Listeria monocytogenes: Identification by high-throughput real-time PCR of 30 clonal complexes prevalent circulating in food, animal, environment and human in Europe

Listeria monocytogenes (Lm) is a bacterium that causes a food-borne illness, the Listeriosis. Typing studies using Multi Locus Sequence Typing (MLST) reveal that most strains are gathered into few major Clonal Complexes (CCs) that account for a majority of human cases worldwide. CC language is harmonized internationally and provides crucial information on strain virulence. To date, the CC identification is based on sequencing of the strain genome and requires between 3 and 5 days depending on labs. Thus here is a need of a front-line typing method for rapid identification of the major circulating CCs. Such a method would aim at screening the wide amount of food strains collected for surveillance and research purposes. At European level, the EU Reference Laboratory for Lm, Anses is coordinating a network of 40 National Reference Laboratories (NRLs) in charge, amongst other duties, of Lm monitoring in food products. The ANSES Laboratory for food safety in collaboration with its French and EU partners (technical food centers, NRLs) has developed a new highthroughput real-time PCR assay based on a microfluidic system that enables simultaneous analysis of 39 real-time PCR arrays on 44 strains in one test. This test provides accurate identification of 30 CCs, including the most prevalent CCs recently reported from human cases and food in Europe [1], the serogroup of the strains and subdivision among CCs. This one-day method is suitable for routine analysis and developed for multiplex fast and conventional real time PCR. We describe here (i) its design from a wide panel of 2299 strain genomes from human, food, animal and environment (ii) its validation according to the standard EN-ISO16140 on a various panel of 402 strains whole sequenced and collected from 18 European countries and (iii) its assessment for the typing of nearly thousand field strains collected during monitoring activities. This assay will represent a key tool to assist surveillance laboratories to establish strain relatedness with human clinical strains, during outbreak investigations. Moreover, this test can assist the food sectors in their microbiological management plans to target contamination sources on the production lines. [1]: Painset et al. 2019 Microb. Genom.