High-Quality Complete Genome Sequences of Three Bovine Shiga Toxin-Producing Escherichia coli O177:H- (fliC_H25) Isolates Harboring Virulent stx2 and Multiple Plasmids

Shiga toxin (Stx)-producing Escherichia coli (STEC) can cause hemorrhagic colitis and hemolytic uremic syndrome (1–3). Cattle are major reservoirs of STEC and are the most common source for foodborne infections (4–6). During a longitudinal survey of STEC in healthy cattle, we isolated three non-O157 STEC strains that share phenotypic similarity with the O157 serogroup (7). Because these strains were nonmotile when isolated, DNA microarray for STEC serotyping (8) confirmed that these strains (SMN152SH1, SMN013SH2, and SMN197SH3, formerly named SH1, SH2 and SH3 respectively.) belonged to serogroup O177:H25. Based on these data we reassigned the serotype of these strains to O177:H- (fliC_H25: fliC genotype H25). Among the surveyed cattle in Washington and Idaho, US, and Alberta, Canada, the O177:H- serotype was more prevalent than the O157:H7 serotype (7). We report here the availability of three high-quality complete genomes of STEC O177:H- assembled using a hybrid approach that combines PacBio and Illumina reads.

Genomic DNA was prepared using a GenElute bacterial Genomic DNA kit (Sigma-Aldrich, St. Louis, MO) per the manufacturer’s instructions. Whole-genome sequencing was carried out on a PacBio RS II (Pacific Biosciences, Menlo Park, CA) platform with 6-kb and 20-kb insert libraries by using P6 polymerase-binding and C4-sequencing kits for 240-min acquisition. Short-read sequencing was done at the IBEST Genomics Resources Core at the University of Idaho, using an Illumina MiSeq and v2 500-cyles kit (PE250) following construction of barcoded Illumina libraries using the IntegenX Apollo 324 PrepX ILM DNA library kit and custom Illumina TruSeq barcoded adapters. The Illumina reads were cleaned using HTStream (https://github.com/ibest/HTStream) to trim adapter sequences and low-quality ends and to overlap paired-end reads. Long reads were assembled with Canu v1.6 using default parameters (9). Gaps between contigs were sealed with GapBlaster (10). The small plasmids of SMN152SH1 and SMN197SH3 were assembled with Unicycler (11). The assembled genomes were oriented and trimmed using Circlator v1.5.2 (12), then corrected with Pilon v1.22 using cleaned Illumina reads (13). The resulting assemblies were verified using restriction enzyme NcoI whole-genome maps generated according to the OpGen protocol (OpGen, Inc., Gaithersburg, MD).

The genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/). Features of the three O177:H- isolates include an stx_2c variant gene which is 100% identical to the stx_2c carried by a clinical isolate E. coli O177 (14); nearly identical pO157-like 88-kb and 48-kb plasmids, a 6.7-kb colicinogenic plasmid in SMN152SH1 and SMN197SH3; and chromosomally encoded AmpC type β-lactamase.

A detailed report on additional analysis of these genomes will be included in a future publication.

This work was funded by the National Institute of General Medical Sciences of the National Institutes of Health under award number P30GM103324 through the IBEST Genomics and Computational Resources Cores and P20GM103408 (Idaho INBRE Program) and the USDA National Institute for Food and Agriculture, Hatch Project numbers IDA01467 (C.J.H.) and IDA01406 (S.A.M.), with additional support from the University of Idaho Agriculture Experiment Station and New Mexico INBRE NM-INBRE P20 GM103451.