Escherichia coli O157:H7 survives within human macrophages: global gene expression profile and involvement of the Shiga toxins

Escherichia coli O157:H7 is an important food-borne pathogen that specifically binds to the follicle-associated epithelium in the intestine, which rapidly brings this bacterial pathogen in contact with underlying human macrophages. Very little information is available about the interaction between E. coli O157:H7 and human macrophages. We evaluated the uptake and survival of strain EDL933 during infection of human macrophages. Surprisingly, EDL933 survived and multiplied in human macrophages at 24 h postinfection. The global gene expression profile of this pathogen during macrophage infection was determined. Inside human macrophages, upregulation of E. coli O157:H7 genes carried on O islands (such as pagC, the genes for both of the Shiga toxins, and the two iron transport system operons fit and chu) was observed. Genes involved in acid resistance and in the SOS response were upregulated. However, genes of the locus of enterocyte effacement or genes involved in peroxide resistance were not differentially expressed. Many genes with putative or unknown functions were upregulated inside human macrophages and may be newly discovered virulence factors. As the Shiga toxin genes were upregulated in macrophages, survival and cytotoxicity assays were performed with isogenic Shiga toxin mutants. The initial uptake of Shiga toxins mutants was higher than that of the wild type; however, the survival rates were significantly lower at 24 h postinfection. Thus, Shiga toxins are implicated in the interaction between E. coli O157:H7 and human macrophages. Understanding the molecular mechanisms used by E. coli to survive within macrophages may help in the identification of targets for new therapeutic agents.     

Escherichia coli EDL933 Requires Gluconeogenic Nutrients To Successfully Colonize the Intestines of Streptomycin-Treated Mice Precolonized with E. coli Nissle 1917

Escherichia coli MG1655, a K-12 strain, uses glycolytic nutrients exclusively to colonize the intestines of streptomycin-treated mice when it is the only E. coli strain present or when it is confronted with E. coli EDL933, an O157:H7 strain. In contrast, E. coli EDL933 uses glycolytic nutrients exclusively when it is the only E. coli strain in the intestine but switches in part to gluconeogenic nutrients when it colonizes mice precolonized with E. coli MG1655 (R. L. Miranda et al., Infect Immun 72:1666–1676, 2004, http://dx.doi.org/10.1128/IAI.72.3.1666-1676.2004). Recently, J. W. Njoroge et al. (mBio 3:e00280-12, 2012, http://dx.doi.org/10.1128/mBio.00280-12) reported that E. coli 86-24, an O157:H7 strain, activates the expression of virulence genes under gluconeogenic conditions, suggesting that colonization of the intestine with a probiotic E. coli strain that outcompetes O157:H7 strains for gluconeogenic nutrients could render them nonpathogenic. Here we report that E. coli Nissle 1917, a probiotic strain, uses both glycolytic and gluconeogenic nutrients to colonize the mouse intestine between 1 and 5 days postfeeding, appears to stop using gluconeogenic nutrients thereafter in a large, long-term colonization niche, but continues to use them in a smaller niche to compete with invading E. coli EDL933. Evidence is also presented suggesting that invading E. coli EDL933 uses both glycolytic and gluconeogenic nutrients and needs the ability to perform gluconeogenesis in order to colonize mice precolonized with E. coli Nissle 1917. The data presented here therefore rule out the possibility that E. coli Nissle 1917 can starve the O157:H7 E. coli strain EDL933 of gluconeogenic nutrients, even though E. coli Nissle 1917 uses such nutrients to compete with E. coli EDL933 in the mouse intestine.     

Identification and distribution of the enterohemorrhagic Escherichia coli factor for adherence (efa1) gene in sorbitol-fermenting Escherichia coli O157: H-

Sorbitol-fermenting (SF) Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H- strains are emerging as causes of hemorrhagic colitis and the hemolytic-uremic syndrome in Europe. Using subtractive hybridization between SF STEC O157:H- strain 493/89 and STEC O157:H7 strain EDL933, three different fragments, of approximately 700 bp in length, were identified. Each demonstrated > 99% homology to genes encoding the enterohemorrhagic E. coli factor for adherence (efa1) and lymphostatin (lifA). Therefore, a cosmid library was constructed from SF STEC O157:H- strain 493/89, and one clone containing these fragments was sequenced. This sequencing demonstrated a 9669-bp open reading frame (ORF) that had 99.9% sequence homology to efa1 of STEC O111:H- strain E45035 and to lifA of an enteropathogenic E. coli O127:H6 strain E2348/69. In STEC O157:H7 strain EDL933, only small (ca. 3 kb) initial and terminal fragments of this ORF are present. PCR analysis with primers complementary to the efa1/lifA sequence of strain 493/89 indicated that the complete sequence is present in each of 10 SF STEC O157:H- isolates but in none of 10 STEC O157:H7 strains investigated. The presence of the complete efa1/lifA also in both tested E. coli O55:H7 strains supports the hypothesis that SF STEC O157:H- are phylogenetically closer to the proposed E. coli O55:H7 ancestor than STEC O157:H7. Our data demonstrate the presence of a potential virulence gene in SF STEC O157:H- that is only rudimentarily present in STEC O157:H7.     

Escherichia coli Pathotypes Occupy Distinct Niches in the Mouse Intestine

Since the first step of the infection process is colonization of the host, it is important to understand how Escherichia coli pathogens successfully colonize the intestine. We previously showed that enterohemorrhagic O157:H7 strain E. coli EDL933 colonizes a niche in the streptomycin-treated mouse intestine that is distinct from that of human commensal strains, which explains how E. coli EDL933 overcomes colonization resistance imparted by some, but not all, commensal E. coli strains. Here we sought to determine if other E. coli pathogens use a similar strategy. We found that uropathogenic E. coli CFT073 and enteropathogenic E. coli E2348/69 occupy intestinal niches that are distinct from that of E. coli EDL933. In contrast, two enterohemorrhagic strains, E. coli EDL933 and E. coli Sakai, occupy the same niche, suggesting that strategies to prevent colonization by a given pathotype should be effective against other strains of the same pathotype. However, we found that a combination of commensal E. coli strains that can prevent colonization by E. coli EDL933 did not prevent colonization by E. coli CFT073 or E. coli E2348/69. Our results indicate that development of probiotics to target multiple E. coli pathotypes will be problematic, as the factors that govern niche occupation and hence stable colonization vary significantly among strains.     

Molecular Evolution of a Pathogenicity Island from Enterohemorrhagic Escherichia coli O157:H7

We report the complete 43,359-bp sequence of the locus of enterocyte effacement (LEE) from EDL933, an enterohemorrhagic Escherichia coli O157:H7 serovar originally isolated from contaminated hamburger implicated in an outbreak of hemorrhagic colitis. The locus was isolated from the EDL933 chromosome with a homologous-recombination-driven targeting vector. Recent completion of the LEE sequence from enteropathogenic E. coli (EPEC) E2348/69 afforded the opportunity for a comparative analysis of the entire pathogenicity island. We have identified a total of 54 open reading frames in the EDL933 LEE. Of these, 13 fall within a putative P4 family prophage designated 933L. The prophage is not present in E2348/69 but is found in a closely related EPEC O55:H7 serovar and other O157:H7 isolates. The remaining 41 genes are shared by the two complete LEEs, and we describe the nature and extent of variation among the two strains for each gene. The rate of divergence is heterogeneous along the locus. Most genes show greater than 95% identity between the two strains, but other genes vary more than expected for clonal divergence among E. coli strains. Several of these highly divergent genes encode proteins that are known to be involved in interactions with the host cell. This pattern suggests recombinational divergence coupled with natural selection and has implications for our understanding of the interaction of both pathogens with their host, for the emergence of O157:H7, and for the evolutionary history of pathogens in general.     

Antagonistic effects of probiotic Escherichia coli Nissle 1917 on EHEC strains of serotype O104:H4 and O157:H7

The largest EHEC outbreak up to now in Germany occurred in 2011. It was caused by the non-O157:H7 Shiga-toxinogenic enterohemorrhagic E. coli strain O104:H4. This strain encodes in addition to the Shiga toxin 2 (Stx2), responsible for the hemolytic uremic syndrome (HUS), several adhesins such as aggregative adherence fimbriae. Currently, there is no effective prophylaxis and treatment available for EHEC infections in humans. Especially antibiotics are not indicated for treatment as they may induce Stx production, thus worsening the symptoms. Alternative therapeutics are therefore desperately needed. We tested the probiotic Escherichia coli strain Nissle 1917 (EcN) for antagonistic effects on two O104:H4 EHEC isolates from the 2011 outbreak and on the classical O157:H7 EHEC strain EDL933. These tests included effects on adherence, growth, and Stx production in monoculture and co-culture together with EcN. The inoculum of each co-culture contained EcN and the respective EHEC strain either at a ratio of 1:1 or 10:1 (EcN:EHEC). Adhesion of EHEC strains to Caco-2 cells and to the mucin-producing LS-174T cells was reduced significantly in co-culture with EcN at the 1:1 ratio and very dramatically at the 10:1 ratio. This inhibitory effect of EcN on EHEC adherence was most likely not due to occupation of adhesion sites on the epithelial cells, because in monocultures EcN adhered with much lower bacterial numbers than the EHEC strains. Both EHEC strains of serotype O104:H4 showed reduced growth in the presence of EcN (10:1 ratio). EHEC strain EDL933 grew in co-culture with EcN only during the first 2 h of incubation. Thereafter, EHEC counts declined. At 24 h, the numbers of viable EDL933 was at or slightly below the numbers at the time of inoculation. The amount of Stx2 after 24 h co-incubation with EcN (EcN:EHEC ratio 10:1) was for all 3 EHEC strains tested significantly reduced in comparison to EHEC monocultures.     

Urease of Enterohemorrhagic Escherichia coli: Evidence for Regulation by Fur and a trans-Acting Factor

Recent genomic analyses of Escherichia coli O157:H7 strain EDL933 revealed two loci encoding urease gene homologues (ureDABCEFG), which are absent in nonpathogenic E. coli strain K-12. This report demonstrates that the cloned EDL933 ure gene cluster is capable of synthesizing urease in an E. coli DH5alpha background. However, when the gene fragment is transformed back into the native EDL933 background, the enzymatic activity of the cloned determinants is undetectable. We speculate that an unidentified trans-acting factor in enterohemorrhagic E. coli (EHEC) is responsible for this regulation of ure expression. In addition, Fur-like recognition sites are present in three independent O157:H7 isolates upstream of ureD and ureA. Enzymatic assays confirmed a difference in urease expression of cloned EHEC ure clusters in E. coli MC3100Deltafur. Likewise, interruption of fur in O157:H7 isolate IN1 significantly diminished urease activity. We propose that, similar to the function of Fur in regulating the acid response of Salmonella enterica serovar Typhimurium, it modulates urease expression in EHEC, perhaps contributing to the acid tolerance of the organism.     

Genetic diversity among Escherichia coli O157:H7 isolates and identification of genes linked to human infections

An Escherichia coli oligonucleotide microarray based on three sequenced genomes was validated for comparative genomic microarray hybridization and used to study the diversity of E. coli O157 isolates from human infections and food and animal sources. Among 26 test strains, 24 (including both Shiga toxin [Stx]-positive and -negative strains) were found to be related to the two sequenced E. coli O157:H7 strains, EDL933 and Sakai. However, these strains showed much greater genetic diversity than those reported previously, and most of them could not be categorized as either lineage I or II. Some genes were found more often in isolates from human than from nonhuman sources; e.g., ECs1202 and ECs2976, associated with stx2AB and stx1AB, were in all isolates from human sources but in only 40% of those from nonhuman sources. Some (but not all) lineage I-specific or -dominant genes were also more frequently associated with isolates from human. The results suggested that it might be more effective to concentrate our efforts on finding markers that are directly related to infection rather than those specific to certain lineages. In addition, two Stx-negative O157 cattle isolates (one confirmed to be H7) were significantly different from other Stx-positive and -negative E. coli O157:H7 strains and were more similar to MG1655 in their gene content. This work demonstrates that not all E. coli O157:H7 strains belong to the same clonal group, and those that were similar to E. coli K-12 might be less virulent.     

Pathogenesis of Escherichia coli O157:H7 strain 86-24 following oral infection of BALB/c mice with an intact commensal flora

Escherichia coli O157:H7 is a food-borne pathogen that can cause hemorrhagic colitis and, occasionally, hemolytic uremic syndrome, a sequela of infection that can result in renal failure and death. Here we sought to model the pathogenesis of orally-administered E. coli O157:H7 in BALB/c mice with an intact intestinal flora. First, we defined the optimal dose that permitted sustained fecal shedding of E. coli O157:H7 over 7 days (～10⁹ colony forming units). Next, we monitored the load of E. coli O157:H7 in intestinal sections over time and observed that the cecum was consistently the tissue with the highest E. coli O157:H7 recovery. We then followed the expression of two key E. coli O157:H7 virulence factors, the adhesin intimin and Shiga toxin type 2, and detected both proteins early in infection when bacterial burdens were highest. Additionally, we noted that during infection, animals lost weight and ～30% died. Moribund animals also exhibited elevated levels of blood urea nitrogen, and, on necropsy, showed evidence of renal tubular damage. We conclude that conventional mice inoculated orally with high doses of E. coli O157:H7 can be used to model both intestinal colonization and subsequent development of certain extraintestinal manifestations of E. coli O157:H7 disease.     

Hemolytic Uremic Syndrome in a 65-Year-Old Male Linked to a Very Unusual Type of stx2e- and eae-Harboring O51:H49 Shiga Toxin-Producing Escherichia coli

We report on a 65-year-old male patient with a Shiga toxin-producing Escherichia coli O51:H49 gastrointestinal infection and sepsis associated with hemolytic uremic syndrome (HUS) with a fatal outcome. The strains isolated harbored stx_2e and eae, a very unusual and new virulence profile for an HUS-associated enterohemorrhagic E. coli.     

Human Microbiota-Secreted Factors Inhibit Shiga Toxin Synthesis by Enterohemorrhagic Escherichia coli O157:H7

Escherichia coli O157:H7 is a food-borne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome, especially in children. The main virulence factor responsible for the more serious disease is the Shiga toxin 2 (Stx2), which is released in the gut after oral ingestion of the organism. Although it is accepted that the amount of Stx2 produced by E. coli O157:H7 in the gut is critical for the development of disease, the eukaryotic or prokaryotic gut factors that modulate Stx2 synthesis are largely unknown. In this study, we examined the influence of prokaryotic molecules released by a complex human microbiota on Stx2 synthesis by E. coli O157:H7. Stx2 synthesis was assessed after growth of E. coli O157:H7 in cecal contents of gnotobiotic rats colonized with human microbiota or in conditioned medium having supported the growth of complex human microbiota. Extracellular prokaryotic molecules produced by the commensal microbiota repress stx₂ mRNA expression and Stx2 production by inhibiting the spontaneous and induced lytic cycle mediated by RecA. These molecules, with a molecular mass of below 3 kDa, are produced in part by Bacteroides thetaiotaomicron, a predominant species of the normal human intestinal microbiota. The microbiota-induced stx₂ repression is independent of the known quorum-sensing pathways described in E. coli O157:H7 involving SdiA, QseA, QseC, or autoinducer 3. Our findings demonstrate for the first time the regulatory activity of a soluble factor produced by the complex human digestive microbiota on a bacterial virulence factor in a physiologically relevant context.     

Prevalence of iron transport gene on pathogenicity-associated island of uropathogenic Escherichia coli in E. coli O157:H7 containing Shiga toxin gene

Uropathogenc Escherichia coli (UPEC) CFT073 has a pathogenicity-associated island (PAI(CFT073)), which causes pyelonephritis and cystitis. Using PCR method, we found the prrA gene of PAI(CFT073) in E. coli O157:H7 EDL933. Further detailed PCR screening of 38 open reading frames, the right and left junction sequences of PAI(CFT073), revealed that it is the prrA-modD-yc73-fepC gene cluster but not the PAI(CFT073) present in E. coli O157:H7 EDL933. A rapid preliminary analysis suggested that the prrA-modD-yc73-fepC gene cluster of the PAI(CFT073), is present in 43 strains of E. coli O157:H7 containing Shiga toxin (Stx) gene but absent in 19 strains of E. coli O157:H7 without Stx gene. A strict co-occurrence of the prrA-modD-yc73-fepC gene cluster and Stx genes was observed, regardless of their origin. The prrA-modD-yc73-fepC gene cluster encode proteins probably involved in iron uptake system, which strongly suggests the importance of iron metabolism in the Stx-mediated virulence. In addition, the prrA-modD-yc73-fepC gene cluster may be used as a diagnostic marker to distinguish E. coli O157:H7 strains containing Stx gene from that without Stx gene, and possibly to quickly detect other pathogenic gram-negative bacteria containing the Stx gene.     

Analysis of the Genome of the Escherichia coli O157:H7 2006 Spinach-Associated Outbreak Isolate Indicates Candidate Genes That May Enhance Virulence

In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. Differences in HUS frequency among E. coli O157:H7 outbreaks have been noted, but our understanding of bacterial factors that promote HUS is incomplete. In 2006, in an outbreak of E. coli O157:H7 caused by consumption of contaminated spinach, there was a notably high frequency of HUS. We sequenced the genome of the strain responsible (TW14359) with the goal of identifying candidate genetic factors that contribute to an enhanced ability to cause HUS. The TW14359 genome contains 70 kb of DNA segments not present in either of the two reference O157:H7 genomes. We identified seven putative virulence determinants, including two putative type III secretion system effector proteins, candidate genes that could result in increased pathogenicity or, alternatively, adaptation to plants, and an intact anaerobic nitric oxide reductase gene, norV. We surveyed 100 O157:H7 isolates for the presence of these putative virulence determinants. A norV deletion was found in over one-half of the strains surveyed and correlated strikingly with the absence of stx₁. The other putative virulence factors were found in 8 to 35% of the O157:H7 isolates surveyed, and their presence also correlated with the presence of norV and the absence of stx₁, indicating that the presence of norV may serve as a marker of a greater propensity for HUS, similar to the correlation between the absence of stx₁ and a propensity for HUS.Escherichia coli O157:H7 is a human pathogen that infects more than 73,000 North Americans per year (39). Although infection by this organism typically causes symptoms such as watery or bloody diarrhea, it may also lead to the development of hemolytic-uremic syndrome (HUS), an infection sequela characterized by hemolysis and renal failure that can result in long-lasting kidney damage. Variables that contribute to the development of HUS include host factors, such as age (51), and the genetic background of the enterohemorrhagic E. coli (EHEC) isolate. Currently, no effective prophylaxis exists for HUS (45). Antibiotic treatment of E. coli O157:H7 infections is contraindicated as it is associated with increased infection sequelae (45, 58).Humans become infected with EHEC by consuming contaminated food. EHEC are noninvasive pathogens that primarily colonize the human colon. Serotype O157:H7 is the predominant EHEC serotype in North America. The other commonly isolated EHEC serotypes include O26:H11, O103:H2, O111:NM, and O113:H21 (34). The systemic absorption of Shiga toxins produced by intestinal EHEC is thought to damage endothelial cells and to cause HUS (31). Shiga toxins are A-B-type toxins that inhibit protein synthesis. The genes encoding these potent toxins are borne on prophages that are related to phage λ. There are two main variants of Shiga toxin, Stx1 and Stx2. Stx2 is more cytotoxic than Stx1 in cell culture and animal models (27, 46, 48), and epidemiologic observations have revealed that the risk of developing HUS following an EHEC infection is heightened if the isolate produces Stx2 (4). Several variants of Stx2 exist, and Stx2c is the variant most commonly found in O157:H7 strains. Stx2 and Stx2c have the same biological function and possess identical A subunits and B subunits that share at least 97% identity (10).Although important for virulence, Stx2 does not appear to be the only EHEC factor that significantly influences whether patients infected with EHEC develop HUS. A comparison of statistics for several outbreaks caused by Stx2-producing O157:H7 strains showed that the rate of HUS can vary from less than 1% to 26% (23), indicating that strain-specific factors of stx₂-carrying O157:H7 strains are involved in determining clinical outcomes. To date, the most significant factor identified as a factor contributing to the variability is the presence of the stx₁ gene. O157:H7 strains that lack stx₁ but carry one or two stx₂ alleles are more likely to cause infections resulting in HUS (11, 35, 36).A comparison of the genome sequences of O157:H7 outbreak isolates that have resulted in different HUS rates may provide further insight into genetic factors that contribute to this severe sequela of EHEC infection. The genome sequences of two O157:H7 strains that caused low frequencies of HUS are available. The Sakai strain, the cause of the 1996 outbreak in Japan, caused ∼8,000 infections in people, the majority of whom were children, and the rate of HUS was 1.2% (32). In 1982, EDL933 caused the first diarrhea outbreak linked to the O157:H7 serotype and involved 44 individuals but no recorded HUS cases (41).Sakai shares 4.1 Mb of DNA with the commensal E. coli K-12 strain MG1655 and has 296 novel DNA segments more than 19 bp long, termed S-loops, that account for 1.39 Mb. EDL933 shares 4.1 Mb with E. coli K-12 strain MG1655 and has 177 unique sequence segments more than 50 bp long, termed O-islands, that account for 1.34 Mb (19). For both the Sakai and EDL933 genomes there is significant evidence of horizontal transfer due to the presence of numerous prophage-related elements and the pO157 virulence plasmid. The virulence factors carried on the O157:H7-specific DNA segments, as well as pO157, include stx₁, stx₂, the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions on enterocytes and, notably, encodes a type III secretion system (TTSS) (22), at least 39 TTSS effectors encoded either on the LEE or at other chromosomal locations (49), numerous fimbrial and nonfimbrial adhesins, and more than one hemolysin (56).No genome sequence is available yet for an O157:H7 outbreak isolate that has caused an outbreak resulting in a significantly higher HUS rate. One O157:H7 isolate, TW14359, caused an outbreak associated with contaminated spinach that sickened 205 individuals in September and October of 2006. A total of 15% of the afflicted individuals developed HUS (5, 28). This rate is significantly higher than the average annual rate of 4.1% for O157:H7 cases that develop HUS (39). The relatively high percentage of adults, ∼8%, who developed HUS in the TW14359 outbreak also likely reflects the greater virulence of this strain (6). Furthermore, Manning et al. performed a phylogenetic analysis of TW14359 utilizing 96 single-nucleotide polymorphisms (SNPs) and demonstrated that this isolate belongs to a more virulent clade of O157:H7 strains (clade 8); the majority of these isolates lack stx₁ and carry stx₂ (28). A partial genome sequence consisting of 200 contigs of the TW14359 genome was also reported by Manning et al., which was found to contain stx₂ and stx_2c. While an analysis of these sequence data identified the genes of the two reference isolates that were also present in TW14359 and identified backbone SNPs, it did not provide a list of novel genetic features or provide assembled DNA segments containing repetitive DNA elements, such as phage-like elements. Here we describe the entire genome sequence of this isolate and, focusing on novel genetic material, identify potential genetic features of TW14359 that may promote this strain''s outstanding pathogenicity.     

Inhibition of Water Absorption and Selective Damage to Human Colonic Mucosa Are Induced by Subtilase Cytotoxin Produced by Escherichia coli O113:H21

Shiga toxin-producing Escherichia coli O157:H7 (STEC) is by far the most prevalent serotype associated with hemolytic uremic syndrome (HUS) although many non-O157 STEC strains have been also isolated from patients with HUS. The main virulence factor of STEC is the Shiga toxin type 2 (Stx2) present in O157 and non-O157 strains. Recently, another toxin, named subtilase cytotoxin (SubAB), has been isolated from several non-O157 strains and may contribute to the pathogenesis of HUS. Here, we have demonstrated that an O113:H21 STEC strain expressing SubAB and Stx2 inhibits normal water absorption across human colon and causes damage to the surface epithelium, necrosis, mononuclear inflammatory infiltration, edema, and marked mucin depletion. This damage was less marked, but nevertheless significant, when purified SubAB or E. coli O113:H21 expressing only SubAB was assayed. This is the first study showing that SubAB may directly participate in the mechanisms of diarrhea in children infected with non-O157 STEC strains.     

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.     

Genotypes and virulence characteristics of Shiga toxin-producing Escherichia coli O104 strains from different origins and sources

Sixty-two Escherichia coli strains carrying the wzx_O104-gene from different sources, origins and time periods were analyzed for their serotypes, virulence genes and compared for genomic similarity by pulsed-field gel-electrophoresis (PFGE). The O104 antigen was present in 55 strains and the structurally and genetically related capsular antigen K9 in five strains. The presence of 49 genes associated with enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC) was investigated. Fifty-four strains of serotypes O104:H2 (n = 1), O104:H4 (n = 37), O104:H7 (n = 5) and O104:H21 (n = 11) produced Shiga-toxins (Stx). Among STEC O104, a close association between serotype, virulence gene profile and genomic similarity was found. EAEC virulence genes were only present in STEC O104:H4 strains. EHEC-O157 plasmid-encoded genes were only found in STEC O104:H2, O104:H7 and O104:H21 strains. None of the 62 O104 or K9 strains carried an eae-gene involved in the attaching and effacing phenotype.     

Complete sequence of the large virulence plasmid pSFO157 of the sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- strain 3072/96

The large virulence plasmid pSFO157 of sorbitol-fermenting E. coli O157:H(-) strain 3072/96 has a size of 121,239bp and contains 96 open reading frames >50bp. It is therefore 29,162bp larger (ca. 32%) than plasmid pO157 of E. coli O157:H7 strain EDL933. Major differences between the plasmids are the absence of katP, espP, and toxB in pSFO157 and, instead of these, the presence of the sfp fimbriae gene cluster and a large part of an F-plasmid transfer region, the latter accounting for most of the additional DNA. The differences in the order of the genes and their composition, as well as the presence of a number of replication-associated genes and mobile genetic elements suggests that the large E. coli O157 virulence plasmids have a complex evolutionary origin.     

Molecular Characterization of Human Atypical Sorbitol-Fermenting Enteropathogenic Escherichia coli O157 Reveals High Diversity

Alongside the well-characterized enterohemorrhagic Escherichia coli (EHEC) O157:H7, serogroup O157 comprises sorbitol-fermenting typical and atypical enteropathogenic E. coli (EPEC/aEPEC) strains that carry the intimin-encoding gene eae but not Shiga toxin-encoding genes (stx). Since little is known about these pathogens, we characterized 30 clinical isolates from patients with hemolytic uremic syndrome (HUS) or uncomplicated diarrhea with respect to their flagellin gene (fliC) type and multilocus sequence type (MLST). Moreover, we applied whole-genome sequencing (WGS) to determine the phylogenetic relationship with other eae-positive EHEC serotypes and the composition of the rfbO157 region. fliC typing resulted in five fliC types (H7, H16, H34, H39, and H45). Isolates of each fliC type shared a unique ST. In comparison to the 42 HUS-associated E. coli (HUSEC) strains, only the stx-negative isolates with fliCH7 shared their ST with EHEC O157:H7/H⁻ strains. With the exception of one O157:H⁻_fliCH16 isolate, HUS was exclusively associated with fliCH7. WGS corroborated the separation of the fliCH7 isolates, which were closely related to the EHEC O157:H7/H⁻ isolates, and the diverse group of isolates exhibiting different fliC types, indicating independent evolution of the different serotypes. This was also supported by the heterogeneity within the rfbO157 region that exhibited extensive recombinations. The genotypic subtypes and distribution of clinical symptoms suggested that the stx-negative O157 strains with fliCH7 were originally EHEC strains that lost stx. The remaining isolates form a distinct and diverse group of atypical EPEC isolates that do not possess the full spectrum of virulence genes, underlining the importance of identifying the H antigen for clinical risk assessment.