Virulence Gene Sequencing Highlights Similarities and Differences in Sequences in Listeria monocytogenes Serotype 1/2a and 4b Strains of Clinical and Food Origin From 3 Different Geographic Locations

Abstract

The prfA-virulence gene cluster (pVGC) is the main pathogenicity island in Listeria monocytogenes, comprising the prfA, plcA, hly, mpl, actA, and plcB genes. In this study, the pVGC of 36 L. monocytogenes isolates with respect to different serotypes (1/2a or 4b), geographical origin (Australia, Greece or Ireland) and isolation source (food-associated or clinical) was characterized. The most conserved genes were prfA and hly, with the lowest nucleotide diversity (π) among all genes (P < 0.05), and the lowest number of alleles, substitutions and non-synonymous substitutions for prfA. Conversely, the most diverse gene was actA, which presented the highest number of alleles (n = 20) and showed the highest nucleotide diversity. Grouping by serotype had a significantly lower π value (P < 0.0001) compared to isolation source or geographical origin, suggesting a distinct and well-defined unit compared to other groupings. Among all tested genes, only hly and mpl were those with lower nucleotide diversity in 1/2a serotype than 4b serotype, reflecting a high within-1/2a serotype divergence compared to 4b serotype. Geographical divergence was noted with respect to the hly gene, where serotype 4b Irish strains were distinct from Greek and Australian strains. Australian strains showed less diversity in plcB and mpl relative to Irish or Greek strains. Notable differences regarding sequence mutations were identified between food-associated and clinical isolates in prfA, actA, and plcB sequences. Overall, these results indicate that virulence genes follow different evolutionary pathways, which are affected by a strain's origin and serotype and may influence virulence and/or epidemiological dominance of certain subgroups.

Keywords: Listeria monocytogenes; actA; diversity; gene sequencing; hly; prfA; virulence.

Figure 1

3-D scatter-plot illustration of nucleotide diversity parameters (π, θ) and d_N/d_S ratio (ω) for the pVGC (A), prfA (B), plcA (C), hly (D), mpl (E), actA (F), and plcB (G) genes. Within each gene, colored dots represent the L. monocytogenes population grouping based on serotype (4b and 1/2a), geographical origin (Australian, Greek, and Irish strains), source of isolation (clinical or food environment), and as a whole (All strains).

Figure 2

Phylogenetic network applied to virulence gene cluster prfA (pVGC; concatenated genes prfA, plcA, hly, mpl, actA, and plcB) using the Neighbor-Net algorithm. L. monocytogenes strains represented food environment isolates (green color) or clinical isolates (red color), isolated in Ireland (IR), Greece (GR), and Australia (AU). Strains clustered together in a box represent identical nucleotide sequence.

Figure 3

Phylogenetic network applied to virulence gene prfA using the Neighbor-Net algorithm. L. monocytogenes strains represented food environment isolates (green color) or clinical isolates (red color), isolated in Ireland (IR), Greece (GR), and Australia (AU). Strains clustered together in a box represent identical nucleotide sequence.

Figure 4

Phylogenetic network applied to virulence gene plcA using the Neighbor-Net algorithm. L. monocytogenes strains represented food environment isolates (green color) or clinical isolates (red color), isolated in Ireland (IR), Greece (GR), and Australia (AU). Strains clustered together in a box represent identical nucleotide sequence.

Figure 5

Phylogenetic network applied to virulence gene hly using the Neighbor-Net algorithm. L. monocytogenes strains represented food environment isolates (green color) or clinical isolates (red color), isolated in Ireland (IR), Greece (GR), and Australia (AU). Strains clustered together in a box represent identical nucleotide sequence.

Figure 6

Phylogenetic network applied to virulence gene mpl using the Neighbor-Net algorithm. L. monocytogenes strains represented food environment isolates (green color) or clinical isolates (red color), isolated in Ireland (IR), Greece (GR), and Australia (AU). Strains clustered together in a box represent identical nucleotide sequence.

Figure 7

Phylogenetic network applied to virulence gene actA using the Neighbor-Net algorithm. L. monocytogenes strains represented food environment isolates (green color) or clinical isolates (red color), isolated in Ireland (IR), Greece (GR), and Australia (AU). Strains clustered together in a box represent identical nucleotide sequence.

Figure 8

Phylogenetic network applied to virulence gene plcB using the Neighbor-Net algorithm. L. monocytogenes strains represented food environment isolates (green color) or clinical isolates (red color), isolated in Ireland (IR), Greece (GR), and Australia (AU). Strains clustered together in a box represent identical nucleotide sequence.