Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli

Multilocus sequence typing of 169 non-O157 enterohemorrhagic Escherichia coli (EHEC) isolated from patients with hemolytic uremic syndrome (HUS) demonstrated 29 different sequence types (STs); 78.1% of these strains clustered in 5 STs. From all STs and serotypes identified, we established a reference panel of EHEC associated with HUS (HUSEC collection).</p

Phylogeny and disease association of Shiga toxin-producing Escherichia coli O91

The diversity and relatedness of 100 Shiga toxin–producing Escherichia coli O91 isolates from different patients were examined by multilocus sequence typing. We identified 10 specific sequence types (ST) and 4 distinct clonal groups. ST442 was significantly associated with hemolytic uremic syndrome

Biophysikalische und biochemische Analyse von Protein-Ligand-Interaktionen in der Antiinfektiva-Wirkstoffentwicklung

The increasing number of antibiotic-resistant pathogens has created an urgent demand for novel treatment options to combat infectious diseases. In this thesis, test systems have been established enabling hit identification, lead optimization, and characterization to contribute to the development of potent and innovative anti-infectives, which overcome existing bacterial resistances. In the first approach, inhibitors of the RNA polymerase ‘switch region’ were studied. Using biochemical and biophysical techniques the exact binding site and ligand binding mode of the ureidothiophene-2-carboxylic acid inhibitors were elucidated. Mode of action studies revealed that congeners of this structural class inhibit the bacterial transcription in the initiation phase. Moreover, the ureidothiophene-2-carboxylic acid inhibitors do not show any cross-resistances with rifampicin or myxopyronin and possess good antibacterial activity in clinically relevant multidrug-resistant MRSA strains. In the second approach, inhibitors of the CsrA-RNA interaction were discovered by using a screening and a ligand-based approach. CsrA is an mRNA-binding regulatory protein being essentiell for full virulence of bacteria and thus, it represents an attractive target for anti infective drug discovery. For this purpose, a test system based on biophysical methods was established, which enabled the identification and characterization of first small molecule and ligand-derived inhibitors of the CsrA-RNA interaction.Aufgrund zunehmender Antibiotika-Resistenzen sind neue effiziente Therapiemöglichkeiten dringend erforderlich um eine erfolgreiche Behandlung von Infektionskrankheiten zu gewährleisten. Um die Entwicklung potenter und innovativer Anti-Infektiva zu unterstützen, wurden im Rahmen dieser Arbeit Testsysteme etabliert, welche die Identifizierung von Hitverbindungen sowie die Lead-Optimierung und Charakterisierung ermöglichen. Im ersten Ansatz wurden Hemmstoffe der RNA-Polymerase ‘switch region’ untersucht. Unter Anwendung biochemischer und biophysikalischer Methoden konnte deren genaue Bindestelle sowie der Ligandbindungsmodus aufgeklärt werden. Wirkmechanistische Studien haben gezeigt, dass die untersuchten Inhibitoren die Initiationsphase der Transkription hemmen. Die Inhibitoren wiesen keine Kreuzresistenz mit Rifampicin oder Myxopyronin auf und waren zudem wirksam gegen klinisch relevante MRSA Stämme. Der zweite Ansatz hatte die Entdeckung von Inhibitoren der CsrA-RNA Interaktion zum Ziel, welches mit einem Screening und einem Ligand-basierten Ansatz verfolgt wurde. Bei CsrA handelt es sich um ein mRNA bindendes, regulatorisches Protein, das essentiell für volle bakterielle Virulenz ist und somit ein interessantes Target für die Entwicklung neuer Anti Infektiva darstellt. Zu diesem Zweck wurde ein auf biophysikalischen Methoden basierendes Testsystem etabliert, mit dem erste ‚Small Molecule’ sowie Ligand-basierte Hemmstoffe identifiziert und charakterisiert werden konnten

Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study

Background: In an ongoing outbreak of haemolytic uraemic syndrome and bloody diarrhoea caused by a virulent Escherichia coli strain O104:H4 in Germany (with some cases elsewhere in Europe and North America), 810 cases of the syndrome and 39 deaths have occurred since the beginning of May, 2011. We analysed virulence profiles and relevant phenotypes of outbreak isolates recovered in our laboratory. Methods: We analysed stool samples from 80 patients that had been submitted to the National Consulting Laboratory for Haemolytic Uraemic Syndrome in Münster, Germany, between May 23 and June 2, 2011. Isolates were screened with standard PCR for virulence genes of Shiga-toxin-producing E coli and a newly developed multiplex PCR for characteristic features of the outbreak strain (rfbO104, fliCH4, stx2, and terD). Virulence profiles of the isolates were determined with PCR targeting typical virulence genes of Shiga-toxin-producing E coli and of other intestinal pathogenic E coli. We sequenced stx with Sanger sequencing and measured Shiga-toxin production, adherence to epithelial cells, and determined phylogeny and antimicrobial susceptibility. Findings: All isolates were of the HUSEC041 clone (sequence type 678). All shared virulence profiles combining typical Shiga-toxin-producing E coli (stx2, iha, lpfO26, lpfO113) and enteroaggregative E coli (aggA, aggR, set1, pic, aap) loci and expressed phenotypes that define Shiga-toxin-producing E coli and enteroaggregative E coli, including production of Shiga toxing 2 and aggregative adherence to epithelial cells. Isolates additionally displayed an extended-spectrum β-lactamase phenotype absent in HUSEC041. Interpretation: Augmented adherence of the strain to intestinal epithelium might facilitate systemic absorption of Shiga toxin and could explain the high progression to haemolytic uraemic syndrome. This outbreak demonstrates that blended virulence profiles in enteric pathogens, introduced into susceptible populations, can have extreme consequences for infected people

Attack of the clones: whole genome-based characterization of two closely related enterohemorrhagic Escherichia coli O26 epidemic lineages

Background: Enterohemorrhagic Escherichia coli (EHEC) O26:H11/H−, the most common non-O157 serotype causing hemolytic uremic syndrome worldwide, are evolutionarily highly dynamic with new pathogenic clones emerging rapidly. Here, we investigated the population structure of EHEC O26 isolated from patients in several European countries using whole genome sequencing, with emphasis on a detailed analysis of strains of the highly virulent new European clone (nEC) which has spread since 1990s. Results: Genome-wide single nucleotide polymorphism (SNP)-based analysis of 32 EHEC O26 isolated in the Czech Republic, Germany, Austria and Italy demonstrated a split of the nEC (ST29C2 clonal group) into two distinct lineages, which we termed, based on their temporal emergence, as “early” nEC and “late” nEC. The evolutionary divergence of the early nEC and late nEC is marked by the presence of 59 and 70 lineage-specific SNPs (synapomorphic mutations) in the genomes of the respective lineages. In silico analyses of publicly available E. coli O26 genomic sequences identified the late nEC lineage worldwide. Using a PCR designed to target the late nEC synapomorphic mutation in the sen/ent gene, we identified the early nEC decline accompanied by the late nEC rise in Germany and the Czech Republic since 2004 and 2013, respectively. Most of the late nEC strains harbor one of two major types of Shiga toxin 2a (Stx2a)-encoding prophages. The type I stx2a-phage is virtually identical to stx2a-phage of EHEC O104:H4 outbreak strain, whereas the type II stx2a-phage is a hybrid of EHEC O104:H4 and EHEC O157:H7 stx2a-phages and carries a novel mutation in Stx2a. Strains harboring these two phage types do not differ by the amounts and biological activities of Stx2a produced. Conclusions: Using SNP-level analyses, we provide the evidence of the evolutionary split of EHEC O26:H11/H− nEC into two distinct lineages, and a recent replacement of the early nEC by the late nEC in Germany and the Czech Republic. PCR targeting the late nEC synapomorphic mutation in ent/sen enables the discrimination of early nEC strains and late nEC strains in clinical and environmental samples, thereby facilitating further investigations of their geographic distribution, prevalence, clinical significance and epidemiology.Peer Reviewe

Treatment effect on biases in size estimation in spider phobia

Background: The current study investigates biases in size estimations made by spider-phobic and healthy participants before and after treatment. Method: Forty-one spider-phobic and 20 healthy participants received virtual reality (VR) exposure treatment and were then asked to rate the size of a real spider immediately before and, on average, 15 days after the treatment. During the VR exposure treatment skin conductance response was assessed. Results: Prior to the treatment, both groups tended to overestimate the size of the spider, but this size estimation bias was significantly larger in the phobic group than in the control group. The VR exposure treatment reduced this bias, which was reflected in a significantly smaller size rating post treatment. However, the size estimation bias was unrelated to the skin conductance response. Conclusion: Our results confirm the hypothesis that size estimation by spider-phobic patients is biased. This bias is not stable over time and can be decreased with adequate treatment. (C) 2016 Elsevier B.V. All rights reserved

Expanding the scaffold for bacterial RNA polymerase inhibitors: design, synthesis and structure–activity relationships of ureido-heterocyclic-carboxylic acids

A series of novel RNA polymerase (RNAP) inhibitors with antibacterial activity was designed and synthesized.</jats:p

Phenotypic and Genotypic Analyses of Enterohemorrhagic Escherichia coli O145 Strains from Patients in Germany

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O145 are emerging as causes of diarrhea and the hemolytic-uremic syndrome. However, there have been few genetic analyses of this EHEC group. We investigated the serotypes, virulence genes, plasmid profiles, pulsed-field gel electrophoresis (PFGE) patterns, and genetic variability of the fliC and eae genes in 120 EHEC O145 strains isolated from cases of hemolytic-uremic syndrome (n = 24) or diarrhea (n = 96) in Germany between 1996 and 2002. Three isolates belonged to serotype O145:H28, one to serotype O145:H25, and 116 were nonmotile (O145:H(−)). One hundred fourteen of the nonmotile strains shared fliC restriction fragment length polymorphism (RFLP) patterns identical to that of the O145:H28 strains. The remaining two nonmotile strains displayed a fliC-RFLP pattern identical to that of the O145:H25 strain. Each of the 117 strains with the fliC-RFLP(H28) pattern harbored eae γ, whereas the three strains with the fliC-RFLP(H25) pattern possessed eae β. Five different stx genotypes, six combinations of plasmid-encoded putative virulence genes, 29 plasmid profiles, and 47 PFGE types were identified. Strains within some of the PFGE types could be further subtyped by means of distinct plasmid profiles. These data demonstrate that the EHEC O145 serogroup is comprised of two different serotypes that possess distinct eae types. The heterogeneity of EHEC O145 strains at the chromosomal and plasmid level, in particular the high diversity in PFGE patterns, provides a basis for molecular subtyping of these pathogens

Heteropathogenic virulence and phylogeny reveal phased pathogenic metamorphosis in Escherichia coli O2:H6

Extraintestinal pathogenic and intestinal pathogenic (diarrheagenic) Escherichia coli differ phylogenetically and by virulence profiles. Classic theory teaches simple linear descent in this species, where non-pathogens acquire virulence traits and emerge as pathogens. However, diarrheagenic Shiga toxin-producing E.coli (STEC) O2:H6 not only possess and express virulence factors associated with diarrheagenic and uropathogenic E.coli but also cause diarrhea and urinary tract infections. These organisms are phylogenetically positioned between members of an intestinal pathogenic group (STEC) and extraintestinal pathogenic E.coli. STEC O2:H6 is, therefore, a 'heteropathogen,' and the first such hybrid virulent E.coli identified. The phylogeny of these E.coli and the repertoire of virulence traits they possess compel consideration of an alternate view of pathogen emergence, whereby one pathogroup of E.coli undergoes phased metamorphosis into another. By understanding the evolutionary mechanisms of bacterial pathogens, rational strategies for counteracting their detrimental effects on humans can be developed

Characterization of Cytolethal Distending Toxin Genes and Expression in Shiga Toxin-Producing Escherichia coli Strains of Non-O157 Serogroups

We identified cytolethal distending toxin and its gene (cdt) in 17 of 340 non-O157 Shiga toxin-producing Escherichia coli (STEC) strains (serotypes O73:H18, O91:H21, O113:H21, and O153:H18), all of which were eae negative. cdt is either chromosomal and homologous to cdt-V (serotypes O73:H18, O91:H21, and O113:H21) or plasmidborne and identical to cdt-III (serotype O153:H18). Among eae-negative STEC, cdt was associated with disease (P = 0.003)