Listeria monocytogenes Isolated from Illegally Imported Food Products into the European Union Harbor Different Virulence Factor Variants

Abstract

Unregulated international flow of foods poses a danger to human health, as it may be contaminated with pathogens. Recent studies have investigated neglected routes of pathogen transmission and reported the occurrence of Listeria monocytogenes in food illegally imported into the European Union (EU), either confiscated at four international airports or sold illegally on the Romanian black market. In this study we investigated the genotype diversity and the amino acid sequence variability of three main virulence factors of 57 L. monocytogenes isolates. These isolates were derived from 1474 food samples illegally imported into the EU and originated from 17 different countries. Multilocus sequence typing revealed 16 different sequence types (STs) indicating moderate genotype diversity. The most prevalent STs were ST2, ST9, and ST121. The pulsed-field gel electrophoresis (PFGE) analysis resulted in 34 unique pulsotypes. PFGE types assigned to the most prevalent STs (ST2, ST9, and ST121) were highly related in their genetic fingerprint. Internalin A (InlA) was present in 20 variants, including six truncated InlA variants, all harbored by isolates of ST9 and ST121. We detected eight ST-specific listeriolysin O (LLO) variants, and among them, one truncated form. The actin-assembly-inducing protein ActA was present in 15 different ST-specific variants, including four ActA variants with an internal truncation. In conclusion, this study shows that L. monocytogenes, isolated from illegally imported food, have moderate genotype diversity, but diverse virulence factors variants, mainly of InlA.

Keywords: ActA; European Union; Listeria monocytogenes; food; genotyping; illegal; internalin A; listeriolysin O; neglected routes; virulence factors.

Figure 1

Maximum likelihood phylogenetic tree of Listeria monocytogenes strains based on multilocus sequence typing (MLST) loci. The maximum likelihood phylogenetic tree is based on concatenated full-length MLST gene sequences and was calculated with MEGA7 using the Tamura–Nei model. Bootstrap values (100× resampling) are indicated at the respective nodes. The analysis involved 57 nucleotide sequences. All positions containing gaps were eliminated. There was a total of 3288 positions in the final dataset. The bar represents the number of substitutions per site. L. monocytogenes sequence types (STs), clonal complexes (CCs), source, and the origin country, are indicated.

Figure 2

Combined pulsed-field gel electrophoresis (PFGE) cluster analysis of 57 L. monocytogenes isolates. Combined PFGE cluster analysis of L. monocytogenes isolates from this study (restriction enzymes AscI & ApaI). The TIFF images were compared using BioNumerics 6.6 software (Applied Math NV, Sint-Martens-Latem, Belgium), and normalized using the PFGE global standard Salmonella ser. Braenderup H9812. Pattern clustering was performed using the unweighted pair group method using arithmetic averages (UPGMA) and the Dice correlation coefficient was applied with a position tolerance of 1.5%.

Figure 3

Internalin A variants. Analysis of InlA amino acid sequence of 57 L. monocytogenes isolates revealed 20 different variants. InlA functional domains are represented as distinct blocks: signal peptide (SP), leucine rich repeats (LRR), intragenic repeat (IR), B-repeats, pre-anchor domain (PA), and cell wall anchor (CWA). Different variants are presented by one representative strain. Red indicates premature stop codon (PMSC).

Figure 4

Phylogenetic analysis of internalin A. The molecular phylogenetic analysis by the maximum likelihood method is based on InlA amino acid sequences, and was calculated with MEGA7 using the Jones–Taylor–Thornton (JTT) matrix-based model [22,26]. The tree is drawn to scale, with branch lengths measured by the number of substitutions per site. Bootstrap values (100× resampling) are indicated at the respective nodes. The analysis involved 57 amino acid sequences. All positions with less than 75% site coverage were eliminated, resulting in 800 positions in the final dataset.

Figure 5

Phylogenetic analysis of listeriolysin O (LLO). The molecular phylogenetic analysis by the maximum likelihood method is based on InlA amino acid sequences, and was calculated with MEGA7 using the JTT matrix-based model [22,26]. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Bootstrap values (100× resampling) are indicated at the respective nodes. The analysis involved 57 amino acid sequences. All positions with less than 75% site coverage were eliminated, resulting in 529 positions in the final dataset.

Figure 6

ActA variants. Analysis of ActA amino acid sequence of 57 L. monocytogenes isolates revealed 15 different variants. ActA functional domains are represented as distinct blocks. SP: signal peptide, PRR: proline reach repeat; Different variants are presented by isolates of the same ST with two exceptions. ST37 and ST155 isolates harbor the same ActA variant. Red indicates internal truncation. ^a start and ^b end of the internal truncation.

Figure 7

Phylogenetic analysis of ActA. Molecular phylogenetic analysis, based on the maximum likelihood method using ActA sequence, was calculated with MEGA7 using the JTT matrix-based model [22,26]. The tree is drawn to scale, with branch lengths measured by the number of substitutions per site. Bootstrap values (100× resampling) are indicated at the respective nodes. The analysis involved 57 amino acid sequences. All positions with less than 75% site coverage were eliminated resulting in 633 positions in the final dataset.