Detection and Serotyping of Salmonella and Escherichia coli in Wheat Flour by a Quasimetagenomic Approach Assisted by Magnetic Capture, Multiple Displacement Amplification and Real-Time Sequencing.

Food safety is a new area for novel applications of metagenomics analysis, which can not only detect and subtype foodborne pathogens in a single workflow but may also produce additional information with in depth analysis capabilities. In this study we applied a quasimetagenomic approach by combining short-term enrichment, immunomagnetic separation (IMS), multiple displacement amplification (MDA) and nanopore sequencing real-time analysis for simultaneous detection of Salmonella and Escherichia coli in wheat flour. Tryptic soy broth was selected for the 12-h enrichment of samples at 42°C. Enrichments were subjected to IMS using beads capable of capturing both Salmonella and E. coli. The MDA was performed on harvested beads and amplified DNA fragments were subjected to DNA library preparation for sequencing. Sequencing was performed on a portable device with real-time basecalling adaptability and resulted sequences were subjected to two parallel pipelines for further analysis. After 1 h of sequencing the quasimetagenomic approach could detect all targets inoculated at approx. 1 CFU/g flour to the species level. Discriminatory power was determined by simultaneous detection of dual inoculums of Salmonella and E. coli, absence of detection in control samples and consistency in microbial flora composition of the same flour samples over several rounds of experiment. The total turnaround time for detection was approximately 20 h. Longer sequencing for up to 15 h enabled serotyping for many of the samples with more than 99% genome coverage which could be subjected to other appropriate genetic analysis pipelines in less than a total of 36 h. [ABSTRACT FROM AUTHOR]