Application of Next Generation Sequencing for the Identification of L. monocytogenes Predictive Genome Features and the Epigenome
Listeria monocytogenes is a gram positive, intracellular pathogen that infects immune-compromised populations and has ~50% mortality rate. It is responsible for numerous food-borne outbreaks worldwide each year and commonly persists in the environment. While L.monocytogenes is the only pathogenic species in this genus it is increasingly important to define specific genomic features that are predictive for Listeria and the pathogenic species.As part of the 100KGenomeProject >1000 isolates from food, the environment, and humans are being sequenced to better understand the pan-genome of Listeria so as to enable more robust detection methods for routine testing as well as in formatic analysis for inclusion and exclusion of isolates from outbreaks as well as differentiation from non-pathogenic species quickly and accurately. To facilitate detection and outbreak identification in food-borne outbreaks, select L.monocytogenes genomes were sequenced to produce a closed genome and identify epigenetic modifications using Pacific Biosciences SMRT cell technology. This sequencing approach visualizes polymerase progression along the genome, creating a pattern of nucleotide recognition and identification of DNA modification sites correlating with the duration of polymerase stalling times. Specific modifications are identified by polymerase stalling times and patterns. Results from this study revealed that the L.monocytogenes genome contains multiple sites for DNA methylation. Methylation patterns were strain-specific with certain strains exhibiting multiple methylation patterns with no correlation to serotype or isolation source. Unexpectedly, a novel DNA modification was detected in an isolate that originated from animals that belongs to serotype 1/2a . The signal observed during sequencing was unlike any previously identified, but was repeatable and measurable in multiple locations in the genome. While the novel L.monocytogenes DNA modification was detected with SMRT cell technology the specific chemical group remains to be characterized. These results illustrate the diversity of bacterial epigenetic events, novel enzymes to catalyze the modification, unknown gene regulation strategies, and highlight the importance of understanding the role of epigenetics in the survival, host association, and pathogenesis of food-borne diseases.