Whole-Genome Sequencing of Recent Listeria monocytogenes Isolates from Germany Reveals Population Structure and Disease Clusters

Abstract

Listeria monocytogenes causes foodborne outbreaks with high mortality. For improvement of outbreak cluster detection, the German consiliary laboratory for listeriosis implemented whole-genome sequencing (WGS) in 2015. A total of 424 human L. monocytogenes isolates collected in 2007 to 2017 were subjected to WGS and core-genome multilocus sequence typing (cgMLST). cgMLST grouped the isolates into 38 complexes, reflecting 4 known and 34 unknown disease clusters. Most of these complexes were confirmed by single nucleotide polymorphism (SNP) calling, but some were further differentiated. Interestingly, several cgMLST cluster types were further subtyped by pulsed-field gel electrophoresis, partly due to phage insertions in the accessory genome. Our results highlight the usefulness of cgMLST for routine cluster detection but also show that cgMLST complexes require validation by methods providing higher typing resolution. Twelve cgMLST clusters included recent cases, suggesting activity of the source. Therefore, the cgMLST nomenclature data presented here may support future public health actions.

Keywords: core genome; listeriosis; outbreak detection; pathogen surveillance; subtyping.

FIG 1

Basic epidemiological characteristics of the 424 L. monocytogenes strains sequenced in this study. Distribution of isolates according to their year of isolation (A), their molecular serogroup (B), and their MLST sequence type (C) is shown. Isolates for which no ST could be determined (e.g., due to missing alleles or sequencing problems) were collectively grouped into a separate category labeled with a question mark.

FIG 2

Population structure of human L. monocytogenes isolates as determined by cgMLST. (A) Minimum spanning tree (MST) showing relatedness of 424 human L. monocytogenes isolates. Individual isolates were labeled according to their CTs. Putative complexes (with ≤10 different alleles between neighbored isolates) are highlighted in gray. Molecular serogroups are indicated. (B) Size (number of isolates) and diversity (number of genotypes) of the 38 cgMLST complexes.

FIG 3

Maximum allelic differences of L. monocytogenes typing methods. Box plot showing the maximum number of allelic differences after 1,701-locus cgMLST observed between any two isolates of a certain molecular serogroup, MLST sequence type, PFGE profile, or cgMLST CT. Values were determined from allele distance matrices obtained from pairwise comparisons of cgMLST typing data.

FIG 4

Reconstruction of L. monocytogenes population structure by SNP calling. (A) Unrooted neighbor-joining tree illustrating the population structure of the L. monocytogenes isolates sequenced in this study. Sequencing reads of all isolates were mapped against the L. monocytogenes EGD-e reference genome, and consensus sequences were aligned using an in-house pipeline. Isolates for which the genome sequences covered less than 80% of the reference sequence were removed from the alignment. Nucleotide positions that were invariable, containing gaps or ambiguities, were stripped from the alignment before tree calculation, which was performed using the Geneious Tree builder (Biomatters, Ltd.). Isolates are color coded according to their molecular serogroup, their MLST sequence type, and their cgMLST complex number. Complexes that do not cluster in unique branches by the SNP-calling approach are labeled in red. (B) Box plot showing the number of SNPs between any two isolates within each of the 38 cgMLST complexes. Median values for SNP distances are indicated. Isolates within the cgMLST complexes that are further differentiated within the complex by the SNP-calling approach are shown in red.

FIG 5

Differentiation of PFGE profiles by cgMLST and vice versa. (A) Box plot showing the number of different CTs that can be detected within each of the 83 distinct PFGE profiles (PFGE → CT) and the number of different PFGE profiles that are observed among isolates with one of the 171 different CTs (CT → PFGE). Relevant PFGE profile designations and CTs are indicated. (B) Minimum spanning tree showing further discrimination of L. monocytogenes strains with the identical PFGE type AscI/ApaI 23/27 by cgMLST into 15 different CTs. Isolates are colored according to their CTs, which are also indicated.

FIG 6

Further discrimination of CT90 isolates by PFGE. (A) Minimum spanning tree of 15 L. monocytogenes CT90 isolates. Isolates are colored according to their PFGE profiles. (B) Pulsed-field gel electrophoresis of CT90 isolates with PFGE profiles 3/8var2 and 3/23 after ApaI macrorestriction. Bands that are shifted in size due to phage insertions are boxed. (C) Virtual ApaI digest of pseudochromosomes obtained from assembled sequencing reads of strain 16-01401 and 16-01911 chromosomal DNA. (D) Position of prophages (labeled in yellow) in the 426-kb and 284-kb ApaI fragments of the AscI/ApaI 3/23 strain 16-01911, which are absent in the corresponding 378-kb and 243-kb fragments of the AscI/ApaI 3/8var2 strain 16-01401.