Retrospective investigation of listeriosis outbreaks in small ruminants using different analytical approaches for whole genome sequencing-based typing of Listeria monocytogenes.

Listeria monocytogenes is the causative agent of listeriosis, a serious disease affecting both humans and animals. While listeriosis outbreaks in humans are commonly investigated in detail, routine typing of L. monocytogenes is generally not performed in animal outbreaks. Here, seven presumable listeriosis outbreaks in small ruminants were retrospectively identified based on the pulsed-field gel electrophoresis (PFGE) profiles. Outbreaks were further characterised using three different analytical approaches based on the whole-genome sequencing (WGS) data: core-genome multilocus sequence typing (cgMLST), whole-genome MLST (wgMLST) and whole-genome single nucleotide polymorphism (wgSNP) typing. A monoclonal pattern of all seven outbreaks was identified using all three approaches, indicating common-source outbreaks. The outbreak strains belonged to sequence types (STs) 1 (n = 3), ST18 (n = 1), ST21 (n = 2) and ST184 (n = 1). Two epidemiologically linked ST1 outbreaks with indistinguishable PFGE profiles showed a polyphyletic nature and differed in >78 SNPs; thus, they were classified as separate outbreaks according to WGS. In ST184, the outbreak strain was also found in faeces of apparently healthy ruminants, silage and water collected from the trough, which were the most likely source(s) of infection. The outbreak-associated isolates differed in 0-7 cgMLST alleles, 0-12 wgMLST alleles and 1-13 SNPs. The minimum genetic diversity between outbreak-associated isolates and epidemiologically unrelated isolates of the same ST was low in all analysed cases, approaching the maximum diversity within the outbreak cluster. The results suggest that a fixed threshold to define the outbreak cluster should only be considered as a guide and highlight the role of epidemiological data for outbreak confirmation. The identified cgMLST clusters may be further investigated by wgMLST and/or wgSNP typing to increase confidence during investigations of outbreaks caused by highly clonal L. monocytogenes groups. This study gives an overview of the inter- and intra-outbreak genetic diversity of L. monocytogenes strains involved in animal outbreaks, hence improving their investigation.