Short-term genome evolution of Listeria monocytogenes in a non-controlled environment

Abstract

Background: While increasing data on bacterial evolution in controlled environments are available, our understanding of bacterial genome evolution in natural environments is limited. We thus performed full genome analyses on four Listeria monocytogenes, including human and food isolates from both a 1988 case of sporadic listeriosis and a 2000 listeriosis outbreak, which had been linked to contaminated food from a single processing facility. All four isolates had been shown to have identical subtypes, suggesting that a specific L. monocytogenes strain persisted in this processing plant over at least 12 years. While a genome sequence for the 1988 food isolate has been reported, we sequenced the genomes of the 1988 human isolate as well as a human and a food isolate from the 2000 outbreak to allow for comparative genome analyses.

Results: The two L. monocytogenes isolates from 1988 and the two isolates from 2000 had highly similar genome backbone sequences with very few single nucleotide (nt) polymorphisms (1 - 8 SNPs/isolate; confirmed by re-sequencing). While no genome rearrangements were identified in the backbone genome of the four isolates, a 42 kb prophage inserted in the chromosomal comK gene showed evidence for major genome rearrangements. The human-food isolate pair from each 1988 and 2000 had identical prophage sequence; however, there were significant differences in the prophage sequences between the 1988 and 2000 isolates. Diversification of this prophage appears to have been caused by multiple homologous recombination events or possibly prophage replacement. In addition, only the 2000 human isolate contained a plasmid, suggesting plasmid loss or acquisition events. Surprisingly, besides the polymorphisms found in the comK prophage, a single SNP in the tRNA Thr-4 prophage represents the only SNP that differentiates the 1988 isolates from the 2000 isolates.

Conclusion: Our data support the hypothesis that the 2000 human listeriosis outbreak was caused by a L. monocytogenes strain that persisted in a food processing facility over 12 years and show that genome sequencing is a valuable and feasible tool for retrospective epidemiological analyses. Short-term evolution of L. monocytogenes in non-controlled environments appears to involve limited diversification beyond plasmid gain or loss and prophage diversification, highlighting the importance of phages in bacterial evolution.

Figure 1

Circular map of genomes analyzed. The F6854 pseudochromosome is shown in red except for the comK prophage which is shown in blue. The genomic contigs of F6900, J0161 and J2818 are shown in blue, purple and gray, respectively. The blocks of aligned sequence in the refined alignment of F6854, F6900, J0161 and J2818 are shown in green. All sequences were aligned against the pseudochromosome of F6854, which was used as the reference.

Figure 2

Schematic of recombination events in comK prophage. The figure represents an alignment of the 1892 polymorphic sites in the comK prophage (including coding and non-coding regions) as well as 13 polymorphic sites immediately upstream the prophage and 41 polymorphic sites immediately downstream of the prophage. Sites representing nt residues found in F6854 and F6900 (1988 isolates) are shown in black and sites representing nt residues found in FSL J1-194 (a L. monocytogenes serotype 1/2b isolate with the comK prophage most closely related to the prophage found in the 2000 isolates) are shown in gray. The nt sites in J0161 and J2818 (2000 isolates) are shaded so that nt identical to the 1988 isolates are in black, nt identical to FSL J1-194 are in gray, and nt that match neither the 1988 isolates nor FSL J1-194 are in white. Apparent darker shades of gray in this figure represent adjacent sites with black and gray or white. R1, R2, and R3 (recombination 1 to 3) represent three main recombinant blocks that were likely introduced from a lineage I strain similar to FSL J1-194 into a descended of the 1988 isolates, yielding the 2000 isolate genotype. The two shorter mixed gray and white blocks at the 5' end, marked as ER1 and ER 2 (ER = external recombination) are likely to have been introduced from a L. monocytogenes genotype not included in this alignment ("external") into a descendent of the 1988 isolates.

Figure 3

Schematic of the putative evolutionary history of the L. monocytogenes strain in the food facility between 1988 and 2000. Numbers on the arrows represent new mutations. Ancestor A is the ancestor of F6854 and F6900 (the food and human isolate, respectively, from the sporadic case in 1988) and Ancestor B is the ancestor of J0161 and J2818 (the food and human isolate, respectively, from the outbreak in 2000).