Multilocus Sequence Typing of Escherichia coli O26:H11 Isolates Carrying stx in Canada Does Not Identify Genetic Diversity

Abstract

Multilocus sequence typing of 31 stx-carrying Escherichia coli O26:H11 strains isolated in Canada between 1999 and 2003 revealed a high degree of genetic relatedness at 10 loci, suggesting either that this is a clonal serotype (similar to O157:H7) or that additional genetic loci need to be examined.Shiga toxin-producing Escherichia coli (STEC) serotype O26:H11 is the second most common serotype of STEC identified in Canada, behind O157:H7 (12). STEC O26:H11 naturally occurs in cattle, diarrheic calves, and feedlots and has been reported globally in countries including, but not limited to, Japan, England, Argentina, and Canada (2,5,7,9,11). In humans, STEC O26:H11 causes sporadic cases and outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome; and the incidence of disease resulting from this serotype may outnumber that resulting from O157:H7 in particular regions (3, 10). The discrepancy between incidence and clinical reporting is partly due to a lack of accessible and standardized methods for differentiation of this pathogen. Nucleotide sequence data for this serotype are limited in comparison to those for O157:H7; however, the virulence determinants which have been consistently observed in O26:H11 isolates include the prophage-encoded stx 1 and stx 2 Shiga toxin genes, the hlyAencoded hemolysin, high-molecular-weight plasmids, and the locus for enterocyte effacement pathogenicity island, which encodes a type III secretion system and the eae-␤1 intimin gene (3,14). Multilocus sequencing typing (MLST) has previously been performed with strains of the O157:H7 and O78 serotypes (1, 8), and it was our objective to develop a MLST scheme to determine the genetic relatedness of stx 1 -carrying E. coli O26:H11 and potentially reveal epidemiological relationships.The National Microbiology Laboratory (NML) in Winnipeg, Manitoba, Canada, received isolates of E. coli O26:H11 from provincial public health laboratories in Alberta, British Columbia, Saskatchewan, Manitoba, and Nova Scotia, a collection that represents a latitudinal cross-section of Canadian regions (Table 1). We sought to develop an alternative to pulsed-field gel electrophoresis (PFGE) in order to reveal epidemiological relationships, and MLST was a candidate methodology because of the ease of standardization and its potentially robust discriminatory power. Sequence analysis of the mdh, gnd, gcl, ppk, metA, fliC, ftsZ, relA, recN, and metG genes from strains isolated from 1999 to 2003 in the different Canadian provinces was completed. Included in this study were two non-H11 O26 isolates to identify serotype-specific genetic relatedness. Additionally, the sole stx 1 -and stx 2 -carrying O26:H11 isolate reported to NML was used to identify the genetic relatedness to stx 1 -carrying strains at the loci examined. The PFGE patterns obtained with XbaI digestion were also determined for each strain.The oligonucleotide primers used to amplify mdh (580 bp), gnd (590 bp), gcl (758 bp), ppk (758 bp), and metA (601 bp) were described previously (1, 8)...

Identification of Genetic Markers for Differentiation of Shiga Toxin-Producing, Enteropathogenic, and Avirulent Strains ofEscherichia coliO26

“…High prevalence rates of EHEC O26 in patients with HUS and diarrhea were reported from other European countries, such as the Czech Republic (3), Denmark (15), Finland (13), Italy (41), and Spain (7). Reports on an association between EHEC O26 and HUS or diarrhea from North America, including the United States (8,22,25), South America (39,42), Australia (14), and Asia (23,24), provide further evidence for the worldwide significance of EHEC O26 strains.…”

Potentially Human-Pathogenic Escherichia coli O26 in Norwegian Sheep Flocks

“…A newer method, like multilocus sequence typing (MLST), would be the method of choice to assess the relatedness of all E. coli O26 isolates regardless of H type. However, MLST has been shown to differentiate poorly between E. coli bacteria that are clonally highly conserved but epidemiologically unlinked, such as isolates of serotype O26:H11 (20). For STEC belonging to serotype O157:H7, multilocus variable number tandem repeat analysis (MLVA) has been shown to be a rapid and relatively simple method with a high level of coclustering with the PFGE method (22,30,35).…”

Use of the espZ Gene Encoded in the Locus of Enterocyte Effacement for Molecular Typing of Shiga Toxin-Producing Escherichia coli

“…The bias towards O157 in clinical settings may be a result of the established methods for identification and the availability of selective media (39). To develop novel molecular methods specifically for non-O157 STEC, we had previously performed multilocus sequence typing on Canadian O26:H11 STEC isolates but were unable to identify significant genetic diversity to subtype this collection of strains (10). Whereas the goal of multilocus sequence typing is to identify polymorphisms between related strains, other typing methodologies such as serotyping can identify more global characteristics that are indicative of corresponding genetic traits (i.e., serotype O157:H7 strains produce Shiga toxins).…”