Genome wide characterization of 40 enterotoxigenic Escherichia coli serogroup O6 strains of multiple outbreaks from 1975-2016

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea in children under the age of five in developing countries and are the leading bacterial agent of traveler’s diarrhea. ETEC strains secrete... [ view full abstract ]

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea in children under the age of five in developing countries and are the leading bacterial agent of traveler’s diarrhea. ETEC strains secrete heat-labile (LT) and/or heat-stable (ST) enterotoxins that induce diarrhea by water and electrolyte imbalance. In this study, we characterized 40 ETEC serogroup O6 isolates from multiple outbreaks from 1975 to 2016 by whole genome sequencing (WGS) and present the findings on virulence factors, plasmids, antimicrobial resistance, whole genome high-quality single nucleotide polymorphisms (WG-hqSNPs) and core genome (CG) analyses. The closed genome of ETEC O6 strain 2011EL-1370-2 (USA, 2011) was the reference strain. DNA libraries were prepared using the Nextera XT kit (Illumina Inc.) and sequenced on the MiSeq (Illumina Inc.) using 2 x 250-bp chemistry. Raw reads were trimmed and filtered using PRINSEQ and assembled with SPAdes. Plasmids, resistance and virulence factors were identified with PlasmidFinder, ResFinder and VirulenceFinder tools from the Center for Genomic Epidemiology (CGE). Additionally, the quinolone resistance determining regions (QRDRs) of gyrA and parC were examined for mutations associated with resistance. All isolates were tested for antibiotic susceptibility to a panel of 14 antibiotics. WG-hqSNP analysis was performed with Lyve-SET 1.1.4f on raw reads with the following options: 20x minimum coverage, 95% read support, phage-masking and clustered SNPs within 5 base pairs of each other were filtered. CG analysis was performed using Parsnp. Results from CGE prediction conveyed that 33/40 genomes harbored the IncFII (pCoo) plasmid which codes for the colonization factor CS1, 24/40 genomes encoded the type IV pilus (lngA), 36/40 genomes encoded the autotransporter gene (eatA), and all 40 isolates were positive for LT and ST. Resistance determinants for streptomycin, ampicillin, tetracycline, sulfonamides and trimethoprim were detected in 11/40 isolates which correlated with phenotypic antimicrobial resistance (AR) while resistance determinants  to nalidixic acid (gyrA mutation) was detected in 10/40 isolates which correlated with resistance to nalidixic acid (>32 µg/ml) by phenotypic AR testing.  Results from WG-hqSNP and CG analyses revealed similar evolutionary relationships and significant diversity of ETEC O6 strains. A subclade in WG-hqSNP phylogeny also branched off to its own clade in CG phylogeny. The longer branch length in CG phylogeny in this subclade was attributed to 871 SNPs. In summary, the ETEC strains characterized in this study carried key virulence factors and multidrug resistance plasmids that contributed to their pathogenicity.  WG-hqSNP and CG analyses have revealed genomic similarities, differences and an overall diversity of the ETEC O6 strains that can be utilized when WGS is implemented for surveillance.