Rita Prager

Publications of Rita Prager

• Clonal Dissemination of Salmonella enterica Serovar Infantis in Germany.

  Authors: Elisabeth Hauser, Erhard Tietze, Reiner Helmuth, Ernst Junker, Rita Prager, Andreas Schroeter, Wolfgang Rabsch, Angelika Fruth, Anne Toboldt, Burkhard Malorny

  Foodborne pathogens and disease. 03/2012; 9(4):352-60.

  Abstract Salmonella enterica serovar Infantis (Salmonella Infantis) is consistently isolated from broiler chickens, pigs, and humans worldwide. This study investigated 93 epidemiologically unrelatedAbstract Salmonella enterica serovar Infantis (Salmonella Infantis) is consistently isolated from broiler chickens, pigs, and humans worldwide. This study investigated 93 epidemiologically unrelated Salmonella Infantis strains isolated in Germany between 2005 and 2008 in respect to their transmission along the food chain. Various phenotypic and genotypic methods were applied, and the pathogenicity and resistance gene repertoire was determined. Phenotypically, 66% of the strains were susceptible to all 17 antimicrobials tested, while the others were almost all multidrug-resistant (two or more antimicrobial resistances), with different resistance profiles and preferentially isolated from broiler chickens. A number of phage types (PTs) were shared by strains from pigs, broiler chickens, and humans (predominated by PT 29). One, PT 1, was only detected in strains from pigs/pork and humans. Pulsed-field gel electrophoresis (PFGE) subdivided strains in seven different clusters, named A-G, consisting of 35 various XbaI profiles with coefficient of similarity values of 0.73-0.97. The majority of XbaI profiles were assigned to clusters A and C, and two predominant XbaI profiles were common in strains isolated from all sources investigated. Multi-locus sequence typing (MLST) analysis of selected strains representing the seven PFGE clusters revealed that they all belonged to ST32. The pathogenicity gene repertoire of 37 representative Salmonella Infantis strains analyzed by microarray was also identical. The resistance gene repertoire correlated perfectly with the phenotypic antimicrobial resistance profiles, and multidrug-resistant strains were associated with class 1 integrons. Overall, this study showed that two major closely related genotypes of Salmonella Infantis can transmit in Germany to humans through contaminated broiler meat or pork, and consequently presents a hazard for human health.

• Identifying risk factors for shiga toxin-producing Escherichia coli by payment information.

  Authors: Hendrik Wilking, Udo Götsch, Helma Meier, Detlef Thiele, Mona Askar, Manuel Dehnert, Christina Frank, Angelika Fruth, Gérard Krause, Rita Prager, Klaus Stark, Boris Böddinghaus, Oswald Bellinger, René Gottschalk

  Emerging infectious diseases. 01/2012; 18(1):169-70.

• EHEC/EAEC O104:H4 strain linked with the 2011 German outbreak of haemolytic uremic syndrome enters into the viable but non-culturable state in response to various stresses and resuscitates upon stress relief.

  Authors: Philipp Aurass, Rita Prager, Antje Flieger

  Environmental microbiology. 09/2011; 13(12):3139-48.

  Various non-spore forming bacteria, including Escherichia coli, enter a dormant-like state, the viable but non-culturable (VBNC) state, characterized by the presence of viable cells but the inabilityVarious non-spore forming bacteria, including Escherichia coli, enter a dormant-like state, the viable but non-culturable (VBNC) state, characterized by the presence of viable cells but the inability to grow on routine laboratory media. Upon resuscitation, these VBNC cells recover both culturability and pathogenicity. In 2011, a large outbreak involving more than 3000 cases of bloody diarrhoea and haemolytic uremic syndrome was caused by an E. coli O104:H4 strain expressing genes characteristic of both enterohaemorrhagic (EHEC) and enteroaggregative E. coli (EAEC). The ability of the outbreak strain to enter the VBNC state may have complicated its detection in the suspected sources. In this paper, we investigated the ability of the outbreak strain to enter and subsequently recover from the VBNC state. We found that in a nutrient-poor micro-environment, various stresses such as toxic concentrations of copper ions or certain types of tap water are able to render the bacteria unculturable within a few days. Without copper ion stress, the majority of cells remained culturable for at least 40 days. Incubation with the stressors at 23°C compared with 4°C hastened this observed loss of culturability. The integrity of a considerable fraction of copper ion- and tap water 1-stressed bacteria was demonstrated by live/dead staining and microscopy. Relieving stress by copper-ion chelation facilitated resuscitation of these bacteria while preserving their fitness, major virulence gene markers (stx2, aggR, aggA genes) and specific phenotypes (ESBL resistance, autoaggregation typical for EAEC strains).

• Diversity of Salmonella enterica serovar Derby isolated from pig, pork and humans in Germany.

  Authors: Elisabeth Hauser, Franka Hebner, Erhard Tietze, Reiner Helmuth, Ernst Junker, Rita Prager, Andreas Schroeter, Wolfgang Rabsch, Angelika Fruth, Burkhard Malorny

  International journal of food microbiology. 08/2011; 151(2):141-9.

  Salmonella enterica serovar Derby (S. Derby) is one of the most prevalent serovars in pigs in Europe and in the U.S. and ranks among the 10 most frequently isolated serovars in humans. Therefore, aSalmonella enterica serovar Derby (S. Derby) is one of the most prevalent serovars in pigs in Europe and in the U.S. and ranks among the 10 most frequently isolated serovars in humans. Therefore, a set of 82 epidemiologically unrelated S. Derby strains isolated between 2006 and 2008 from pigs, pork and humans in Germany was selected and investigated in respect to the transmission of clonal groups of the serovar along the food chain. Various phenotypic and genotypic methods were applied and the pathogenicity and resistance gene repertoire was determined. Phenotypically 72% of the strains were susceptible to all 17 antimicrobials tested while the others were monoresistant to tetracycline or multi-resistant with different resistance profiles. Four major clonal groups were identified based on PFGE, sequence data of the virulence genes sopA, sopB and sopD, VNTR-locus STTR5 and MLST revealing also the new sequence type ST774. Thirty different PFGE profiles were detected resulting in four clusters representing the four groups. The pathogenicity gene repertoire of 32 representative S. Derby strains analyzed by microarray showed six types with differences in the Salmonella pathogenicity islands, pathogenicity genes on smaller islets or prophages and fimbriae coding genes. The pathogenicity gene repertoire of the predominant types PAT DE1 and DE2 were most similar to the ones of S. Paratyphi B (dT+, O5-) and to a minor degree to S. Infantis and S. Virchow PATs. Overall this study showed that in Germany currently one major S. Derby clone is frequently isolated from pigs and humans. Contaminated pork was identified as one vehicle and consequently is a risk for human health. To prevent this serovar from entering the food chain, control measurements should be applied at the farm level.

• Epidemic profile of Shiga-toxin-producing Escherichia coli O104:H4 outbreak in Germany.

  Authors: Christina Frank, Dirk Werber, Jakob P Cramer, Mona Askar, Mirko Faber, Matthias an der Heiden, Helen Bernard, Angelika Fruth, Rita Prager, Anke Spode [......] Alexander Zoufaly, Sabine Jordan, Markus J Kemper, Per Follin, Luise Müller, Lisa A King, Bettina Rosner, Udo Buchholz, Klaus Stark, Gérard Krause

  The New England journal of medicine. 06/2011; 365(19):1771-80.

  We describe an outbreak of gastroenteritis and the hemolytic-uremic syndrome caused by Shiga-toxin-producing Escherichia coli in Germany in May, June, and July, 2011. The consumption of sprouts wasWe describe an outbreak of gastroenteritis and the hemolytic-uremic syndrome caused by Shiga-toxin-producing Escherichia coli in Germany in May, June, and July, 2011. The consumption of sprouts was identified as the most likely vehicle of infection. We analyzed data from reports in Germany of Shiga-toxin-producing E. coli gastroenteritis and the hemolytic-uremic syndrome and clinical information on patients presenting to Hamburg University Medical Center (HUMC). An outbreak case was defined as a reported case of the hemolytic-uremic syndrome or of gastroenteritis in a patient infected by Shiga-toxin-producing E. coli, serogroup O104 or serogroup unknown, with an onset of disease during the period from May 1 through July 4, 2011, in Germany. A total of 3816 cases (including 54 deaths) were reported in Germany, 845 of which (22%) involved the hemolytic-uremic syndrome. The outbreak was centered in northern Germany and peaked around May 21 to 22. Most of the patients in whom the hemolytic-uremic syndrome developed were adults (88%; median age, 42 years), and women were overrepresented (68%). The estimated median incubation period was 8 days, with a median of 5 days from the onset of diarrhea to the development of the hemolytic-uremic syndrome. Among 59 patients prospectively followed at HUMC, the hemolytic-uremic syndrome developed in 12 (20%), with no significant differences according to sex or reported initial symptoms and signs. The outbreak strain was typed as an enteroaggregative Shiga-toxin-producing E. coli O104:H4, producing extended-spectrum beta-lactamase. In this outbreak, caused by an unusual E. coli strain, cases of the hemolytic-uremic syndrome occurred predominantly in adults, with a preponderance of cases occurring in women. The hemolytic-uremic syndrome developed in more than 20% of the identified cases.

• Babies and bearded dragons: sudden increase in reptile-associated Salmonella enterica serovar Tennessee infections, Germany 2008.

  Authors: Bettina Weiss, Wolfgang Rabsch, Rita Prager, Erhard Tietze, Judith Koch, Frank Mutschmann, Peter Roggentin, Christina Frank

  Vector borne and zoonotic diseases (Larchmont, N.Y.). 05/2011; 11(9):1299-301.

  In 2008 a marked increase in Salmonella enterica serovar Tennessee infections in infants occurred in Germany. In March and April 2008, eight cases were notified compared to a median of 0-1 cases inIn 2008 a marked increase in Salmonella enterica serovar Tennessee infections in infants occurred in Germany. In March and April 2008, eight cases were notified compared to a median of 0-1 cases in 2001-2006. We carried out an investigation including a case-control study to identify the source of infection. A patient was a child < 3 years of age with Salmonella Tennessee isolated from stool from September 1, 2007, through December 31, 2008, identified through the national surveillance system. A control was a child with a notified rotavirus infection in the matching district, frequency matched by age group. We conducted telephone interviews on feeding, herbal infusions, and animal contact. Matched odds ratios (mOR) were calculated using exact conditional logistic regression. For Salmonella Tennessee isolates, pulsed-field gel electrophoresis and multiple-locus variable number tandem repeat analysis were performed. Further cloacal swab samples of reptiles kept in case households were investigated. We identified 18 cases < 3 years. Ten children were male; median age was 3 months (1-32 months). In 8 of 16 case households reptiles were kept. Direct contact between child and reptile was denied. Other forms of reptile contact were reported in four of the remaining eight households. Ten case- and 21 control-patients were included in the study. Only keeping of a reptile and "any reptile contact" were associated with Salmonella Tennessee infection (mOR 29.0; 95% CI 3.1 ± ∞ and mOR 119.5; 95% CI 11.7 - ∞). Identical Salmonella Tennessee strains of child and reptile kept in the same household could be shown in 2 cases. Reptiles were the apparent source of Salmonella Tennessee infection in these infants. Indirect contact between infants and reptiles seems to be sufficient to cause infection and should therefore be avoided.

• Comparative analysis of virulence genes, genetic diversity, and phylogeny of Shiga toxin 2g and heat-stable enterotoxin STIa encoding Escherichia coli isolates from humans, animals, and environmental sources.

  Authors: Rita Prager, Angelika Fruth, Ulrich Busch, Erhard Tietze

  International journal of medical microbiology : IJMM. 03/2011; 301(3):181-91.

  An analysis for stx(2) variants among the 2010 human stx(2)-positive Shiga toxin-producing Escherichia coli (STEC) strains from Germany collected at the National Reference Centre 1999-2008 revealedAn analysis for stx(2) variants among the 2010 human stx(2)-positive Shiga toxin-producing Escherichia coli (STEC) strains from Germany collected at the National Reference Centre 1999-2008 revealed 0.6% to possess the recently described stx(2g) gene. Sequencing of the whole stx(2g) operons showed new alleles and pseudogenes. The further molecular, phenotypic, and phylogenetic comparison of 12 human stx(2g)-harbouring isolates with 12 stx(2g)-harbouring isolates from animals or environmental sources demonstrated that both groups are closely related, indicating the human infections as a potential zoonotic disease. Although originating from various different sources, the stx(2g)-containing strains belong to only 3 phylogenetic lineages, represented by 4 serovars belonging to 4 sequence types. In view of the huge diversity among other STEC, this suggests the emergence of the stx(2g) variant as a rather recent microevolutionary event. Interestingly, in the strains under investigation, Stx2g was not expressed. However, all of them contained the estIa gene which typically is associated with enterotoxin-producing E. coli and did express STIa. By this combination of virulence genes of different pathotypes of intestinal pathogenic E. coli, these strains represent a new, intermediate pathotype and emerging pathogens. Given a rising number of intermediate pathotypes becoming described among E. coli, a wider range of virulence markers should be included in the regular pathotype diagnostics.

• 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.

• 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.

• Large listeriosis outbreak linked to cheese made from pasteurized milk, Germany, 2006-2007.

  Authors: Judith Koch, Regine Dworak, Rita Prager, Biserka Becker, Stefan Brockmann, Amal Wicke, Heidi Wichmann-Schauer, Herbert Hof, Dirk Werber, Klaus Stark

  Foodborne pathogens and disease. 12/2010; 7(12):1581-4.

  A commercial cheese (acid curd) made from pasteurized milk caused a large listeriosis outbreak in Germany from October 2006 through February 2007. The Listeria monocytogenes outbreak strain wasA commercial cheese (acid curd) made from pasteurized milk caused a large listeriosis outbreak in Germany from October 2006 through February 2007. The Listeria monocytogenes outbreak strain was identified in humans and in cheese samples from a patient's home and from the production plant. During the outbreak period, 189 patients were affected, which was 97% above the mean case number for the respective time period of the years 2002 to 2005. Of patients with available detailed information on cheese consumption (n=47), 70% reported to have consumed the incriminated cheese product. Recent European food safety alerts due to Listeria-contaminated cheeses more often concerned products made from pasteurized or heat-treated milk than from raw milk. The findings should be considered in prevention guidelines addressing vulnerable populations.

• Pork contaminated with Salmonella enterica serovar 4,[5],12:i:-, an emerging health risk for humans.

  Authors: Elisabeth Hauser, Erhard Tietze, Reiner Helmuth, Ernst Junker, Kathrin Blank, Rita Prager, Wolfgang Rabsch, Bernd Appel, Angelika Fruth, Burkhard Malorny

  Applied and environmental microbiology. 07/2010; 76(14):4601-10.

  Salmonella enterica subsp. enterica serovar 4,[5],12:i:- is a monophasic variant of S. enterica serovar Typhimurium (antigenic formula 4,[5],12:i:1,2). Worldwide, especially in several EuropeanSalmonella enterica subsp. enterica serovar 4,[5],12:i:- is a monophasic variant of S. enterica serovar Typhimurium (antigenic formula 4,[5],12:i:1,2). Worldwide, especially in several European countries and the United States, it has been reported among the 10 most frequently isolated serovars in pigs and humans. In the study reported here, 148 strains of the monophasic serovar isolated from pigs, pork, and humans in 2006 and 2007 in Germany were characterized by various phenotypic and genotypic methods. This characterization was done in order to investigate their clonality, the prevalence of identical subtypes in pigs, pork, and humans, and the genetic relatedness to other S. enterica serovar Typhimurium subtypes in respect to the pathogenic and resistance gene repertoire. Two major clonal lineages of the monophasic serovar were detected which can be differentiated by their phage types and pulsed-field gel electrophoresis (PFGE) profiles. Seventy percent of the strains tested belonged to definite phage type DT193, and those strains were mainly assigned to PFGE cluster B. Nineteen percent of the strains were typed to phage type DT120 and of these 86% belonged to PFGE cluster A. Sixty-five percent of the isolates of both lineages carried core multiresistance to ampicillin, streptomycin, tetracycline, and sulfamethoxazole encoded by the genes bla(TEM1-like), strA-strB, tet(B), and sul2. No correlation to the source of isolation was observed in either lineage. Microarray analysis of 61 S. enterica serovar 4,[5],12:i:- and 20 S. enterica serovar Typhimurium isolates tested determining the presence or absence of 102 representative pathogenicity genes in Salmonella revealed no differences except minor variations in single strains within and between the serovars, e.g., by presence of the virulence plasmid in four strains. Overall the study indicates that in Germany S. enterica serovar 4,[5],12:i:- strains isolated from pig, pork, and human are highly related, showing their transmission along the food chain. Since the pathogenicity gene repertoire is highly similar to that of S. enterica serovar Typhimurium, it is essential that interventions are introduced at the farm level in order to limit human infection.

• 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.

• Escherichia coli encoding Shiga toxin 2f as an emerging human pathogen.

  Authors: Rita Prager, Angelika Fruth, Ute Siewert, Ute Strutz, Helmut Tschäpe

  International journal of medical microbiology : IJMM. 02/2009;

  Escherichia coli harbouring the stx2f gene have been previously reported in pigeons. Here we demonstrate the presence of this allele in human diarrhoeagenic E. coli strains originally classified asEscherichia coli harbouring the stx2f gene have been previously reported in pigeons. Here we demonstrate the presence of this allele in human diarrhoeagenic E. coli strains originally classified as atypical enteropathogenic E. coli (aEPEC). Thirty-two stx2f-positive E. coli serotyped as O63:H6, O128:H2, O132:H34, O145:H34, and O178:H7 were found to belong to a large number of clonal groups due to their different MLST-, PFGE- and virulence patterns. The appearance of various stx2f-positive clonal lineages among E. coli reveals emerging clinical significance. Therefore, it seems to be prudent to include stx2f into the diagnostic scope employed for laboratory investigation of enteric infections.

• Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli.

  Authors: Rolf Reissbrodt, Walter P Hammes, Fabio Dal Bello, Rita Prager, Angelika Fruth, Klaus Hantke, Alexander Rakin, Marjanca Starcic Erjavec, Peter H Williams

  FEMS microbiology letters. 12/2008;

  Abstract During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxinAbstract During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.

• Serine protease espP subtype alpha, but not beta or gamma, of Shiga toxin-producing Escherichia coli is associated with highly pathogenic serogroups.

  Authors: Abdul Basit Khan, Asma Naim, Dorothea Orth, Katharina Grif, Mashkoor Mohsin, Rita Prager, Manfred P Dierich, Reinhard Würzner

  International journal of medical microbiology : IJMM. 12/2008;

  Besides Shiga toxins (Stx), Stx-producing Escherichia coli (STEC) harbour several other putative virulence factors, including the serine protease EspP. We have investigated 214 STEC strains fromBesides Shiga toxins (Stx), Stx-producing Escherichia coli (STEC) harbour several other putative virulence factors, including the serine protease EspP. We have investigated 214 STEC strains from Austria belonging to 61 different serotypes from humans, animals, and food for the presence of this serine protease gene and have determined the espP subtypes and their association with clinical outcome. espP was detected in 121 (57%) out of 214 strains. Sixty-five of 68 strains (96%) of non-sorbitol-fermenting (NSF) O157:H7/NM (NM, non-motile) were positive for espP, while none of 8 SF E. coli O157:NM isolates contained this gene. All 9 strains of serotype O145:NM and 17 of 21 strains (81%) of serotype O26:H11/NM were positive for espP. Nineteen STEC serogroups including O103 and O111 serogroups - considered to be highly pathogenic - were completely negative for espP. Only 5 of 12 strains isolated from patients suffering from haemolytic uraemic syndrome (HUS) were espP-positive (all serogroup NSF O157) as well as 28 of 39 strains from patients with bloody diarrhoea, 40 of 63 strains from patients with non-bloody diarrhoea, and 15 of 19 strains from asymptomatic patients. In O157:H7/NM, O26:H11/NM, and O145:NM only espP subtype alpha was found, whereas in most of the other non-O157 serogroups, subtypes beta and gamma were found. Subtype delta was not detected in our strain collection. Regarding the espP subtypes, only subtype alpha, but not beta and gamma, were found in HUS patients. Moreover, we could demonstrate that espP, and in particular subtype alpha, is associated with highly pathogenic serogroups.

• 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.

• 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.

• Shiga toxin-producing Escherichia coli infection in Germany: different risk factors for different age groups.

  Authors: Dirk Werber, Susanne C Behnke, Angelika Fruth, Roswitha Merle, Susanne Menzler, Sabine Glaser, Lothar Kreienbrock, Rita Prager, Helmut Tschäpe, Peter Roggentin, Jochen Bockemühl, Andrea Ammon

  American journal of epidemiology. 02/2007; 165(4):425-34.

  The authors conducted a matched case-control study in Germany to identify risk factors for sporadic illness associated with Shiga toxin-producing Escherichia coli (STEC) infection, regardless ofThe authors conducted a matched case-control study in Germany to identify risk factors for sporadic illness associated with Shiga toxin-producing Escherichia coli (STEC) infection, regardless of serogroup. From April 2001 through March 2003, cases were prospectively enrolled through a laboratory-based sentinel surveillance system located in 14 of the 16 German federal states. One control was identified per case, matched by age and region. Conditional logistic regression was used in the analysis, which was conducted separately for three age groups (<3 years, 3-9 years, and > or =10 years). The median age of the 202 enrolled cases was 2.5 years (range, 3 months-89 years). Hemolytic uremic syndrome developed in five patients. Non-O157 strains accounted for 85% of the isolated STEC. In children under 3 years of age, having touched a ruminant had the highest odds of disease, and raw milk was the only food identified as a risk factor. In contrast, in persons aged 10 years or older, only food items (i.e., lamb meat, raw spreadable sausages) were significantly associated with illness. In this study, risk factors were age-specific. Direct transmission through food played a lesser role in children under 3 years of age, the population at greatest risk of both acquiring STEC infection and developing hemolytic uremic syndrome.

• Cytolethal distending toxin in Escherichia coli O157:H7: spectrum of conservation, structure, and endothelial toxicity.

  Authors: Alexander W Friedrich, Shan Lu, Martina Bielaszewska, Rita Prager, Phillip Bruns, Jian-Guo Xu, Helmut Tschäpe, Helge Karch

  Journal of clinical microbiology. 06/2006; 44(5):1844-6.

  We identified the cytolethal distending toxin V (CDT-V) gene cluster in 19 (4.9%) of 391 enterohemorrhagic Escherichia coli O157:H7. cdt-V+ strains belonged to five phage types (PTs) and were mostWe identified the cytolethal distending toxin V (CDT-V) gene cluster in 19 (4.9%) of 391 enterohemorrhagic Escherichia coli O157:H7. cdt-V+ strains belonged to five phage types (PTs) and were most frequent within PTs 14 and 34. CDT-V was expressed in all but two cdt-V+ strains and was lethal to cultured endothelial cells. Subtyping schemes should include cdt-V as a marker to differentiate E. coli O157:H7 even within the same phage type.

• Chromosomal dynamism in progeny of outbreak-related sorbitol-fermenting enterohemorrhagic Escherichia coli O157:NM.

  Authors: Martina Bielaszewska, Rita Prager, Wenlan Zhang, Alexander W Friedrich, Alexander Mellmann, Helmut Tschäpe, Helge Karch

  Applied and environmental microbiology. 03/2006; 72(3):1900-9.

  Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-definedSorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-defined clusters of cases, we have observed significant variability in pulsed-field gel electrophoresis (PFGE) patterns which could indicate coinfection by different strains. An analysis of randomly selected progeny colonies of an outbreak strain after subcultivation demonstrated that they displayed either the cognate PFGE outbreak pattern or one of four additional patterns and were <89% similar. These profound alterations were associated with changes in the genomic position of one of two Shiga toxin 2-encoding genes (stx2) in the outbreak strain or with the loss of this gene. The two stx2 alleles in the outbreak strain were identical but were flanked with phage-related sequences with only 77% sequence identity. Neither of these phages produced plaques, but one lysogenized E. coli K-12 and integrated in yecE in the lysogens and the wild-type strain. The presence of two stx2 genes which correlated with increased production of Stx2 in vitro but not with the clinical outcome of infection was also found in 14 (21%) of 67 SF EHEC O157:NM isolates from sporadic cases of human disease. The variability of PFGE patterns for the progeny of a single colony must be considered when interpreting PFGE patterns in SF EHEC O157-associated outbreaks.