Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Whole-Genome Sequence-Based Typing of Listeria monocytogenes

Abstract

Whole-genome sequencing (WGS) has emerged today as an ultimate typing tool to characterize Listeria monocytogenes outbreaks. However, data analysis and interlaboratory comparability of WGS data are still challenging for most public health laboratories. Therefore, we have developed and evaluated a new L. monocytogenes typing scheme based on genome-wide gene-by-gene comparisons (core genome multilocus the sequence typing [cgMLST]) to allow for a unique typing nomenclature. Initially, we determined the breadth of the L. monocytogenes population based on MLST data with a Bayesian approach. Based on the genome sequence data of representative isolates for the whole population, cgMLST target genes were defined and reappraised with 67 L. monocytogenes isolates from two outbreaks and serotype reference strains. The Bayesian population analysis generated five L. monocytogenes groups. Using all available NCBI RefSeq genomes (n = 36) and six additionally sequenced strains, all genetic groups were covered. Pairwise comparisons of these 42 genome sequences resulted in 1,701 cgMLST targets present in all 42 genomes with 100% overlap and ≥90% sequence similarity. Overall, ≥99.1% of the cgMLST targets were present in 67 outbreak and serotype reference strains, underlining the representativeness of the cgMLST scheme. Moreover, cgMLST enabled clustering of outbreak isolates with ≤10 alleles difference and unambiguous separation from unrelated outgroup isolates. In conclusion, the novel cgMLST scheme not only improves outbreak investigations but also enables, due to the availability of the automatically curated cgMLST nomenclature, interlaboratory exchange of data that are crucial, especially for rapid responses during transsectorial outbreaks.

FIG 1

Minimum-spanning tree based on cgMLST allelic profiles of 9 L. monocytogenes isolates (all share ST1) from the jellied pork outbreak (21) and two outgroup isolates L2708 (ST249) and L7508 (ST4) in comparison to reference strains F2365 (GenBank accession number NC_002973) and LL195 (NC_019556) (both ST1) exhibiting the same serotype 4b. Each circle represents an allelic profile based on sequence analysis of 1,701 cgMLST target genes. The numbers on the connecting lines illustrate the numbers of target genes with differing alleles. The different groups of strains are distinguished by the colors of the circles. Closely related genotypes (≤10 allele difference) are shaded in gray. NCBI RefSeq strains are marked with an asterisk.

FIG 2

Minimum-spanning tree illustrating the phylogenetic relationship based on the cgMLST allelic profiles of 33 L. monocytogenes isolates from the outbreak associated with acid curd cheese (ACCO) (22, 23) consisting of two clones (ACCO I and ACCO II). Three outgroup isolates per outbreak (with identical PFGE profiles and serotypes) are shown in comparison to the reference strain EGD-e (GenBank accession number NC_003210; ST35). ACCO I isolates L27-09, L31-09, L32-09, L35-09, and L68-09 were ST777; the remaining isolates, including the three ACCO I outgroup isolates were ST403. ACCO II isolates, including the three ACCO II outgroup isolates were all ST398. Each circle represents an allelic profile based on sequence analysis of 1,701 genes. The numbers on the connecting lines illustrate the numbers of target genes with differing alleles. The different groups of strains are distinguished by the colors of the circles. Closely related genotypes (≤10 allele difference) are shaded in gray. The NCBI RefSeq strain is marked with an asterisk.