Dorothea Orth

Shiga Toxin Activates Complement and Binds Factor H: Evidence for an Active Role of Complement in Hemolytic Uremic Syndrome

Infections with enterohemorrhagic Escherichia coli (EHEC) are a major cause of hemolytic uremic syndrome (HUS). Shiga toxins (Stxs), especially Stx2, are believed to represent major virulence factors of EHEC, contributing to HUS pathogenesis. Beside EHEC-associated HUS, there are hereditary atypical forms of HUS, which are mostly caused by mutations of complement regulators. The aim of the present study was to investigate whether or not complement is also involved in the pathogenesis of EHEC-induced typical HUS, by being activated either directly or indirectly by involvement of its inhibitors. Purified Stx2 markedly activated complement via the alternative pathway and was found to bind to factor H (FH), however, only when it was active. No apparent cleavage or destruction of FH was visible, and cofactor activity in fluid phase was unaffected, but clearly delayed for surface-attached FH, where it is essential for host cell protection. Binding studies using FH constructs revealed that Stx2 binds to short consensus repeats (SCRs) 6–8 and SCRs18–20, but not to SCRs16–17, i.e., to regions involved in the surface recognition function of FH. In conclusion, complement, and in particular FH, not only plays an important role in atypical HUS, but most probably also in EHEC-induced HUS.

EspP, a Serine Protease of Enterohemorrhagic Escherichia coli, Impairs Complement Activation by Cleaving Complement Factors C3/C3b and C5

ABSTRACT Hemolytic-uremic syndrome (HUS) is a life-threatening disorder characterized by hemolytic anemia, thrombocytopenia, and renal insufficiency. It is caused mainly by infections with enterohemorrhagic Escherichia coli (EHEC). Recently, Shiga toxin 2, the best-studied virulence factor of EHEC, was reported to interact with complement, implying that complement may be involved in the pathogenesis of EHEC-induced HUS. The aim of the present study was to investigate whether or not the serine protease EspP, an important virulence factor of EHEC, interacts with complement proteins. EspP did not have any effect on the integrity of factor H or factor I. However, EspP was shown to cleave purified C3/C3b and C5. Cleavage of the respective complement proteins also occurred in normal human serum (NHS) as a source of C3/C3b or C5 or when purified complement proteins were added to the supernatant of an EspP-producing wild-type strain. Edman degradation allowed unequivocal mapping of all three main C3b fragments but not of the three main C5 fragments. Complement activation was significantly downregulated in all three pathways for C5-depleted serum to which C5, preincubated with EspP, was added (whereas C5 preincubated with an EspP mutant was able to fully reconstitute complement activation). This indicates that EspP markedly destroyed the functional activity, as measured by a commercial total complement enzyme-linked immunosorbent assay (Wieslab). Downregulation of complement by EspP in vivo may influence the colonization of EHEC bacteria in the gut or the disease severity of HUS.

Sorbitol-fermenting Shiga toxin-producing Escherichia coli O157: indications for an animal reservoir

This study investigates a sorbitol-fermenting enterohaemorrhagic Escherichia coli (SF EHEC) O157 infection in a farmers family in the Austrian province of Salzburg. The investigation commenced after a 10-month-old boy was admitted to hospital with the clinical diagnosis of a haemolytic-uraemic syndrome (HUS) and his stool specimen grew SF EHEC O157:H-. In a subsequent environmental survey, a stool specimen of the 2-year-old brother and faecal samples of two cattle from the familys farm were also found to be positive for SF EHEC O157:H-. All four isolates had indistinguishable phenotypic and molecular characteristics and were identical to the first strain detected in Bavaria in 1988. Despite identical isolates being demonstrated in Bavaria after 1988, and until this report, increased surveillance in neighbouring Austria had not found this organism. We propose that the strain may have recently spread from Bavaria to Austria. Although SF EHEC O157:H- strains are still rare, they may represent a considerable health threat as they can spread from farm animals to humans and between humans.

Improvement of Detection of Bacterial Pathogens in Normally Sterile Body Sites with a Focus on Orthopedic Samples by Use of a Commercial 16S rRNA Broad-Range PCR and Sequence Analysis

ABSTRACT A new commercially available universal 16S and 18S rRNA gene PCR test, which is followed by sequence analysis of amplicons (SepsiTest), was evaluated for rapid identification of pathogens in the diagnosis of bone and joint infections. Eighty-three orthopedic samples and 21 specimens from other normally sterile body sites collected from 84 patients were analyzed in parallel by culture and PCR for detection of bacteria and fungi. Compared to culture, the diagnostic sensitivity and specificity of PCR were 88.5% and 83.5%, respectively. The detection rate of PCR (34.6%) was higher than that of bacterial culture (25.0%) as a consequence of the presence of fastidious and noncultivable species in samples and antibiotic treatment of patients. Thirteen culture-negative infections were identified by PCR, and PCR was able to detect culture-proven polymicrobial infections. On the other hand, three samples were culture positive but PCR negative. SepsiTest was demonstrated to be a valuable supplemental tool in the rapid detection of bacteria, especially for fastidious and noncultivable organisms, allowing earlier initiation of pathogen-adapted therapy in patients with bone and joint infections.

Diminished response to tick-borne encephalitis vaccination in thymectomized children

In order to analyze the clinical impact of immunological alterations in thymectomized children after exposure to a new antigen (tick-borne encephalitis virus (TBEV) vaccine), 17 thymectomized children completed a three-dose immunization regimen. Thymectomized children showed significantly lower TBEV IgG antibody levels after the second vaccination when compared to healthy age-matched controls (n=30) (p=0.03), but a normal response after the third vaccination. Age at thymectomy correlated significantly with the TBEV IgG antibody levels (p=0.04). Thymectomized children also showed significantly lower total counts and percentages for naïve T cells correlating with time after thymectomy (p=0.02), than observed for controls. These changes in T cell subsets and the decreased ability to respond to new antigens in thymectomized children, as observed here, may precede more striking effects such as higher infection rates or autoimmune conditions as they age.

Prevention and treatment of enterohemorrhagic Escherichia coli infections in humans.

Infections with enterohemorrhagic Escherichia coli (EHEC) result in various clinical symptoms and outcomes ranging from watery or bloody diarrhea to the life-threatening hemolytic–uremic syndrome (HUS). Shiga toxins (Stxs) are supposed to play a major role in the pathogenesis of EHEC infections; however, the role of other putative virulence factors is not fully elucidated. So far, there is only supportive therapy available for the treatment of both EHEC-associated diarrhea and HUS. Antibiotic therapy for the treatment of EHEC-associated diarrhea is discussed. In recent years other therapeutic strategies have been developed, including Gb3 receptor analogues, that bind Stx in the gut or in the circulation, passive immunization with Stx-neutralizing monoclonal antibodies, or active immunization with stx1 and stx2 toxoids as a preventive procedure. These approaches have been demonstrated to be effective in animal models but clinical trials are lacking.

Complement in Typical Hemolytic Uremic Syndrome

Hemolytic uremic syndrome (HUS) is a severe disease characterized by the clinical triad of hemolytic anemia, thrombocytopenia, and acute renal failure. HUS exists in two forms: the atypical diarrhea-negative HUS, which is often associated with complement disorders, and the more frequent diarrheal-associated typical HUS, which is caused by infections with enterohemorrhagic ESCHERICHIA COLI. The virulence factors of the latter have been studied well, and Shiga toxin (Stx)2 is reported to represent the most important one. In contrast, risk factors on the host side have not been intensively studied until recently: Complement activation products have been detected in the serum and plasma of HUS patients, and an in vitro study could show that Stx2 not only damages the kidney directly but also indirectly via complement, in two ways. First, it activates complement, and second, it delays the functions of its control protein factor H on the cell surface, both known to damage the kidney.

Emergence of VIM-1-carbapenemase-producing Enterobacter cloacae in Tyrol, Austria.

The rapid emergence and dissemination of carbapenemase-producing Enterobacter species and other members of the Enterobacteriaceae poses a considerable threat to the care of hospitalized patients and to public health. In this study, Enterobacter isolates demonstrating decreased susceptibility to carbapenems detected at the Division of Hygiene and Medical Microbiology, Innsbruck Medical University, between January 2006 and December 2010 were tested for bla(VIM-1), bla(NDM-1), bla(IMP), bla(KPC) and bla(OXA-48) using a multiplex PCR with published primers. PFGE was performed to determine the genetic relatedness. In total, 33 isolates (28 Enterobacter cloacae and 5 Enterobacter aerogenes) were collected during the study period. From 2006 to 2009, between two and seven isolates were found per year. In 2010, a significant increase of carbapenem-resistant strains was observed (n = 12). The bla(VIM-1) gene was detected in all 28 isolates of E. cloacae. Typing of E. cloacae by PFGE revealed three distinct clusters, the biggest of which contained 18 isolates. These findings demonstrate the emergence of VIM-1-producing Enterobacter in Tyrol, western Austria. The clonal relationship confirms the risk of spread of these organisms and their possible persistence over time.

Bactobilia after liver transplantation: Frequency and antibiotic susceptibility

After liver transplantation (LT), bactobilia occurs frequently in patients, leading in some cases to cholangitis and biliary sepsis. The present study is the first to investigate bactobilia after LT, and it gives an overview of predisposing factors for bactobilia, the microbial spectrum in the bile of LT patients, and the antibiotic susceptibility. A total of 172 endoscopic retrograde cholangiography (ERC) procedures were performed in 66 LT patients between 1 month and 5.8 years after LT. Bile samples were examined microbiologically. Sixty‐eight nontransplanted patients without cholestasis, but requiring ERC for other reasons served as a control group. Of 172 samples obtained from LT patients, 126 (73.3%) were positive for microbes. A total of 236 organisms were isolated: 114 (48.3%) gram‐positive bacteria, 92 (39.0%) aerobic gram‐negative, 8 (3.4%) anaerobes, and 22 (9.3%) fungi. Ciprofloxacin and amoxycillin/clavulanic acid showed the best susceptibility results among oral antibiotics and piperacillin/tazobactam and imipenem/cilastatin among intravenous preparations. In contrast, only 15.7% of non‐LT patients showed bactobilia. In conclusion, our study shows that bactobilia is a problem in patients after LT and that it is not only a contamination from endoscopic intervention. Mechanical obstruction, plastic stents, gallstones, and papillotomy increase the risk of bactobilia significantly. In our cohort we had the best antibiotic susceptibility results for positive cultures in LT patients with piperacillin/tazobactam, ciprofloxacin, or amoxycillin/clavulanic acid. Liver Transpl 12:747–753, 2006.

Sorbitol-fermenting Shiga toxin-producing Escherichia coli O157 in Austria.

ZusammenfassungInfektionen mit enterohämorrhagischen Escherichia coli (EHEC) sind die Hauptursache für das hämolytisch urämische Syndrom (HUS), die häufigste Ursache für akutes Nierenversagen im Kindesalter. Shigatoxine stellen den Hauptvirulenzfaktor von EHEC Stämmen dar. Nicht-sorbit-fermentierende EHEC O157:H7 sind zwar immer noch der am häufigsten isolierte Serotyp weltweit, es wird jedoch zunehmend von Sorbit-fermentierenden (SF) O157:H- (H- bezeichnet die fehlende Motilität) berichtet. In Österreich wurden 13SF EHEC O157:H- (11 humanen und zwei tierischen Ursprungs) in den Jahren 2002–2008 isoliert. Unter den 11 humanen Fällen waren sieben Patienten an einem HUS erkrankt, zwei litten an Diarrhö und zwei weitere waren asymptomatisch. Die Mehrheit der SF O157:H- Fälle (n = 7) wurde von Kindern aus Salzburg und vier von Patienten aus Vorarlberg isoliert. Unter den SF O157:H- Fällen wurden drei Ausbrüche mit jedoch nicht mehr als drei Beteiligten und vier sporadische Fälle diagnostiziert. Das Pulsfeld-Gelelektrophorese (PFGE) Bandenmuster der 13 SF O157:H- ergab drei verschiedene Cluster (Gruppen 1, 2 und 3). Die Stämme der drei Ausbrüche zeigten identische Bandenmuster (bzw. in einem Stamm eine Bande Unterschied) untereinander. Der Bayrische Ausbruchstamm zeigte ein anderes Bandenmuster als alle SF O157:H- Stämme, die in Österreich isoliert wurden. Für eine erfolgreiche Detektion von SF EHEC O157:H- ist ein Screening auf Shigatoxine mittels ELISA und/oder Shigatoxin Gene mittels PCR unerlässlich, ein Screening basierend auf phänotypischen Charakteristika, wie die fehlende Sorbitfermentation ist nicht ausreichend. Typisierungsmethoden, die alleine auf dem Nachweis von O157 beruhen, werden zwar diese Stämme identifizieren, sollten aber ebenso verlassen werden, um die prävalenten non-O157 Stämme, die auch HUS verursachen, nicht zu übersehen.SummaryInfections with enterohemorrhagic Escherichia coli (EHEC) are the major cause of hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in childhood. Shiga toxins are considered to be the most important virulence factor of EHEC strains. Non-sorbitol-fermenting EHEC O157:H7 is still the most prevalent serotype isolated worldwide; however, sorbitol-fermenting (SF) EHEC O157:H- (H- indicates nonmotility) strains are increasingly reported. Thirteen SF EHEC O157:H- strains (11 of human origin, two from animals) were detected in Austria between 2002 and 2008. Among the 11 human cases, seven suffered from HUS, two from diarrhea and the remaining two were asymptomatic. Seven of the cases were identified in patients living in or visiting (in one case) the province Salzburg, four were in patients from the province Vorarlberg. Three outbreaks with no more than three persons involved were detected, the other four cases occurred sporadically. The pulsed-field gel-electrophoresis banding patterns of the 13 SF EHEC O157:H- isolates were grouped into three distinct clusters (groups 1, 2 and 3). Strains of the three outbreaks were identical (except for one outbreak strain with one band difference) within each outbreak. In comparison, the Bavarian epidemic strain showed a pattern different from all SF O157:H- strains isolated in Austria. For effective detection of SF EHEC O157:H-, screening for Shiga toxins by ELISA and/or Shiga toxin genes by PCR is absolutely necessary; screening on the basis of phenotypic characteristics such as sorbitol-non-fermentation is not sufficient. Typing methods relying solely on investigation of O157 will detect these strains but should nevertheless also be avoided, so that the prevalent non-O157 strains causing HUS are not missed.