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Helge Karch

Publications and source records attributed to Helge Karch.

At least 37 records · Page 2Linked to original sources
PubMed2005

Cytolethal distending toxin from Shiga toxin-producing Escherichia coli O157 causes irreversible G2/M arrest, inhibition of proliferation, and death of human endothelial cells.

Recently, cytolethal distending toxin V (CDT-V), a new member of the CDT family, was identified in Shiga toxin-producing Escherichia coli (STEC) O157 and particular non-O157 serotypes. Here we investigated the biological effects of CDT-V from STEC O157:H(-) (strain 493/89) on human endothelial cells, which are believed to be major pathogenetic targets in severe STEC-mediated diseases. CDT-V caused dose-dependent G(2)/M cell cycle arrest leading to distension, inhibition of proliferation, and death in primary human umbilical vein endothelial cells (HUVEC) and two endothelial cell lines, EA.hy 926 cells (HUVEC derived) and human brain microvascular endothelial cells (HBMEC). The cell cycle effects of CDT-V were cell type specific. In HUVEC and EA.hy 926 cells, CDT-V caused a slowly developing but persistent G(2)/M block which resulted in delayed nonapoptotic cell death. In contrast, in HBMEC, CDT-V induced a rapidly evolving but transient G(2)/M block which was followed by progressive, mostly apoptotic cell death. In both HBMEC and EA.hy 926 cells, G(2)/M arrest was preceded by the early accumulation of a phosphorylated inactive form of cdc2 kinase. Significant G(2)/M arrest and inhibition of proliferation in both HUVEC and each of the endothelial cell lines were induced by 2 to 15 min of exposure to CDT-V, indicating that the effects of the toxin are irreversible. CDT-V-treated HBMEC and EA.hy 926 cells displayed fragmented nuclei and expressed phosphorylated histone protein H2AX, indicative of DNA damage followed by a DNA repair response. Our data demonstrate that CDT-V causes irreversible damage to human endothelial cells and thus may contribute to the pathogenesis of STEC-mediated diseases.

Apoptosis↗
PubMed2005

Phenotypic and molecular analysis of tellurite resistance among enterohemorrhagic Escherichia coli O157:H7 and sorbitol-fermenting O157:NM clinical isolates.

A total of 66 (98.5%) of 67 enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains had increased potassium tellurite (Te) MICs (32 to 1,024 microg/ml), grew on Te-containing media, and possessed Te resistance (ter) genes, whereas 83 (96.5%) of 86 sorbitol-fermenting (SF) EHEC O157:NM strains had Te MICs of </=4 microg/ml, did not grow on Te-containing media, and lacked ter genes. Optimal detection of SF EHEC O157:NM strains requires Te-independent strategies.

Culture Media↗
PubMed2005

Distribution of the urease gene cluster among and urease activities of enterohemorrhagic Escherichia coli O157 isolates from humans.

Enterohemorrhagic Escherichia coli (EHEC) O157 strains belong to two closely related major groups, which are differentiated by their sorbitol fermentation phenotypes. Here we studied the conservation of urease genes and their expression in sorbitol-fermenting (SF) and non-SF EHEC O157 isolates. PCR targeting ure genes (ureA, -B, -C, -D, -E, -F, and -G) demonstrated that each of these genes was present in 58 of 59 EHEC O157:H7 isolates. In contrast, none of 82 SF EHEC O157:NM (nonmotile) isolates contained any of the ure genes. Hence, the absence of the urease genes distinguishes SF EHEC O157:NM strains from EHEC O157:H7, but this absence demonstrates that the urease genes are not useful genetic targets for the detection of EHEC strains, because SF EHEC O157:NM strains are missed by such a strategy. When examined for urease activity on Christensen agar and in the API 20E system, only one O157:H7 strain displayed urease activity and produced elevated levels of ammonia, which was subsequently confirmed by ammonia electrode measurement. Because the ure genes were absent from each of nine strains of E. coli O55:H7, the proposed progenitor of EHEC O157, we hypothesize that EHEC O157:H7 diverged from the evolutionary pathway at an early stage and then acquired the O islands carrying the ure gene cluster.

Diarrhea↗
PubMed2005

Molecular profiling and phenotype analysis of Escherichia coli O26:H11 and O26:NM: secular and geographic consistency of enterohemorrhagic and enteropathogenic isolates.

Fifty-eight enterohemorrhagic Escherichia coli O26:H11 or O26:NM (nonmotile) strains and 44 atypical enteropathogenic E. coli O26:H11 or O26:NM strains isolated from patients in 11 countries during 52 years share a common pool of non-stx virulence genes, fitness loci, and genotypic and phenotypic diagnostic markers. These findings indicate close relatedness between these pathotypes and provide a basis for their clinical laboratory diagnosis.

Escherichia coli↗
PubMed2005

Consequences of enterohaemorrhagic Escherichia coli infection for the vascular endothelium.

Microvascular endothelial damage underlies the pathological changes in haemorrhagic colitis and the haemolytic uraemic syndrome (HUS) caused by enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) are presently the best characterised EHEC virulence factors that can cause the microvascular endothelium injury. Stxs are released by EHEC in the intestine, absorbed across the gut epithelium into the circulation, and transported to small vessel endothelial cells. Then, they presumably injure the host cell by inhibiting protein synthesis, stimulating prothrombotic messages, or inducing apoptosis. The net result is a multi-organ thrombotic process. Moreover, Stxs stimulate a variety of non-endothelial cells to produce and secrete inflammatory mediators (cytokines, chemokines, adhesion molecules) which could potentiate the effects of Stxs on endothelial cells. The association of HUS with Stx-negative E. coli strains stimulated intensive research on putative non-Stx virulence factors, which might also contribute to the pathogenesis of HUS and haemorrhagic colitis. Based on current data, cytolethal distending toxin, EHEC haemolysin, and subtilase cytotoxin might be such candidates.

Animals↗
PubMed2004

Shiga toxin-encoding bacteriophages--genomes in motion.

Shiga toxins (Stx) represent a group of bacterial toxins that are involved in human and animal disease. Stx are mainly produced by Escherichia coli isolated from human and non-human sources, Shigella dysenteriae type 1, and sporadically, by Citrobacter freundii, Enterobacter cloacae and Shigella flexneri. The genes encoding Stx are encoded in the genome of heterogeneous lambdoid prophages (Stx-converting bacteriophages; Stx-phages). They are located in a similar position in the late region of the prophage genome and stx is under control of phage genes. Therefore, induction of Stx-converting prophages triggers increased production of Stx. Following induction, Stx-phages can infect other bacteria in vivo and in vitro. Stx-phages may be considered to represent highly mobile genetic elements that play an important role in the expression of Stx, in horizontal gene transfer, and hence in genome diversification.

Animals↗
PubMed2004

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).

Animals↗
PubMed2004

Phylogeny, clinical associations, and diagnostic utility of the pilin subunit gene (sfpA) of sorbitol-fermenting, enterohemorrhagic Escherichia coli O157:H-.

The plasmid-borne sfpA gene encodes the pilin subunit in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H-. We investigated the distribution of sfpA among 600 E. coli isolates comprising the complete E. coli standard reference (ECOR) and diarrheagenic E. coli (DEC) strain collections and clinical isolates associated with enteric disease. sfpA was detected in DEC3F SF EHEC O157:H- strain 493/89, each of 107 SF EHEC O157:H- clinical isolates, and 14 Shiga toxin-negative SF E. coli O157:H- strains which contained eae, which encodes gamma-intimin, and fliC, which encodes the H7 antigen. sfpA was absent from all other strains, including the ECOR strain collection, all non-SF EHEC O157:H7 strains, and all E. coli O55:H7 strains (E. coli O55:H7 is the postulated ancestor of Shiga toxin-producing E. coli [STEC] O157). These results suggest that there was a single acquisition of the sfpA gene in the nonmotile SF E. coli O157 branch, presumably after the eae-encoding pathogenicity island (the locus of enterocyte effacement) was acquired and motility was lost. We then applied the sfpA PCR in combination with rfbO157, stx, and eae PCRs to screen 636 stool samples from patients with diarrhea or hemolytic-uremic syndrome for SF STEC O157:H-. In 27 cases, the simultaneous presence of the sfpA, eae, and rfbO157 amplicons indicated the presence of SF E. coli O157:H- strains, and the result was subsequently confirmed by isolation. All but two of these strains possessed stx2. None of the other stool samples was positive by the sfpA PCR; 59 of these stool samples contained EHEC O157:H7. The sfpA gene can be recommended as a target for screening for SF E. coli O157:H-.

Child↗
PubMed2004

Sequence-based typing of flaB is a more stable screening tool than typing of flaA for monitoring of Campylobacter populations.

Different typing schemes for Campylobacter spp. were evaluated with 70 outbreak and sporadic isolates. The discriminatory indexes were 0.944 (by pulsed-field gel electrophoresis), 0.920 (by genotyping of the flagellin A gene), 0.902 (by genotyping of flaB), and 0.886 (by multilocus sequence typing). Cross-classification gave 94.77 or 95.82% (PFGE-flaA or PFGE-flaB) concordance. flaA was overdiscriminatory in three cases, most probably due to intragenomic recombination.

Animals↗
PubMed2004

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 gamma, whereas the three strains with the fliC-RFLP(H25) pattern possessed eae beta. 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.

Electrophoresis, Gel, Pulsed-Field↗
PubMed2004

Cellular prion protein acquires resistance to proteolytic degradation following copper ion binding.

The conversion of cellular prion protein (PrP(C)) into its pathological isoform (PrP(Sc)) conveys an increase in hydrophobicity and induces a partial resistance to proteinase K (PK). Interestingly, co-incubation with high copper ion concentrations also modifies the solubility of PrP(c) and induces a partial PK resistance which was reminiscent of PrP(Sc). However, concerns were raised whether this effect was not due to a copper-induced inhibition of the PK itself. We have therefore analyzed the kinetics of the formation of PK-resistant PrP(C) and excluded possible interference effects by removing unbound copper ions prior to the addition of PK by methanol precipitation or immobilization of PrP(C) followed by washing steps. We found that preincubation of PrPc with copper ions at concentrations as low as 50 microM indeed rendered these proteins completely PK resistant, while control substrates were proteolyzed. No other divalent cations induced a similar effect. However, in addition to this specific stabilizing effect on PrP(C), higher copper ion concentrations in solution (>200 microM) directly blocked the enzymatic activity of PK, possibly by replacing the Ca2+ ions in the active center of the enzyme. Therefore, as a result of this inhibition the proteolytic degradation of PrP(C) as well as PrP(Sc) molecules was suppressed.

Animals↗
PubMed2003

Multifactorial assessment of predictors for prevention of periodontal disease progression.

Univariate approaches have identified single factors influencing periodontal disease progression. The aim of this explorative approach was to assess the influence of various predictive factors responsible for the prevention of periodontal disease progression in the same patient sample. Patients with untreated chronic periodontitis underwent subgingival debridement alone or in combination with adjunctive antimicrobial therapy (systemic amoxicillin and metronidazole/7 days plus supragingival CHX irrigation). Supportive periodontal therapy was performed over a 24-month period. As predictors, clinical, microbial, immunological, and genetic parameters were assessed. The primary outcome variable was the percentage of teeth without attachment loss >/=2 mm over the study period (stability of attachment). At 24 months, multiple regression analysis identified adjunctive antimicrobial therapy for teeth with initially at least one site showing a pocket probing depth of >/=7 mm and IgG(4) reactivity against a 110-kDa protein of A. actinomycetemcomitans at teeth with initial pocket probing depths </=6 mm as main predictors of long-term attachment stability ( p<0.05). Other parameters failed to influence treatment outcome. Adjunctive antimicrobial therapy and antibody reactivity may be dominant factors influencing the prevention of attachment loss in patients receiving periodontal therapy.

Adult↗
PubMed2003

Hemolytic-uremic syndrome associated with enterohemorrhagic Escherichia coli O26:H infection and consumption of unpasteurized cow's milk.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O26 has emerged as a significant cause of hemolytic-uremic syndrome (HUS). The source and the vehicle of contamination with EHEC O26 are not often identified. We report two Austrian cases of HUS due to E. coli O26:H- affecting an 11-month-old boy and a 28-month-old girl in which transmission through unpasteurized cow's milk was positively identified. METHODS AND RESULTS: Using automated ribotyping and pulsed-field gel electrophoresis (PFGE), the isolates (which yielded the virulence genes stx2, eae, and hly) were indistinguishable from each other. An epidemiologic investigation revealed that the children had stayed in the same hotel. Both patients had consumed unpasteurized cow's milk from the breakfast buffet. Fecal samples were taken from the cows of the farm producing the incriminating milk, and one of three cattle EHEC O26:H- isolates had a PFGE pattern indistinguishable from that of the patients' strains. CONCLUSIONS: These two cases of E. coli O26 infection illustrate the hazards associated with the consumption of raw milk, and underline the importance of microbiological diagnostic approaches able to detect sorbitol-fermenting, non-O157 EHEC.

Animals↗
PubMed2003

Cluster of hemolytic-uremic syndrome caused by Shiga toxin-producing Escherichia coli O26:H11.

BACKGROUND: The epidemiology and clinical characteristics of the hemolytic-uremic syndrome (HUS) caused by Escherichia coli O157:H7 are well-known, but HUS attributable to non-O157:H7 Shiga toxin (Stx)-producing E. coli (STEC) are less thoroughly described. Here we report a cluster of HUS cases caused by STEC O26:H11 the most common non-O157:H7 STEC isolated from sporadic cases of HUS in Europe. METHODS: Three children between 13 and 17 months of age, living in the same small town, developed HUS within an interval of 5 days. We present clinical and microbiologic data on the patients and their infecting isolates. RESULTS: The clinical course ranged from mild uncomplicated HUS to severe HUS complicated by multiorgan involvement. Microbiologic investigation demonstrated STEC of serotype O26:H11 in stools of all the patients. The phenotypic and molecular characterization of the STEC O26:H11 isolates demonstrated that these strains were identical and, unusual for STEC O26, they harbored the stx2 but not the stx1 gene. None of the patients had evidence of STEC O157:H7 infection either by culture or by E. coli O157 serology. The source of the STEC O26:H11 infection was undetermined. CONCLUSIONS: Our results demonstrate that diagnostic procedures based on the detection of stx genes and/or Stx production and subsequent subtyping of the isolates using molecular methods are necessary to identify such outbreaks caused by non-O157:H7 STEC.

Cluster Analysis↗
PubMed2003

Multilocus sequence analysis of Porphyromonas gingivalis indicates frequent recombination.

In this study, the genetic relationship of 19 Porphyromonas gingivalis isolates from patients with periodontitis was investigated by multilocus sequence analysis. Internal 400-600 bp DNA fragments of the 10 chromosomal genes ef-tu, ftsQ, hagB, gpdxJ, pepO, mcmA, dnaK, recA, pga and nah were amplified by PCR and sequenced. No two isolates were identical at all 10 loci. Phylogenetic analyses indicated a panmictic population structure of P. gingivalis. Split decomposition analysis, calculation of homoplasy ratios and analyses of clustered polymorphisms all indicate that recombination plays a major role in creating the genetic heterogeneity of P. gingivalis. A standardized index of association of 0.0898 indicates that the P. gingivalis genes analysed are close to linkage equilibrium.

Chromosomes, Bacterial↗
PubMed2003

Cytolethal distending toxin gene cluster in enterohemorrhagic Escherichia coli O157:H- and O157:H7: characterization and evolutionary considerations.

We identified a cytolethal distending toxin (cdt) gene cluster in 87, 6, and 0% of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-), EHEC O157:H7, and E. coli O55:H7/H(-) strains, respectively. The toxin was expressed by the wild-type EHEC O157 strains and by a cdt-containing cosmid from a library of SF EHEC O157:H(-) strain 493/89. The cdt flanks in strain 493/89 were homologous to bacteriophages P2 and lambda. Our data demonstrate that cdt, encoding a potential virulence factor, is present in the EHEC O157 complex and suggest that cdt may have been acquired by phage transduction.

Bacterial Toxins↗
PubMed2003

Analysis of genome plasticity in pathogenic and commensal Escherichia coli isolates by use of DNA arrays.

Genomes of prokaryotes differ significantly in size and DNA composition. Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants. Pathogenic and nonpathogenic E. coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information. To analyze the genetic diversity of pathogenic and commensal E. coli isolates, a whole-genome approach was applied. Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E. coli K-12 strain MG1655 was investigated in 26 E. coli isolates, including various extraintestinal and intestinal pathogenic E. coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains. Additionally, the presence of virulence-associated genes of E. coli was determined using a DNA "pathoarray" developed in our laboratory. The frequency and distributional pattern of genomic variations vary widely in different E. coli strains. Up to 10% of the E. coli K-12-specific ORFs were not detectable in the genomes of the different strains. DNA sequences described for extraintestinal or intestinal pathogenic E. coli are more frequently detectable in isolates of the same origin than in other pathotypes. Several genes coding for virulence or fitness factors are also present in commensal E. coli isolates. Based on these results, the conserved E. coli core genome is estimated to consist of at least 3,100 translatable ORFs. The absence of K-12-specific ORFs was detectable in all chromosomal regions. These data demonstrate the great genome heterogeneity and genetic diversity among E. coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes.

Electrophoresis, Gel, Pulsed-Field↗