Prävalenz und Typisierung von Vibrio an der Nachweisgrenze mittels Next Generation Sequencing (Vibrio-NGS)

• Forschungsstelle: METAS
• Projektnummer: F-5237.30145

Schlüsselwörter (Englisch)

Vibrio, next generation sequencing, digital PCR, quantitative PCR, bioinformatics, meta-genomics, pooled resequencing, measurement challenge, measurement uncertainty, metro-logical traceability, reference material, collaboration

Kurzbeschreibung (Englisch)

Vibrio are salt-tolerant bacteria that are found in marine waters and estuaries (brackish water, lagoons) worldwide. Gastroenteritic infections may occur after eating contaminated seafood. Most foodborne Vibrio infections are caused by the three species Vibrio parahaemolyticus, V. cholerae, and V. vulnificus. In severe cases, sepsis can occur, which can even be fatal under certain circumstances. Another particularly feared disease is cholera, which must be reported in Switzerland and is caused by Vibrio cholerae contaminating drinking water.

Global warming and the associated increased temperatures of seawater raise concerns that there could be an increase in infections with Vibrio worldwide. The FSVO will therefore conduct a study on the prevalence of these bacteria in seafood and fish in order to be able to better estimate the risk for consumers in Switzerland. The standard method for the detection of Vibrio species (ISO 21872-1: 2017) is based on two enrichment steps in selective liquid media, isolation on selective agar plates, and confirmation by means of molecular biological or biochemical reactions. The method is therefore very time-consuming. It would be desirable to have a diagnostic system that can directly detect the pathogen in marine animals, particularly oysters, but also in raw fish.

METAS and the FSVO are striving for closer cooperation in the use of modern molecular biological techniques for the detection and analysis of genetic material (DNA). One possibility of jointly using new detection methods is in the area of so-called next generation sequencing (NGS) technology, which can also be used for Vibrio in seafood. NGS not only allows the species to be clearly identified, but can also identify virulence markers and other relevant marker genes that are present in the genome of pathogenic Vibrio strains. The characterization of Vibrio strains from the FSVO's prevalence study with NGS can therefore make a significant contribution to risk assessment. While the number of living Vibrio in a sample is usually determined microbiologically using the most probable number estimate, NGS can also be used to check whether the number of living Vibrio can be estimated using jointly extracted RNA. The amount of data required for this could also be generated using NGS. Reference materials to be developed at METAS as part of a dissertation for the quantitative use of NGS analyzes can be used here for the first time in real cases and help to determine the detection and quantification limits in the event of Vibrio contamination in food. The work proposed here is directly embedded in the ongoing work at the FSVO and extends and complements the research and development work carried out at METAS. 

Projektziele (Englisch)

The main objectives of this project are: 
- Detect human pathogenic Vibrio species by next generation sequencing from fresh oysters and raw fish;
- Successfully cultivate Vibrio species from samples taken from seafood and fish imported into Switzerland;
- Chracterize these Vibrio strains with regard to virulence markers and other relevant marker genes using NGS
- Assess the risk for consumers and the prevalence in Switzerland
- Develop reference materials and determine the LOD / LOQ in practice  

Abstract (Englisch)

The main objective of this project was to develop and establish a quantitative "next generation sequencing" (NGS)-based method for the detection and characterisation of Vibrio spp. in food samples. The following main goals were achieved:

• A suitable NGS-pipeline including bioinformatics was developed, tested and established at METAS.
• METAS developed six Vibrio-specific DNA-based reference standards that were successfully applied in a quantitative NGS-workflow to precisely quantify Vibrio-derived DNA from different human pathogenic species. Furthermore, the quantitative NGS-pipeline is metrologically traceable through the coupling of NGS and the SI-reference method digital PCR (dPCR).
• A proof-of-concept study illustrating the developed NGS-pipeline and the Vibrio-specific reference standards was published in BMC Genomics (Flütsch et al., 2023).
• The development of a "host depletion" method to reduce the genome of the Vibrio spp. host is currently ongoing. This method will allow the direct sequencing of Vibrio spp. from food samples without the necessity of culturing. Therefore, the method will become unbiased, faster, less labour-intensive and more precise. The development of this method has advanced a lot, but is not yet finalised.

Umsetzung und Anwendungen (Englisch)

NGS is widely applied in clinical institutions and diagnostic laboratories. However, NGS workflows lack standardisation and metrological traceability as they are comprised of multiple subsequent steps and for most of the individual steps, standards or reference materials are lacking. Furthermore, there are also no reference measurement systems in place. The results achieved within this project not only provide a valuable contribution to a more standardised application of NGS, but they also contribute to a larger monitoring study concerning the consumption of raw uncooked seafood and fish in Switzerland. The methods and materials developed within this project lay the basis for more metrological research and work towards standardised application of NGS.

Publikationen / Ergebnisse (Englisch)

Flütsch S, Wiestner F, Butticaz L, Moor D, Stölting KN. Vibrio-Sequins - dPCR-traceable DNA standards for quantitative genomics of Vibrio spp. BMC Genomics. 2023 Jul 4;24(1):375. doi: 10.1186/s12864-023-09429-8.

Data from the above publication were presented in the form of a poster with the same title as the above publication at the conference for analytical chemistry "EuroAnalysis" in August 2023 in Geneva, as well as in the form of a lecture entitled "Sequins - dPCR-traceable DNA Standards for Quantitative Genomics" at the "NGS-Workshop" of the PTB on September 28, 2023 in Berlin.