High-throughput genome sequencing of two Listeria monocytogenes clinical isolates during a large foodborne outbreak

Abstract

Background: A large, multi-province outbreak of listeriosis associated with ready-to-eat meat products contaminated with Listeria monocytogenes serotype 1/2a occurred in Canada in 2008. Subtyping of outbreak-associated isolates using pulsed-field gel electrophoresis (PFGE) revealed two similar but distinct AscI PFGE patterns. High-throughput pyrosequencing of two L. monocytogenes isolates was used to rapidly provide the genome sequence of the primary outbreak strain and to investigate the extent of genetic diversity associated with a change of a single restriction enzyme fragment during PFGE.

Results: The chromosomes were collinear, but differences included 28 single nucleotide polymorphisms (SNPs) and three indels, including a 33 kbp prophage that accounted for the observed difference in AscI PFGE patterns. The distribution of these traits was assessed within further clinical, environmental and food isolates associated with the outbreak, and this comparison indicated that three distinct, but highly related strains may have been involved in this nationwide outbreak. Notably, these two isolates were found to harbor a 50 kbp putative mobile genomic island encoding translocation and efflux functions that has not been observed in other Listeria genomes.

Conclusions: High-throughput genome sequencing provided a more detailed real-time assessment of genetic traits characteristic of the outbreak strains than could be achieved with routine subtyping methods. This study confirms that the latest generation of DNA sequencing technologies can be applied during high priority public health events, and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology.

Figure 1

AscI pulsed-field gel electrophoresis of two Listeria monocytogenes PFGE patterns associated with a large foodborne outbreak. A: LMACI.0040; B: LMACI.0001. Restriction fragment sizes were estimated using BioNumerics relative to the control standard. Unmatched bands are highlighted (red arrow).

Figure 2

Circular map and genetic features of Listeria monocytogenes isolate 08-5578. The outer ring denotes genetic coordinates, and prophage and the novel 50 kbp Listeria genomic island (LGI1) are indicated in grey text. Prophage ϕLMC1 is not encoded within isolate 08-5923. Light blue bars (2^nd and 3^rd rings) denote coding sequences on the positive and negative strands, respectively. Red bars (4^th ring) denote those coding sequences present in 08-5578 but absent in the genome sequence of strain EGDe. Dark blue bars (5^th ring) indicate confirmed single nucleotide polymorphisms between isolate 08-5578 and 08-5923. The black/grey and blue/green plots indicate G+C content and G+C skew, respectively.

Figure 3

Maximum likelihood phylogenetic trees determined for Listeria genome sequences (A) and MLST loci (B). L. monocytogenes lineages and serotypes are indicated (grey text). 'CC' denotes clonal complexes and 'ST' denotes sequence types. Strain F2365 was isolated from a 1985 California cheese outbreak; H7858 from a 1998-9 Multistate hotdog oubreak; F6854 from a 1988 Oklahoma turkey hot dog sporadic case; EGDe is a laboratory strain passaged from an animal isolate from 1924.

Figure 4

Schematic of a 33 kbp prophage unique to Listeria monocytogenes isolate 08-5578. Blue-colored loci represent a contiguous segment within the isolate 08-5923 genome. Similarly, regions flanking the prophage of isolate 08-5578 are also denoted in blue. The interrupting contiguous coding sequences (red) represent prophage ϕLMC1 in 08-5578. The tRNA-Ser gene is denoted with a green box. Putative phage-related functions or structures are indicated above the locus tag identifiers. A nucleotide scale bar for size estimation is included.

Figure 5

Genetic organization and predicted functions of pLM5578 (A) and the Listeria genomic island 1 (LGI1) (B). Both sequences represent contiguous genetic regions but are split onto two lines for visual clarity, with the site of the artificial segmentation denoted with angled lines. Locus tags are as denoted above the CDS map and predicted gene names (italics) denoted below. Coding sequences are color-coded based on predicted function, with the legend included in the inset. Black-colored coding sequences are similar to L. monocytogenes EGDe (locus tags, lmo) and the imperfect inverted 16 bp repeats surrounding the genomic island are indicated. A nucleotide scale bar for size estimation is included.

Figure 6

Evolutionary model for the Listeria monocytogenes isolates recovered during the 2008 nation-wide foodborne outbreak. Predicted mutational events are indicated on the diagonal lines, genotypes of the resulting lineages are denoted within circles, and isolates representative of those lineages are indicated to the right of solid dots. Sequenced isolates are denoted with bold text.