Population structure and genetic diversity of non-O157 Shiga toxin-producing Escherichia coli (STEC) clinical isolates from Michigan

Abstract

Non-O157 STEC are increasingly linked to foodborne infections, yet little is known about the diversity and molecular epidemiology across locations. Herein, we used whole genome sequencing to examine genetic variation in 894 isolates collected from Michigan patients between 2001 and 2018. In all, 67 serotypes representing 69 multilocus sequence types were identified. Serotype diversity increased from an average of four (2001-2006) to 17 (2008-2018) serotypes per year. The top six serogroups reported nationally caused > 60% of infections in 16 of the 18 years; serogroups O111 and O45 were associated with hospitalization as were age ≥ 65 years, diarrhea with blood and female sex. Phylogenetic analyses of seven multilocus sequence typing (MLST) loci identified three clades as well as evidence of parallel evolution and recombination. Most (95.5%) isolates belonged to one clade, which could be further differentiated into seven subclades comprising isolates with varying virulence gene profiles and serotypes. No association was observed between specific clades and the epidemiological data, suggesting that serogroup- and serotype-specific associations are more important predictors of disease outcomes than lineages defined by MLST. Molecular epidemiological studies of non-O157 STEC are important to enhance understanding of circulating strain distributions and traits, genetic variation, and factors that may impact disease risk and severity.

Figure 1

Frequency and number of non-O157 Shiga toxin-producing Escherichia coli serogroups reported in Michigan by year. The y-axis on the left represents the frequency of each serogroup by year and is indicated by the different colored bars. The y-axis on the right shows the number of serogroups per year indicated by the black line. The top six serogroups are indicated with an * in the figure legend. NT non-typeable.

Figure 2

Neighbor-joining phylogeny based on seven multilocus sequence typing loci (3738 bp) among non-O157 Shiga toxin-producing Escherichia coli isolates from Michigan, 2001–2018. The nodes at each branch represent the support percentages after bootstrapping (1000 replicates). The Maximum Composite Likelihood method was used to calculate the evolutionary distances (number of base substitutions per site). Three clades were identified that clustered together with > 70% bootstrap support, while seven subclades, A-G, are shown within clade I. Sequence types (STs) are noted at the end of each branch followed by the serogroup and number (n) of isolates per serogroup. The big six serogroups and a set of additional serogroups that were found on multiple branches of the phylogeny are represented by different colored circles. The frequency of Shiga toxin (stx) genes per subclade and clade is shown in the pie charts with different colors representing the three toxin gene profiles.

Figure 3

The neighbor-net analysis of 194 parsimonious informative sites among 69 non-O157 Shiga toxin-producing Escherichia coli multilocus sequence types (STs). The phylogenetic network was constructed from untransformed distances using Splitstree4. Recombination is indicated as parallelograms and illustrate multiple paths between most STs. The pairwise homoplasy index (PHI), which was used to test for recombination, was significant (p ≤ 0.00001). The network confirms the tree structure of the neighbor-joining phylogeny and clearly delineates the three clades and singleton STs. The five clade I subclades are shaded with light green rectangles. The STs are indicated at the end of specific branches followed by the serogroups represented. Colored squares represent the intimin (eae) allele identified, while the colored triangles are the enterohemolysin (ehxA) subtypes.

Figure 4

Distribution of gene alleles encoding the (A) Shiga toxins (stx), (B) intimin (eae), and (C) enterohemolysin (ehxA) among the predominant non-O157 Shiga toxin-producing Escherichia coli serogroups in Michigan. NT = non-typeable.