Analysis of Multilocus Sequence Typing and Virulence Characterization of Listeria monocytogenes Isolates from Chinese Retail Ready-to-Eat Food

Abstract

Eighty Listeria monocytogenes isolates were obtained from Chinese retail ready-to-eat (RTE) food and were previously characterized with serotyping and antibiotic susceptibility tests. The aim of this study was to characterize the subtype and virulence potential of these L. monocytogenes isolates by multilocus sequence typing (MLST), virulence-associate genes, epidemic clones (ECs), and sequence analysis of the important virulence factor: internalin A (inlA). The result of MLST revealed that these L. monocytogenes isolates belonged to 14 different sequence types (STs). With the exception of four new STs (ST804, ST805, ST806, and ST807), all other STs observed in this study have been associated with human listeriosis and outbreaks to varying extents. Six virulence-associate genes (inlA, inlB, inlC, inlJ, hly, and llsX) were selected and their presence was investigated using PCR. All strains carried inlA, inlB, inlC, inlJ, and hly, whereas 38.8% (31/80) of strains harbored the listeriolysin S genes (llsX). A multiplex PCR assay was used to evaluate the presence of markers specific to epidemic clones of L. monocytogenes and identified 26.3% (21/80) of ECI in the 4b-4d-4e strains. Further study of inlA sequencing revealed that most strains contained the full-length InlA required for host cell invasion, whereas three mutations lead to premature stop codons (PMSC) within a novel PMSCs at position 326 (GAA → TAA). MLST and inlA sequence analysis results were concordant, and different virulence potentials within isolates were observed. These findings suggest that L. monocytogenes isolates from RTE food in China could be virulent and be capable of causing human illness. Furthermore, the STs and virulence profiles of L. monocytogenes isolates have significant implications for epidemiological and public health studies of this pathogen.

Keywords: Listeria monocytogenes; MLST; PMSC; epidemic clones; inlA; virulence genes.

Figure 1

The UPGMA (unweighted pair group method with arithmetic mean) tree of the seven multi-locus sequence typing loci (3288 base pair concatenated length) of RTE L. monocytogenes isolates (Supplementary Table 1). This tree was generated using the S.T.A.R.T (version 2).

Figure 2

Maximum likelihood tree of selected RTE L. monocytogenes isolates based on inlA sequence. The inlA sequences of 36 isolates are shown, including 35 selected isolates in this study and Listeria monocytogenes EGDe.

Figure 3

Internalin A variants in the 16 distinct ST and inlA alleles encountered. Comparison of the amino acid sequences of inlA of 35 L. monocytogenes isolated from Chinese RTE food to the homologous sequence of type strain EGDs. InlA functional domains are represented as distinct blocks: SP, signal peptide, α-domain, alpha-domain linker; LRRs, leucine rich repeats; IR, intragenic repeat, B-repeats, PA, pre-anchor domain; CWA, cell wall anchor; and C, C terminus. Yellow represents replacement with amino-acid and black indicates nonsense mutations leading to a premature stop codon (PMSC).