Whole-Genome Phylogenetic Analysis Reveals a Wide Diversity of Non-O157 STEC Isolated From Ground Beef and Cattle Feces

Abstract

Shiga toxin-producing Escherichia coli (STEC) causes foodborne outbreaks that can lead to complications such as hemolytic uremic syndrome. Their main reservoir is cattle, and ground beef has been frequently associated with disease and outbreaks. In this study, we attempted to understand the genetic relationship among STEC isolated in Chile from different sources, their relationship to STEC from the rest of the world, and to identify molecular markers of Chilean STEC. We sequenced 62 STEC isolated in Chile using MiSeq Illumina. In silico typing was determined using tools of the Center Genomic Epidemiology, Denmark University (CGE/DTU). Genomes of our local STEC collection were compared with 113 STEC isolated worldwide through a core genome MLST (cgMLST) approach, and we also searched for distinct genes to be used as molecular markers of Chilean isolates. Genomes in our local collection were grouped based on serogroup and sequence type, and clusters were formed within local STEC. In the worldwide STEC analysis, Chilean STEC did not cluster with genomes of the rest of the world suggesting that they are not phylogenetically related to previously described STEC. The pangenome of our STEC collection was 11,650 genes, but we did not identify distinct molecular markers of local STEC. Our results showed that there may be local emerging STEC with unique features, nevertheless, no molecular markers were detected. Therefore, there might be elements such as a syntenic organization that might explain differential clustering detected between local and worldwide STEC.

Keywords: STEC; WGS; diversity; genomics; non-O157 E. coli.

FIGURE 1

Minimum Spanning Tree of STEC isolated in Chile inferred from the cgMLST analysis. Reference genome was E. coli K-12 (Accession number NC_000913.3). The STEC core genome inclueded 1,974 genes. The number on the branches represent the allele difference between isolates. Clusters were defined as genomes with fewer than 10 allele differences and are identified as blue colors surrounding a group of genomes. Colors indicate isolation source: yellow, red, etc.

FIGURE 2

Dendrogram of 62 STEC isolated in Chile. Dendrogram calculated by the maximum likelihood method using the GTR + CAT model with 1000 bootstrap repetitions. The perimeter line color indicates serotype, stx gene, eae presence, and Sequence Type. Bootstrap value is indicated over each branch. Background color on branches indicates phylogroups.

FIGURE 3

Minimum Spanning Tree of STEC isolated from different locations in the world (n = 113) and STEC isolated in Chile (n = 62) inferred from the cgMLST analysis. Reference genome was E. coli K-12 (Number accession NC_000913.3) and the clusters (highly related genomes) were defined as genomes with 10 or fewer allele differences. The size of each circle depends on the number of genomes determined as clones. All the genomes were grouped based on serotype and sequence type. Local STEC genomes (in green) were not closely related to 113 genomes isolated from other countries and grouped at the center of the figure, except by isolates in cluster 11 (E7-2 and E6-4) and isolate P2-2-8, all at the bottom of the figure.

FIGURE 4

Pangenome of our collection of STEC isolated in Chile. Input data was generated with get_homologues, and visualization with graph tool of Microsoft, excel. The estimated pangenome was 11,650 genes. Each point corresponds to one genome; after the consecutive analysis of each one, the number of estimated genes of the STEC pangenome increases.