Bacteriophage 2851 is a prototype phage for dissemination of the Shiga toxin variant gene 2c in Escherichia coli O157:H7

The production of Shiga toxin (Stx) (verocytotoxin) is a major virulence factor of Escherichia coli O157:H7 strains (Shiga toxin-producing E. coli [STEC] O157). Two types of Shiga toxins, designated Stx1 and Stx2, are produced in STEC O157. Variants of the Stx2 type (Stx2, Stx2c) are associated with high virulences of these strains for humans. A bacteriophage designated 2851 from a human STEC O157 encoding the Stx2c variant was described previously. Nucleotide sequence analysis of the phage 2851 genome revealed 75 predicted coding sequences and indicated a mosaic structure typical for lambdoid phages. Analyses of free phages and K-12 phage 2851 lysogens revealed that upon excision from the bacterial chromosome, the loss of a phage-encoded IS629 element leads to fusion of phage antA and antB genes, with the generation of a recombined antAB gene encoding a strong antirepressor. In wild-type E. coli O157 as well as in K-12 strains, phage 2851 was found to be integrated in the sbcB locus. Additionally, phage 2851 carries an open reading frame which encodes an OspB-like type III effector similar to that found in Shigella spp. Investigation of 39 stx_2c E. coli O157 strains revealed that all except 1 were positive for most phage 2851-specific genes and possessed a prophage with the same border sequences integrated into the sbcB locus. Phage 2851-specific sequences were absent from most stx_2c-negative E. coli O157 strains, and we suggest that phage 2851-like phages contributed significantly to the dissemination of the Stx2c variant toxin within this group of E. coli.     

Shiga toxin 2 overexpression in Escherichia coli O157:H7 strains associated with severe human disease

Variation in disease severity among Escherichia coli O157:H7 infections may result from differential expression of Shiga toxin 2 (Stx2). Eleven strains belonging to four prominent phylogenetic clades, including clade 8 strains representative of the 2006 U.S. spinach outbreak, were examined for stx2 expression by real-time PCR and western blot analysis. Clade 8 strains were shown to overexpress stx2 basally, and following induction with ciprofloxacin when compared to strains from clades 1–3. Differences in stx2 expression generally correlated with Stx2 protein levels. Single-nucleotide polymorphisms identified in regions upstream of stx2AB in clade 8 strains were largely absent in non-clade 8 strains. This study concludes that stx2 overexpression is common to strains from clade 8 associated with hemolytic uremic syndrome, and describes SNPs which may affect stx2 expression and which could be useful in the genetic differentiation of highly-virulent strains.     

Identification and characterisation of Escherichia coli strains of O157 and non-O157 serogroups containing three distinct Shiga toxin genes

Three Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from patients with diarrhoea were identified, each of which contained three distinct stx genes (stx1, stx2 and stx2c). The strains belonged to the serotypes O52:H19, O75:H- and O157:H- and harboured eae and EHEC-hly sequences. Colony-blot immunoassay was used to demonstrate that both major types of Stx were expressed. The association of stx genes with either phage or phage DNA was demonstrated in all three strains. Isolated phage DNA from all strains contained stx1 sequences, but stx2 sequences were found only in phage DNA of two of these strains. The presence of three distinct stx genes may enhance the virulence of STEC strains and should be monitored. The observations demonstrate not only the potential of stx genes to spread within different serotypes, but also their capacity to accumulate within a single strain.     

Identification, characterization, and distribution of a Shiga toxin 1 gene variant (stx(1c)) in Escherichia coli strains isolated from humans

By using sequence analysis of Shiga toxin 1 (Stx 1) genes from human and ovine Stx-producing Escherichia coli (STEC) strains, we identified an Stx1 variant in STEC of human origin that was identical to the Stx1 variant from ovine STEC, but demonstrated only 97.1 and 96.6% amino acid sequence identity in its A and B subunits, respectively, to the Stx1 encoded by bacteriophage 933J. We designated this variant "Stx1c" and developed stxB(1) restriction fragment length polymorphism and stx(1c)-specific PCR strategies to determine the frequency and distribution of stx(1c) among 212 STEC strains isolated from humans. stx(1c) was identified in 36 (17.0%) of 212 STEC strains, 19 of which originated from asymptomatic subjects and 16 of which were from patients with uncomplicated diarrhea. stx(1c) was most frequently (in 23 STEC strains [63.9%]) associated with stx(2d), but 12 (33.3%) of the 36 STEC strains possessed stx(1c) only. A single STEC strain possessed stx(1c) together with stx(2) and was isolated from a patient with hemolytic-uremic syndrome. All 36 stx(1c)-positive STEC strains were eae negative and belonged to 10 different serogroups, none of which was O157, O26, O103, O111, or O145. Stx1c was produced by all stx(1c)-containing STEC strains, but reacted weakly with a commercial immunoassay. We conclude that STEC strains harboring the stx(1c) variant account for a significant proportion of human STEC isolates. The procedures developed in this study now allow the determination of the frequency of STEC strains harboring stx(1c) among clinical STEC isolates and their association with human disease in prospective studies.     

Comparative pathogenicity of Escherichia coli O157 and intimin-negative non-O157 Shiga toxin-producing E coli strains in neonatal pigs

We compared the pathogenicity of intimin-negative non-O157:H7 Shiga toxin (Stx)-producing Escherichia coli (STEC) O91:H21 and O104:H21 strains with the pathogenicity of intimin-positive O157:H7 and O157:H(-) strains in neonatal pigs. We also examined the role of Stx2d-activatable genes and the large hemolysin-encoding plasmid of O91:H21 strain B2F1 in the pathogenesis of STEC disease in pigs. We found that all E. coli strains that made wild-type levels of Stx caused systemic illness and histological lesions in the brain and intestinal crypts, whereas none of the control Stx-negative E. coli strains evoked comparable central nervous system signs or intestinal lesions. By contrast, the absence of intimin, hemolysin, or motility had little impact on the overall pathogenesis of systemic disease during STEC infection. The most striking differences between pigs inoculated with non-O157 STEC strains and pigs inoculated with O157 STEC strains were the absence of attaching and effacing intestinal lesions in pigs inoculated with non-O157:H7 strains and the apparent association between the level of Stx2d-activatable toxin produced by an STEC strain and the severity of lesions.     

Escherichia coli strains producing a novel Shiga toxin 2 subtype circulate in China

Shiga toxin (Stx) is the key virulence factor in Shiga toxin producing Escherichia coli (STEC), which can cause diarrhea and hemorrhagic colitis with life-threatening complications. Stx comprises two toxin types, Stx1 and Stx2. Several Stx1/Stx2 subtypes have been identified in E. coli, which are variable in sequences, toxicity and host specificity. Here, we report the identification of a novel Stx2 subtype, designated Stx2k, in E. coli strains widely detected from diarrheal patients, animals, and raw meats in China over time. Stx2k exhibits varied cytotoxicity in vitro among individual strains. The Stx2k converting prophages displayed considerable heterogeneity in terms of insertion site, genetic content and structure. Whole genome analysis revealed that the stx_2k-containing strains were genetically heterogeneous with diverse serotypes, sequence types, and virulence gene profiles. The nine stx_2k-containing strains formed two major phylogenetic clusters closely with strains belonging to STEC, enterotoxigenic E. coli (ETEC), and STEC/ETEC hybrid. One stx_2k-containing strain harbored one plasmid-encoded heat-stable enterotoxin sta gene and two identical copies of chromosome-encoded stb gene, exhibiting STEC/ETEC hybrid pathotype. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of STEC strains. Given the wide distribution of the Stx2k-producing strains in diverse sources and their pathogenic potential, Stx2k should be taken into account in epidemiological surveillance of STEC infections and clinical diagnosis.     

Purification and some properties of a Vero toxin from Escherichia coli O157:H7 that is immunologically unrelated to Shiga toxin

A cytotoxin to Vero cells (Vero toxin) was purified from Escherichia coli O157:H7 isolated from a patient with hemorrhagic colitis by ammonium sulfate fractionation, DEAE-cellulose column chromatography, repeated chromatofocusing column chromatography and repeated high performance liquid chromatography. About 440 micrograms of purified Vero toxin was obtained from 12 liters of culture with a yield of about 22%. The purified Vero toxin showed similar cytotoxic activity to that of Shiga toxin to Vero cells and killed about 50% of the Vero cells at 1 pg. The activity was lost on heating the toxin at 80 degrees C for 10 minutes, but not at 60 degrees C for 10 minutes. The toxin also showed lethal toxicity to mice when injected intraperitoneally, the LD50 being 1 ng per mouse. The purified Vero toxin consisted of A and B subunits with molecular weights of about 35,000 and 10,700, respectively, which were slightly larger than those of Shiga toxin. On polyacrylamide gel disc electrophoresis, the mobility of the purified Vero toxin differed from that of Shiga toxin. The isoelectric point of the toxin was 4.1, which was also different from that of Shiga toxin (pI = 7.0). Furthermore, Vero toxin and Shiga toxin were found to be immunologically unrelated; anti-Vero toxin did not react with Shiga toxin, and similarly anti-Shiga toxin did not react with the Vero toxin in either the Ouchterlony double gel diffusion test or enzyme-linked immunosorbent assay. The Vero toxin purified in this work was found to be immunologically identical to VT2 and Shiga-like toxin II reported previously.     

Purification and some properties of Shiga-like toxin from Escherichia coli O157:H7 that is immunologically identical to Shiga toxin

A cytotoxin to Vero cells (Shiga-like toxin), which was neutralized by antibody against purified Shiga toxin produced by Shigella dysenteriae 1, was purified from Escherichia coli O157:H7, isolated from a patient with hemorrhagic colitis. The purification procedure consisted of ammonium sulfate fractionation, DEAE-cellulose column chromatography, chromatofocusing column chromatography and high performance liquid chromatography. About 200 micrograms of purified Shiga-like toxin was obtained from cell extracts of 14 liters of culture with a yield of about 15%. The purified Shiga-like toxin showed identical physicochemical, biological and immunological properties to those of Shiga toxin. Purified Shiga-like toxin and Shiga toxin also had the same mobilities on polyacrylamide disc gel electrophoresis and polyacrylamide gel isoelectrofocusing. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, purified Shiga-like toxin migrated as two bands corresponding to the A and B subunits, and these migrated to the same positions as A and B subunits of Shiga toxin. The amino acid composition of the purified Shiga-like toxin was also similar to that of Shiga toxin. The purified Shiga-like toxin showed various biological activities: lethal toxicity to mice when injected intraperitoneally, the LD50 being 30 ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 6 pg; and fluid accumulation in rabbit ileal loops at concentrations of more than 1.25 micrograms/loop. These values are comparable with those obtained with Shiga toxin. In an Ouchterlony double gel diffusion test, the lines formed by the purified Shiga-like toxin and Shiga toxin fused, indicating that the two toxins were immunologically identical.     

Comparative virulence characterization of the Shiga toxin phage-cured Escherichia coli O104:H4 and enteroaggregative Escherichia coli

Escherichia coli O104:H4 (E. coli O104:H4), which caused in 2011 a massive foodborne outbreak in Germany, is characterized by an unusual combination of virulence traits. E. coli O104:H4 contains a prophage-encoded Shiga toxin (Stx) gene, which is the cardinal virulence factor of enterohemorrhagic E. coli (EHEC). However, the outbreak strain shares highest DNA sequence similarity with enteroaggregative E. coli (EAEC) and displays the EAEC-characteristic tight adherence to epithelial cells. The virulence potential of the underlying EAEC background has not been investigated and it is therefore not clear whether E. coli O104:H4 displays distinct virulence characteristics in comparison to prototypical EAEC. In this study, we performed a detailed comparative phenotypic characterization of the Stx phage-cured E. coli O104:H4 strain C227-11φcu, the closely related EAEC strain 55989 and two other well-characterized EAEC strains 042 and 17-2 with focus on virulence traits. C227-11φcu displayed superior aggregative adherence phenotype to cultured HCT-8 epithelial cells, adhering with 3–6 times more bacteria per epithelial cells than the tested EAEC strains. Otherwise, C227-11φcu showed similar virulence characteristics to its closest relative 55989, i.e. strong acid resistance, good biofilm formation and cytotoxic culture supernatants. Furthermore, C227-11φcu was characterized by significantly weaker motility and pro-inflammatory properties than 55989 and 042, nevertheless stronger than 17-2. Taken together, C227-11φcu displayed mostly robust, but not outstanding virulence characteristics in comparison to the tested EAEC. Therefore, it appears likely that the combination of Stx production and EAEC characteristics in general, rather than an exceptionally potent EAEC background resulted in the unusual virulence of the E. coli O104:H4. Thus, the emergence of such hypervirulent strains in the future might be more likely than previously anticipated.     

Characterization of Shiga toxin-producing Escherichia coli O26 strains and establishment of selective isolation media for these strains

We characterized the carbohydrate-fermenting ability of 31 strains of Shiga toxin-producing Escherichia coli (STEC) O26 isolated from diarrhea patients in Aichi Prefecture, Japan, in order to establish selective isolation media for these strains. None of the 31 STEC O26 strains (24 O26:H11, 7 O26:H-) fermented rhamnose, whereas all of the other 108 STEC strains (100 O157, 8 O111) and all of the non-STEC strains except one (i.e., 58 of 59) fermented rhamnose. The great majority of the STEC O26 strains (96.8% [30 of 31]) showed very high resistance to potassium tellurite (MIC > or = 50 microg/ml), whereas the majority of the non-STEC strains (72.9% [43 of 59]) showed very high sensitivity (MIC < or = 1.56 microg/ml) to this compound. Accordingly, we developed a rhamnose-MacConkey (RMAC) medium in which the lactose in MacConkey medium was replaced by rhamnose, and cefixime-tellurite-RMAC (CT-RMAC) medium in which potassium tellurite (2.5 mg/liter) and cefixime (0.05 mg/liter) were added to RMAC. All of the STEC O26 strains generated colorless (rhamnose-nonfermented) colonies on both media; the vast majority of selected E. coli strains (95.7% [89 of 93; including 26 STEC O157, 8 STEC O111]), other than STEC O26, generated red colonies on RMAC, and most of the non-STEC strains (84.7% [50 of 59]) did not grow on CT-RMAC. We demonstrate that both the RMAC and the CT-RMAC media can be used for the isolation of STEC O26 and that CT-RMAC has better specificity for the routine isolation for STEC O26 in a laboratory.     

Commensal bacteria influence Escherichia coli O157:H7 persistence and Shiga toxin production in the mouse intestine

The presence of commensal flora reduced colonization of Escherichia coli O157:H7 and production of Shiga toxin (Stx) in the murine intestine. Stx production was not detected in mice colonized with E. coli that were resistant to the Shiga toxin phage, but it was detected in mice colonized with phage-susceptible E. coli.     

Antimicrobial resistance of Shiga toxin (verotoxin)-producing Escherichia coli O157:H7 and non-O157 strains isolated from humans, cattle, sheep and food in Spain

A total of 722 Shiga toxin-producing Escherichia coli (STEC) isolates recovered from humans, cattle, ovines and food during the period from 1992 to 1999 in Spain were examined to determine antimicrobial resistance profiles and their association with serotypes, phage types and virulence genes. Fifty-eight (41%) out of 141 STEC O157:H7 strains and 240 (41%) out of 581 non-O157 STEC strains showed resistance to at least one of the 26 antimicrobial agents tested. STEC O157:H7 showed a higher percentage of resistant strains recovered from bovine (53%) and beef meat (57%) than from human (23%) and ovine (20%) sources, whereas the highest prevalence of antimicrobial resistance in non-O157 STEC was found among isolates recovered from beef meat (55%) and human patients (47%). Sulfisoxazole (36%) had the most common antimicrobial resistance, followed by tetracycline (32%), streptomycin (29%), ampicillin (10%), trimethoprim (8%), cotrimoxazole (8%), chloramphenicol (7%), kanamycin (7%), piperacillin (6%), and neomycin (5%). The multiple resistance pattern most often observed was that of streptomycin, sulfisoxazole, and tetracycline. Ten (7%) STEC O157:H7 and 71 (12%) non-O157 strains were resistant to five or more antimicrobial agents. Most strains showing resistance to five or more antimicrobial agents belonged to serotypes O4:H4 (4 strains), O8:H21 (3 strains), O20:H19 (6 strains), O26:H11 (8 strains eae-beta1), O111:H- (3 strains eae-gamma2), O118:H- (2 strains eae-beta1), O118:H16 (5 strains eae-beta1), O128:H- (2 strains), O145:H8 or O145:H- (2 strains eae-gamma1), O157:H7 (10 strains eae-gamma1), O171:H25 (3 strains), O177:H11 (5 strains eae-beta1), ONT:H- (3 strains/1 eae-beta1) and ONT:H21 (2 strains). Interestingly, most of these serotypes, i.e., those indicated in bold) were found among human STEC strains isolated from patients with hemolytic uremic-syndrome (HUS) reported in previous studies. We also detected, among non-O157 strains, an association between a higher level of multiple resistance to antibiotics and the presence of the virulence genes eae and stx(1). Moreover, STEC O157:H7, showed an association between certain phage types, PT21/28 (90%), PT23 (75%), PT34 (75%), and PT2 (54%), with a higher number of resistant strains. We conclude that the high prevalence of antimicrobial resistance detected in our study is a source of concern, and cautious use of antibiotics in animals is highly recommended.     

Isolation and genetic characterization of a coinfection of non-O157 Shiga toxin-producing Escherichia coli

A coinfection of O177:NM and O55:H7 Shiga toxin-producing Escherichia coli (STEC) was identified for a child with acute bloody diarrhea and hemolytic uremic syndrome by using culture and serotype-specific molecular reagents. The profile of O157-related genetic islands revealed that the O55:H7 isolate was highly similar to O157 STEC whereas the O177:NM isolate lacked several fimbrial O islands and non-locus-of-enterocyte-effacement effector determinants. However, both STEC serotypes are known to cause serious disease, and the significant repertoire of virulence determinants in both strains made it impossible to determine their individual contributions to the clinical symptoms.     

Phage types and genotypes of Shiga toxin-Producing Escherichia coli O157 in Finland

This study examined Shiga toxin-producing Escherichia coli (STEC) O157, using phage typing, pulsed-field gel electrophoresis, and typing of Shiga toxin variant genes by PCR with restriction fragment length polymorphism in an epidemiological survey of STEC O157 isolated from humans in Finland between 1990 and 1999.     

Isogenic strain of Escherichia coli O157:H7 that has lost both Shiga toxin 1 and 2 genes.

An Escherichia coli O157:H7 strain isolated from a patient with hemorrhagic colitis was found to exhibit two slightly different colony morphology types on differential medium. Each morphological type, designated TT12A and TT12B, was isolated, and serological testing using various assays confirmed that both strains carried the O157 and the H7 antigens. Biochemical testing showed that the strains had identical profiles on AP120E analysis and, like typical O157:H7 strains, did not ferment sorbitol or exhibit beta-glucuronidase activity. Analysis with a multiplex PCR assay showed that TT12B did not carry the gene for either Shiga toxin 1 (Stx1) or Stx2, whereas these genes were present in TT12A and the toxins were produced. Apart from that, both strains carried the +93 gusA mutation, the cluster I ehxA gene for enterohemolysin, and the eae gene for gamma-intimin, which are all characteristics of the O157:H7 serotype. Phenotypic assays confirmed that both strains exhibited enterohemolysin activity and the attachment and effacing lesion on HeLa cells. Multilocus enzyme electrophoresis analysis showed that the strains are closely related genetically and belong in the same clonal group. Pulsed-field gel electrophoresis (PFGE) typing of XbaI-digested genomic DNA revealed that the two strains differed by two bands but shared 90% similarity and clustered in the same clade. All other non-Stx-producing O157:H7 strains examined clustered in a major clade that was distinct from that of Stx-producing O157:H7 strains. The findings that TT12B was identical to TT12A, except for Stx production, and its PFGE profile is also more closely related to that of Stx-producing O157:H7 strains suggest that TT12B was derived from TT12A by the loss of both stx genes.     

Shiga toxin 1c-producing Escherichia coli strains: phenotypic and genetic characterization and association with human disease

The distribution of the stx(1c) allele among Shiga toxin (Stx)-producing Escherichia coli (STEC) and the virulence characteristics of stx(1c)-harboring STEC are unknown. In this study, we identified stx(1c) in 76 (54.3%) of 140 eae-negative, but in none of 155 eae-positive, human STEC isolates (P < 0.000001). The 76 stx(1c)-harboring E. coli isolates belonged to 22 serotypes, and each produced Stx1c as demonstrated by latex agglutination. Characterization of putative virulence factors demonstrated the presence of the locus of proteolysis activity (LPA) and the high-pathogenicity island in 65.8 and 21.1%, respectively, of the 76 Stx1c-producing E. coli isolates. Moreover, all but three of these strains contained saa, the gene encoding an STEC autoagglutinating adhesin. The virulence profiles of Stx1c-producing E. coli isolates were mostly serotype independent and heterogeneous. This enabled us to subtype the isolates within the same serotype. The individuals infected with Stx1c-producing E. coli strains were between 3 months and 72 years old (median age, 23.5 years) and usually had uncomplicated diarrhea or were asymptomatic. We conclude that Stx1c-producing E. coli strains represent a significant subset of eae-negative human STEC isolates, which belong to various serotypes and frequently possess LPA and saa as their putative virulence factors. The phenotypic and molecular characteristics determined in this study allow the subtyping of Stx1c-producing STEC in epidemiological and clinical studies.     

Identification and characterization of a new variant of Shiga toxin 1 in Escherichia coli ONT:H19 of bovine origin

A new variant of Shiga toxin 1 (Stx1), designated Stx1d, which deviates considerably more than any other known variant from Stx1 encoded by phage 933J, was identified in an Escherichia coli strain, ONT:H19, isolated from bovine feces. The complete stx(1) gene of this strain was amplified and sequenced. Nucleotide sequence homology with stx(1) from phage 933J was only 91%, resulting in the substitution of 20 amino acids in the A subunit and 7 amino acids in the B subunit of the protein. Cell culture supernatant of this strain, which was negative for stx(2) by PCR testing, was cytotoxic to Vero cells and gave positive results in two commercial enzyme-linked immunosorbent assays for Stx. PCR primers were constructed for the specific detection of the new variant. The findings of this study suggest that Stx1 is not as conserved as thought before and that there might be more variants which cannot be detected by commonly used PCR methods.     

Detection in Escherichia coli of the genes encoding the major virulence factors,the genes defining the O157:H7 serotype,and components of the type 2 Shiga toxin family by multiplex PCR

Strains of Shiga toxin-producing Escherichia coli (STEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of Shiga toxin 1 (Stx1), Stx2 or combinations of these toxins. Other major virulence factors include enterohemorrhagic E. coli hemolysin (EHEC hlyA), and intimin, the product of the eaeA gene that is involved in the attaching and effacing adherence phenotype. In this study, a series of multiplex-PCR assays were developed to detect the eight most-important E. coli genes associated with virulence, two that define the serotype and therefore the identity of the organism, and a built-in gene detection control. Those genes detected were stx(1), stx(2), stx(2c), stx(2d), stx(2e), stx(2f), EHEC hlyA, and eaeA, as well as rfbE, which encodes the E. coli O157 serotype; fliC, which encodes the E. coli flagellum H7 serotype; and the E. coli 16S rRNA, which was included as an internal control. A total of 129 E. coli strains, including 81 that were O157:H7, 10 that were O157:non-H7, and 38 that were non-O157 isolates, were investigated. Among the 129 samples, 101 (78.3%) were stx positive, while 28 (21.7%) were lacked stx. Of these 129 isolates, 92 (71.3%) were EHEC hlyA positive and 96 (74.4%) were eaeA positive. All STEC strains were identified by this procedure. In addition, all Stx2 subtypes, which had been initially identified by PCR-restriction fragment length polymorphism, were identified by this method. A particular strength of the assay was the identification of these 11 genes without the need to use restriction enzyme digestion. The proposed method is a simple, reliable, and rapid procedure that can detect the major virulence factors of E. coli while differentiating O157:H7 from non-O157 isolates.     

Geographically Distinct Escherichia coli O157 Isolates Differ by Lineage,Shiga Toxin Genotype,and Total Shiga Toxin Production

While the differential association of Escherichia coli O157 genotypes with animal and human hosts has recently been well documented, little is known about their distribution between countries and how this might affect regional disease rates. Here, we used a 48-plex single nucleotide polymorphism (SNP) assay to segregate 148 E. coli O157 isolates from Australia, Argentina, and the United States into 11 SNP lineages. We also investigated the relationship between SNP lineages, Shiga toxin (Stx) gene profiles, and total Stx production. E. coli O157 isolates clearly segregated into SNP lineages that were differentially associated with each country. Of the 11 SNP lineages, seven were detected among isolates from a single country, two were detected among isolates from all three countries, and another two were detected only among U.S. and Argentinean isolates. A number of Australian (30%) and Argentinean (14%) isolates were associated with novel, previously undescribed SNP lineages that were unique to each country. Isolates within SNP lineages that were strongly associated with the carriage of stx_2a produced comparatively more Stx on average than did those lacking the stx_2a subtype. Furthermore, the proportion of isolates in stx_2a-associated SNP lineages was significantly higher in Argentina and the United States than Australia (P < 0.05). This study provides evidence for the geographic divergence of E. coli O157 and for a prominent role of stx_2a in total Stx production. These results also highlight the need for more comprehensive studies of the global distribution of E. coli O157 lineages and the impacts of regionally predominant E. coli O157 lineages on the prevalence and severity of disease.