Martin Bitzan

Epidemiology and diagnosis of Shiga toxin-producing Escherichia coli infections.

Shiga toxin-producing Escherichia coli (STEC) have been identified as a worldwide cause of serious human gastrointestinal disease and the life-threatening hemolytic uremic syndrome. The most common serotype implicated is E. coli O157: H7, but infections involving various non-O157 serotypes have been found with increasing frequency in many countries. Food-borne outbreaks caused by STEC can affect large numbers of people and cause serious morbidity, making the bacteria one of the most important emerging pathogens. Because there is no specific treatment of the disease currently available, there is an urgent need for effective preventive measures based on a detailed understanding of the epidemiology of STEC infections. Such measures will also be dependent on the availability of rapid, sensitive, and simple procedures for the detection of the pathogens both in human samples and in samples of nonhuman origin such as food. This review summarizes the current knowledge on the epidemiology of STEC infection and presents a survey of laboratory methods currently available for diagnosis of STEC. Special attention is given to new diagnostic procedures for the less readily detectable non-O157 STEC strains and to simple procedures, usually based on commercially available kits, that can be used in routine clinical microbiological laboratories.

Shiga Toxin-Producing Escherichia coli Infection and Antibodies against Stx2 and Stx1 in Household Contacts of Children with Enteropathic Hemolytic-Uremic Syndrome

ABSTRACT Ninety-five household contacts (aged 2 months to 73 years) of patients with enteropathic hemolytic-uremic syndrome (HUS) were investigated for the presence of immunoglobulin (Ig) G antibodies to Shiga toxins Stx2 and Stx1 by Western blot assay. Thirty-one percent of the household contacts and 19% of 327 controls had anti-Stx2 IgG (heavy and light chain [H + L]), 5 and 8%, respectively, had anti-Stx1 IgG (H + L), and 3 and 2%, respectively, had both anti-Stx2 and anti-Stx1 IgG (H + L). The incidence of infections with Stx-producing Escherichia coli (STEC) was determined based on the following diagnostic criteria: STEC isolation, detection of stx gene sequences, free fecal Stx in stool filtrates, and serum IgM antibodies against E. coli O157 lipopolysaccharide. Evidence of STEC infection was observed in 25 household contacts, of whom 18 (72%) were asymptomatic and represented a potential source of infection. Six of 13 (46%) household contacts with Stx2-producing E. coli O157:H7 in stool culture developed anti-Stx2 IgG (H + L), compared to 71% of Stx2-associated HUS cases. In individuals showing anti-Stx2 IgG (H + L), the antibody response was directed against the B subunit in 69% of household contacts and 71% of controls, in contrast to 28% of HUS patients. In this investigation controls had a significant increase of the median of IgM antibodies to O157 lipopolysaccharide (LPS) with age, up to the fifth decade. The lack of disease in household contacts with B subunit-specific antibodies, as well as the significantly higher median of anti-O157 LPS IgM antibodies in controls beyond 4.9 years of age, suggests a protective role for anti-Stx and anti-O157 LPS antibodies.

Purified verotoxins of Escherichia coli O157:H7 decrease prostacyclin synthesis by endothelial cells

Two immunologically distinct verotoxins purified from Escherichia coli C600, lysogenized with distinct temperate phages from E. coli strain 933 of serotype O157:H7, were compared by SDS-PAGE and different biological assays. The two toxins termed verotoxin 1 (VT1) and verotoxin 2 (VT2) differing in molecular weight exhibited similar biological activities. Both preparations were toxic for HeLa cells and lethal for mice. Epidemiological evidence of verotoxinogenesis in some cases of hemolytic-uremic syndrome (HUS) and the recent observations of inadequate prostacyclin production by endothelial cells associated with HUS prompted us to study the effect of purified verotoxins on prostacyclin synthesis in rat aortic tissue. Our results demonstrate a significant reduction of prostacyclin by both toxins at picomolar levels. The suppression of prostacyclin release by a lower concentration of VT2 as compared with VT1 reflects the relative potencies of these toxins in HeLa cell toxicity and mouse lethality. The results suggest an effect of verotoxins on endothelial cells and support the concept of these toxins as virulence factors in E. coli.

Differences in verotoxin neutralizing activity of therapeutic immunoglobulins and sera from healthy controls

SummaryIntestinal infection byEscherichia coli O157 and other verotoxin (VT) producingE. coli has been increasingly recognized as an important factor for the causation of classic (enteropathic) hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Toxins most frequently involved are VT1 and VT2. As with other toxin-mediated diseases, administration of immunoglobulin (Ig) may be beneficial. However, little is known about the immune response elicited by the toxin(s), and the prevalence of VT neutralizing antibodies in the healthy population. We studied the capacity of seven Igs and a commercial plasma preparation to neutralize four different VTs (VT1, VT2, VT2c and VT2e). The results were compared with the neutralization titers (NT50%) of normal human serum samples from various age groups. Plasma products and normal sera were separated by protein G affinity chromatography to investigate the factor(s) responsible for VT neutralization. All Igs neutralized VT1 (8 to 96 NT50%). None of them inhibited VT2, VT2c or VT2e effectively. In contrast, none of 40 pediatric, and only one of 20 adult control sera (starting dilution 1 : 4) neutralized VT1 (25 NT50%). All 60 samples as well as the plasma preparation blocked VT2 (22 to 446 NT50%, median 137), but not VT2c and VT2e. The VT1 neutralizing activity was eluted with the IgG fraction. The VT2 neutralizing activity was not bound by protein G, but was recovered in the IgG-free effluent. In conclusion, therapeutic Igs significantly neutralize VT1, but are largely ineffective against other VTs. In contrast, all control sera inhibited VT2, but rarely VT1. Different principles, notably IgG and non-IgG (probably non-immunoglobulin) factors, respectively, appear to be responsible for the reduction of VT1 and VT2 cytotoxicityin vitro. Patients with VTEC disease are rarely expected to benefit from the use of presently available Igs.ZusammenfassungEnterale Infektionen durch Verotoxin-(VT-)produzierendeEscherichia coli werden zunehmend als Ursache des klassischen hämolytisch-urämischen Syndroms (HUS) erkannt. Das HUS ist häufig mit VT2 allein, seltener mit VT1 und VT2 assoziiert. Wie bei anderen bakteriell-toxischen Erkrankungen könnten Immunglobuline (Ig) von therapeutischem Nutzen sein. Die VT-induzierte Immunantwort und die Prävalenz neutralisierender Antikörper in der gesunden Bevölkerung sind jedoch nur unzureichend bekannt. Wir untersuchten die VT-Neutralisationstiter (NT50%) von sieben Ig- und einem kommerziellen Plasmapräparat gegenüber VT1, VT2, VT2c und VT2e und verglichen sie mit normalen Serumproben (NHS) aus verschiedenen Altersstufen. Zur näheren Charakterisierung der neutralisierenden Aktivität wurden das Plasmapräparat und NHS über eine Protein-G-Säule chromatographisch aufgetrennt. Alle Ig-neutralisierten VT1 (8–96 NT50%), jedoch nicht VT2, VT2c oder VT2e. Andererseits wurde VT1 von keinem der 40 pädiatrischen und nur von einem von 20 adulten Kontrollseren neutralisiert (25 NT50%; Anfangsverdünnung 1 : 4). Alle 60 Serumproben und die Plasmakonserve blockierten VT2 (22–446 NT50%, Median 137), aber nicht VT2c oder VT2e. Die VT1-neutralisierende Aktivität wurde mit der IgG-Fraktion eluiert, während die VT2-neutralisierende Aktivität im IgG-freien Durchlauf lokalisiert wurde. Therapeutische Ig neutralisieren VT1in vitro, sind aber weitgehend unwirksam gegen andere VT. Im Gegensatz dazu inhibieren Seren aller Altersgruppen VT2, aber selten VT1. Wahrscheinlich sind unterschiedliche Wirkprinzipien für die Neutralisation von Verotoxinen durch normale Seren verantwortlich, nämlich IgG für VT1 und eine Nicht-Immunglobulinfraktion für VT2. Bei der Mehrzahl der HUS-Patienten ist von der Gabe derzeit verfügbarer Ig-Präparate ein VT-spezifischer therapeutischer Effekt nicht zu erwarten.

Escherichia coli O157 Fails to Induce a Long-Lasting Lipopolysaccharide- Specific, Measurable Humoral Immune Response in Children with Hemolytic- Uremic Syndrome

Escherichia coli O157 lipopolysaccharide (LPS)-specific antibodies were measured in sequential serum samples from 131 children with serologically defined E. coli O157-associated hemolytic-uremic syndrome (HUS), using an enzyme immunoassay. On the basis of evaluation of 66 children with culture-proven E. coli O157 infection and serum samples from 132 age-matched control subjects, the assay showed a sensitivity of 95%, 88%, and 74% and a specificity of 99%, 99%, and 98% for IgM, IgA, and IgG, respectively. Anti-O157 LPS antibodies decreased below the cut-off levels in >50% of the children at 11 (IgM), 5 (IgA), and 11 weeks (IgG) after onset of diarrhea and 10, 4, and 10 weeks, respectively, after the onset of HUS. Children with enteropathic HUS fail to develop a long-lasting humoral immune response to the O157 antigen. Incomplete immunity to E. coli O157 may signal a risk for recurrent infections and has implications for serodiagnostic studies.

Clinical and Genetic Aspects of Shiga-like Toxin Production in Traditional Enteropathogenic Escherichia coli

Cell culture tests, DNA colony blot hybridization and polymerase chain reaction were used to examine classical enteropathogenic Escherichia coli (EPEC) for the presence of Shiga-like toxin (SLT). Fifteen of 155 strains from West Germany, originally identified as EPEC on the basis of serotyping, were shown to harbor either SLT-I or SLT-II genes. All strains that hybridized with the 20-base oligonucleotide probes which are complementary to slt-IA or slt-IIA sequences derived from the genomic DNA of enterohemorrhagic E. coli O157:H7 strain 933 produced moderate or high levels of cytotoxin in Vero and HeLa cell assays. Four additional strains of low to moderate cytotoxicity did not hybridize with either probe. Five different serogroups producing SLTs were identified: O26, O55, O111, O119 and O128. All three SLT-positive E. coli O26:H11 and four of five E. coli O111:H- isolates hybridized with a 3.4 kilobase fragment (CVD 419 probe) derived from the 60-megadalton plasmid of EHEC O157:H7. Seven of the 15 SLT-gene positive strains were associated with bloody diarrhea, six isolates were from patients with hemolytic uremic syndrome (HUS). Based on their clinical, epidemiological, pathogenic and genetic features SLT-producing E. coli among classical EPEC mimic enterohemorrhagic E. coli O157:H7 and might be considered as EHEC.

Saliva IgM and IgA Are a Sensitive Indicator of the Humoral Immune Response to Escherichia coli O157 Lipopolysaccharide in Children with Enteropathic Hemolytic Uremic Syndrome

Saliva antibodies to Escherichia coli O157 were investigated as markers of the immune response in children with enteropathic hemolytic uremic syndrome (HUS). Paired serum and saliva samples were collected from 22 children with HUS during acute disease and convalescence and were tested for E. coli O157 lipopolysaccharide (LPS)-specific IgM and IgA antibodies by ELISA. Serum and saliva samples from 44 age-matched controls were used to establish the cut-off values. Elevated levels of IgM and/or IgA antibodies to O157 LPS were detected in saliva of 13/13 HUS patients with Shiga toxin-producing E. coli (STEC) O157 in stool culture and from 4 of 5 HUS patients in whom STEC were not detected. These results closely mirrored the results obtained with paired serum samples. In contrast, saliva and serum samples from four children with STEC isolates belonging to O-groups O26, O145 (n = 2), and O165 lacked detectable O157 LPS-specific antibodies. The specificity of the ELISA was confirmed by western blotting. In STEC O157 culture-confirmed cases, the sensitivity of the ELISA was 92% for saliva IgM and IgA, based on the first available sample, and 100% and 92%, respectively, when subsequent samples were included. The specificity was 98% for IgM and 100% for IgA. Children with E. coli O157 HUS demonstrate a brisk, easily detectable immune response as reflected by the presence of specific antibodies in their saliva. Saliva-based immunoassays offer a reliable, noninvasive method for the diagnosis of E. coli O157 infection in patients with enteropathic HUS.

Silencing of Bak Ameliorates Apoptosis of Human Proximal Tubular Epithelial Cells by Escherichia coli–Derived Shiga Toxin 2

Background:Escherichia coli–derived Shiga toxin (Stx), the cause of the enteropathic hemolytic uremic syndrome, is a potent inducer of apoptotic cell death. The present study was performed to examine the hypothesis that Stx initiates apoptosis by activating the mitochondrial pathway involving mitochondrial–associated, pro–apoptotic Bcl–2 family proteins Bax and Bak.Materials and Methods:To determine if Stx2–mediated apoptosis is dependent on Bax or Bak, a gene–silencing approach was employed using sequence–specific small interfering (si)RNA duplexes. Silencing of Bax and Bak protein expression in human renal proximal tubular epithelial (HK–2) cells and its effect on Shiga toxicity was assessed by immunofluorescence microscopy and Western blotting.Results:Transfection of HK–2 cells, shown to be exquisitely sensitive to Stx, with siRNA duplexes successfully diminished Bak, but not Bax protein expression. In order to determine if silencing of pro–apoptotic gene expression affects Stx–induced apoptosis, HK–2 cells were transfected with Bak–specific or control siRNA, exposed to lethal concentrations of Stx2 and assessed for cleavage of poly(ADPribose) polymerase–1 (PARP) as a marker of apoptosis, using Western blot technology. We observed that siRNA–induced reduction of Bak expression levels correlated with decreased PARP cleavage.Conclusion:Results suggest that Stx–induced cell death involves pro–apoptotic Bak and that silencing of Bak gene expression affords partial protection against Stx–mediated apoptosis.

Purification and characterization of a phage-encoded cytotoxin from an Escherichia coli O111 strain associated with hemolytic-uremic syndrome

Cytotoxin production by Escherichia coli O111:H-strain HUS-2 (Hamburg) is associated with a temperate toxin-converting bacteriophage (Tcp-111). E. coli laboratory strain C600 transduced and subsequently lysed by the phage produced and liberated large amounts of cytotoxin (CT111) which was purified by sequential chromatography. When compared with published procedures for toxin release from viable cells, lysis of the C600 culture by the phage was most effective. By SDS-PAGE CT111 as Shiga toxin from Shigella dysenteriae 1 were shown to consist of two polypeptides of MW 31 kd and 4-5 kd. Both toxins share common antigenic epitopes as revealed by immunoblotting and neutralization studies. With rabbit anti-CT111 toxic activity of only 5 out of 8 clinical E. coli O111 isolates was neutralized suggesting the presence of different cytotoxins in E. coli serogroup O111. Taken together, our data established CT111 as a potent cytotoxin with significant enterotoxic and neurotoxic properties similar or identical to Shiga toxin and to Shiga-like toxin I from E. coli O26:H11 and O157:H7 strains.

137 SHIGA-LIKE TOXIN (SLT) ASSOCIATED HEMOLYTIC-UREMIC SYNDROME (HUS), A 4-YEARS PROSPECTIVE STUDY

Patients with classical HUS (n=22; median age 2;2 yrs) were prospectively studied at our institution since 1986. SLT-association was established by cytotoxicity assays of fecal filtrates, bacterial isolation or colony-blot-hybridization with SLT-specific DNA probes. Serial serum samples from 12 pts and appropriate controls were studied for SLT I and II neutralizing antibodies (NAB) and for hemagglutinating antibodies (HA) using erythocytes coated with purified LPS from E.coli 0157:H7 and other serovars.SLT association was demonstrated in 13/22 children (59%); E.coli isolates (n=6 pts) included serogroups 026, 055, 0111 and 0157; fecal SLT alone was present in 7 additional cases. 4 pts developed rising NAB-titers against SLT I or II; 9 pts (75%) presented with high LPS-0157-HA at the time of the diagnosis of HUS that rapidly declined during convalescence.Thus in Northern Germany an association of the classical HUS with in-vivo SLT production has been confirmed. Detection of LPS-0157-AB appears to be a new, valuable serological marker for this subgroup of HUS. Further studies are needed to elucidate a possible protective or pathogenic role of SLT- and LPS-specific AB.