|BERGHOLZ, TERESA - North Dakota State University|
|DEN BAKKER, HENK - Cornell University - New York|
|KATZ, LEE - Centers For Disease Control And Prevention (CDCP) - United States|
|SILK, BENJAMIN - Centers For Disease Control And Prevention (CDCP) - United States|
|JACKSON, KELLY - Centers For Disease Control And Prevention (CDCP) - United States|
|KUCEROVA, ZUZANA - Centers For Disease Control And Prevention (CDCP) - United States|
|JOSEPH, LAVIN - Centers For Disease Control And Prevention (CDCP) - United States|
|TURNSEK, MARYANN - Centers For Disease Control And Prevention (CDCP) - United States|
|GLADNEY, LORI - Centers For Disease Control And Prevention (CDCP) - United States|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/29/2015
Publication Date: 5/29/2015
Citation: Bergholz, T.M., Den Bakker, H.C., Katz, L.S., Silk, B.J., Jackson, K.A., Kucerova, Z., Joseph, L.A., Turnsek, M., Gladney, L.M., Ward, T.J. 2015. Evolutionary relationships of outbreak-associated Listeria monocytogenes strains of serotypes 1/2a and 1/2b determined by whole genome sequencing [abstract].
Technical Abstract: Listeria monocytogenes (Lm) is a bacterial pathogen that is almost exclusively transmitted by food. Although listeriosis is relatively rare (~1600 cases occur annually in the U.S.), ~20% of cases are fatal and outbreaks are not uncommon. Molecular subtyping differentiates Lm into four lineages (LI – LIV), and strains of serotypes 1/2a (belonging to LI), and 1/2b and 4b (belonging to LII) are most frequently associated with human illness. Within LI and LII, prevalent clonal complexes (CCs) have been identified using a 7-gene Multi-Locus Sequence Typing scheme (MLST, Pasteur Institute). Some prevalent CCs correspond to the epidemic clones (ECs) that have been designated in Lm; ECs are groups of strains that have been implicated in multiple unrelated outbreaks. Traditional subtyping methods such as pulsed-field gel electrophoresis (PFGE) cannot be used for evolutionary inference, so the genetic relationship of some historical outbreak strains to the globally disseminated CCs/ECs is unknown. In 2011, strains of serotypes 1/2a and 1/2b were implicated in the largest listeriosis outbreak in U.S. history; the large outbreak associated with cantaloupe consumption provided the impetus to use whole genome sequencing (WGS) to reconstruct the evolutionary relationships among outbreak-associated strains of serotypes 1/2a and 1/2b. We used WGS to determine the evolutionary relationships among 69 Lm strains, including 20 outbreak-associated isolates of serotypes 1/2a and 1/2b. We used a GAIIx and standard Illumina chemistry to generate 76-bp reads, which were assembled using Velvet v. 18.104.22.168 and VelvetOptimiser-2.2.4. Single nucleotide variants (SNVs) were called using kSNP v2.0 with a k-mer size of 19; the resulting SNV matrix was input into RAxML v7.4.2 for phylogenetic analysis. The seven MLST genes were extracted from WGS and isolates were assigned to CCs. We used various comparative genomics tools [Lyve-SET v0.7 for hqSNPs, SnpEff , a Markov Clustering (MCL) method, MAUVE, PHAST, MEGA5] to perform detailed comparisons of WGS from epidemiologically-related isolate pairs within CCs. Two key findings emerged from WGS analyses: first, epidemic potential in Lm was not confined to prevalent CCs/ECs. The 20 isolates of serotype 1/2a and 1/2b fell into 10 distinct phylogenetic groups, but six of these groups fell outside of the large CCs that have been previously associated with outbreaks, suggesting that epidemic potential may be widespread in Lm. Second, genetic divergence among isolates occurred rapidly via recombination within the production environment. Pairwise comparisons showed that genome-level polymorphism varied by two orders of magnitude between epidemiologically related isolates; divergence in one CC was driven primarily by recombination in phage regions, which could be attributed to a diverse bacterial community present in the production environment associated with the implicated product. Understanding how both sporadic and outbreak isolates are distributed into clonal groups provides a framework for further investigation into the biological characteristics of more successful groups, with the goal of identifying the genetic basis for the traits that enable higher rates of transmission and virulence. The insights provided by examining diversity in an evolutionary framework may ultimately lead to better control methods for Lm.