employed methods are 'fit-for-purpose' provide high-quality results. A harmonized framework with guidelines WGS...

Whole genome sequencing (WGS) has proven to be the ultimate tool for bacterial isolate characterization and relatedness determination. However, standardized harmonized workflows, e.g. DNA extraction, are required ensure robust exchangeable WGS data. Data sharing between (inter)national laboratories is essential support foodborne pathogen control, including outbreak investigation. This study evaluated eight commercial preparation kits their potential influence on: (i) quality Nextera XT...

Linezolid is a critically important antibiotic used to treat human infections caused by MRSA and VRE. While linezolid not licensed for food-producing animals, linezolid-resistant (LR) isolates have been reported in European countries, including Belgium.To: (i) assess LR occurrence staphylococci enterococci isolated from different Belgian animals 2019 through selective monitoring; (ii) investigate the genomes relatedness of these isolates.Faecal samples (n = 1325) nasal swab 148) were...

The influx of whole genome sequencing (WGS) data in the public health and clinical diagnostic sectors has created a need for analysis methods bioinformatics expertise, which can be bottleneck many laboratories. At Sciensano, Belgian national institute, an intuitive user-friendly tool portal was implemented using Galaxy, open-source platform workflow creation. Galaxy @Sciensano instance is available to both internal external scientists offers wide range tools provided by community,...

The Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) networks 'Seq4AMR' and 'B2B2B AMR Dx' were established to promote collaboration between microbial whole genome sequencing (WGS) antimicrobial resistance (AMR) stakeholders. A key topic discussed was the frequent variability in results obtained different WGS-related gene prediction workflows. Further, comparative benchmarking studies are difficult perform due differences accuracy a lack of agreement naming genes (semantic...

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 standardization, variation regarding bioinformatics workflows parameters, however, complicates interoperability among (inter)national laboratories. We present a validation strategy applied to workflow Illumina data that performs Shiga toxin-producing Escherichia coli (STEC)...

Gallibacterium anatis is an opportunistic pathogen, previously associated with deaths in poultry, domestic birds, and occasionally humans. We obtained G. isolates from bronchoalveolar lavage samples of 10 calves bronchopneumonia unresponsive to antimicrobial therapy. Collected were multidrug-resistant extensively drug-resistant, exhibiting resistance against 5-7 classes drugs. Whole-genome sequencing revealed 24 different antimicrobial-resistance determinants, including genes not described...

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 WGS, mainly because significant investments required to generate and analyze data a limited amount samples. To facilitate this process incite policy makers support its implementation, Shiga toxin-producing Escherichia coli (STEC) O157:H7 (stx1+, stx2+, eae+)...

The use of whole-genome sequencing (WGS) for routine typing bacterial isolates has increased substantially in recent years. For Mycobacterium tuberculosis (MTB), particular, WGS the benefit drastically reducing time required to generate results compared most conventional phenotypic methods. Consequently, a multitude solutions analyzing MTB data have been developed, but their successful integration clinical and national reference laboratories is hindered by requirement validation, which...

Whole genome sequencing (WGS) has become the reference standard for bacterial outbreak investigation and pathogen typing, providing a resolution unattainable with conventional molecular methods. Data generated Illumina sequencers can however only be analysed after run finished, thereby losing valuable time during emergency situations. We evaluated both effect of decreasing overall time, also protocol to transfer convert intermediary files by enabling real-time data analysis multiple samples...

Abstract Recently, the unexpected presence of a viable unauthorized genetically modified bacterium in commercialized food enzyme (protease) product originating from microbial fermentation process has been notified at European level (RASFF 2019.3332). This finding was made possible thanks to use next-generation sequencing technology, as reported this study. Whole-genome used characterize genetic modification comprising sequence pUB110 shuttle vector (GenBank: M19465.1), harbouring...

Genetically modified microorganisms (GMM) are frequently employed for manufacturing microbial fermentation products such as food enzymes or vitamins. Although the product is required to be pure, GMM contaminations have repeatedly been reported in numerous commercial produce types, leading several rapid alerts at European level. The aim of this study was investigate added value shotgun metagenomic high-throughput sequencing confirm and extend results classical analysis methods genomic...

Through staphylococcal enterotoxin (SE) production, Staphylococcus aureus is a common cause of food poisoning. Detection poisoning (SFP) mostly performed using immunoassays, which, however, only detect five 27 SEs described to date. Polymerase chain reactions are, therefore, frequently used in complement identify bigger arsenal SE at the gene level (se) but are labor-intensive. Complete se profiling isolates from different sources, i.e., and human cases, is, important provide an indication...

In recent years, there has been an increase in the number of reported cases Mycobacterium microti infection various animals, which can interfere with ante-mortem diagnosis animal tuberculosis caused by bovis . this study, whole genome sequencing (WGS) was used to search for protein-coding genes distinguish M. from addition, population structure available genomic WGS datasets is described, including three novel Belgian isolates infections alpacas. Candidate were identified examining presence...

Bacillus cereus is a spore-forming bacterium that occurs as contaminant in food and feed, occasionally resulting poisoning through the production of various toxins. In this study, we retrospectively characterized viable B. sensu lato ( s.l. ) isolates originating from commercial vitamin B 2 feed additives collected between 2016 2022 by Belgian Federal Agency for Safety Food Chain products sold on market. total, 75 product samples were cultured general medium and, case bacterial growth, two...

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

Since January 2019, the Belgian National Reference Center for Mycobacteria (NRC) has switched from conventional typing to prospective whole-genome sequencing (WGS) of all submitted Mycobacterium tuberculosis complex (MTB) isolates. The ISO17025 validated procedure starts with semi-automated extraction and purification gDNA directly MGIT tubes, without preceding subculturing. All samples are then sequenced on an Illumina MiSeq sequencer analyzed using in-house developed bioinformatics...

The increasing worldwide prevalence of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli constitutes a serious threat to global public health. Surgical site infections are associated with high morbidity and mortality rates in developing countries, fueled by the limited availability effective antibiotics. We used whole-genome sequencing (WGS) evaluate antimicrobial resistance phylogenomic relationships 19 ESBL-positive E. isolates collected from surgical patients across...

Abstract Background Carbapenem-resistant Acinetobacter baumannii is considered a top priority pathogen by the World Health Organization for combatting increasing antibiotic resistance and development of new drugs. Since it was originally reported in Klebsiella pneumoniae 2009, quick spread bla NDM-1 gene encoding New-Delhi metallo-beta-lactamase-1 (NDM-1) increasingly recognized as serious threat. This usually carried large plasmids has already been documented diverse bacterial species,...

Seasonal influenza epidemics are associated with high mortality and morbidity in the human population. Influenza surveillance is critical for providing information to national programmes making vaccine composition predictions. Vaccination prevents viral infections, but rapid evolution results emerging mutants that differ antigenically from strains. Current relies on Sanger sequencing of haemagglutinin (HA) gene. Its classification according World Health Organization (WHO) European Centre...

Abstract The phylogenetically closely related Shigella species and enteroinvasive Escherichia coli (EIEC) are responsible for millions of episodes bacterial dysenteriae worldwide. Given its distinct epidemiology public health relevance, only Shigellae subject to mandatory reporting follow‐up by authorities. However, many clinical laboratories struggle differentiate non‐EIEC, EIEC, in their current workflows, leading inaccuracies surveillance rising numbers misidentified E. samples at the...

Shiga toxin-producing