On June 1, 2011, a 67-year-old Canadian man sought treatment at an Ontario hospital with a 3-day history of bloody diarrhea. He had returned from Germany on May 27. He had no signs of HUS, and E. coli O157:H7 was not detected by routine testing. Clinical specimens from this patient were referred to the Public Health Ontario Laboratories for testing. Shiga toxin was detected by enzyme immunoassay (Meridian Biosciences, Inc., Cincinnati, OH, USA), and real–time PCR confirmed that the strain, named ON-2011, was positive for the stx2 gene and negative for the eae gene (3,6). Biochemical and serologic testing confirmed that the isolate was E. coli serogroup O104:H4. The patient recovered uneventfully (4).

Before the May 2011 outbreak in Germany, a single isolate of E. coli O104:H4 had been identified in Ontario. That isolate, ON-2010, was recovered in June 2010 from a 10-month-old boy who had returned from the Philippines 2 days earlier. He was brought to the hospital with a 1-day history of vomiting and nonbloody diarrhea. A sorbitol-nonfermenting colony was recovered from a stool specimen, but it did not react with E. coli O157 antiserum. The specimen was referred to the Canadian National Microbiology Laboratory, which confirmed E. coli O104:H4. Retrospective PCR-based testing showed that this isolate was negative for stx and eae. The infant made a full and uneventful recovery.

Etest (AB BIODISK, Solna, Sweden) susceptibility testing was performed by using a standard inoculum (0.5–McFarland standard); agar dilution indicated that ON-2010 was pansusceptible, whereas ON-2011 was resistant to amikacin, tetracycline, trimethoprim/sulfamethoxazole, and extended-spectrum β–lactams. PCR results for CTX–M–15 and TEM–1 were negative for ON-2010 and positive for ON-2011 (7). The gene for tellurite resistance was also absent in ON-2010, but present in ON-2011. Although both isolates were of multilocus sequence type 678, pulsed-field gel electrophoresis profiles were distinct (Figure, panel A) (8). Patterns for ON-2011 (ENXAI.0024/ENBNI.0022, PulseNet Canada designations) were identical with those reported for the current outbreak strain from Germany, whereas the ON-2010 profiles (ECXAI.2585/ECBNI.0922) were distinct (9).

Circular, highresolution NcoI restriction maps of the ON-2010 (≈5.1 Mbp) and ON-2011 (≈5.25 Mbp) genomes were generated by optical mapping (Argus Optical Mapper, OpGen, Inc, Gaithersburg, MD, USA). These were compared with an in silico map of E. coli 55989, an O104:H4 strain that was isolated in Africa and sequenced in France (10). The extensive synteny observed and the absence of large-scale genomic rearrangements (e.g., inversions, translocations) suggest that ON-2010 and ON-2011 descended from an E. coli 55989–like ancestor (Figure, panel B).

The evolutionary relationship of the 2 isolates from Ontario was further assessed by whole-genome sequencing (Roche GS–FLX Titanium; Roche Diagnostics, Laval, QC, Canada). This confirmed the genomic similarity of ON-2010 to E. coli 55989 and uncovered a 72-kb plasmid, pON-2010. Virulence genes ON-2010 and ON-2011 were compared by Bielaszewska et al. (11) (Table) on the basis of virulence factor analysis of the 2011 outbreak strain of E. coli O104:H4 from Germany. The plasmid exhibits >99% identity with p55989 and encodes the aggregative adhesion fimbriae cluster that is a defining features of enteroaggregative E. coli (EAEC) (12). In contrast, ON-2011 contains 3 plasmids. Reference-based mapping of raw sequencing reads against publicly available genome and plasmid scaffolds (HPA, BGI) confirmed that ON-2011 is a German outbreak clone (13). Except for a small number of single nucleotide polymorphisms, the clone isolated in Canada is virtually identical to those from Germany and the United Kingdom.

The O104:H4 outbreak strain from Germany exhibits key features of classic EAEC, but also contains numerous horizontally acquired virulence factors. An stx2-encoding prophage is responsible for the toxigenic properties of this strain, and multidrug-resistance loci are present, including a chromosomal tetR loci, and the plasmid-encoded CTX-M-15, TEM-1, and tellurite resistance–encoding loci (ter) (14). In contrast, the ON-2010 strain does not encode Shiga toxin, and no resistance genes have been identified. Even the tetracycline-resistance genes, independently acquired by both ON-2011 and strain 55989, are absent (Figure, panel B). However, the plasmid-encoding aggregative adhesion fimbriae cluster is present. Preliminary comparison of the 3 genomes has also shown some small-scale rearrangement events. One of these rearrangements eliminates siderophore biosynthesis genes from ON-2010 and may compromise bacterial iron acquisition. However, ON-2010 does have a strain-specific insertion that contains the hydroxyphenylacetate (hpa) operon, which may provide a nutritional advantage by enabling the organism to catabolize the phenolic and aromatic compounds abundant in the gut. This insertion is absent from the other O104:H4 genomes (15).

Since 2010, 2 cases of travel-associated E. coli O104:H4 infection have been identified in Ontario, Canada. Our analysis of these isolates could inform genomic studies on the emergence and evolution of the O104 clone first observed in Europe. The relationship of these strains was assessed by a combination of traditional, molecular, and genomic approaches. Optical mapping and sequencing indicate that ON-2010 and ON-2011 exhibit extensive synteny and are derived from a common EAEC ancestor. However, a series of horizontal gene transfer events has contributed to genotypic and phenotypic divergence. The outbreak clone from Germany, ON-2011, is an antimicrobial drug–resistant STEC isolate, whereas ON-2010 is pansusceptible and nontoxigenic.

These differences are clinically important but are not detected by diagnostic strategies that use serotype as a proxy for pathogenic capacity. Given the potential for severe clinical sequelae of STEC infections, clinical testing should use improved and affordable methods for identification of Shiga toxin that can be easily integrated into routine clinical microbiology laboratories (4,9). Future capacity to detect additional virulence factors, such as aggregative adhesion fimbriae, would enable expanded diagnostic capacity to detect pathogenic E. coli subtypes that cause human disease.