Large Multicountry Outbreak of Invasive Listeriosis by a Listeria monocytogenes ST394 Clone Linked to Smoked Rainbow Trout, 2020 to 2021

Abstract

Whole-genome sequencing (WGS) has revolutionized surveillance of infectious diseases. Disease outbreaks can now be detected with high precision, and correct attribution of infection sources has been improved. Listeriosis, caused by the bacterium Listeria monocytogenes, is a foodborne disease with a high case fatality rate and a large proportion of outbreak-related cases. Timely recognition of listeriosis outbreaks and precise allocation of food sources are important to prevent further infections and to promote public health. We report the WGS-based identification of a large multinational listeriosis outbreak with 55 cases that affected Germany, Austria, Denmark, and Switzerland during 2020 and 2021. Clinical isolates formed a highly clonal cluster (called Ny9) based on core genome multilocus sequence typing (cgMLST). Routine and ad hoc investigations of food samples identified L. monocytogenes isolates from smoked rainbow trout filets from a Danish producer grouping with the Ny9 cluster. Patient interviews confirmed consumption of rainbow trout as the most likely infection source. The Ny9 cluster was caused by a MLST sequence type (ST) ST394 clone belonging to molecular serogroup IIa, forming a distinct clade within molecular serogroup IIa strains. Analysis of the Ny9 genome revealed clpY, dgcB, and recQ inactivating mutations, but phenotypic characterization of several virulence-associated traits of a representative Ny9 isolate showed that the outbreak strain had the same pathogenic potential as other serogroup IIa strains. Our report demonstrates that international food trade can cause multicountry outbreaks that necessitate cross-border outbreak collaboration. It also corroborates the relevance of ready-to-eat smoked fish products as causes for listeriosis. IMPORTANCE Listeriosis is a severe infectious disease in humans and characterized by an exceptionally high case fatality rate. The disease is transmitted through consumption of food contaminated by the bacterium Listeria monocytogenes. Outbreaks of listeriosis often occur but can be recognized and stopped through implementation of whole-genome sequencing-based pathogen surveillance systems. We here describe the detection and management of a large listeriosis outbreak in Germany and three neighboring countries. This outbreak was caused by rainbow trout filet, which was contaminated by a L. monocytogenes clone belonging to sequence type ST394. This work further expands our knowledge on the genetic diversity and transmission routes of an important foodborne pathogen.

Keywords: Europe; Ny; Ny9; ST394; cgMLST; epidemiology.

FIG 1

Identification of the Ny9 cluster by core genome multilocus sequence typing (cgMLST) and single nucleotide polymorphism (SNP) calling. (A) Minimum spanning tree of ST394 strains deduced from cgMLST data. Isolates are colored according to their origin, and clusters are highlighted by colored backgrounds. A pair of isolates was considered part of a cluster when they differed in ≤7 alleles. (B) Neighbor-joining tree for the same set of isolates calculated after read mapping to the closed genome of strain 20-05651 as the reference, which generated an alignment with a length of 201 SNPs after SNP filtering. Isolates are colored according to their origin. Scale is expressed as substitutions per site.

FIG 2

Chronology of outbreak progression and geographical distribution of cases. (A) Epidemic curve based on the notification week (sampling week for Denmark). The last Ny9 case from calendar week 2 of 2022, which was not considered during the epidemiological investigations, is shown separately. (B) Geographic distribution of the Ny9 outbreak cases between September 2020 and January 2022.

FIG 3

Phylogenetic classification of the Ny9 outbreak strain. Neighbor-joining tree based on core genome multi locus sequencing (cgMLST) data showing the position of the Ny9 (ST394) reference strain 20-05651 within a tree of representative serogroup IIa strains from a previously published nonredundant model population of clinical L. monocytogenes isolates from Germany (32). ST394 forms a deeply branching clade within the IIa strains and has ST8 (black arcs), ST1462 (purple), ST16 (orange), and ST120 (green) strains as next relatives. Of these, the ST8 strains are most closely related. The tree was calculated from data from 1,701-locus cgMLSTs and annotated with iTOL (65).

FIG 4

Comparison of the Ny9 genome with closed genomes of related subtypes. The genome map shows the Ny9 reference strain 20-05651 (gray circle). The positions of coding sequences (CDS), ribosomal RNAs (rRNAs), insertion sequences (IS), and putative phage regions are indicated. The presence/absence of Ny9 sequences in three available closed ST394 genomes, as well as in selected ST199, ST398, ST8 (Sigma1), and ST35 (EGD-e) genomes is illustrated by the several outer rings. ST394-specific regions I to IV are marked. The map was generated using Proksee (66). GC, guanine cytosine.

FIG 5

In vitro virulence profile of the Ny9 outbreak strain. (A) CAMP test showing background hemolysis and hemolytic synergism of L. monocytogenes strains EGD-e (wild type [wt]) and 20-5651 (Ny9) with S. aureus SG511. Strain LMS250 (Δhly) was included as a control. (B) Phospholipolytic activity of L. monocytogenes strains EGD-e (wt) and 20-05651 (Ny9) on egg-yolk agar. L. monocytogenes strain ΔprfA was chosen as a control. (C) Motility of L. monocytogenes strains EGD-e (wt) and 20-05651 (Ny9) on soft agar. L. monocytogenes strain LMS3 (ΔfliI) was used as a control. (D) Intracellular growth of L. monocytogenes strain 20-05651 (Ny9) in J774 mouse macrophages. Strains EGD-e (wt), LMS250 (Δhly), and 18-04415 (Sigma1 outbreak) are included for comparison. 18-04415 is one of the closest available ST8 relatives of strain 20-05651. The experiment was performed in triplicate, and average values and standard deviations are shown. The values labeled “-2 h” correspond to the inoculum. (E) Plaque formation assay to compare cell-to-cell spread of the Ny9 strain 20-05651 with strain 18-04415 (Sigma1 outbreak).