Wenlan Zhang

Publications of Wenlan Zhang

• Epidemic Escherichia coli O104:H4: Effects of antibiotics on Shiga toxin 2 production and bacteriophage induction.

  Authors: Martina Bielaszewska, Evgeny A Idelevich, Wenlan Zhang, Andreas Bauwens, Frieder Schaumburg, Alexander Mellmann, Georg Peters, Helge Karch

  Antimicrobial agents and chemotherapy. 03/2012;

  The role of antibiotics in treatment of enterohemorrhagic Escherichia coli (EHEC) infections is controversial because of concerns about triggering hemolytic uremic syndrome (HUS) by increasing ShigaThe role of antibiotics in treatment of enterohemorrhagic Escherichia coli (EHEC) infections is controversial because of concerns about triggering hemolytic uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicated in some patients. We tested a diverse panel of antibiotics to which the outbreak strain is susceptible to interrogate the effects of subinhibitory antibiotic concentrations on induction of stx(2)-harboring bacteriophages, stx(2) transcription and Stx2 production in this emerging pathogen. Ciprofloxacin significantly increased (P < 0.001 to P < 0.05), fosfomycin, gentamicin, and kanamycin insignificantly influenced (P > 0.1), and chloramphenicol, meropenem, azithromycin, rifaximin, and tigecycline significantly decreased (P ≤ 0.05) stx(2)-phage induction and Stx2 production in outbreak isolates. Ciprofloxacin and chloramphenicol significantly upregulated and downregulated, respectively, stx(2) transcription (P < 0.01); the other antibiotics had insignificant effects (P > 0.1). Meropenem, azithromycin, and rifaximin, which had been used for necessary therapeutic or prophylactic interventions during the EHEC O104:H4 outbreak, as well as tigecycline, neither induce stx(2)-harboring phages nor increase stx(2) transcription and Stx2 production in the outbreak strain. These antibiotics might represent therapeutic options in patients with EHEC O104:H4 infection if antibiotic treatment is inevitable. We await further analysis of the epidemic to determine if usage of these agents was associated with an altered risk of developing HUS.

• Real-time multiplex polymerase chain reaction for detecting Shiga toxin 2-producing Escherichia coli O104:H4 in human stools.

  Authors: Wenlan Zhang, Martina Bielaszewska, Andreas Bauwens, Angelika Fruth, Alexander Mellmann, Helge Karch

  Journal of clinical microbiology. 02/2012;

  A real-time multiplex PCR targeting stx(2), wzy(O104), and fliC(H4) of enterohemorrhagic Escherichia coli (EHEC) O104:H4 correctly determined the presence or absence of these genes in 253 EHECA real-time multiplex PCR targeting stx(2), wzy(O104), and fliC(H4) of enterohemorrhagic Escherichia coli (EHEC) O104:H4 correctly determined the presence or absence of these genes in 253 EHEC isolates and 132 patients' stool enrichment cultures. It is a rapid, sensitive and specific tool for detecting EHEC O104:H4 in human stools.

• Promiscuous Shiga toxin 2e and its intimate relationship to Forssman.

  Authors: Johannes Müthing, Iris Meisen, Wenlan Zhang, Martina Bielaszewska, Michael Mormann, Rolf Bauerfeind, M Alexander Schmidt, Alexander W Friedrich, Helge Karch

  Glycobiology. 01/2012;

  Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) represents the major virulence factor responsible for the pig edema disease which is characterized by hemorrhagic lesions, neurologicalShiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) represents the major virulence factor responsible for the pig edema disease which is characterized by hemorrhagic lesions, neurological disorders, and often fatal outcome. Stx2e-producing strains from the intestine of slaughtered pigs (n=3), feces of piglets with postweaning diarrhea or edema disease (n=12) and feces of humans with asymptomatic infections or mild diarrhea (n=13) were comparatively analyzed for the binding specificities of Stx2e to glycosphingolipids (GSLs) of the globo-series. Besides equivalent binding towards globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) we could demonstrate specific interaction of Stx2e preparations from human and porcine STEC isolates with Forssman GSL. Notably, Forssman GSL was recognized neither by structurally closely related Stx2 nor by Stx1 derived from human STEC isolates conferring Stx2e a unique recognition feature. Noteworthy, 7 (54%) of the 13 human and 8 (53%) of the 15 pig Stx2e samples exhibited cytotoxic action towards human brain microvascular endothelial cells. Our findings provide a basis for further exploring the functional role of the promiscuous receptor repertoire of Stx2e and the exact nature of the mechanisms that underlie different pathological outcomes of Stx2e-producing STEC in humans and pigs.

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

  Authors: Martina Bielaszewska, Alexander Mellmann, Wenlan Zhang, Robin Köck, Angelika Fruth, Andreas Bauwens, Georg Peters, Helge Karch

  The Lancet infectious diseases. 06/2011; 11(9):671-6.

  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), 810In 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. 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 (rfb(O104), fliC(H4), stx(2), 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. All isolates were of the HUSEC041 clone (sequence type 678). All shared virulence profiles combining typical Shiga-toxin-producing E coli (stx(2), iha, lpf(O26), lpf(O113)) 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. 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. German Federal Ministry of Education and Research, Network Zoonoses.

• Chromosomal instability in enterohaemorrhagic Escherichia coli O157:H7: impact on adherence, tellurite resistance and colony phenotype.

  Authors: Martina Bielaszewska, Barbara Middendorf, Phillip I Tarr, Wenlan Zhang, Rita Prager, Thomas Aldick, Ulrich Dobrindt, Helge Karch, Alexander Mellmann

  Molecular microbiology. 02/2011; 79(4):1024-44.

  Tellurite (Tel) resistant enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a global pathogen. In strain EDL933 Tel resistance (Tel(R) ) is encoded by duplicate ter cluster in O islands (OI) 43Tellurite (Tel) resistant enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a global pathogen. In strain EDL933 Tel resistance (Tel(R) ) is encoded by duplicate ter cluster in O islands (OI) 43 and 48, which also harbour iha, encoding the adhesin and siderophore receptor Iha. We identified five EHEC O157:H7 strains that differentiate into large (L) colonies and small (S) colonies with high and low Tel minimal inhibitory concentrations (MICs) respectively. S colonies (Tel-MICs ≤ 4 µg ml⁻¹) sustained large internal deletions within the Tel(R) OIs via homologous recombination between IS elements and lost ter and iha. Moreover, complete excision of the islands occurred by site-specific recombination between flanking direct repeats. Complete excision of OI 43 and OI 48 occurred in 1.81 × 10⁻³ and 1.97 × 10⁻⁴ cells in culture, respectively; internal deletion of OI 48 was more frequent (9.7 × 10⁻¹ cells). Under iron limitation that promotes iha transcription, iha-negative derivatives adhered less well to human intestinal epithelial cells and grew slower than did their iha-positive counterparts. Experiments utilizing iha deletion and complementation mutants identified Iha as the major factor responsible for these phenotypic differences. Spontaneous deletions affecting Tel(R) OIs contribute to EHEC O157 genome plasticity and might impair virulence and/or fitness.

• Enterohaemorrhagic Escherichia coli haemolysin is cleaved and inactivated by serine protease EspPα.

  Authors: Jens Brockmeyer, Thomas Aldick, Jens Soltwisch, Wenlan Zhang, Philip I Tarr, André Weiss, Klaus Dreisewerd, Johannes Müthing, Martina Bielaszewska, Helge Karch

  Environmental microbiology. 02/2011; 13(5):1327-41.

  The haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly) and the serine protease EspPα are putative virulence factors of EHEC. We investigated the interplay between these secreted factorsThe haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly) and the serine protease EspPα are putative virulence factors of EHEC. We investigated the interplay between these secreted factors and demonstrate that EspPα cleaves the 107 kDa large EHEC-Hly. Degradation was observed when purified EspPα was added to a growing culture of an EHEC-Hly-expressing strain, with isolated proteins and with coexpressing strains, and was independent of the EHEC serotype. EHEC-Hly breakdown occurred as a multistage process with the formation of characteristic fragments with relative molecular masses of ~82 kDa and/or ~84 kDa and ~34 kDa. The initial cleavage occurred in the N-terminal hydrophobic domain of EHEC-Hly between Leu(235) and Ser(236) and abolished its haemolytic activity. In a cellular infection system, the cytolytic potential of EHEC-Hly-secreting recombinant strains was abolished when EspPα was coexpressed. EHEC in contact with human intestinal epithelial cells simultaneously upregulated their EHEC-Hly and EspP indicating that both molecules might interact under physiological conditions. We propose the concept of bacterial effector molecule interference (BEMI), reflecting the concerted interplay of virulence factors. Interference between effector molecules might be an additional way to regulate virulence functions and increases the complexity of monomolecular phenotypes.

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

  Authors: Alexander Mellmann, Dag Harmsen, Craig A Cummings, Emily B Zentz, Shana R Leopold, Alain Rico, Karola Prior, Rafael Szczepanowski, Yongmei Ji, Wenlan Zhang, Stephen F McLaughlin, John K Henkhaus, Benjamin Leopold, Martina Bielaszewska, Rita Prager, Pius M Brzoska, Richard L Moore, Simone Guenther, Jonathan M Rothberg, Helge Karch

  PloS one. 01/2011; 6(7):e22751.

  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 deathsAn 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.

• Distribution and phylogeny of immunoglobulin-binding protein G in Shiga toxin-producing Escherichia coli and its association with adherence phenotypes.

  Authors: Viktor Merkel, Barbara Ohder, Martina Bielaszewska, Wenlan Zhang, Angelika Fruth, Christian Menge, Erika Borrmann, Barbara Middendorf, Johannes Müthing, Helge Karch, Alexander Mellmann

  Infection and immunity. 08/2010; 78(8):3625-36.

  eibG in Shiga toxin-producing Escherichia coli (STEC) O91 encodes a protein (EibG) which binds human immunoglobulins G and A and contributes to bacterial chain-like adherence to human epithelialeibG in Shiga toxin-producing Escherichia coli (STEC) O91 encodes a protein (EibG) which binds human immunoglobulins G and A and contributes to bacterial chain-like adherence to human epithelial cells. We investigated the prevalence of eibG among STEC, the phylogeny of eibG, and eibG allelic variations and their impact on the adherence phenotype. eibG was found in 15.0% of 240 eae-negative STEC strains but in none of 157 eae-positive STEC strains. The 36 eibG-positive STEC strains belonged to 14 serotypes and to eight multilocus sequence types (STs), with serotype O91:H14/H(-) and ST33 being the most common. Sequences of the complete eibG gene (1,527 bp in size) from eibG-positive STEC resulted in 21 different alleles with 88.11% to 100% identity to the previously reported eibG sequence; they clustered into three eibG subtypes (eibG-alpha, eibG-beta, and eibG-gamma). Strains expressing EibG-alpha and EibG-beta displayed a mostly typical chain-like adherence pattern (CLAP), with formation of long chains on both human and bovine intestinal epithelial cells, whereas strains with EibG-gamma adhered in short chains, a pattern we termed atypical CLAP. The same adherence phenotypes were displayed by E. coli BL21(DE3) clones containing the respective eibG-alpha, eibG-beta, and eibG-gamma subtypes. We propose two possible evolutionary scenarios for eibG in STEC: a clonal development of eibG in strains with the same phylogenetic background or horizontal transfer of eibG between phylogenetically unrelated STEC strains.

• Application of thin-layer chromatography/infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry to structural analysis of bacteria-binding glycosphingolipids selected by affinity detection.

  Authors: Anne Müsken, Jamal Souady, Klaus Dreisewerd, Wenlan Zhang, Ute Distler, Jasna Peter-Katalinić, Halina Miller-Podraza, Helge Karch, Johannes Müthing

  Rapid communications in mass spectrometry : RCM. 03/2010; 24(7):1032-8.

  Glycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin-layer chromatography (TLC) overlay assay represents one of the mostGlycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin-layer chromatography (TLC) overlay assay represents one of the most powerful approaches for the detection of GSL receptors of microorganisms. Here we report on the direct structural characterization of microbial GSL receptors by employment of the TLC overlay assay combined with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). The procedure includes TLC separation of GSL mixtures, overlay of the chromatogram with GSL-specific bacteria, detection of bound microbes with primary antibodies against bacterial surface proteins and appropriate alkaline phosphatase labeled secondary antibodies, and in situ MS analysis of bacteria-specific GSL receptors. The combined method works on microgram scale of GSL mixtures and is advantageous in that it omits laborious and time-consuming GSL extraction from the silica gel layer. This technique was successfully applied to the compositional analysis of globo-series neutral GSLs recognized by P-fimbriated Escherichia coli bacteria, which were used as model microorganisms for infection of the human urinary tract. Thus, direct TLC/IR-MALDI-o-TOF-MS adds a novel facet to this fast and sensitive method offering a wide range of applications for the investigation of carbohydrate-specific pathogens involved in human infectious diseases.

• Genetic structure and distribution of the colibactin genomic island among Enterobacteriaceae.

  Authors: Johannes Putze, Claire Hennequin, Jean-Philippe Nougayrède, Wenlan Zhang, Stefan Homburg, Helge Karch, Marie-Agnés Bringer, Corinne Fayolle, Elisabeth Carniel, Wolfgang Rabsch, Tobias A Oelschlaeger, Eric Oswald, Christiane Forestier, Jörg Hacker, Ulrich Dobrindt

  Infection and immunity. 09/2009;

  A genomic island encoding the biosynthesis and secretion pathway of putative hybrid non-ribosomal peptide-polyketide colibactin has been recently described in Escherichia coli. Colibactin acts as aA genomic island encoding the biosynthesis and secretion pathway of putative hybrid non-ribosomal peptide-polyketide colibactin has been recently described in Escherichia coli. Colibactin acts as a cyclomodulin and blocks the eukaryotic cell cycle. The origin and prevalence of the colibactin island among enterobacteria are unknown. We therefore screened 1565 isolates of different genera and species related to the Enterobacteriaceae by PCR for the presence of this DNA element. The island was detected not only in E.coli, but also in Klebsiella pneumoniae, Enterobacter aerogenes and Citrobacter koseri isolates. It was highly conserved among these species and was always associated with the yersiniabactin determinant. Structural variations between individual strains were only observed in an intergenic region containing variable numbers of tandem repeats. In E.coli, the colibactin island was usually restricted to isolates of phylogenetic group B2 and inserted at the asnW tRNA locus. Interestingly, in K.pneumoniae, E.aerogenes, C.koseri and three E.coli strains of phylogenetic group B1, the functional colibactin determinant was associated with a genetic element similar to the integrative and conjugative elements ICEEc1 and ICEKp1 and to several enterobacterial plasmids. Different asn-tRNA genes served as chromosomal insertion sites of the ICE-associated colibactin determinant: asnU in the three E.coli strains of ECOR group B1, and different asn-tRNA loci in K.pneumoniae. The detection of the colibactin genes associated with an ICE-like element in several enterobacteria provides new insights into the spread of this gene cluster and its putative mode of transfer. Our results shed light on the mechanisms of genetic exchange between Enterobacteriaceae.

• High sensitivity detection of the glial fibrillary acidic protein as indicator for TSE risk material in meat products using an immuno-PCR.

  Authors: Thorsten Kuczius, Karsten Becker, Helge Karch, Wenlan Zhang

  Molecular nutrition & food research. 09/2009;

  The emergence of prion diseases in cattle during the bovine spongiform encephalopathy (BSE) epidemic and the transmission to humans causing variant Creutzfeldt-Jakob disease by consume ofThe emergence of prion diseases in cattle during the bovine spongiform encephalopathy (BSE) epidemic and the transmission to humans causing variant Creutzfeldt-Jakob disease by consume of BSE-contaminated meat has focused attention on the use of tissues from the central nervous system (CNS) in food. To avoid food contamination, it is regulated by law that specified risk material has to be removed from food chains. Detection of well-expressed CNS indicator proteins such as the glial fibrillary acidic protein (GFAP) assumes an important role in detection of food contamination; however, the sensitivity of detection performed on basis of ELISAs is limited. Consequently, there is an urgent need for high-sensitivity detection methods. We describe the development of an immuno-PCR assay with enhancement of the immunoreaction by amplification of a DNA fragment linked to the antigen-antibody complex. This immuno-PCR assay shows enhanced sensitivity in GFAP detection of at least two orders of magnitude compared to the ELISA technique carried out under identical conditions. The immuno-PCR proves to be a suitable tool for the high-sensitivity detection of marker proteins in food imported by food chain processes. It is applicable for use by food and veterinary facilities, and institutes involved in food monitoring.

• Shiga Toxin, Cytolethal Distending Toxin and Hemolysin Repertoirs in Clinical Escherichia coli O91 Isolates.

  Authors: Martina Bielaszewska, Franziska Stoewe, Angelika Fruth, Wenlan Zhang, Rita Prager, Jens Brockmeyer, Alexander Mellmann, Helge Karch, Alexander W Friedrich

  Journal of clinical microbiology. 05/2009;

  Shiga toxin (Stx)-producing Escherichia coli (STEC) of serogroup O91 are the most common human pathogenic eae-negative STEC. To facilitate diagnosis and subtyping of these pathogens, we genotypicallyShiga toxin (Stx)-producing Escherichia coli (STEC) of serogroup O91 are the most common human pathogenic eae-negative STEC. To facilitate diagnosis and subtyping of these pathogens, we genotypically and phenotypically characterized 100 clinical STEC O91 isolates. Motile strains expressed flagellar antigens H8 (n = 1), H10 (n = 2), H14 (n = 52) or H21 (n = 20), or were H nontypeable (Hnt; n = 10); 15 strains were nonmotile. All nonmotile and Hnt strains possessed the flagellin subunit-encoding gene fliCH14. Most STEC O91 possesed EHEC-hlyA and expressed an enterohemolytic phenotype. Among seven stx alleles identified, stx2d-activatable encoding mucus- and elastase-activatable Stx2d was solely present in STEC O91:H21, whereas most strains in the other serotypes possessed stx1. Moreover, only STEC O91:H21 possessed the cdt-V cluster encoding cytolethal distending toxin V; the toxin was regularly expressed and was lethal to human microvascular endothelial cells. Infection with STEC O91:H21 was associated with hemolytic-uremic syndrome (P = 0.0015), whereas strains of the other serotypes mostly originated in patients with nonbloody diarrhea. We conclude that STEC O91 clinical isolates belong to at least four lineages that differ by H antigens/fliC types, stx genotypes and non-stx putative virulence factors, with accumulation of virulence determinants in the O91:H21 lineage. Isolation of STEC O91 from patients stools on enterohemolysin agar and their rapid initial subtyping using fliC genotyping facilitates identification of these emerging pathogens in clinical and epidemiological studies and enables prediction of the risk of a severe clinical outcome.

• Phenotypic and Genotypic Traits of Shiga Toxin-Negative E. coli O157:H7/H(-) Bovine and Porcine Strains.

  Authors: Roger Stephan, Wenlan Zhang, Martina Bielaszewska, Alexander Mellmann, Helge Karch

  Foodborne pathogens and disease. 04/2009; 6(2):235-43.

  Abstract Enterohemorrhagic E. coli (EHEC) O157:H7/H(-) (nonmotile) exist as Shiga toxin gene (stx)-positive and stx-negative variants in patients and the environment. We analyzed molecularAbstract Enterohemorrhagic E. coli (EHEC) O157:H7/H(-) (nonmotile) exist as Shiga toxin gene (stx)-positive and stx-negative variants in patients and the environment. We analyzed molecular characteristics, phenotypes, and the phylogenetic background of three stx-negative E. coli O157:H7/H(-) strains isolated from cattle and a pig and compared them with those of human EHEC and stx-negative E. coli O157:H7/H(-). All three animal strains contained fliCH7 and two contained eae. One eae-positive strain (O157:H(-)) was sorbitol-fermenting (SF) and the other (O157:H7) was non-sorbitol-fermenting (nSF). These two strains shared a spectrum of non-stx putative virulence and fitness genes with human nSF and SF EHEC and stx-negative E. coli O157:H7/H(-) and belonged, similar to the vast majority of human isolates, to sequence type (ST) 11 in multilocus sequence typing. In contrast, the eae-negative O157:H7 animal isolate, which was SF, differed in spectrum of virulence genes and also differed phylogenetically (ST717) from the two eae-positive strains and the human EHEC and stx-negative E. coli O157:H7/H(-). In contrast to efforts with human stx-negative E. coli O157:H(-), attempts to transduce the two stx-negative/eae-positive animal O157:H7/H(-) strains with stx(2)-encoding phages from human SF and nSF EHEC O157:H7/H(-) failed, despite the animal strains having intact loci where such phages integrate in human EHEC O157 (wrbA and yecE). The role of animal stx-negative/eae-positive and stx-negative/eae-negative E. coli O157:H7/H(-) in their natural source and in human infections needs further investigation.

• Detection and Characterization of Fimbrial sfp Cluster in Enterohemorrhagic Escherichia coli O165:H25/NM from Humans and Cattle.

  Authors: Martina Bielaszewska, Rita Prager, Liz Vandivinit, Anne Müsken, Alexander Mellmann, Nicholas J Holt, Phillip I Tarr, Helge Karch, Wenlan Zhang

  Applied and environmental microbiology. 10/2008;

  The sfp cluster encoding Sfp fimbriae which is located in the large plasmid of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157 (pSFO157) has been considered a uniqueThe sfp cluster encoding Sfp fimbriae which is located in the large plasmid of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157 (pSFO157) has been considered a unique characteristic of these organisms. We discovered, and then characterized, the sfp cluster in EHEC O165:H25/NM (nonmotile) of human and bovine origin. All seven strains investigated harbored a complete sfp cluster (sfpA, sfpH, sfpC, sfpD, sfpJ, sfpF, sfpG) of 6,838 bp with >99% nucleotide sequence homology to sfp of SF EHEC O157:NM. The sfp cluster in EHEC O165:H25/NM was located in approximately 80 (six strains) or approximately 120 (one strain) kb plasmids which differed in structure, virulence genes and sfp flanks from pSFO157. All O165:H25/NM strains belonged to the same multilocus sequence type (ST119), and were only distantly phylogenetically related to SF EHEC O157:NM (ST11). The highly conserved sfp cluster in different clonal backgrounds suggests that this segment was acquired independently by EHEC O165:H25 and SF EHEC O157:NM. Its presence in an additional EHEC serotype extends the diagnostic utility of PCR targeting sfpA as an easy and efficient approach to seek EHEC in patients' stools. The reasons for convergence of pathogenic EHEC on a suite of virulence loci remain unknown.

• New immuno-PCR assay for detection of low concentrations of shiga toxin 2 and its variants.

  Authors: Wenlan Zhang, Martina Bielaszewska, Matthias Pulz, Karsten Becker, Alexander W Friedrich, Helge Karch, Thorsten Kuczius

  Journal of clinical microbiology. 05/2008; 46(4):1292-7.

  Shiga toxin (Stx)-producing Escherichia coli (STEC) strains secrete toxins that are major virulence factors and diagnostic targets, but some STEC strains secrete Stx in amounts that cannot beShiga toxin (Stx)-producing Escherichia coli (STEC) strains secrete toxins that are major virulence factors and diagnostic targets, but some STEC strains secrete Stx in amounts that cannot be detected using conventional cell cytotoxicity or immunological assays. Therefore, there is an urgent need for more-sensitive Stx detection methods. We describe the development of an assay that can detect low concentrations of Stx2 and its variants. An immuno-PCR Stx2 assay was developed based on an enzyme immunoassay (EIA) combining antibody capture and DNA amplification to increase the signal. The immuno-PCR assay detected 10 pg/ml of purified Stx2, compared to 1 ng/ml Stx2 detected by commercial EIA. Consequently, immuno-PCR detected Stx2 and its variants in STEC strains that produce the toxins at levels that are nondetectable by using the EIA, as well as the Stx2 in EIA-negative enriched stool cultures from patients. Our data demonstrate that the immuno-PCR developed here is a highly sensitive and specific method for the detection of trace amounts of Stx2 and Stx2 variants. It is therefore suitable for use by clinical microbiological laboratories to improve the toxin detection in clinical samples.

• Anaerobic conditions promote expression of Sfp fimbriae and adherence of sorbitol-fermenting enterohemorrhagic Escherichia coli O157:NM to human intestinal epithelial cells.

  Authors: Anne Müsken, Martina Bielaszewska, Lilo Greune, Christian H Schweppe, Johannes Müthing, Herbert Schmidt, M Alexander Schmidt, Helge Karch, Wenlan Zhang

  Applied and environmental microbiology. 02/2008; 74(4):1087-93.

  The sfp gene cluster, unique to sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM strains, encodes fimbriae that mediate mannose-resistant hemagglutination in laboratory E.The sfp gene cluster, unique to sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM strains, encodes fimbriae that mediate mannose-resistant hemagglutination in laboratory E. coli strains but are not expressed in wild-type SF EHEC O157:NM strains under standard laboratory conditions. We investigated whether Sfp fimbriae are expressed under conditions that mimic the intestinal environment and whether they contribute to the adherence of SF EHEC O157:NM strains to human intestinal epithelial cells. The transcription of sfpA (encoding the major fimbrial subunit) was upregulated in all strains investigated, and all expressed SfpA and possessed fimbriae that reacted with an anti-SfpA antibody when the strains were grown on solid media under anaerobic conditions. Sfp expression was absent under aerobic conditions and in liquid media. Sfp upregulation under anaerobic conditions was significantly higher on blood agar and a medium simulating the colonic environment than on a medium simulating the ileal environment (P < 0.05). The induction of Sfp fimbriae in SF E. coli O157:NM strains correlates with increased adherence to Caco-2 and HCT-8 cells. Our data indicate that the expression of Sfp fimbriae in SF E. coli O157:NM strains is induced under conditions resembling those of the natural site of infection and that Sfp fimbriae may contribute to the adherence of the organisms to human intestinal epithelium.

• Prevalence, virulence profiles, and clinical significance of Shiga toxin-negative variants of enterohemorrhagic Escherichia coli O157 infection in humans.

  Authors: Alexander W Friedrich, Wenlan Zhang, Martina Bielaszewska, Alexander Mellmann, Robin Köck, Angelika Fruth, Helmut Tschäpe, Helge Karch

  Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 07/2007; 45(1):39-45.

  BACKGROUND: Escherichia coli O157, of the H7 clone, exists in humans and in the environment as Shiga toxin (Stx)-positive and Stx-negative variants. Stx production by infecting organisms isBACKGROUND: Escherichia coli O157, of the H7 clone, exists in humans and in the environment as Shiga toxin (Stx)-positive and Stx-negative variants. Stx production by infecting organisms is considered to be a critical requirement for the development of hemolytic uremic syndrome (HUS), which occurs in approximately 15% of E. coli O157-infected patients. It is unknown if loss of the stx gene during the early stage of an enterohemorrhagic E. coli infection prevents HUS, or if absence of the stx gene from E. coli O157 reduces or ablates virulence. METHODS: We determined the frequency of stx-positive and stx-negative E. coli O157 isolates in stool samples obtained from patients who experienced sporadic cases of diarrhea or HUS, as well as the frequency in samples obtained during outbreaks, and investigated the clinical course of the disease. RESULTS: Among E. coli O157 isolates obtained from samples related to sporadic cases of diarrhea, stx-negative strains accounted for 4%. The proportion of stx-negative strains was significantly higher among sorbitol-fermenting, nonmotile E. coli O157 isolates (12.7%) than among non-sorbitol-fermenting E. coli O157:H7 or nonmotile isolates (0.8%; P<.001). stx-Negative sorbitol-fermenting E. coli O157 isolates were also observed in samples related to 3 HUS outbreaks and 1 outbreak of diarrhea caused by sorbitol-fermenting, nonmotile enterohemorrhagic E. coli O157; additionally, they were the only pathogens that were isolated in 2 other outbreaks of diarrhea without HUS. CONCLUSIONS: Strains of stx-negative E. coli O157 isolated from stool samples of patients are either inherently stx-negative strains that cause mostly uncomplicated diarrhea, or strains that descended from enterohemorrhagic E. coli O157 by the loss of the stx gene during infection; the latter strains may still cause severe disease.

• Shiga toxin gene loss and transfer in vitro and in vivo during enterohemorrhagic Escherichia coli O26 infection in humans.

  Authors: Martina Bielaszewska, Rita Prager, Robin Köck, Alexander Mellmann, Wenlan Zhang, Helmut Tschäpe, Phillip I Tarr, Helge Karch

  Applied and environmental microbiology. 05/2007; 73(10):3144-50.

  Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHECEscherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx2 loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.

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

  Authors: Thomas Aldick, Martina Bielaszewska, Wenlan Zhang, Jens Brockmeyer, Herbert Schmidt, Alexander W Friedrich, Kwang S Kim, M Alexander Schmidt, Helge Karch

  Microbes and infection / Institut Pasteur. 04/2007; 9(3):282-90.

  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).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.

• Structural and functional differences between disease-associated genes of enterohaemorrhagic Escherichia coli O111.

  Authors: Wenlan Zhang, Alexander Mellmann, Anne K Sonntag, Lothar Wieler, Martina Bielaszewska, Helmut Tschäpe, Helge Karch, Alexander W Friedrich

  International journal of medical microbiology : IJMM. 02/2007; 297(1):17-26.

  We analysed 72 clinical isolates of enterohaemorrhagic Escherichia coli (EHEC) O111 from patients with diarrhoea or haemolytic uraemic syndrome (HUS) isolated during the period from 1955 to 2005 andWe analysed 72 clinical isolates of enterohaemorrhagic Escherichia coli (EHEC) O111 from patients with diarrhoea or haemolytic uraemic syndrome (HUS) isolated during the period from 1955 to 2005 and identified six motile strains (flagellar antigens 8, 10 and 11); the remaining 66 (92%) were nonmotile (NM) and could not be typed by conventional H serotyping. To improve subtyping methodologies, we determined genotypes of the flagellin-encoding fliC. Three fliC genotypes were found which were identical to those of motile EHEC O111 with H antigens 8, 10 and 11 and designated fliC(H8), fliC(H10) and fliC(H11). The IS629 insertion element was present, identically located, in six epidemiologically unrelated isolates with fliC(H8). The prevalence of the fliC genotypes in the 72 EHEC O111 strains were fliC(H8) (89%), fliC(H10) (7%) and fliC(H11) (4%). Within these fliC genotypes, a high degree of homogeneity for the presence of disease-associated genes was found. The adhesins-encoding genes eae and efa-1 were present in all strains with fliC(H8) and fliC(H11), but absent from strains with fliC(H10). The latter strains have not been reported previously. Strains with fliC(H10) and fliC(H11), but not those with fliC(H8), retained intact cadA and cadC loci and decarboxylated lysine. Three different stx genotypes including stx(1), stx(2) and stx(1)/stx(2) were determined among the 72 EHEC O111. We observed a significant increase over time in the frequency of strains harbouring both stx(1) and stx(2). The presence of stx(2) both alone and in combination with stx(1) was significantly (chi(2)=23.16, P<0.00001, CI(95) [2.29; 9.76]) associated with HUS. Therefore, the emergence of EHEC O111 should be monitored carefully. We conclude that EHEC O111 strains can be differentiated using specific loci required for motility, adherence, Stx production, and lysine decarboxylation. The divergence within EHEC O111 makes it possible to subtype these emerging pathogens in the laboratory thereby providing a basis for further investigations into their ecological niches and survival capabilities.