Abstract
Whole genome sequencing (WGS) analyses have been instrumental in traceback investigations of Listeria monocytogenes (Lm). To demonstrate how long-read sequencing analysis can capture and describe relationships among isolates from clinical, food, and environmental sources, we analyzed 366 long-read- and shotgun-sequenced isolates from 16 Lm outbreak strains associated with cantaloupe, leafy green, stone fruit, caramel apple, mung bean sprout, multiple cheese products, multiple ice cream products, and their production environments. The analyses demonstrated that outbreak strains could be distributed in different areas and zones of food production environments through persistent or repeated contamination. Multi-strain and multi-clone contamination were common. Further, WGS could differentiate among isolates collected at different time points or from different production lines in the same facility, revealing microevolution events in processing environments. Our comparison between complete and shotgun genomes showed that isolates of the same outbreak strain diversified mostly by gain/loss of plasmids and chromosome-borne prophages that constitute 2 to 5% of the chromosome. In contrast, other genes missing in the shotgun genomes were randomly scattered, constituting ~0.5% of the chromosome. Among different outbreak strains of the same CC, most gene-scale differences were due to gain/loss of mobile genetic elements, such as plasmids, chromosome-borne prophages, a Tn916 like transposon, and Listeria Genomic Island 2. The nucleotide variations in the same prophage and the same plasmid shared among isolates of the same outbreak strain were limited, which enabled different WGS tools to unambiguously cluster isolates of the same outbreak strain. In some outbreak strains, correlation between prophage gain/loss and single nucleotide polymorphism (SNP) accumulations in the genome backbone were observed.
Highlights
Listeria monocytogenes (Lm) is a foodborne pathogen that can persist in or become repeatedly introduced to food-processing facilities (Orsi et al, 2008), causing food contamination and subsequent listeriosis, a potentially fatal illness
It is unlikely that 72 genes in an entire 47 Kb region were not covered by shotgun sequencing, so we considered these genes to be genuinely missing in those isolates
It is possible that the prophage ends predicted by PHASTER are slightly different from the actual prophage insertion sites (Chen et al, 2017b); this possibility applies to all analyses and we do not mention this again from this point on
Summary
Listeria monocytogenes (Lm) is a foodborne pathogen that can persist in or become repeatedly introduced to food-processing facilities (Orsi et al, 2008), causing food contamination and subsequent listeriosis, a potentially fatal illness. Whole genome sequencing (WGS) has been valuable for identifying and describing genomic variations in prophages, other mobile genomic islands, and core genomes of Lm during both long-term and short-term evolution (Kuenne et al, 2013). In long-term evolution scenarios, especially those involving multiple serotypes and genetic lineages, the majority of gene-scale differences (i.e., presence/absence of genes) in Lm occurred in mobile genetic elements (MGEs), such as hypervariable hotspots, prophages, transposons, and mobile genomic islands (Kuenne et al, 2013). A nomenclature system to define Lm clones is clonal complex (CC), which includes a group of multilocus sequence typing-defined sequence types (STs) in which any ST differs from at least one other ST by no more than one allele (Ragon et al, 2008). A singleton includes a group of isolates that differed from any existing STs of the species by at least 2 alleles (Ragon et al, 2008)
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