Chromosomal dynamism in progeny of outbreak-related sorbitol-fermenting enterohemorrhagic Escherichia coli O157:NM

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-defined clusters of cases, we have observed significant variability in pulsed-field gel electrophoresis (PFGE) patterns which could indicate coinfection by different strains. An analysis of randomly selected progeny colonies of an outbreak strain after subcultivation demonstrated that they displayed either the cognate PFGE outbreak pattern or one of four additional patterns and were <89% similar. These profound alterations were associated with changes in the genomic position of one of two Shiga toxin 2-encoding genes (stx2) in the outbreak strain or with the loss of this gene. The two stx2 alleles in the outbreak strain were identical but were flanked with phage-related sequences with only 77% sequence identity. Neither of these phages produced plaques, but one lysogenized E. coli K-12 and integrated in yecE in the lysogens and the wild-type strain. The presence of two stx2 genes which correlated with increased production of Stx2 in vitro but not with the clinical outcome of infection was also found in 14 (21%) of 67 SF EHEC O157:NM isolates from sporadic cases of human disease. The variability of PFGE patterns for the progeny of a single colony must be considered when interpreting PFGE patterns in SF EHEC O157-associated outbreaks.     

Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

Using colony blot hybridization with stx₂ and eae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated stx₂-positive and eae-positive sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens and stx-negative and eae-positive SF E. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The stx-negative isolates from each patient shared with the corresponding stx₂-positive isolates fliC_H7, non-stx virulence traits, and multilocus sequence types, which indicates that they arose from the stx₂-positive strains by loss of stx₂ during infection. Analysis of the integrity of the yecE gene, a possible stx phage integration site in EHEC O157, in the consecutive stx₂-positive and stx-negative isolates demonstrated that yecE was occupied in stx₂-positive but intact in stx-negative strains. It was possible to infect and lysogenize the stx-negative E. coli O157 strains in vitro using an stx₂-harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the stx₂-containing phage resulted in the occupation of yecE and production of biologically active Shiga toxin 2. We conclude that the yecE gene in SF E. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their stx-negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of stx₂-harboring phages. The ability to recycle stx₂, a critical virulence trait, makes SF E. coli O157:NM strains ephemeral EHEC that can exist as stx-negative variants during certain phases of their life cycle.     

Characterization of Shiga Toxin-Producing Escherichia coli Isolates Associated with Two Multistate Food-Borne Outbreaks That Occurred in 2006

Shiga toxin-producing Escherichia coli isolates from two 2006 outbreaks were compared to other O157:H7 isolates for virulence genotype, biofilm formation, and stress responses. Spinach- and lettuce-related-outbreak strains had similar pulsed-field gel electrophoresis patterns, and all carried both stx₂ and stx_2c variant genes. Cooperative biofilm formation involving an E. coli O157:H7 strain and a non-O157:H7 strain was also demonstrated.     

Shiga Toxin Gene Loss and Transfer In Vitro and In Vivo during Enterohemorrhagic Escherichia coli O26 Infection in Humans

Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx₂ loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.     

Detection and Characterization of the Fimbrial sfp Cluster in Enterohemorrhagic Escherichia coli O165:H25/NM Isolates from Humans and Cattle

The sfp cluster, encoding Sfp fimbriae and located in the large plasmid of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157 (pSFO157), has been considered a unique characteristic of this organism. We discovered and then characterized the sfp cluster in EHEC O165:H25/NM (nonmotile) isolates of human and bovine origin. All seven strains investigated harbored a complete sfp cluster (carrying sfpA, sfpH, sfpC, sfpD, sfpJ, sfpF, and sfpG) of 6,838 bp with >99% nucleotide sequence homology to the sfp cluster of SF EHEC O157:NM. The sfp cluster in EHEC O165:H25/NM strains was located in an ~80-kb (six strains) or ~120-kb (one strain) plasmid which differed in structure, virulence genes, and sfp flanks from pSFO157. All O165:H25/NM strains belonged to the same multilocus sequence type (ST119) and were only distantly phylogenetically related to SF EHEC O157:NM (ST11). The highly conserved sfp cluster in different clonal backgrounds suggests that this segment was acquired independently by EHEC O165:H25 and SF EHEC O157:NM. Its presence in an additional EHEC serotype extends the diagnostic utility of PCR targeting sfpA as an easy and efficient approach to seek EHEC in patients' stools. The reasons for the convergence of pathogenic EHEC strains on a suite of virulence loci remain unknown.     

Virulence Genes and Molecular Typing of Different Groups of Escherichia coli O157 Strains in Cattle

Characterization of an Escherichia coli O157 strain collection (n = 42) derived from healthy Hungarian cattle revealed the existence of diverse pathotypes. Enteropathogenic E. coli (EPEC; eae positive) appeared to be the most frequent pathotype (n = 22 strains), 11 O157 strains were typical enterohemorrhagic E. coli (EHEC; stx and eae positive), and 9 O157 strains were atypical, with none of the key stx and eae virulence genes detected. EHEC and EPEC O157 strains all carried eae-gamma, tir-gamma, tccP, and paa. Other virulence genes located on the pO157 virulence plasmid and different O islands (O island 43 [OI-43] and OI-122), as well as espJ and espM, also characterized the EPEC and EHEC O157 strains with similar frequencies. However, none of these virulence genes were detected by PCR in atypical O157 strains. Interestingly, five of nine atypical O157 strains produced cytolethal distending toxin V (CDT-V) and carried genes encoding long polar fimbriae. Macro-restriction fragment enzyme analysis (pulsed-field gel electrophoresis) revealed that these E. coli O157 strains belong to four main clusters. Multilocus sequence typing analysis revealed that five housekeeping genes were identical in EHEC and EPEC O157 strains but were different in the atypical O157 strains. These results suggest that the Hungarian bovine E. coli O157 strains represent at least two main clones: EHEC/EPEC O157:H7/NM (nonmotile) and atypical CDT-V-producing O157 strains with H antigens different from H7. The CDT-V-producing O157 strains represent a novel genogroup. The pathogenic potential of these strains remains to be elucidated.Escherichia coli O157:H7 is a food- and waterborne zoonotic pathogen with serious effects on public health. E. coli O157:H7 causes diseases in humans ranging from uncomplicated diarrhea to hemorrhagic colitis and hemolytic-uremic syndrome (HUS) (30). Typically, enterohemorrhagic E. coli (EHEC) strains express two groups of important virulence factors: one or more Shiga toxins (Stx; also called verotoxins), encoded by lambda-like bacteriophages, and a pathogenicity island called the locus of enterocyte effacement (LEE) encoding all the proteins necessary for attaching and effacing lesions of epithelial cells (41). Comparative genomic studies of E. coli O157:H7 strains revealed extensive genomic diversity related to the structures, positions, and genetic contents of bacteriophages and the variability of putative virulence genes encoding non-LEE effector proteins (29, 43).Ruminants and, in particular, healthy cattle are the major reservoir of E. coli O157:H7, although the prevalence of O157:H7 strains in cattle may vary widely, as reviewed by Caprioli et al. (12). E. coli O157:H7 has been found to persist and remain infective in the environment for a long time, e.g., for at least 6 months in water trough sediments, which may be an important environmental niche.In Hungary, infections with E. coli O157 and other Shiga toxin-producing E. coli (STEC) strains in humans in cases of “enteritidis infectiosa” have been notifiable since 1998 on a case report basis. Up to now, the disease has been sporadic, and fewer than 100 (n = 83) cases of STEC infection among 2,700 suspect cases have been reported since 2001. However, until the present study, no systematic, representative survey of possible animal sources had been performed.In this study, our aim was to investigate healthy cattle in Hungary for the presence of strains of E. coli O157 and the genes encoding Shiga toxins (stx₁ and stx₂) and intimin (eae) and a wide range of putative virulence genes found in these strains. In addition, the phage type (PT) was determined, and pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to further compare the strains at the molecular level. Shiga toxin and cytolethal distending toxin (CDT) production was also examined, and phage induction experiments were conducted. The high incidence of enteropathogenic E. coli (EPEC; eae-positive) O157:H7 strains and atypical (eae- and stx-negative) O157 strains indicates that cattle are a major reservoir of not only EHEC O157 but also EPEC O157 and atypical E. coli O157 strains. These atypical, non-sorbitol-fermenting O157 strains frequently produced CDT-V and may represent a novel O157 clade as demonstrated by MLST and PFGE.     

Presence and Characterization of a Mosaic Genomic Island Which Distinguishes Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H? from E. coli O157:H7

A mosaic genomic island comprising Shigella resistance locus (SRL) sequences flanked by segments of Escherichia coli O157:H7 strain EDL933 O islands 43, 81, and 82 was identified in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H⁻ strain 493/89. This mosaic island is absent from strain EDL933. PCR targeting the SRL-related sequence is a useful tool to distinguish SF EHEC O157:H⁻ from EHEC O157:H7.     

Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7

Background

Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases.

Results

We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains.

Conclusions

Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a BsuBI/PstI methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.     

Virulence Profiling of Shiga Toxin-Producing Escherichia coli O111:NM Isolates from Cattle

Shiga toxin-producing Escherichia coli (STEC) O111:NM is an important serotype that has been incriminated in disease outbreaks in the United States. This study characterized cattle STEC O111:NM for virulence factors and markers by PCR. Major conclusions are that STEC O111:NM characterized in this study lacks stx₂ and the full spectrum of nle gene markers, and it has an incomplete OI-122.     

Genomic Diversity and Virulence Profiles of Historical Escherichia coli O157 Strains Isolated from Clinical and Environmental Sources

Escherichia coli O157:H7 is, to date, the major E. coli serotype causing food-borne human disease worldwide. Strains of O157 with other H antigens also have been recovered. We analyzed a collection of historic O157 strains (n = 400) isolated in the late 1980s to early 1990s in the United States. Strains were predominantly serotype O157:H7 (55%), and various O157:non-H7 (41%) serotypes were not previously reported regarding their pathogenic potential. Although lacking Shiga toxin (stx) and eae genes, serotypes O157:H1, O157:H2, O157:H11, O157:H42, and O157:H43 carried several virulence factors (iha, terD, and hlyA) also found in virulent serotype E. coli O157:H7. Pulsed-field gel electrophoresis (PFGE) showed the O157 serogroup was diverse, with strains with the same H type clustering together closely. Among non-H7 isolates, serotype O157:H43 was highly prevalent (65%) and carried important enterohemorrhagic E. coli (EHEC) virulence markers (iha, terD, hlyA, and espP). Isolates from two particular H types, H2 and H11, among the most commonly found non-O157 EHEC serotypes (O26:H11, O111:H11, O103:H2/H11, and O45:H2), unexpectedly clustered more closely with O157:H7 than other H types and carried several virulence genes. This suggests an early divergence of the O157 serogroup to clades with different pathogenic potentials. The appearance of important EHEC virulence markers in closely related H types suggests their virulence potential and suggests further monitoring of those serotypes not implicated in severe illness thus far.     

Highly Virulent Non-O157 Enterohemorrhagic Escherichia coli (EHEC) Serotypes Reflect Similar Phylogenetic Lineages,Providing New Insights into the Evolution of EHEC

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of bloody diarrhea and extraintestinal sequelae in humans, most importantly hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Besides the bacteriophage-encoded Shiga toxin gene (stx), EHEC harbors the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions. Currently, the vast majority of EHEC infections are caused by strains belonging to five O serogroups (the “big five”), which, in addition to O157, the most important, comprise O26, O103, O111, and O145. We hypothesize that these four non-O157 EHEC serotypes differ in their phylogenies. To test this hypothesis, we used multilocus sequence typing (MLST) to analyze a large collection of 250 isolates of these four O serogroups, which were isolated from diseased as well as healthy humans and cattle between 1952 and 2009. The majority of the EHEC isolates of O serogroups O26 and O111 clustered into one sequence type complex, STC29. Isolates of O103 clustered mainly in STC20, and most isolates of O145 were found within STC32. In addition to these EHEC strains, STC29 also included stx-negative E. coli strains, termed atypical enteropathogenic E. coli (aEPEC), yet another intestinal pathogenic E. coli group. The finding that aEPEC and EHEC isolates of non-O157 O serogroups share the same phylogeny suggests an ongoing microevolutionary scenario in which the phage-encoded Shiga toxin gene stx is transferred between aEPEC and EHEC. As a consequence, aEPEC strains of STC29 can be regarded as post- or pre-EHEC isolates. Therefore, STC29 incorporates phylogenetic information useful for unraveling the evolution of EHEC.     

Presence and Characterization of Shiga Toxin-Producing Escherichia coli and Other Potentially Diarrheagenic E. coli Strains in Retail Meats

To determine the presence of Shiga toxin-producing Escherichia coli (STEC) and other potentially diarrheagenic E. coli strains in retail meats, 7,258 E. coli isolates collected by the U.S. National Antimicrobial Resistance Monitoring System (NARMS) retail meat program from 2002 to 2007 were screened for Shiga toxin genes. In addition, 1,275 of the E. coli isolates recovered in 2006 were examined for virulence genes specific for other diarrheagenic E. coli strains. Seventeen isolates (16 from ground beef and 1 from a pork chop) were positive for stx genes, including 5 positive for both stx₁ and stx₂, 2 positive for stx₁, and 10 positive for stx₂. The 17 STEC strains belonged to 10 serotypes: O83:H8, O8:H16, O15:H16, O15:H17, O88:H38, ONT:H51, ONT:H2, ONT:H10, ONT:H7, and ONT:H46. None of the STEC isolates contained eae, whereas seven carried enterohemorrhagic E. coli (EHEC) hlyA. All except one STEC isolate exhibited toxic effects on Vero cells. DNA sequence analysis showed that the stx₂ genes from five STEC isolates encoded mucus-activatable Stx2d. Subtyping of the 17 STEC isolates by pulsed-field gel electrophoresis (PFGE) yielded 14 distinct restriction patterns. Among the 1,275 isolates from 2006, 11 atypical enteropathogenic E. coli (EPEC) isolates were identified in addition to 3 STEC isolates. This study demonstrated that retail meats, mainly ground beef, were contaminated with diverse STEC strains. The presence of atypical EPEC strains in retail meat is also of concern due to their potential to cause human infections.Escherichia coli is an important component of the intestinal microflora of humans and warm-blooded mammals. While E. coli typically harmlessly colonizes the intestinal tract, several E. coli clones have evolved the ability to cause a variety of diseases within the intestinal tract and elsewhere in the host. Those strains that cause enteric infections are generally called diarrheagenic E. coli strains, and their pathogenesis is associated with a number of virulence attributes, which vary according to pathotype (54). Currently, diarrheagenic E. coli strains are classified into six main pathotypes based on their distinct virulence determinants and pathogenic features, including enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC)/Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusively adherent E. coli (DAEC) (37).Among diarrheagenic E. coli strains, STEC strains are distinguished by the ability to cause severe life-threatening complications, such as hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) (30). Other symptoms of STEC infection include watery diarrhea, bloody diarrhea, and hemorrhagic colitis (HC). STEC strains that cause HC and HUS are also called EHEC. Although individuals of all ages are at risk of STEC infection, children younger than 5 years of age and the elderly are more likely to suffer from severe complications (51). Outbreaks and sporadic cases of STEC infections have been reported frequently worldwide.The pathogenesis of STEC infection in humans is not fully understood. The major virulence factors implicated in STEC infection are potent Shiga toxins, which are classified into two groups: Stx1 and Stx2 (23). Additional factors that contribute to virulence have also been described, including intimin (encoded by the eae gene), an outer membrane protein involved in the attachment of E. coli to the enterocyte, and EHEC hemolysin (encoded by EHEC hlyA), which acts as a pore-forming cytolysin and causes damage to cells (41).The first STEC O157 infections were reported in 1982, when E. coli O157:H7 was involved in outbreaks associated with two fast food chain restaurants in the United States (44). Since then, ever-increasing numbers of cases and outbreaks due to STEC O157 have been reported worldwide. Although non-O157 STEC strains have also been associated with human cases and outbreaks, few laboratories have been looking for them, and their potential in causing human infections may be underestimated (2). Recently, though, the significance of non-O157 STEC strains as human pathogens has become more recognized. In the United States alone, there were 23 reported outbreaks of non-O157 STEC infection between 1990 and 2007 (10).Shiga toxin-producing E. coli can be transmitted through different routes, including food and water, person-to-person contact, and animal-to-person contact (9). Most human infections are caused by consumption of contaminated foods (16). Domestic and wild ruminant animals, in particular cattle, are considered the main reservoir of STEC and the main source for contamination of the food supply. Retail meats derived from animals could potentially act as transmission vehicles for STEC and other diarrheagenic E. coli strains. However, there is limited information about STEC contamination in retail meats, and fewer data exist about the presence of other diarrheagenic E. coli strains in retail meats. In the present study, we investigated 7,258 E. coli isolates from four types of meat samples (beef, chicken, pork, and turkey) collected during 2002 to 2007 to assess STEC contamination of retail meats. In addition, the presence of other potentially diarrheagenic E. coli strains was examined by detecting specific virulence determinants among E. coli isolates collected in 2006.     

Shiga Toxin-Producing Escherichia coli in Yaks (Bos grunniens) from the Qinghai-Tibetan Plateau,China

Shiga toxin (Stx)-producing Escherichia coli (STEC) are recognized as important human pathogens of public health concern. Many animals are the sources of STEC. In this study we determined the occurrence and characteristics of the STEC in yaks (Bos grunniens) from the Qinghai-Tibetan plateau, China. A total of 728 yak fecal samples was collected from June to August, 2012 and was screened for the presence of the stx ₁ and stx ₂ genes by TaqMan real-time PCR after the sample was enriched in modified Tryptone Soya Broth. Of the 138 (18.96%) stx ₁ and/or stx ₂-positive samples, 85 (61.59%) were confirmed to have at least 1 STEC isolate present by culture isolation, from which 128 STEC isolates were recovered. All STEC isolates were serotyped, genotyped by pulsed-field gel electrophoresis (PFGE) and characterized for the presence of 16 known virulence factors. Fifteen different O serogroups and 36 different O:H serotypes were identified in the 128 STEC isolates with 21 and 4 untypable for the O and H antigens respectively. One stx ₁ subtype (stx _1a) and 5 stx ₂ subtypes (stx _2a, stx _2b, stx _2c, stx _2d and stx _2g) were present in these STEC isolates. Apart from lpfA _O157/OI-141, lpfA _O157/OI-154, lpfA _O113, katP and toxB which were all absent, other virulence factors screened (eaeA, iha, efa1, saa, paa, cnf1, cnf2, astA, subA, exhA and espP) were variably present in the 128 STEC isolates. PFGE were successful for all except 5 isolates and separated them into 67 different PFGE patterns. For the 18 serotypes with 2 or more isolates, isolates of the same serotypes had the same or closely related PFGE patterns, demonstrating clonality of these serotypes. This study was the first report on occurrence and characteristics of STEC isolated from yaks (Bos grunniens) from the Qinghai-Tibetan plateau, China, and extended the genetic diversity and reservoir host range of STEC.     

Combined Use of Bacteriophage Typing and Pulsed-Field Gel Electrophoresis in the Epidemiological Analysis of Japanese Isolates of Enterohemorrhagic Escherichia coli O157:H7

A total of 236 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates in Japan were investigated by bacteriophage typing, and the results were compared with those of pulsed-field gel electrophoresis (PFGE). Seven phage types (PTs) were observed in 71 isolates which were derived from 22 outbreaks. All of the isolates from ten outbreaks in the Kinki region (midwestern part of Japan) in July-August 1996 were grouped into the same PFGE type (IIa) and PT 32, while among total isolates, there were such varieties as PFGE type IIa containing five phage types and PT32 containing two PFGE types. These results suggest that the ten outbreaks should be considered to be a single outbreak, and show that the combined use of bacteriophage typing and PFGE enhances reliability in epidemiological surveys.     

Detection of virulent Escherichia coli O157 strains using multiplex PCR and single base sequencing for SNP characterization

Aims: To compare 167 Norwegian human and nonhuman Escherichia coli O157:H7/NM (nonmotile) isolates with respect to an A/T single nucleotide polymorphism (SNP) in the tir gene and to detect specific SNPs that differentiate STEC O157 into distinct virulence clades (1–3 and 8). Methods and Results: We developed a multiplex PCR followed by single base sequencing for detection of the SNPs, and examined the association among SNP genotype, virulence profile (stx and eae status), multilocus variable number of tandem repeats analysis (MLVA) profile and clinical outcome. We found an over‐representation of the T allele among human strains compared to nonhuman strains, including 5/6 haemolytic‐uraemic syndrome cases. Fourteen strains belonged to clade 8, followed by two clade 2 strains. No clade 1 nor 3 isolates were observed. stx1 in combination with either stx2_EDL933 or stx2c were frequently observed among human strains, whereas stx2c was dominating in nonhuman strains. MLVA indicated that only single cases or small outbreaks with E. coli O157 have been observed in Norway through the years 1993–2008. Conclusion: We observed that the tir‐255 A/T SNP and the stx status were different between human and nonhuman O157 strains. No major outbreaks were observed, and only a few strains were differentiated into the virulence clades 2 and 8. Significance and Impact of the Study: The detection of virulence clade‐specific SNPs enables the rapid designation of virulent E. coli O157 strains, especially in outbreak situations.     

Heterogeneity of Shiga Toxin-Producing Escherichia coli Strains Isolated from Hemolytic-Uremic Syndrome Patients, Cattle, and Food Samples in Central France

A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products. The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time. Five typing methods were used; some of these were used to explore potentially mobile elements like the stx genes or the plasmids (stx₂-restriction fragment length polymorphism [RFLP], stx₂ gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis [PFGE]). The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France. A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping. However, stx₂-RFLP and stx₂ variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef. The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain. Overall, our results indicate that the combination of stx₂-RFLP, stx₂ variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity. Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area. The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.     

Shiga Toxin and Shiga Toxin-Encoding Phage Do Not Facilitate Escherichia coli O157:H7 Colonization in Sheep

Isogenic strains of Escherichia coli O157:H7, missing either stx₂ or the entire Stx2-encoding phage, were compared with the parent strain for their abilities to colonize sheep. The absence of the phage or of the Shiga toxin did not significantly impact the magnitude or duration of shedding of E. coli O157:H7.     

Transduction of Enteric Escherichia coli Isolates with a Derivative of Shiga Toxin 2-Encoding Bacteriophage φ3538 Isolated from Escherichia coli O157:H7

We investigated the ability of a detoxified derivative of a Shiga toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize enteric Escherichia coli strains and to develop infectious progeny from such lysogenized strains. The stx₂ gene of the patient E. coli O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184. Phage 3538(Δstx₂::cat) was isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Stx-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and E. coli from the physiological stool microflora were infected with 3538(Δstx₂::cat), and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of one EIEC strain, none of the E. coli strains supported the formation of plaques when used as indicators for 3538(Δstx₂::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6 E. coli isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of 3538(Δstx₂::cat) that formed plaques on a lawn of E. coli laboratory strain C600. The results of our study demonstrate that 3538(Δstx₂::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced infectious phage progeny. Stx-encoding bacteriophages are able to spread stx genes among enteric E. coli strains.     

Serotyping, stx2 Subtyping, and Characterization of the Locus of Enterocyte Effacement Island of Shiga Toxin-Producing Escherichia coli and E. coli O157:H7 Strains Isolated from the Environment in France

Twenty-seven Shiga toxin-producing Escherichia coli (STEC) strains were isolated from 207 stx-positive French environmental samples. Ten of these strains were positive for stx₁, and 24 were positive for stx₂ (10 were positive for stx_2vh-a or stx_2vh-b, 19 were positive for stx_2d, and 15 were positive for stx_2e). One strain belonged to serotype O157:H7, and the others belonged to serogroups O2, O8, O11, O26, O76, O103, O113, O121, O141, O166, and O174. The environment is a reservoir in which new clones of STEC that are pathogenic for humans can emerge.     

Persistence of Escherichia coli O157:H7 and its mutants in soils

The persistence of Shiga toxin-producing E. coli O157:H7 in the environment poses a serious threat to public health. However, the role of Shiga toxins and other virulence factors in the survival of E. coli O157:H7 is poorly defined. The aim of this study was to determine if the virulence factors, stx ₁, stx ₂, stx _1–2, and eae in E. coli O157:H7 EDL933 play any significant role in the growth of this pathogen in rich media and in soils. Isogenic deletion mutants that were missing one of four virulence factors, stx ₁, stx ₂, stx _1–2, and eae in E. coli O157:H7 EDL933 were constructed, and their growth in rich media and survival in soils with distinct texture and chemistry were characterized. The survival data were successfully analyzed using Double Weibull model, and the modeling parameters of the mutant strains were not significantly different from those of the wild type. The calculated T_d (time needed to reach the detection limit, 100 CFU/g soil) for loamy sand, sandy loam, and silty clay was 32, 80, and 110 days, respectively. It was also found that T_d was positively correlated with soil structure (e.g. clay content), and soil chemistry (e.g. total nitrogen, total carbon, and water extractable organic carbon). The results of this study showed that the possession of Shiga toxins and intimin in E. coli O157:H7 might not play any important role in its survival in soils. The double deletion mutant of E. coli O157:H7 (stx ₁ ⁻ stx ₂ ⁻) may be a good substitute to use for the investigation of transport, fate, and survival of E. coli O157:H7 in the environment where the use of pathogenic strains are prohibited by law since the mutants showed the same characteristics in both culture media and environmental samples.