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Whole Genome Sequence Analysis Using JSpecies Tool Establishes Clonal Relationships between Listeria monocytogenes Strains from Epidemiologically Unrelated Listeriosis Outbreaks - PubMed

  • ️Fri Jan 01 2016

Whole Genome Sequence Analysis Using JSpecies Tool Establishes Clonal Relationships between Listeria monocytogenes Strains from Epidemiologically Unrelated Listeriosis Outbreaks

Laurel S Burall et al. PLoS One. 2016.

Abstract

In an effort to build a comprehensive genomic approach to food safety challenges, the FDA has implemented a whole genome sequencing effort, GenomeTrakr, which involves the sequencing and analysis of genomes of foodborne pathogens. As a part of this effort, we routinely sequence whole genomes of Listeria monocytogenes (Lm) isolates associated with human listeriosis outbreaks, as well as those isolated through other sources. To rapidly establish genetic relatedness of these genomes, we evaluated tetranucleotide frequency analysis via the JSpecies program to provide a cursory analysis of strain relatedness. The JSpecies tetranucleotide (tetra) analysis plots standardized (z-score) tetramer word frequencies of two strains against each other and uses linear regression analysis to determine similarity (r2). This tool was able to validate the close relationships between outbreak related strains from four different outbreaks. Included in this study was the analysis of Lm strains isolated during the recent caramel apple outbreak and stone fruit incident in 2014. We identified that many of the isolates from these two outbreaks shared a common 4b variant (4bV) serotype, also designated as IVb-v1, using a qPCR protocol developed in our laboratory. The 4bV serotype is characterized by the presence of a 6.3 Kb DNA segment normally found in serotype 1/2a, 3a, 1/2c and 3c strains but not in serotype 4b or 1/2b strains. We decided to compare these strains at a genomic level using the JSpecies Tetra tool. Specifically, we compared several 4bV and 4b isolates and identified a high level of similarity between the stone fruit and apple 4bV strains, but not the 4b strains co-identified in the caramel apple outbreak or other 4b or 4bV strains in our collection. This finding was further substantiated by a SNP-based analysis. Additionally, we were able to identify close relatedness between isolates from clinical cases from 1993-1994 and a single case from 2011 as well as links between two isolates from over 30 years ago. The identification of these potential links shows that JSpecies Tetra analysis can be a useful tool in rapidly assessing genetic relatedness of Lm isolates during outbreak investigations and for comparing historical isolates. Our analyses led to the identification of a highly related clonal group involved in two separate outbreaks, stone fruit and caramel apple, and suggests the possibility of a new genotype that may be better adapted for certain foods and/or environment.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Evolutionary relationships of taxa.

The evolutionary history was inferred using the Neighbor-Joining method based on the data from the BLAST-SNP analysis [22]. The optimal tree with the sum of branch length = 30986.27311707is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches [32]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the number of differences method [33] and are in the units of the number of base differences per site. The analysis involved 49 nucleotide sequences of 4b and 4bV strains indicated in Table 1. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair. There were a total of 23545 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [27]. The strains highlighted in Table 1 are similarly noted here with cluster 1 in purple, cluster 2 in green, and cluster 3 in blue.

Fig 2
Fig 2. An optimal tree focusing on cluster 3 containing the stone fruit and apple 4bV isolates.

The tree was generated as in Fig 1 with a sum of branch length = 129.46875000 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [32]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the number of differences method [33] and are in the units of the number of base differences per sequence. The analysis involved 13 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair. There were a total of 23545 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

Fig 3
Fig 3. Neighbor-Joining trees based on the CFSAN SNP pipeline results.

A) This tree shows the evolutionary relationships between the 4b and 4bV strains, using LS642 as the reference strain. B) This tree examines 4b and 4bV strains, using F2365 as the reference strain. Clusters 1, 2 and 3 are highlighted in purple, green and blue, respectively, in both. The use of arrows used in 3a show the alteration that can occur using a more distantly related reference strain.

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Grants and funding

This project was supported in part by an appointment to the Internship/Research Participation Program at the Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy and FDA (http://orise.orau.gov/). This funding supported the appointment of CJG. ORISE had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. All other funding came from the U.S. Food and Drug Administration.