Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology

An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM? sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM? within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.     

肠出血性大肠杆菌O157感染防治研究进展

肠出血性大肠杆菌 (EHEC)感染是一种重要的新发传染病 ,O15 7是EHEC的一个主要菌型 ,感染该菌可使人患腹泻、出血性结肠炎 (HC)、溶血性尿毒综合征 (HUS)等 ,死亡率较高。EHECO15 7感染在许多国家包括我国都有暴发流行。EHECO15 7产生的粘附因子Intimin可引起粘附擦拭 (A/E)损伤 ,并可产生致死性的毒素Stx。抗生素治疗可使患者并发HUS危险性增加 ,临床上无特效的治疗药物 ,疫苗研究将对EHECO15 7的控制起重要作用。     

Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium

We identified Shiga toxin gene (stx)-negative Escherichia coli O26:H11 and O26:NM (nonmotile) strains as the only pathogens in the stools of five patients with hemolytic-uremic syndrome (HUS). Because the absence of stx in E. coli associated with HUS is unusual, we examined the strains for potential virulence factors and interactions with microvascular endothelial cells which are the major targets affected during HUS. All five isolates possessed the enterohemorrhagic E. coli (EHEC)-hlyA gene encoding EHEC hemolysin (EHEC-Hly), expressed the enterohemolytic phenotype, and were cytotoxic, in dose- and time-dependent manners, to human brain microvascular endothelial cells (HBMECs). Significantly reduced cytotoxicity in an EHEC-Hly-negative spontaneous derivative of one of these strains, and a dose- and time-dependent cytotoxicity of recombinant E. coli O26 EHEC-Hly to HBMECs, suggest that the endothelial cytotoxicity of these strains was mediated by EHEC-Hly. The toxicity of EHEC-Hly to microvascular endothelial cells plausibly contributes to the virulence of the stx-negative E. coli O26 strains and to the pathogenesis of HUS.     

Interactions with M cells and macrophages as key steps in the pathogenesis of enterohemorrhagic Escherichia coli infections

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens that can cause serious infections ranging from diarrhea to hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Translocation of Shiga-toxins (Stx) from the gut lumen to underlying tissues is a decisive step in the development of the infection, but the mechanisms involved remain unclear. Many bacterial pathogens target the follicle-associated epithelium, which overlies Peyer's patches (PPs), cross the intestinal barrier through M cells and are captured by mucosal macrophages. Here, translocation across M cells, as well as survival and proliferation of EHEC strains within THP-1 macrophages were investigated using EHEC O157:H7 reference strains, isogenic mutants, and 15 EHEC strains isolated from HC/HUS patients. We showed for the first time that E. coli O157:H7 strains are able to interact in vivo with murine PPs, to translocate ex vivo through murine ileal mucosa with PPs and across an in vitro human M cell model. EHEC strains are also able to survive and to produce Stx in macrophages, which induce cell apoptosis and Stx release. In conclusion, our results suggest that the uptake of EHEC by M cells and underlying macrophages in the PP may be a critical step in Stx translocation and release in vivo. A new model for EHEC infection in humans is proposed that could help in a fuller understanding of EHEC-associated diseases.     

Presence of virulence and fitness gene modules of enterohemorrhagic Escherichia coli in atypical enteropathogenic Escherichia coli O26

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O26 cause hemolytic-uremic syndrome (HUS) whereas atypical enteropathogenic E. coli (aEPEC) O26 typically cause uncomplicated diarrhea but have been also isolated from HUS patients. To gain insight into the virulence of aEPEC O26, we compared the presence of O island (OI) 122, which is associated with enhanced virulence in EHEC strains, among aEPEC O26 and EHEC O26 clinical isolates. We also tested these strains for the high pathogenicity island (HPI) which is a fitness island. All 20 aEPEC O26 and 20 EHEC O26 investigated contained virulence genes located within OI-122 (efa1/lifA, nleB, nleE, ent). In both aEPEC O26 and EHEC O26, OI-122 was linked to the locus for enterocyte effacement, forming a mosaic island which was integrated in pheU. Moreover, strains of these two pathotypes shared a conserved HPI. These data support a close relatedness between aEPEC O26 and EHEC O26 and have evolutionary implications. The presence of OI-122 in aEPEC O26 might contribute to their pathogenic potential.     

Role of bacterial toxins in pathogenesis of hemolytic-uremic syndrome, caused by enterohemorrhagic Escherichia

The pathogenesis of the hemolytico-uremic syndrome (HUS) caused by enterohemorrhagic E. coli (EHEC) has been studied previously rather completely. HUS is characterized by the signs of microangiopathic hemolytic anemia, thrombocytopenia with renal lesions and manifestations of transient disturbances in the functions of the central nervous system. The adherence of EHEC to enterocytes was found to occur in the terminal section of the ileum and the large intestine. This process is realized with involvement intimin, EHEC outer membrane protein. Shiga-like toxins (SLT) produced by EHEC are the leading factor of their pathogenicity. The mechanism of the toxin translocation through enterocytes is not yet clear, still there is no doubt that SLT penetrates into the systemic blood stream. This is indicated by the results of histopathological studies it possible to find the toxin traces on the membranes of endothelial cells of blood vessels. The study reveals that the cells of the vascular epithelium are highly sensitive to SLT. These cells carry receptors Gb3, also known as CD77, on their membranes. Enterohemolysin, serine protease, causing disturbances in the barrier function of the intestine, can be regarded in the pathogenesis of hemorrhagic colitis which develops as the result of the damaging action of EHEC and the above-mentioned toxins. This leads to the increased level of blood systemic bacterial lipopolysaccharides, which may play, in combination with the action of SLT, an important role in the development of multi-organ pathology in HUS patients.     

Interactions with M Cells and Macrophages as Key Steps in the Pathogenesis of Enterohemorragic Escherichia coli Infections

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens that can cause serious infections ranging from diarrhea to hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Translocation of Shiga-toxins (Stx) from the gut lumen to underlying tissues is a decisive step in the development of the infection, but the mechanisms involved remain unclear. Many bacterial pathogens target the follicle-associated epithelium, which overlies Peyer''s patches (PPs), cross the intestinal barrier through M cells and are captured by mucosal macrophages. Here, translocation across M cells, as well as survival and proliferation of EHEC strains within THP-1 macrophages were investigated using EHEC O157:H7 reference strains, isogenic mutants, and 15 EHEC strains isolated from HC/HUS patients. We showed for the first time that E. coli O157:H7 strains are able to interact in vivo with murine PPs, to translocate ex vivo through murine ileal mucosa with PPs and across an in vitro human M cell model. EHEC strains are also able to survive and to produce Stx in macrophages, which induce cell apoptosis and Stx release. In conclusion, our results suggest that the uptake of EHEC by M cells and underlying macrophages in the PP may be a critical step in Stx translocation and release in vivo. A new model for EHEC infection in humans is proposed that could help in a fuller understanding of EHEC-associated diseases.     

Fenotypic and genotypic characterization of Shiga toxin-producing Escherichia coli O111 strain isolated from patient with hemolytic-uremic syndrome

Shiga toxin-producing E. coli O157 and non-O157 are important emergance pathogens that can cause diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome (HUS). A few cases of EHEC infections are documented per year in Poland. Among them only one patient with EHEC O157 infection developed HUS. We characterized the first VTEC non-O157 strain isolated from child with HUS in Poland. The VTEC O111 strain produced Stx2 which was cytotoxic for Vero cell. Using DNA microarray analysis we have found set of virulence genes in VTEC O111 strain as: stx2A, stx2B, ehly, eae, tir tccP espA, espJ, cif nleA, nleB, lpfA, iha, efa1, cba. The strain was fenotypic resistant to streptomycin, tetracyclin and sulphonamides (strA, tetA, sul2 genes were detected).     

Enterohemorrhagic Escherichia coli trivalent recombinant vaccine containing EspA, intimin and Stx2 induces strong humoral immune response and confers protection in mice

Enterohemorrhagic Escherichia coli (EHEC) is an important food-borne pathogen, which causes a wide spectrum of diseases ranging from hemorrhagic colitis to life-threatening hemolytic uremic syndrome (HUS). Currently, insufficient measures to prevent and treat EHEC infection make a vaccine against EHEC in great demand. EspA (E. coli secreted protein A), intimin, and Stx2 (Shiga toxin 2) are three predominant virulence factors of EHEC, and each of them has proved to be capable of inducing partial protective immunity. In this study, we constructed a trivalent recombinant protein designated EIS that is composed of EspA (E), C-terminal 300 amino acids of intimin (I) and B subunit of Stx2 (S), and tested it as vaccine using a mouse model. Our results showed that immunization of EIS induced strong humoral response to EspA, intimin and Stx2 and protected mice against the challenges with live EHEC or EHEC sonicated lysate. Moreover, it enhanced clearance of intestinally colonized bacteria. This work suggests that for EHEC vaccines using a combination of EspA, intimin and Stx2 antigens appears to be more effective than using any of these immunogens alone.     

E. coli O104:H4: social media and the characterization of an emerging pathogen

The 2011 German E. coli O104:H4 outbreak resulted in thousands of cases of enterohaemorrhagic illness, with approximately 25% of these progressing to develop haemolytic uraemic syndrome (HUS). This high rate of progression to HUS was the first indicator that the bacterial cause of illness was not a typical enterohaemorrhagic E. coli (EHEC) strain. Collaborative bioinformatic analysis while the outbreak was still in progress indicated that the O104:H4 strain was in fact an enteroaggregative E. coli (EAEC) strain which had acquired genes for the production of Shiga - like toxin.     

Symptoms and Clinical Course of EHEC O104 Infection in Hospitalized Patients: A Prospective Single Center Study

Objectives

Shiga-toxin producing O157:H7 Entero Haemorrhagic E. coli (STEC/EHEC) is one of the most common causes of Haemolytic Uraemic Syndrome (HUS) related to infectious haemorrhagic colitis. Nearly all recommendations on clinical management of EHEC infections refer to this strain. The 2011 outbreak in Northern Europe was the first to be caused by the serotype O104:H4. This EHEC strain was found to carry genetic features of Entero Aggregative E. coli (EAEC) and extended spectrum β lactamase (ESBL). We report symptoms and complications in patients at one of the most affected centres of the 2011 EHEC O104 outbreak in Northern Germany.

Methods

The courses of patients admitted to our hospital due to bloody diarrhoea with suspected EHEC O104 infection were recorded prospectively. These data include the patients’ histories, clinical findings, and complications.

Results

EHEC O104 infection was confirmed in 61 patients (female = 37; mean age: 44±2 years). The frequency of HUS was 59% (36/61) in our cohort. An enteric colonisation with co-pathogens was found in 57%. Thirty-one (51%) patients were treated with plasma-separation/plasmapheresis, 16 (26%) with haemodialysis, and 7 (11%) with Eculizumab. Patients receiving antibiotic treatment (n = 37; 61%) experienced no apparent change in their clinical course. Twenty-six (43%) patients suffered from neurological symptoms. One 83-year-old patient died due to comorbidities after HUS was successfully treated.

Conclusions

EHEC O104:H4 infections differ markedly from earlier reports on O157:H7 induced enterocolitis in regard to epidemiology, symptomatology, and frequency of complications. We recommend a standard of practice for clinical monitoring and support the renaming of EHEC O104:H4 syndrome as “EAHEC disease”.     

Inflammatory and immunological parameters in children with haemolytic uremic syndrome (HUS) and gastroenteritis-pathophysiological and diagnostic clues

The objective of this study was to identify parameters indicating a risk for developing typical haemolytic uremic syndrome (D+HUS) during the prodromal phase of diarrhea caused by enterohaemorrhagic Escherichia coli (EHEC). Forty-eight children were studied prospectively with regard to inflammatory serum factors on admission to hospital. Ten patients developed D+HUS (group I), 15 suffered from viral-gastroenteritis (group IIa) and 23 from other types of bacterial gastroenteritis (group IIb). Mean levels of IL-8 tended to be elevated in group I compared to groups IIa and IIb. Neopterin and IL-10 levels particularly were significantly decreased in HUS in comparison to both gastroenteritis groups. Low IL-10 levels indicate a substantial disregulation of the immune response in HUS, as IL-10 downregulates the pro-inflammatory response and suppresses pro-coagulant activity in experimental endotoxemia. Our results suggest low neopterin, high IL-8 and especially low IL-10 levels are indicators of a high risk for developing HUS.     

First isolation of the enterohaemorrhagic <Emphasis Type="Italic">Escherichia coli</Emphasis> O145:H- from cattle in feedlot in Argentina

Background  

Enterohaemorrhagic Escherichia coli (EHEC) is considered to be common cause of haemorrhagic colitis (HC), thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome (HUS) in humans. In a previous paper, we have demonstrated that EHEC are commonly found in the intestines of livestock. Infections in humans are, in part, a consequence of consumption of undercooked meat or raw milk. Argentina has one of the highest records of HUS (300–400 cases/year; 22/100,000 children under 4 years of age). The aim of this work is to communicate the first isolation of O145:H-from cattle in this country and characterize the virulence cassette, providing useful information to evaluate the risk of foodborne transmission of this emergent non-O157:H7 serotype.     

Protection from Hemolytic Uremic Syndrome by Eyedrop Vaccination with Modified Enterohemorrhagic E. coli Outer Membrane Vesicles

We investigated whether eyedrop vaccination using modified outer membrane vesicles (mOMVs) is effective for protecting against hemolytic uremic syndrome (HUS) caused by enterohemorrhagic E. coli (EHEC) O157:H7 infection. Modified OMVs and waaJ-mOMVs were prepared from cultures of MsbB- and Shiga toxin A subunit (STxA)-deficient EHEC O157:H7 bacteria with or without an additional waaJ mutation. BALB/c mice were immunized by eyedrop mOMVs, waaJ-mOMVs, and mOMVs plus polymyxin B (PMB). Mice were boosted at 2 weeks, and challenged peritoneally with wild-type OMVs (wtOMVs) at 4 weeks. As parameters for evaluation of the OMV-mediated immune protection, serum and mucosal immunoglobulins, body weight change and blood urea nitrogen (BUN)/Creatinin (Cr) were tested, as well as histopathology of renal tissue. In order to confirm the safety of mOMVs for eyedrop use, body weight and ocular histopathological changes were monitored in mice. Modified OMVs having penta-acylated lipid A moiety did not contain STxA subunit proteins but retained non-toxic Shiga toxin B (STxB) subunit. Removal of the polymeric O-antigen of O157 LPS was confirmed in waaJ-mOMVs. The mice group vaccinated with mOMVs elicited greater humoral and mucosal immune responses than did the waaJ-mOMVs and PBS-treated groups. Eyedrop vaccination of mOMVs plus PMB reduced the level of humoral and mucosal immune responses, suggesting that intact O157 LPS antigen can be a critical component for enhancing the immunogenicity of the mOMVs. After challenge, mice vaccinated with mOMVs were protected from a lethal dose of wtOMVs administered intraperitoneally, conversely mice in the PBS control group were not. Collectively, for the first time, EHEC O157-derived mOMV eyedrop vaccine was experimentally evaluated as an efficient and safe means of vaccine development against EHEC O157:H7 infection-associated HUS.     

Shiga Toxin and Lipopolysaccharide Induce Platelet-Leukocyte Aggregates and Tissue Factor Release,a Thrombotic Mechanism in Hemolytic Uremic Syndrome

Background

Aggregates formed between leukocytes and platelets in the circulation lead to release of tissue factor (TF)–bearing microparticles contributing to a prothrombotic state. As enterohemorrhagic Escherichia coli (EHEC) may cause hemolytic uremic syndrome (HUS), in which microthrombi cause tissue damage, this study investigated whether the interaction between blood cells and EHEC virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS) led to release of TF.

Methodology/Principal Findings

The interaction between Stx or LPS and blood cells induced platelet-leukocyte aggregate formation and tissue factor (TF) release, as detected by flow cytometry in whole blood. O157LPS was more potent than other LPS serotypes. Aggregates formed mainly between monocytes and platelets and less so between neutrophils and platelets. Stimulated blood cells in complex expressed activation markers, and microparticles were released. Microparticles originated mainly from platelets and monocytes and expressed TF. TF–expressing microparticles, and functional TF in plasma, increased when blood cells were simultaneously exposed to the EHEC virulence factors and high shear stress. Stx and LPS in combination had a more pronounced effect on platelet-monocyte aggregate formation, and TF expression on these aggregates, than each virulence factor alone. Whole blood and plasma from HUS patients (n = 4) were analyzed. All patients had an increase in leukocyte-platelet aggregates, mainly between monocytes and platelets, on which TF was expressed during the acute phase of disease. Patients also exhibited an increase in microparticles, mainly originating from platelets and monocytes, bearing surface-bound TF, and functional TF was detected in their plasma. Blood cell aggregates, microparticles, and TF decreased upon recovery.

Conclusions/Significance

By triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.     

Effect of antibiotics, levofloxacin and fosfomycin, on a mouse model with Escherichia coli O157 infection

There have been some reservations about the treatment of enterohemorrhagic Escherichia coli (EHEC) infection with antibiotics to prevent the occurrence of hemolytic uremic syndrome (HUS). However, the administration of antimicrobial agents for EHEC infection is under discussion. Therefore, we used an experimental mouse model to assess the advantage/disadvantage of two major antibiotics, levofloxacin (LVFX) and fosfomycin (FOM). Germ-free IQI mice were inoculated with EHEC O157 strain EDL931 or #7. Bacteria colonized feces at 10(9)-10(10) CFU/g, and Shiga toxins (STXs) were detected in the feces. From 1 day after infection, mice were assigned to LVFX (20 mg/kg) once daily or FOM (400 mg/kg) once daily. A significant decrease in overall mortality was observed after treatment of LVFX, with EHEC disappearing immediately from the feces of mice. FOM also reduced mortality for one strain, the STX level decreased gradually. LVFX exhibited higher therapeutic efficacy than FOM. Strain differences were observed in the model during the treatment.     

EHEC 的新型粘附因子研究进展*

摘要：肠出血性大肠杆菌（enterohemorrhage ，EHEC）是一种重要的人畜共患病，世界各地包括中国都有不同规模的暴发流 行。EHEC有多种血清型，其中毒力最强血清型是O157：H7。EHEC O157：H7 感染除可使人发生常规腹泻外，还可在5%-10%的病 例中引发严重并发症，甚至死亡。该菌是重要的食源性致病菌，危害严重，缺乏有效的防治手段，而抗生素治疗可能会加剧溶血性 尿毒症（haemolutic uraemic syndrome，HUS）。由于以上特点EHEC O157：H7 成为世界公共卫生问题，引起微生物学家和公共卫生 工作者的广泛关注。目前，临床针对EHEC 感染只是对症治疗和适当的抗菌治疗。粘附是EHEC感染宿主细胞的第一步，没有这 一步，细菌和宿主肠道细胞之间不会发生任何的相互作用，而且对于许多病原菌来说，粘附具有宿主特异性。本文概述了EHEC 的流行病学及粘附机理，并对近年在EHEC 研究中的发现一些新型粘附因子和一些假设的定植因子的研究背景及作用机理作一 综述。     

New system for multilocus variable-number tandem-repeat analysis of the enterohemorrhagic Escherichia coli strains belonging to three major serogroups: O157, O26, and O111

Enterohemorrhagic Escherichia coli (EHEC), a food- and waterborne pathogen, causes diarrhea, hemorrhagic colitis, and life-threatening HUS. MLVA is a newly developed and widely accepted genotyping tool. An MLVA system for EHEC O157 involving nine genomic loci has already been established. However, the present study revealed that the above-mentioned MLVA system cannot analyze EHEC O26 and O111 isolates-the second and third most dominant EHEC serogroups in Japan, respectively. Therefore, with several modifications to the O157 system and the use of nine additional loci, we developed an expanded MLVA system applicable to EHEC O26, O111, and O157. Our MLVA system had a relatively high resolution power for each of the three serogroups: Simpson's index of diversity was 0.991 (95% CI = 0.989-0.993), 0.988 (95% CI, 0.986-0.990), and 0.986 (95% CI, 0.979-0.993) for O26, O111, and O157, respectively. This system also detected outbreak-related isolates; the isolates collected during each of the 12 O26 and O111 outbreaks formed unique clusters, and most of the repeat copy numbers among the isolates collected during the same outbreak exhibited no or single-locus variations. These results were comparable to those of cluster analyses based on PFGE profiles. Therefore, our system can complement PFGE analysis-the current golden method. Because EHEC strains of three major serogroups can be rapidly analyzed on a single platform with our expanded MLVA system, this system could be widely used in molecular epidemiological studies of EHEC infections.     

EspP,an Extracellular Serine Protease from Enterohemorrhagic E. coli,Reduces Coagulation Factor Activities,Reduces Clot Strength,and Promotes Clot Lysis

Background

EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7, a causative agent of diarrhea-associated Hemolytic Uremic Syndrome (D+HUS). The mechanism by which EHEC induces D+HUS has not been fully elucidated.

Objectives

We investigated the effects of EspP on clot formation and lysis in human blood.

Methods

Human whole blood and plasma were incubated with EspP^WT at various concentrations and sampled at various time points. Thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT), coagulation factor activities, and thrombelastgraphy (TEG) were measured.

Results and Conclusions

Human whole blood or plasma incubated with EspP^WT was found to have prolonged PT, aPTT, and TT. Furthermore, human whole blood or plasma incubated with EspP^WT had reduced activities of coagulation factors V, VII, VIII, and XII, as well as prothrombin. EspP did not alter the activities of coagulation factors IX, X, or XI. When analyzed by whole blood TEG, EspP decreased the maximum amplitude of the clot, and increased the clot lysis. Our results indicate that EspP alters hemostasis in vitro by decreasing the activities of coagulation factors V, VII, VIII, and XII, and of prothrombin, by reducing the clot strength and accelerating fibrinolysis, and provide further evidence of a functional role for this protease in the virulence of EHEC and the development of D+HUS.