Hypervirulent Listeria monocytogenes clones’ adaption to mammalian gut accounts for their association with dairy products

Listeria monocytogenes (Lm) is a major human and animal foodborne pathogen. Here we show that hypervirulent Lm clones, particularly CC1, are strongly associated with dairy products, whereas hypovirulent clones, CC9 and CC121, are associated with meat products. Clone adaptation to distinct ecological niches and/or different food products contamination routes may account for this uneven distribution. Indeed, hypervirulent clones colonize better the intestinal lumen and invade more intestinal tissues than hypovirulent ones, reflecting their adaption to host environment. Conversely, hypovirulent clones are adapted to food processing environments, with a higher prevalence of stress resistance and benzalkonium chloride tolerance genes and a higher survival and biofilm formation capacity in presence of sub-lethal benzalkonium chloride concentrations. Lm virulence heterogeneity therefore reflects the diversity of the ecological niches in which it evolves. These results also have important public health implications and may help in reducing food contamination and improving food consumption recommendations to at-risk populations.


Statistics
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Software and code
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Data collection
No software was used for data collection Data analysis Bionumerics version 7.6.3 was used for single linkage clustering based on cgMLST profiles of isolates and for deduction of MLST clones based on PFGE profiles of isolates. CLC Assembly Cell version 4.3.0 and SPADES version 3.11.0 were used to assemble genomic sequences. BLAST+ v. 2.6.0 was used to detect BC tolerance genes and stress resistance genes. Prokka version 1.12 was used to annotate genome sequences. Roary version 3.6 was used for pangenome definition. Scoary version 1.6.10 was used to look for genes associated with dairy or meat origins. R was used for statistical analyzes (Mann-Whitney U test, AUC calculations, non-parametric tests) and graphical representations.
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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The authors declare that all the data supporting the findings of this study are available within the article and its supplementary information files. Genome data analyzed in this study were generated in the context of the epidemiological surveillance of listeriosis in France and should not be made publicly available for regulatory issues, but can be available from the corresponding author upon reasonable request.

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Study description
We studied the totality of the food (n = 3,333) and clinical (n = 3,308) non-redundant isolates prospectively collected for 12 consecutive years (from 2005 and 2016) in the context of the surveillance of listeriosis in France. With this, we analysed the distribution of Lm sub-populations (clones) in different food products. We then tested some phenotypes in-vitro and in-vivo in order to understand the differences of distribution of clones in the different types of food products. Seven genetically diverse strains per clone were tested in each experiement; each analysis was repeated at least three times for each isolate.
We also performed genomic analyzes by using all available genomes in our database (n = 2,928) in order to detect an unrichement of disinfectant and stress resistance genes in some clones.

Research sample
We analyze a collection of Listeria monocytogenes isolates collected in the context of the epidemiological surveillance of listeriosis in France. This collection has a high level of exhaustiveness due to mandatory declaration of listeriosis in France and is therefore highly representative of the listeriosis cases that occurred in France during the study period. Regarding the food isolates, 2.623 were collected in the context of food alerts, which are triggered when contaminated food products are on the market. The remaining 710 isolates (21.3%) were collected in the context of own-checks performed by food industries or in case of investigations following neurolisteriosis cases. Therefore, they largely represent the Lm isolates circulating in France, to which the population is exposed.

Sampling strategy
For the 3,308 clinical isolates included, they correspond to all clinical isolates collected at the CNRL between January 2005 and May 2016. This collection has a high level of exhaustiveness due to mandatory declaration of listeriosis in France and is therefore highly representative of the listeriosis cases that occurred in France during the study period. Regarding the food isolates, 2.623 were collected in the context of food alerts, which are triggered when contaminated food products are on the market. The remaining 710 isolates (21.3%) were collected in the context of own-checks performed by food industries or in case of investigations following neurolisteriosis cases. Therefore, they largely represent the Lm isolates circulating in France, to which the population is exposed.

Data collection
Isolates were collected at the National Reference Centre for Listeria (NRCL) in the context of the French surveillance of listeriosis between January 2005 and May 2016.
Timing and spatial scale Isolates were collected between January 2005 and May 2016 with no interruption.

Data exclusions
This collection of isolates was deduplicated in order to avoid any bias in the analyses. More specifically, only one isolate was considered in case of maternal-neonatal listeriosis (mother's isolates were kept). Regarding the food isolates, only one was considered when several had identical cgMLST type or CC (cf. methods below) and identical food alert number or precise food product.

Reproducibility
Regarding experimental procedures, each clone of interest was represented by 7 genetically diverse isolates, which were tested independently at least three times. Field-collected samples The study did not involve field-collected animals

Ethics oversight
All the procedures used in this study are in agreement with the guidelines of the European Commission for the handling of laboratory animals, directive 86/609/EEC. They were approved by the ethical committee of Institut Pasteur (CETEA-C2EA no. 89) under the number dap170057 and received an agreement from the ministry of higher education, research and innovation under the number APAFIS#14644-2018041116183944 v1.
Note that full information on the approval of the study protocol must also be provided in the manuscript.