Metagenomics-based tracing of genetically modified microorganism contaminations in commercial fermentation products.

Genetically modified microorganisms (GMM) are frequently employed for the production of microbial fermentation products such as food enzymes. Although presence of the GMM or its recombinant DNA in the final product is not authorized, contaminations occur frequently. Insight into the contamination source of a GMM is of crucial importance to allow the competent authorities to take appropriate action.

BenchAMRking: a Galaxy-based platform for illustrating the major issues associated with current antimicrobial resistance (AMR) gene prediction workflows.

Background: 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) and antimicrobial resistance (AMR) stakeholders. A key topic discussed was the frequent variability in results obtained between different microbial WGS-related AMR gene prediction workflows. Further, comparative benchmarking studies are difficult to perform due to differences in AMR gene prediction accuracy and a lack of agreement in the naming of AMR genes (semantic conformity) for the results obtained.

Galaxy @Sciensano: a comprehensive bioinformatics portal for genomics-based microbial typing, characterization, and outbreak detection.

The influx of whole genome sequencing (WGS) data in the public health and clinical diagnostic sectors has created a need for data analysis methods and bioinformatics expertise, which can be a bottleneck for many laboratories. At Sciensano, the Belgian national public health institute, an intuitive and user-friendly bioinformatics tool portal was implemented using Galaxy, an open-source platform for data analysis and workflow creation. The Galaxy @Sciensano instance is available to both internal and external scientists and offers a wide range of tools provided by the community, complemented by over 50 custom tools and pipelines developed in-house.

An interlaboratory proficiency test using metagenomic sequencing as a diagnostic tool for the detection of RNA viruses in swine fecal material.

Unlabelled: Metagenomic shotgun sequencing (mNGS) can serve as a generic molecular diagnostic tool. An mNGS proficiency test (PT) was performed in six European veterinary and public health laboratories to detect porcine astroviruses in fecal material and the extracted RNA. While different mNGS workflows for the generation of mNGS data were used in the different laboratories, the bioinformatic analysis was standardized using a metagenomic read classifier as well as read mapping to selected astroviral reference genomes to assess the semiquantitative representation of astrovirus species mixtures.

Benchmarking bacterial taxonomic classification using nanopore metagenomics data of several mock communities.

Taxonomic classification is crucial in identifying organisms within diverse microbial communities when using metagenomics shotgun sequencing. While second-generation Illumina sequencing still dominates, third-generation nanopore sequencing promises improved classification through longer reads. However, extensive benchmarking studies on nanopore data are lacking.

Phage-Mediated Digestive Decolonization in a Gut-On-A-Chip Model: A Tale of Gut-Specific Bacterial Prosperity.

Infections due to antimicrobial-resistant bacteria have become a major threat to global health. Some patients may carry resistant bacteria in their gut microbiota. Specific risk factors may trigger the conversion of these carriages into infections in hospitalized patients.

Towards facilitated interpretation of shotgun metagenomics long-read sequencing data analyzed with KMA for the detection of bacterial pathogens and their antimicrobial resistance genes.

Metagenomic sequencing is a promising method that has the potential to revolutionize the world of pathogen detection and antimicrobial resistance (AMR) surveillance in food-producing environments. However, the analysis of the huge amount of data obtained requires performant bioinformatics tools and databases, with intuitive and straightforward interpretation. In this study, based on long-read metagenomics data of chicken fecal samples with a spike-in mock community, we proposed confidence levels for taxonomic identification and AMR gene detection, with interpretation guidelines, to help with the analysis of the output data generated by KMA, a popular mer read alignment tool.

Closing the gap: Oxford Nanopore Technologies R10 sequencing allows comparable results to Illumina sequencing for SNP-based outbreak investigation of bacterial pathogens.

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.

Pilot market surveillance of GMM contaminations in alpha-amylase food enzyme products: A detection strategy strengthened by a newly developed qPCR method targeting a GM producing alpha-amylase.

Using high-throughput metagenomics on commercial microbial fermentation products, DNA from a new unauthorized genetically modified microorganism (GMM), namely the GM strain producing alpha-amylase (GMM alpha-amylase2), was recently discovered and characterized. On this basis, a new qPCR method targeting an unnatural association of sequences specific to the GMM alpha-amylase2 strain was designed and developed in this study, allowing to strengthen the current GMM detection strategy. The performance of the newly developed qPCR method was assessed for its specificity and sensitivity to comply with the minimum performance requirements established by the European Network of GMO Laboratories for GMO analysis.

Development of a portable on-site applicable metagenomic data generation workflow for enhanced pathogen and antimicrobial resistance surveillance.

Rapid, accurate and comprehensive diagnostics are essential for outbreak prevention and pathogen surveillance. Real-time, on-site metagenomics on miniaturized devices, such as Oxford Nanopore Technologies MinION sequencing, could provide a promising approach. However, current sample preparation protocols often require substantial equipment and dedicated laboratories, limiting their use.

Comparison of 6 DNA extraction methods for isolation of high yield of high molecular weight DNA suitable for shotgun metagenomics Nanopore sequencing to detect bacteria.

Background: Oxford Nanopore Technologies (ONT) offers an accessible platform for long-read sequencing, which improves the reconstruction of genomes and helps to resolve complex genomic contexts, especially in the case of metagenome analysis. To take the best advantage of long-read sequencing, DNA extraction methods must be able to isolate pure high molecular weight (HMW) DNA from complex metagenomics samples, without introducing any bias. New methods released on the market, and protocols developed at the research level, were specifically designed for this application and need to be assessed.

Transforming Shiga toxin-producing surveillance through whole genome sequencing in food safety practices.

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.

Retrospective surveillance of viable group contaminations in commercial food and feed vitamin B products sold on the Belgian market using whole-genome sequencing.

is a spore-forming bacterium that occurs as a contaminant in food and feed, occasionally resulting in food poisoning through the production of various toxins. In this study, we retrospectively characterized viable () isolates originating from commercial vitamin B feed and food additives collected between 2016 and 2022 by the Belgian Federal Agency for the Safety of the Food Chain from products sold on the Belgian market. In total, 75 collected product samples were cultured on a general medium and, in case of bacterial growth, two isolates per product sample were collected and characterized using whole-genome sequencing (WGS) and subsequently characterized in terms of sequence type (ST), virulence gene profile, antimicrobial resistance (AMR) gene profile, plasmid content, and phylogenomic relationships.

Targeted High-Throughput Sequencing Enables the Detection of Single Nucleotide Variations in CRISPR/Cas9 Gene-Edited Organisms.

Similar to genetically modified organisms (GMOs) produced by classical genetic engineering, gene-edited (GE) organisms and their derived food/feed products commercialized on the European Union market fall within the scope of European Union Directive 2001/18/EC. Consequently, their control in the food/feed chain by GMO enforcement laboratories is required by the competent authorities to guarantee food/feed safety and traceability (2003/1829/EC; 2003/1830/EC). However, their detection is potentially challenging at both the analytical and interpretation levels since this requires methodological approaches that can target and detect a specific single nucleotide variation (SNV) introduced into a GE organism.

Using a combination of short- and long-read sequencing to investigate the diversity in plasmid- and chromosomally encoded extended-spectrum beta-lactamases (ESBLs) in clinical and isolates in Belgium.

For antimicrobial resistance (AMR) surveillance, it is important not only to detect AMR genes, but also to determine their plasmidic or chromosomal location, as this will impact their spread differently. Whole-genome sequencing (WGS) is increasingly used for AMR surveillance. However, determining the genetic context of AMR genes using only short-read sequencing is complicated.

Metagenomic Characterization of Multiple Genetically Modified Contaminations in Commercial Microbial Fermentation Products.

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

Metagenomics to Detect and Characterize Viruses in Food Samples at Genome Level? Lessons Learnt from a Norovirus Study.

In this proof-of-concept study on food contaminated with norovirus, we investigated the feasibility of metagenomics as a new method to obtain the whole genome sequence of the virus and perform strain level characterization but also relate to human cases in order to resolve foodborne outbreaks. We tested several preparation methods to determine if a more open sequencing approach, i.e.

A general approach to identify low-frequency variants within influenza samples collected during routine surveillance.

Influenza viruses exhibit considerable diversity between hosts. Additionally, different quasispecies can be found within the same host. High-throughput sequencing technologies can be used to sequence a patient-derived virus population at sufficient depths to identify low-frequency variants (LFV) present in a quasispecies, but many challenges remain for reliable LFV detection because of experimental errors introduced during sample preparation and sequencing.

A shotgun metagenomics approach to detect and characterize unauthorized genetically modified microorganisms in microbial fermentation products.

The presence of a genetically modified microorganism (GMM) or its DNA, often harboring antimicrobial resistance (AMR) genes, in microbial fermentation products on the market is prohibited by European regulations. GMMs are currently screened for through qPCR assays targeting AMR genes and vectors, and then confirmed by targeting known specific GM constructs/events. However, when the GMM was not previously characterized and an isolate cannot be obtained, its presence cannot be proven.