A clade of Listeria monocytogenes serotype 4b variant strains linked to recent listeriosis outbreaks associated with produce from a defined geographic region in the US

Abstract

Four listeriosis incidences/outbreaks, spanning 19 months, have been linked to Listeria monocytogenes serotype 4b variant (4bV) strains. Three of these incidents can be linked to a defined geographical region, while the fourth is likely to be linked. In this study, whole genome sequencing (WGS) of strains from these incidents was used for genomic comparisons using two approached. The first was JSpecies tetramer, which analyzed tetranucleotide frequency to assess relatedness. The second, the CFSAN SNP Pipeline, was used to perform WGS SNP analyses against three different reference genomes to evaluate relatedness by SNP distances. In each case, unrelated strains were included as controls. The analyses showed that strains from these incidents form a highly related clade with SNP differences of ≤101 within the clade and >9000 against other strains. Multi-Virulence-Locus Sequence Typing, a third standardized approach for evaluation relatedness, was used to assess the genetic drift in six conserved, known virulence loci and showed a different clustering pattern indicating possible differences in selection pressure experienced by these genes. These data suggest a high degree of relatedness among these 4bV strains linked to a defined geographic region and also highlight the possibility of alterations related to adaptation and virulence.

Fig 1. Maximum likelihood tree obtained from an alignment of the SNP file generated by the CFSAN Pipeline from the 96 strains initially used in the study with bootstrapping (n = 1000), using CFSAN023463 as the reference strain.

The scale bar indicates distance as assessed by the Tamura-Nei Method [32].

Fig 2. Maximum likelihood tree obtained from an alignment of the SNP file generated by the CFSAN Pipeline from the strains associated with the four outbreaks with bootstrapping (n = 1000), using CFSAN023463 as the reference strain.

The scale bar indicates distance as assessed by the Tamura-Nei Method [32].

Fig 3

Maximum likelihood trees (bootstrapping n = 1000) obtained of the 4b isolates using CFSAN023463 (A) or F2365 (B) as the reference strain for the CFSAN Pipeline analysis. The scale bars indicate distance as assessed by the Tamura-Nei Method [32].

Fig 4. Maximum likelihood trees (bootstrapping n = 1000) obtained via MVLST analysis of selected strains.

Pertinent EC and CC are identified as well as the new EC, ECIX. The scale bar indicates distance as assessed by the Tamura-Nei Method [32].