Ruminant rhombencephalitis-associated Listeria monocytogenes alleles linked to a multilocus variable-number tandem-repeat analysis complex

Abstract

Listeria monocytogenes is among the most important food-borne pathogens and is well adapted to persist in the environment. To gain insight into the genetic relatedness and potential virulence of L. monocytogenes strains causing central nervous system (CNS) infections, we used multilocus variable-number tandem-repeat analysis (MLVA) to subtype 183 L. monocytogenes isolates, most from ruminant rhombencephalitis and some from human patients, food, and the environment. Allelic-profile-based comparisons grouped L. monocytogenes strains mainly into three clonal complexes and linked single-locus variants (SLVs). Clonal complex A essentially consisted of isolates from human and ruminant brain samples. All but one rhombencephalitis isolate from cattle were located in clonal complex A. In contrast, food and environmental isolates mainly clustered into clonal complex C, and none was classified as clonal complex A. Isolates of the two main clonal complexes (A and C) obtained by MLVA were analyzed by PCR for the presence of 11 virulence-associated genes (prfA, actA, inlA, inlB, inlC, inlD, inlE, inlF, inlG, inlJ, and inlC2H). Virulence gene analysis revealed significant differences in the actA, inlF, inlG, and inlJ allelic profiles between clinical isolates (complex A) and nonclinical isolates (complex C). The association of particular alleles of actA, inlF, and newly described alleles of inlJ with isolates from CNS infections (particularly rhombencephalitis) suggests that these virulence genes participate in neurovirulence of L. monocytogenes. The overall absence of inlG in clinical complex A and its presence in complex C isolates suggests that the InlG protein is more relevant for the survival of L. monocytogenes in the environment.

Fig. 1.

Minimum-spanning-tree analysis of 183 L. monocytogenes isolates based on eight genetic markers. Circles represent all isolates, and their size is proportional to the number of isolates. Colored zones surrounding the circles delineate the different clonal complexes. Clonal complexes were created based on the maximum neighbor distance of changes at two loci and the minimum size of five types. The length of the branches represents genetic distances (changes in loci are represented with numbers) between two neighboring types. The color codes indicate the clinical or nonclinical origin of the isolates (a), the specific origin of clinical isolates (b), the human and ruminant species (c), and the serovar or the PCR-based serovar group (d).

Fig. 1.

Minimum-spanning-tree analysis of 183 L. monocytogenes isolates based on eight genetic markers. Circles represent all isolates, and their size is proportional to the number of isolates. Colored zones surrounding the circles delineate the different clonal complexes. Clonal complexes were created based on the maximum neighbor distance of changes at two loci and the minimum size of five types. The length of the branches represents genetic distances (changes in loci are represented with numbers) between two neighboring types. The color codes indicate the clinical or nonclinical origin of the isolates (a), the specific origin of clinical isolates (b), the human and ruminant species (c), and the serovar or the PCR-based serovar group (d).

Fig. 2.

Distribution of the actA (a) and inlJ (b) alleles in the minimum spanning tree. Characteristics of the minimum spanning tree are the same as in Fig. 1. (a) actA alleles are indicated with different colors. Schematic differences of the ActA profiles are shown in the inset. PRRs are depicted as gray boxes. (b) inlJ alleles are shown in different colors. The inset shows the schematic differences of the InlJ profiles. The mucin-binding protein (MucBP) domains are depicted as gray boxes. Broken boxes represent partial MucBP; dashed lines indicate deletions. “G+ anchor” indicates the LPXTG-motif cell wall anchor domain of Gram-positive organisms. The first amino acid of the first mucin-binding protein domain corresponds to position 506 for all three alleles.

Fig. 3.

(a) Alignment of the nucleotide sequence-derived amino acid sequences of the variable parts of ActA for the two different actA alleles. PRRs are marked in gray. The dashes indicate the 35 amino acids missing in ActA3, consisting of the 29 amino acids from the long repeat (underlined) and one 6-amino-acid-long PPR marked in gray. L86 (ActA4) and L11 (ActA3) predicted amino acid sequences are compared to ActA of the EGDe strain (accession number CAA42407). The amino acid variations in ActA3 are indicated by the black background. (b) Alignment of the nucleotide sequence-derived amino acid sequences of the variable part of InlJ for the three inlJ alleles. The predicted amino acid sequences of InlJ1, InlJ2, and InlJ3 are compared to InlJ of strain EGDe (accession number NP_466343). The dashes indicate the missing amino acids. The amino acid variations are indicated with a black background. Amino acids belonging to mucin-binding protein (MucBP) domains are marked in gray.