Hemolytic uremic syndrome in renal allografted patients treated with cyclosporin

The classical triad of hemolytic uremic syndrome (microangiopathic hemolytic anemia, severe thrombopenia, and renal failure) developed de novo in three of our renal transplanted patients under cyclosporin A treatment. The predominant morphologic findings in the grafts consisted of glomerular and arteriolar thrombosis as well as arteriolonecrosis, all features of the syndrome. In one instance, ischemic bowel disease supervened after graft removal and was associated with persistent low grade microangiopathic process. De novo hemolytic uremic syndrome has been reported in patients treated with cyclosporin A following bone marrow or liver transplantation as well as in a few renal graft recipients. This peculiar form of cyclosporin A nephrotoxicity should not be confused with acute rejection of the renal transplant.     

Hemolytic uremic syndrome following the infusion of oxaliplatin: case report

Background  

Oxaliplatin is a platinum derivative, which is used in the treatment of colorectal cancer. A small number of oxaliplatin-related hemolytic and/or thrombocytopenic reactions have been reported. We present a case of hemolytic-uremic syndrome that developed during the 4^th cycle of combination chemotherapy with oxaliplatin, 5-fluorouracil and leucovorin.     

Mouse model for hemolytic uremic syndrome induced by outer membrane vesicles of Escherichia coli O157:H7

Hemolytic uremic syndrome (HUS) is characterized by acute renal failure in children and is typically complicated with thrombocytopenia and hemolytic anemia. Although mouse models of HUS have been evaluated using Shiga toxin (STx) combined with or without lipopolysaccharide (LPS), no HUS model has been tested using purified outer membrane vesicles (OMVs) from STx-producing Escherichia coli (STEC) O157:H7. Accordingly, we investigated whether OMVs of STEC O157:H7 conveying STx2 and LPS can cause HUS-like symptoms in mice inoculated intraperitoneally. Three types of OMVs differing in LPS acylation status and STx2 amount were used to compare their ability to induce HUS-like symptoms. Native OMVs (nOMV) with fully hexa-acylated LPS caused HUS-like symptoms at 72-96?h after dually divided injections of 1?μg nOMV per animal. However, elevated doses of modified OMVs (mOMV) carrying mainly penta-acylated LPS were required to induce similar HUS signs. Moreover, mitomycin-C-induced OMVs (mcOMV) that were enriched with STx2 induced HUS-like symptoms similar to those of nOMV at the same dose. These results suggest that the OMVs of STEC O157:H7 potentiated with STx2 and fully hexa-acylated LPS can be utilized for inducing HUS-like symptoms in mice and could be the causative material involved in the development of HUS.     

Sequence of the Escherichia coli O26 O antigen gene cluster and identification of O26 specific genes

Escherichia coli associated with outbreaks of gastroenteritis and hemolytic uremic syndrome include clones with O antigens O157 and O111. However, O26 has emerged as an O antigen present in pathogenic strains, particularly those implicated in cases of infantile gastroenteritis worldwide. The O26 O antigen gene cluster was sequenced. It was found to contain the genes expected for biosynthesis of nucleotide sugars L-rhamnose, N-acetyl-L-fucosamine and N-acetyl-glucosamine, as well genes for O unit flippase, O antigen polymerase and potential transferase genes. By polymerase chain reaction testing against representative strains for the 166 Escherichia coli O serogroups and some randomly selected Gram-negative bacteria, we identified three O antigen genes that are highly specific to O26. This work provides the basis for a sensitive test for the rapid detection of pathogenic clones with the O26 antigen, which has implications for public health, especially in the control of food-borne outbreaks.     

A novel Escherichia coli O157:H7 clone causing a major hemolytic uremic syndrome outbreak in China

An Escherichia coli O157:H7 outbreak in China in 1999 caused 177 deaths due to hemolytic uremic syndrome. Sixteen outbreak associated isolates were found to belong to a new clone, sequence type 96 (ST96), based on multilocus sequence typing of 15 housekeeping genes. Whole genome sequencing of an outbreak isolate, Xuzhou21, showed that the isolate is phylogenetically closely related to the Japan 1996 outbreak isolate Sakai, both of which share the most recent common ancestor with the US outbreak isolate EDL933. The levels of IL-6 and IL-8 of peripheral blood mononuclear cells induced by Xuzhou21 and Sakai were significantly higher than that induced by EDL933. Xuzhou21 also induced a significantly higher level of IL-8 than Sakai while both induced similar levels of IL-6. The expression level of Shiga toxin 2 in Xuzhou21 induced by mitomycin C was 68.6 times of that under non-inducing conditions, twice of that induced in Sakai (32.7 times) and 15 times higher than that induced in EDL933 (4.5 times). Our study shows that ST96 is a novel clone and provided significant new insights into the evolution of virulence of E. coli O157:H7.     

Molecular and Phenotypic Characterization of Escherichia coli O26:H8 among Diarrheagenic E. coli O26 Strains Isolated in Brazil

Escherichia coli strains of serogroup O26 comprise two distinct groups of pathogens, characterized as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among the several genes related to type III secretion system-secreted effector proteins, espK was found to be highly specific for EHEC O26:H11 and its stx-negative derivative strains isolated in European countries. E. coli O26 strains isolated in Brazil from infant diarrhea, foods, and the environment have consistently been shown to lack stx genes and are thus considered atypical EPEC. However, no further information related to their genetic background is known. Therefore, in this study, we aimed to discriminate and characterize these Brazilian O26 stx-negative strains by phenotypic, genetic, and biochemical approaches. Among 44 isolates confirmed to be O26 isolates, most displayed flagellar antigen H11 or H32. Out of the 13 nonmotile isolates, 2 tested positive for fliC_H11, and 11 were fliC_H8 positive. The identification of genetic markers showed that several O26:H11 and all O26:H8 strains tested positive for espK and could therefore be discriminated as EHEC derivatives. The presence of H8 among EHEC O26 and its stx-negative derivative isolates is described for the first time. The interaction of three isolates with polarized Caco-2 cells and with intestinal biopsy specimen fragments ex vivo confirmed the ability of the O26 strains analyzed to cause attaching-and-effacing (A/E) lesions. The O26:H32 strains, isolated mostly from meat, were considered nonvirulent. Knowledge of the virulence content of stx-negative O26 isolates within the same serotype helped to avoid misclassification of isolates, which certainly has important implications for public health surveillance.     

Immunomagnetic separation of Escherichia coli O26, O111 and O157 from vegetables

AIMS: Raw fruits and vegetables have been increasingly associated with human infections caused by Shiga toxin-producing Escherichia coli. This study evaluates the isolation and detection of E. coli O26, O111 and O157 from vegetable samples using immunomagnetic particles. METHODS AND RESULTS: Standard cultivation and immunomagnetic separation (IMS) procedures were compared. It was found that immunomagnetic particles could efficiently concentrate E. coli cells, detecting significantly more bacteria than with standard cultivation procedures. CONCLUSION: Bacteria were detected in 93-100% of the inoculated samples using the IMS procedure, but only 36-93% samples tested by standard cultivation procedures were found to be positive. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that E. coli O26, O111 and O157 immunomagnetic particles can be a very useful and efficient tool for the detection of E. coli strains in raw vegetables, and could probably be used with samples of animal origin.     

Molecular serotyping of Escherichia coli O26:H11

Serotyping is the foundation of pathogenic Escherichia coli diagnostics; however, few laboratories have this capacity. We developed a molecular serotyping protocol that targets, genetically, the same somatic and flagellar antigens of E. coli O26:H11 used in traditional serotyping. It correctly serotypes strains untypeable by traditional methods, affording primary laboratories serotyping capabilities.     

Molecular Serotyping of Escherichia coli O26:H11

Serotyping is the foundation of pathogenic Escherichia coli diagnostics; however, few laboratories have this capacity. We developed a molecular serotyping protocol that targets, genetically, the same somatic and flagellar antigens of E. coli O26:H11 used in traditional serotyping. It correctly serotypes strains untypeable by traditional methods, affording primary laboratories serotyping capabilities.     

Potentially human-pathogenic Escherichia coli O26 in Norwegian sheep flocks

A national survey of Escherichia coli O26 in Norwegian sheep flocks was conducted, using fecal samples to determine the prevalence. In total, 491 flocks were tested, and E. coli O26 was detected in 17.9% of the flocks. One hundred forty-two E. coli O26 isolates were examined for flagellar antigens (H typing) and four virulence genes, including stx and eae, to identify possible Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC). Most isolates (129 out of 142) were identified as E. coli O26:H11. They possessed eae and may have potential as human pathogens, although only a small fraction were identified as STEC O26:H11, giving a prevalence in sheep flocks of only 0.8%. Correspondingly, the sheep flock prevalence of atypical EPEC (aEPEC) O26:H11 was surprisingly high (15.9%). The genetic relationship between the E. coli O26:H11 isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA), identifying 63 distinct PFGE profiles and 22 MLVA profiles. Although the MLVA protocol was less discriminatory than PFGE and a few cases of disagreement were observed, comparison by partition mapping showed an overall good accordance between the two methods. A close relationship between a few isolates of aEPEC O26:H11 and STEC O26:H11 was identified, but all the E. coli O26:H11 isolates should be considered potentially pathogenic to humans. The present study consisted of a representative sampling of sheep flocks from all parts of Norway. This is the first large survey of sheep flocks focusing on E. coli O26 in general, including results of STEC, aEPEC, and nonpathogenic isolates.     

Murine monoclonal antibodies specific for lipopolysaccharide of Escherichia coli O26 and O111

Monoclonal antibody (MAb) 12F5 reacted with 35 Escherichia coli O26 isolates and cross-reacted with 1 of 365 non-E. coli O26 isolates. MAb 15C4 reacted with 30 E. coli O111 strains and 8 Salmonella O35 strains (possessing identical O antigen) but not with 362 other bacterial strains. Lipopolysaccharide immunoblots confirmed MAb O-antigen specificity.     

Murine Monoclonal Antibodies Specific for Lipopolysaccharide of Escherichia coli O26 and O111

Monoclonal antibody (MAb) 12F5 reacted with 35 Escherichia coli O26 isolates and cross-reacted with 1 of 365 non-E. coli O26 isolates. MAb 15C4 reacted with 30 E. coli O111 strains and 8 Salmonella O35 strains (possessing identical O antigen) but not with 362 other bacterial strains. Lipopolysaccharide immunoblots confirmed MAb O-antigen specificity.     

Detection of Escherichia coli Serogroups O26 and O113 by PCR Amplification of the wzx and wzy Genes

PCR-based assays for detecting enterohemorrhagic Escherichia coli serogroups O26 and O113 were developed by targeting the wzx (O-antigen flippase) and the wzy (O-antigen polymerase) genes found in the O-antigen gene cluster of each organism. The PCR assays were specific for the respective serogroups, as there was no amplification of DNA from non-O26 and non-O113 E. coli serogroups or from other bacterial genera tested. Using the PCR assays, we were able to detect the organisms in seeded apple juice inoculated at concentration levels as low as ≤10 CFU/ml. The O26- and O113-specific PCR assays can potentially be used for typing E. coli O26 and O113 serogroups; these assays will offer an advantage to food and environmental microbiology laboratories in terms of identifying these non-O157 serogroups by replacing antigen-based serotyping.     

Atypical hemolytic uremic syndrome

Hemolytic uremic syndrome (HUS) is defined by the triad of mechanical hemolytic anemia, thrombocytopenia and renal impairment. Atypical HUS (aHUS) defines non Shiga-toxin-HUS and even if some authors include secondary aHUS due to Streptococcus pneumoniae or other causes, aHUS designates a primary disease due to a disorder in complement alternative pathway regulation. Atypical HUS represents 5 -10% of HUS in children, but the majority of HUS in adults. The incidence of complement-aHUS is not known precisely. However, more than 1000 aHUS patients investigated for complement abnormalities have been reported. Onset is from the neonatal period to the adult age. Most patients present with hemolytic anemia, thrombocytopenia and renal failure and 20% have extra renal manifestations. Two to 10% die and one third progress to end-stage renal failure at first episode. Half of patients have relapses. Mutations in the genes encoding complement regulatory proteins factor H, membrane cofactor protein (MCP), factor I or thrombomodulin have been demonstrated in 20-30%, 5-15%, 4-10% and 3-5% of patients respectively, and mutations in the genes of C3 convertase proteins, C3 and factor B, in 2-10% and 1-4%. In addition, 6-10% of patients have anti-factor H antibodies. Diagnosis of aHUS relies on 1) No associated disease 2) No criteria for Shigatoxin-HUS (stool culture and PCR for Shiga-toxins; serology for anti-lipopolysaccharides antibodies) 3) No criteria for thrombotic thrombocytopenic purpura (serum ADAMTS 13 activity > 10%). Investigation of the complement system is required (C3, C4, factor H and factor I plasma concentration, MCP expression on leukocytes and anti-factor H antibodies; genetic screening to identify risk factors). The disease is familial in approximately 20% of pedigrees, with an autosomal recessive or dominant mode of transmission. As penetrance of the disease is 50%, genetic counseling is difficult. Plasmatherapy has been first line treatment until presently, without unquestionable demonstration of efficiency. There is a high risk of post-transplant recurrence, except in MCP-HUS. Case reports and two phase II trials show an impressive efficacy of the complement C5 blocker eculizumab, suggesting it will be the next standard of care. Except for patients treated by intensive plasmatherapy or eculizumab, the worst prognosis is in factor H-HUS, as mortality can reach 20% and 50% of survivors do not recover renal function. Half of factor I-HUS progress to end-stage renal failure. Conversely, most patients with MCP-HUS have preserved renal function. Anti-factor H antibodies-HUS has favourable outcome if treated early.     

Persistence of Shiga toxin-producing Escherichia coli O26 in cow slurry

AIMS: The main objective of this study was to evaluate the growth and survival of Shiga toxin-producing Escherichia coli (STEC) O26 in cow slurry; this serogroup is regarded as an important cause of STEC-associated diseases. METHODS AND RESULTS: Four STEC were examined by polymerase chain reaction (PCR) to determine whether they harbour key virulence determinants and also by pulsed-field gel electrophoresis (PFGE) to obtain overview fingerprints of their genomes. They were transformed with the pGFPuv plasmid and were separately inoculated at a level of 10(6) CFU ml(-1) in 15 l of cow slurry. All STEC O26 strains could be detected for at least 3 months in cow slurry without any genetic changes. The moisture content of the slurry decreased over time to reach a final value of 75% while the pH increased from 8.5 to 9.5 units during the last 50 days. CONCLUSION: STEC O26 strains were able to survive in cow slurry for an extended period. SIGNIFICANCE AND IMPACT OF THE STUDY: Long-term storage of waste slurry should be required to reduce the pathogen load and to limit environmental contamination by STEC O26.