Listeria monocytogenes sequence type 1 is predominant in ruminant rhombencephalitis

Abstract

Listeria (L.) monocytogenes is an opportunistic pathogen causing life-threatening infections in diverse mammalian species including humans and ruminants. As little is known on the link between strains and clinicopathological phenotypes, we studied potential strain-associated virulence and organ tropism in L. monocytogenes isolates from well-defined ruminant cases of clinical infections and the farm environment. The phylogeny of isolates and their virulence-associated genes were analyzed by multilocus sequence typing (MLST) and sequence analysis of virulence-associated genes. Additionally, a panel of representative isolates was subjected to in vitro infection assays. Our data suggest the environmental exposure of ruminants to a broad range of strains and yet the strong association of sequence type (ST) 1 from clonal complex (CC) 1 with rhombencephalitis, suggesting increased neurotropism of ST1 in ruminants, which is possibly related to its hypervirulence. This study emphasizes the importance of considering clonal background of L. monocytogenes isolates in surveillance, epidemiological investigation and disease control.

Figure 1. Minimum spanning tree (MST) of 248 L. monocytogenes isolates based on multilocus sequence typing (MLST) analysis.

Circles represent sequence types (STs) and their size corresponds to the number of isolates present in each ST. The lines between different STs represent phylogenetic relationships, bold lines indicate one mismatch in the seven housekeeping genes, plain lines two mismatches, discontinuous lines three mismatches and light discontinuous lines four or more mismatches. Grey zones surrounding multiple STs, represent clonal complexes (CCs), which contain STs with a single mismatch in the seven loci. The three evolutionary lineages are indicated. Isolates of ruminant rhombencephalitis cases are represented in blue (n = 140), non-encephalitic ruminant clinical cases in red (n = 47), ruminant faecal isolates in brown (n = 5) and isolates of the ruminant farm environment in green (n = 56). (a) MST of cattle isolates. Isolates of rhombencephalitis cases are represented in blue (n = 39), non-encephalitic cattle clinical cases in red (n = 28), cattle faecal isolates in brown (n = 3), isolates of their environment in green (n = 33) and small ruminant-associated isolates in white (n = 145). (b) MST of small ruminant isolates. Isolates of rhombencephalitis cases are represented in blue (n = 101), non-encephalitic clinical cases in red (n = 19), faecal isolates in brown (n = 2), isolates of their environment in green (n = 23) and cattle-associated isolates in white (n = 103).

Figure 2. Frequency of L. monocytogenes isolates according to their source of isolation in the most prevalent sequence types (STs) and clonal complexes (CCs).

(a) Non-homogeneous distribution of STs in ruminant-associated isolates. The 11 most prevalent STs are arranged according to their abundance. Blue = ruminant rhombencephalitis (n = 134), red = ruminant non-encephalitic infections (n = 27), green = ruminant-associated environment (n = 40), brown = faeces (n = 4). (b) Divergent distribution of CCs in ruminant (left) and human clinical isolates from Switzerland (CH, middle) and France (FR, right). Blue = ruminant rhombencephalitis/human central nervous system (CNS) isolates, red = abortions, black = other infection sources, orange = human bacteremia.

Figure 3. CFUs per well of six isolates in the bovine macrophage (BoMac) cell line.

CFUs were enumerated from cell lysates at indicated time points post infection, three independent experiments were performed in triplicates. Error bars indicate 95% SEM. *p-value < 0.05 (2 h: ST1 rhombencephalitis VS. ST18 and ST37; 4 h: ST1 environment and ST4 Vs. ST18, ST1 rhombencephalitis and ST412 Vs. ST37; 24 h: ST1 Vs. ST18, ST4 Vs. ST37), **p-value < 0.01 (2 h: ST1 environment VS. ST18 and ST37; 4 h: ST1 environment and ST4 Vs. ST37; 8 h: ST1 and ST4 Vs. ST18 and ST37; 24 h: ST1 Vs. ST37).

Figure 4. Distribution of polymorphisms in the hfq and actA genes (grey bars).

The scale above the grey bars indicates nucleotide numbers. Below the functional domains, predicted amino acid sequences of the hfq and actA alleles are aligned to the reference strain EGD-e, and scales indicate amino acid number. (a) Polymorphisms within the 234 nucleotides of hfq are indicated as vertical bars in the grey bar. The functional domain LSM (like-Sm) is represented by a white box. While on the nucleotide level every lineage has a specific allele, none of the polymorphisms results in amino-acid changes and the amino-acid sequence is conserved across lineages. (b) Synonymous and non-synonymous nucleotide polymorphisms across the 26 actA alleles are shown as vertical bars in the upper grey bar. The functional domains (signal peptide, SP; proline rich repeats, PRR and transmembrane domain, TM) are shown in white boxes. The scale above the lower grey bar indicates amino-acid numbers and vertical bars correspond to non-synonymous nucleotide polymorphisms. Below, the amino-acid sequences of the actA 21 alleles are aligned to the reference strain EGD-e. Amino acid polymorphisms (or non-synonymous mutations) are represented in colour and gaps by “-”.