PubMed Health⌕ Search

Biomedical subjects

Helge Karch

Publications and source records attributed to Helge Karch.

At least 55 records · Page 3Linked to original sources
PubMed2003

Shiga toxin 1c-producing Escherichia coli strains: phenotypic and genetic characterization and association with human disease.

The distribution of the stx(1c) allele among Shiga toxin (Stx)-producing Escherichia coli (STEC) and the virulence characteristics of stx(1c)-harboring STEC are unknown. In this study, we identified stx(1c) in 76 (54.3%) of 140 eae-negative, but in none of 155 eae-positive, human STEC isolates (P < 0.000001). The 76 stx(1c)-harboring E. coli isolates belonged to 22 serotypes, and each produced Stx1c as demonstrated by latex agglutination. Characterization of putative virulence factors demonstrated the presence of the locus of proteolysis activity (LPA) and the high-pathogenicity island in 65.8 and 21.1%, respectively, of the 76 Stx1c-producing E. coli isolates. Moreover, all but three of these strains contained saa, the gene encoding an STEC autoagglutinating adhesin. The virulence profiles of Stx1c-producing E. coli isolates were mostly serotype independent and heterogeneous. This enabled us to subtype the isolates within the same serotype. The individuals infected with Stx1c-producing E. coli strains were between 3 months and 72 years old (median age, 23.5 years) and usually had uncomplicated diarrhea or were asymptomatic. We conclude that Stx1c-producing E. coli strains represent a significant subset of eae-negative human STEC isolates, which belong to various serotypes and frequently possess LPA and saa as their putative virulence factors. The phenotypic and molecular characteristics determined in this study allow the subtyping of Stx1c-producing STEC in epidemiological and clinical studies.

Adhesins, Bacterial↗
PubMed2002

Clinical course and the role of shiga toxin-producing Escherichia coli infection in the hemolytic-uremic syndrome in pediatric patients, 1997-2000, in Germany and Austria: a prospective study.

Hemolytic-uremic syndrome (HUS) is mainly associated with foodborne infections by Shiga toxin-producing Escherichia coli (STEC). From January 1997 through December 2000, 394 children with HUS were evaluated in a prospective multicenter surveillance study in Germany and Austria (incidences, 0.7/100,000 and 0.4/100,000 children <15 years old, respectively). Blood leukocytosis was associated with increased detection of STEC in stool cultures (P<.01) and a more severe disease course. Risk of death was associated with cerebral involvement (P<.01). Most strikingly, non-O157:H7 STEC were detected in 43% of stool cultures of patients with HUS: O26 was detected in 15%, sorbitol-fermenting O157:H(-) in 10%, O145 in 9%, O103 in 3%, and O111 in 43%. Patients with O157:H7 serotypes required dialysis for a longer time and had bloody diarrhea detected more frequently, compared with patients with non-O157:H7 serotypes (P<.05). This large study in children with HUS underlines the rising importance of non-O157:H7 serotypes, and, despite increased public awareness, the number of patients remained unchanged.

Austria↗
PubMed2002

A multistate outbreak of Shiga toxin-producing Escherichia coli O26:H11 infections in Germany, detected by molecular subtyping surveillance.

In the spring of 2000, a cluster of indistinguishable Shiga toxin-producing Escherichia coli (STEC) O26:H11 was identified in Germany by molecular subtyping surveillance. An investigation was prompted to identify a common source of exposure. A case subject was defined as a person having a polymerase chain reaction-confirmed STEC O26 infection between March and April 2000, irrespective of clinical signs, and whose isolate was indistinguishable from the index strain by use of pulsed-field gel electrophoresis. Eleven case subjects were found in 5 institutions that were supplied by 4 kitchens located in 3 states. The median age was 2 years (range, 2-31 years). No bloody diarrhea was reported, and 5 persons remained asymptomatic. Comparison of invoices revealed a certain type of beef ("Seemerrolle") as possible source of infection. This is, to our knowledge, the first multistate outbreak associated with a non-O157 STEC detected by laboratory-based surveillance. Molecular subtyping was pivotal, as disease occurrence was sporadic or family-related.

Adolescent↗
PubMed2002

Antibody reactivity of a standardized human serum protein solution against a spectrum of microbial pathogens and toxins: comparison with fresh frozen plasma.

In this study, we compared a standardized solution of human serum protein (HSP) and fresh frozen plasma (FFP) with regard to the antibody specificity against a number of microbial pathogens and some important pathogenicity factors of bacterial pathogens. Due to the clinical use of HSP and FFP for therapeutical plasma exchange, we have chosen a spectrum of microbial pathogens for serological analysis that is critical in clinical settings. With the enzyme-linked immunosorbent assay technique, we could show that HSP contains marked IgG antibody reactivity against antigens of Escherichia coli, Campylobacter jejuni, Enterobacter sakazakii, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, S. epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, Chlamydia pneumoniae, and Candida albicans. Although no IgM antibodies against the pathogens tested could be detected in HSP, moderate IgA reactivity was found against 4 of 12 microbial antigens. Immunoblot analysis demonstrated specific IgA and IgG responses against the endoproteinase Glu-C and the superantigens enterotoxin A and B of S. aureus, the IgA-protease of Neisseria gonorrhoeae, and Shiga toxin 2 of enterohemorrhagic E. coli. By using 3 different HSP batches in parallel, we could demonstrate antibody reactivity against important microbial pathogens and toxins. This antibody profile is essentially more homogeneous than that of 3 batches of FFP.

Antibody Specificity↗
PubMed2002

Identification and distribution of the enterohemorrhagic Escherichia coli factor for adherence (efa1) gene in sorbitol-fermenting Escherichia coli O157: H-.

Sorbitol-fermenting (SF) Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H- strains are emerging as causes of hemorrhagic colitis and the hemolytic-uremic syndrome in Europe. Using subtractive hybridization between SF STEC O157:H- strain 493/89 and STEC O157:H7 strain EDL933, three different fragments, of approximately 700 bp in length, were identified. Each demonstrated > 99% homology to genes encoding the enterohemorrhagic E. coli factor for adherence (efa1) and lymphostatin (lifA). Therefore, a cosmid library was constructed from SF STEC O157:H- strain 493/89, and one clone containing these fragments was sequenced. This sequencing demonstrated a 9669-bp open reading frame (ORF) that had 99.9% sequence homology to efa1 of STEC O111:H- strain E45035 and to lifA of an enteropathogenic E. coli O127:H6 strain E2348/69. In STEC O157:H7 strain EDL933, only small (ca. 3 kb) initial and terminal fragments of this ORF are present. PCR analysis with primers complementary to the efa1/lifA sequence of strain 493/89 indicated that the complete sequence is present in each of 10 SF STEC O157:H- isolates but in none of 10 STEC O157:H7 strains investigated. The presence of the complete efa1/lifA also in both tested E. coli O55:H7 strains supports the hypothesis that SF STEC O157:H- are phylogenetically closer to the proposed E. coli O55:H7 ancestor than STEC O157:H7. Our data demonstrate the presence of a potential virulence gene in SF STEC O157:H- that is only rudimentarily present in STEC O157:H7.

Bacterial Proteins↗
PubMed2002

Detection and characterization of class 1 integrons in enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) strains isolated from humans, cattle, and food and belonging to serogroups O26 (7 strains), O111 (19 strains), and O157 (70 strains) were examined for susceptibility to 11 antimicrobial drugs. Fifty-nine strains showing resistance to at least one of the drugs were examined by PCR for the presence of class 1 integrons, which were identified in 17 strains. Integrons were found more frequently in strains belonging to serogroups O111 and O26 than in the O157 isolates. DNA sequence analysis demonstrated that most of the integrons contained the aadA1 gene cassette conferring resistance to streptomycin/ spectinomycin, alone or associated with the drfA1 gene cassette conferring resistance to trimethoprim. One integron, identified in a O157:H7 strain, carried the aadA2 and dfrA12 gene cassettes, conferring resistance to streptomycin/spectinomycin and trimethoprim, and the open reading frame F (OrfF) encoding unknown functions. Most of the integrons were carried by Tn21 derivative transposons and were transferable by conjugation to an E. coli K-12 strain. In conclusion, integrons and antibiotic resistance genes can be frequently found in EHEC strains, particularly E. coli O111 and E. coli O26, and their presence could complicate therapeutic trials.

Animals↗
PubMed2002

Identification, characterization, and distribution of a Shiga toxin 1 gene variant (stx(1c)) in Escherichia coli strains isolated from humans.

By using sequence analysis of Shiga toxin 1 (Stx 1) genes from human and ovine Stx-producing Escherichia coli (STEC) strains, we identified an Stx1 variant in STEC of human origin that was identical to the Stx1 variant from ovine STEC, but demonstrated only 97.1 and 96.6% amino acid sequence identity in its A and B subunits, respectively, to the Stx1 encoded by bacteriophage 933J. We designated this variant "Stx1c" and developed stxB(1) restriction fragment length polymorphism and stx(1c)-specific PCR strategies to determine the frequency and distribution of stx(1c) among 212 STEC strains isolated from humans. stx(1c) was identified in 36 (17.0%) of 212 STEC strains, 19 of which originated from asymptomatic subjects and 16 of which were from patients with uncomplicated diarrhea. stx(1c) was most frequently (in 23 STEC strains [63.9%]) associated with stx(2d), but 12 (33.3%) of the 36 STEC strains possessed stx(1c) only. A single STEC strain possessed stx(1c) together with stx(2) and was isolated from a patient with hemolytic-uremic syndrome. All 36 stx(1c)-positive STEC strains were eae negative and belonged to 10 different serogroups, none of which was O157, O26, O103, O111, or O145. Stx1c was produced by all stx(1c)-containing STEC strains, but reacted weakly with a commercial immunoassay. We conclude that STEC strains harboring the stx(1c) variant account for a significant proportion of human STEC isolates. The procedures developed in this study now allow the determination of the frequency of STEC strains harboring stx(1c) among clinical STEC isolates and their association with human disease in prospective studies.

Animals↗
PubMed2002

Shiga toxin-producing Escherichia coli infection and antibodies against Stx2 and Stx1 in household contacts of children with enteropathic hemolytic-uremic syndrome.

Ninety-five household contacts (aged 2 months to 73 years) of patients with enteropathic hemolytic-uremic syndrome (HUS) were investigated for the presence of immunoglobulin (Ig) G antibodies to Shiga toxins Stx2 and Stx1 by Western blot assay. Thirty-one percent of the household contacts and 19% of 327 controls had anti-Stx2 IgG (heavy and light chain [H + L]), 5 and 8%, respectively, had anti-Stx1 IgG (H + L), and 3 and 2%, respectively, had both anti-Stx2 and anti-Stx1 IgG (H + L). The incidence of infections with Stx-producing Escherichia coli (STEC) was determined based on the following diagnostic criteria: STEC isolation, detection of stx gene sequences, free fecal Stx in stool filtrates, and serum IgM antibodies against E. coli O157 lipopolysaccharide. Evidence of STEC infection was observed in 25 household contacts, of whom 18 (72%) were asymptomatic and represented a potential source of infection. Six of 13 (46%) household contacts with Stx2-producing E. coli O157:H7 in stool culture developed anti-Stx2 IgG (H + L), compared to 71% of Stx2-associated HUS cases. In individuals showing anti-Stx2 IgG (H + L), the antibody response was directed against the B subunit in 69% of household contacts and 71% of controls, in contrast to 28% of HUS patients. In this investigation controls had a significant increase of the median of IgM antibodies to O157 lipopolysaccharide (LPS) with age, up to the fifth decade. The lack of disease in household contacts with B subunit-specific antibodies, as well as the significantly higher median of anti-O157 LPS IgM antibodies in controls beyond 4.9 years of age, suggests a protective role for anti-Stx and anti-O157 LPS antibodies.

Adolescent↗
PubMed2002

Real-time fluorescence PCR assays for detection and characterization of Shiga toxin, intimin, and enterohemolysin genes from Shiga toxin-producing Escherichia coli.

PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx(1) and stx(2)) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx(1), eae, and E-hly genes and 96 and 100%, respectively, for the stx(2) gene. No stx(2) genes were detected from 10 stx(2f)-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains.

Adhesins, Bacterial↗
PubMed2002

Saliva IgM and IgA are a sensitive indicator of the humoral immune response to Escherichia coli O157 lipopolysaccharide in children with enteropathic hemolytic uremic syndrome.

Saliva antibodies to Escherichia coli O157 were investigated as markers of the immune response in children with enteropathic hemolytic uremic syndrome (HUS). Paired serum and saliva samples were collected from 22 children with HUS during acute disease and convalescence and were tested for E. coli O157 lipopolysaccharide (LPS)-specific IgM and IgA antibodies by ELISA. Serum and saliva samples from 44 age-matched controls were used to establish the cut-off values. Elevated levels of IgM and/or IgA antibodies to O157 LPS were detected in saliva of 13/13 HUS patients with Shiga toxin-producing E. coli (STEC) O157 in stool culture and from 4 of 5 HUS patients in whom STEC were not detected. These results closely mirrored the results obtained with paired serum samples. In contrast, saliva and serum samples from four children with STEC isolates belonging to O-groups O26, O145 (n = 2), and O165 lacked detectable O157 LPS-specific antibodies. The specificity of the ELISA was confirmed by western blotting. In STEC O157 culture-confirmed cases, the sensitivity of the ELISA was 92% for saliva IgM and IgA, based on the first available sample, and 100% and 92%, respectively, when subsequent samples were included. The specificity was 98% for IgM and 100% for IgA. Children with E. coli O157 HUS demonstrate a brisk, easily detectable immune response as reflected by the presence of specific antibodies in their saliva. Saliva-based immunoassays offer a reliable, noninvasive method for the diagnosis of E. coli O157 infection in patients with enteropathic HUS.

Adolescent↗
PubMed2001

Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms.

Shiga toxin (Stx)-producing Escherichia coli (STEC) from patients with hemolytic-uremic syndrome (HUS), patients with diarrhea without HUS, or asymptomatic subjects were genotyped to assess associations between stx2 variants and clinical manifestations of infection. Neither stx2d nor stx2e was found in 268 STEC isolates from patients with HUS. Of 262 STEC isolates from patients with diarrhea, stx(2d) was found in 41 (15.6%; P<.000001), and stx2e was found in 12 (4.6%; P=.0004). The stx2c genotype frequency was similar among isolates from patients with HUS (3.7%) and diarrhea (5.0%). The frequencies of stx2c, stx2d, and stx2e among 96 STEC isolates from asymptomatic subjects were comparable to those among isolates from patients with diarrhea. None of the 626 STEC isolates contained stx2f. All stx2d-positive or stx2e-positive STEC isolates were eae negative and originated from subjects older than those with STEC isolates with stx2c. stx2c-positive STEC isolates can cause HUS, but the presence of stx2d or stx2e may predict a milder disease with a minimal risk of HUS.

Adhesins, Bacterial↗
PubMed2000

Identification and characterisation of Escherichia coli strains of O157 and non-O157 serogroups containing three distinct Shiga toxin genes.

Three Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from patients with diarrhoea were identified, each of which contained three distinct stx genes (stx1, stx2 and stx2c). The strains belonged to the serotypes O52:H19, O75:H- and O157:H- and harboured eae and EHEC-hly sequences. Colony-blot immunoassay was used to demonstrate that both major types of Stx were expressed. The association of stx genes with either phage or phage DNA was demonstrated in all three strains. Isolated phage DNA from all strains contained stx1 sequences, but stx2 sequences were found only in phage DNA of two of these strains. The presence of three distinct stx genes may enhance the virulence of STEC strains and should be monitored. The observations demonstrate not only the potential of stx genes to spread within different serotypes, but also their capacity to accumulate within a single strain.

Animals↗
PubMed2000

Antibacterials that are used as growth promoters in animal husbandry can affect the release of Shiga-toxin-2-converting bacteriophages and Shiga toxin 2 from Escherichia coli strains.

Antibiotics are commonly used as growth promoters in animal husbandry worldwide. This practice has been linked to the emergence of particular antibiotic-resistant bacteria, and is now controversial. In this study, the ability of growth-promoting antibiotics to induce Shiga toxin (Stx)-converting bacteriophages from Stx-producing Escherichia coli (STEC) strains was investigated. Subinhibitory concentrations of the antibacterial growth promoters olaquindox, carbadox, tylosin and monensin were used for induction experiments. The amount of mature Stx-converting phage particles released from induced and non-induced cultures was determined, and the production of Stx was simultaneously measured by ELISA. Whereas the quinoxaline-1,4-dioxide-type antibiotics olaquindox and carbadox enhanced the release of Stx-converting phage particles from STEC cells, tylosin and monensin decreased phage induction. The production of Stx increased or decreased simultaneously with the amount of free phages. The results of this study show that particular antibacterial growth promoters can induce Stx phages. In vivo induction of Stx phages from lysogenic STEC may increase the amount of free phages in the intestine and therefore may contribute to the spread of STEC and development of new STEC pathotypes.

Animal Husbandry↗
PubMed1999

Cloning, sequencing and molecular analysis of the Campylobacter jejuni groESL bicistronic operon.

The groESL bicistronic operon from the enteric pathogen Campylobacter jejuni was cloned and sequenced. It consists of two ORFs encoding proteins with molecular masses of 9.5 and 57.9 kDa, which showed a high degree of homology to other bacterial GroES and GroEL proteins. Northern blot analysis suggested that the groESL operon is transcribed as a bicistronic mRNA, and its steady-state level was markedly increased after temperature upshift. By primer extension assay, one potential transcription start point preceding the groESL genes could be demonstrated, and a putative promoter region compatible with both Escherichia coli and C. jejuni sigma70 consensus sequences was identified. A conserved inverted repeat, which is believed to be involved in the regulation of the groESL genes, was found between the -10 promoter box and the groES translation start site. The complete coding region of groEL was fused with pET-22b(+) and expressed in E. coli as a His6-tagged recombinant protein (rCjHsp60-His). After purification, the protein was recognized by an anti-HSP60 monoclonal antibody. ELISA and Western immunoblotting experiments showed that IgG and IgA antibody responses against rCjHsp60-His were not significantly increased in sera from 24 patients with sporadic Campylobacter infection when compared to sera from 16 healthy controls.

Amino Acid Sequence↗
PubMed1999

The large plasmids of Shiga-toxin-producing Escherichia coli (STEC) are highly variable genetic elements.

Shiga-toxin-producing Escherichia coli (STEC) of different serotypes are known to harbour large plasmids. The aim of this study was to investigate, using the example of the plasmid-encoded serine protease EspP, whether these plasmids are a uniform genetic element present in STEC. Examination of 201 diarrhoeagenic E. coli strains using a newly developed espP-specific PCR showed that espP is specific for STEC and present in 57% of STEC belonging to 16 different serotypes. The espP genes of the 16 STEC serotypes varied to a certain extent, as shown by nucleotide sequence and restriction enzyme analyses, but the DNA regions adjacent to the espP gene were completely different. When two further STEC-plasmid markers, the catalase-peroxidase gene katP and the enterohaemorrhagic E. coli-haemolysin gene EHEC-hlyA were included, many combinations of the three markers were found, depending in part on the serotype. In addition, strains possessing none of the three markers still harboured large plasmids. In the most prevalent STEC serogroup, O157, it was observed that the plasmid of sorbitol-fermenting STEC O157:H- lacks the espP and katP genes although both genes are present in the plasmid of the non-sorbitol-fermenting STEC O157:H7. The EHEC-hlyA gene, however, is present in both. In conclusion, this study shows that the large plasmids of STEC are not uniform genetic elements but heterogeneous in both their gene composition and arrangement.

Bacterial Toxins↗