Complete Genome Sequences of 12 Isolates of Listeria monocytogenes Belonging to Serotypes 1/2a, 1/2b, and 4b Obtained from Food Products and Food-Processing Environments in Canada

GENOME ANNOUNCEMENT

Listeriosis, a life-threatening infection caused by Listeria monocytogenes, results in one of the highest mortality rates among foodborne illnesses in Canada, with an average of 44 deaths each year (90% probability intervals: 31-76 deaths) (1). In order to reduce the risk of listeriosis, studies aimed at understanding the nature and behavior of the causative bacterial organism at the genomic level are necessary. L. monocytogenes is a ubiquitous, Gram-positive bacterium commonly found in natural environments such as plants, soil, and surface water, from where it contaminates agricultural products, food-processing environments, and foods such as fresh produce and ready-to-eat meat and milk, leading to possible human exposure (2). More than 95% of human clinical cases are associated with three serotypes of L. monocytogenes: 1/2a, 1/2b, and 4b (3). Here, we report the complete genome sequences of 12 L. monocytogenes isolates belonging to these serotypes. The strains were collected from foods and food-processing environments in Canada during the period from 2002 to 2009 as part of routine testing procedures carried out by the Agriculture and Food Laboratory in Guelph, Ontario, Canada. Strains were selected to represent both predominant and unique genotypes among over 2,400 strains tested during a comprehensive genotypic analysis (4).

Genomic DNA samples were extracted from overnight cultures of the 12 L. monocytogenes strains using the Wizard Genomic DNA purification Kit (Promega, Madison, WI, USA). Each isolate was sequenced using PacBio (Pacific BioSciences Inc., Menlo Park, CA, USA) and Illumina MiSeq (Illumina Inc., San Diego, CA, USA) technologies, as per standard protocols in 2013, and assembled independently. The PacBio reads were assembled with the Celera assembler using the HGAP/Quiver protocol (5) and corrected with Illumina sequence data. The Illumina reads, on the other hand, were assembled using the hybrid approach as previously described (6). The final corrected genome for each chromosome was developed by aligning an in silico map of the Illumina read-corrected PacBio assembly with the map of the corresponding, independently assembled Illumina sequence against an optical, de novo whole-genome map. The map consisted of NheI restriction sites and was developed using the Argus optical mapping system (OpGen Inc., Gaithersburg, MD, USA), and the analysis was done using the MapSolver software. The nucleotide sequences of all detected gaps and misalignments were corrected using Clone Manager software (Professional edition, Scientific and Educational Software, Cary, NC, USA).

The generated genomes range from 2,913,947 to 3,072,093 bp with a GC content of 37.95 ± 0.02% (average ± standard error of the mean [SEM]). We used the Rapid Annotations using Subsystem Technology server (7–9) to annotate each genome. We identified 2,944 ± 21.6 (average ± SEM) protein-coding sequences and 64 ± 1.29 (average ± SEM) tRNAs per genome. Alignment of all 12 genome sequences using the Mauve system of multiple genome alignment (10) demonstrated clustering based on serotypes. Average nucleotide identity analysis (http://enve-omics.ce.gatech.edu/ani ) confirmed similarities within each serotype (1/2a: 98.60 to 98.99%, n = 5; 1/2b: 99.44 to 99.94%, n = 3; 4b: 99.47 to 99.60%, n = 4) and was able to differentiate between serotype 1/2a and the other two serotypes (versus 1/2b: 94.32 to 94.47%; versus 4b: 94.19 to 94.35%); however, serotypes 1/2b and 4b were very similar (99.42 to 99.57%). A complete comparative genomic study of the 12 strains will be presented elsewhere.