Application of MinION sequencing as a tool for the characterization of STEC in raw milk - Anses - Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail Accéder directement au contenu
Communication Dans Un Congrès Année : 2023

Application of MinION sequencing as a tool for the characterization of STEC in raw milk

Résumé

Metagenomics is widely used for the detection and characterization of food-borne pathogens but its efficacy to distinguish STEC that may be eae-positive or -negative in complex samples like raw milk is questionable. STEC harbor various virulence markers, like the eae gene, that are detectable by real-time PCR in complex samples like raw milk. However, it is not resolvable whether these genes belong to the same strain or are present in different strains, and isolation is required to confirm the presence of all virulence factors in a single strain (strain characterization). STEC isolation is laborious, time-consuming and frequently unsuccessful in raw milk, because of the background flora and a lack of a STEC-specific isolation medium. The diversity of STEC emphasizes the need for STEC characterization methods that ideally circumvent the isolation problems posed by current methods. Metagenomics has the potential to accelerate the STEC characterization process in an isolation-free manner. Short-read sequencing metagenomics have been applied for this purpose, but assembly quality and contiguity suffer from the high proportion of mobile genetic elements occurring in STEC strains. The short-read sequences generated consistently leads to highly fragmented assemblies and the detection of context-free virulence factors. In addition, the distance between stx and eae genes on the chromosome can be over 1.7 Mb. The increasing interest in third generation sequencing, with reads reaching over 100 kb, could potentially allow the reconstruction of complete STEC genomes from metagenomics data. The aim of our study was to investigate MinION long-read sequencing potential to identify and characterize STEC from raw milk metagenome without strain isolation. We first optimized the extraction method to obtain high quantities of high molecular weight DNA required for MinION sequencing. Preliminary in-silico analysis revealed the necessity of an enrichment step to characterize eae-positive STEC from metagenomes using long-read assembly. We then optimized the enrichment step to favor STEC growth. An overnight enrichment at 37°C in acriflavine-supplemented buffered peptone water was found to be most effective at preventing the growth of the raw milk background flora. Under the optimized enrichment conditions, raw milk artificially contaminated with an eae-positive STEC strain of serotype O26:H11, at initial concentrations from 0.5×101 to 0.5×103 CFU.mL−1 in raw milk, could be clearly identified by MinION long-read metagenomics. To facilitate data analysis, a pipeline named STECmetadetector was designed to specifically identify the presence of eae-positive STEC and characterize the STEC strain present in the metagenome using an assembly-based approach. The limit that appeared from the method was when multiple E. coli strains were present in the sample. This study demonstrates the potential of long-read metagenomics to efficiently characterize E. coli strain from a complex matrix. However, strain-aware assemblers are still required to improve the sensitivity of the method.

Domaines

Bactériologie
Fichier non déposé

Dates et versions

anses-04206803 , version 1 (14-09-2023)

Identifiants

  • HAL Id : anses-04206803 , version 1

Citer

Sandra Jaudou, Carlus Deneke, Mai-Lan Tran, Elisabeth Schuh, André Goehler, et al.. Application of MinION sequencing as a tool for the characterization of STEC in raw milk. VTEC 2023 - 11th International Symposium on Shiga Toxin (Verocytotoxin) Producing Escherichia coli Infections, May 2023, Banff, Canada. ⟨anses-04206803⟩

Collections

ANSES
56 Consultations
0 Téléchargements

Partager

Gmail Facebook X LinkedIn More