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M Frosch

Publications and source records attributed to M Frosch.

At least 91 records · Page 5Linked to original sources
PubMed1998

A 3.3-kb plasmid of enterohemorrhagic Escherichia coli O157:H7 is closely related to the core region of the Salmonella typhimurium antibiotic resistance plasmid NTP16.

A restriction enzyme map was constructed of a 3.3-kb plasmid derived from enterohemorrhagic Escherichia coli O157:H7 strain 4821 using the enzymes SmaI, HpaI, FokI, HaeIII, StyI, RsaI, Bg/II, ClaI, and EcoRV. The molecular size of p4821 was 3307 bp, determined by nucleotide sequencing. Homology searches in the EMBL database library revealed that the nucleotide sequence of P4821 is similar (> 98%) to the core region of the antibiotic resistance plasmid NTP16 of Salmonella typhimurium strains. Nucleotide sequence analysis showed that p4821 contains all the information necessary for its replication, stability, and mobilization. However, the lack of tra genes indicates that the plasmid is nonconjugative. A possible transposon insertion site was found in p4821 but two such sites were found at the boundaries between the core region and the antibiotic resistance encoding transposons in plasmid NTP16. Using a hybridization assay, we determined that of 50 E. coli O157:H7 strains isolated in 1996 in Würzburg, Germany from patients with diarrhea and hemolytic-uremic syndrome, 4 strains (8%) contained plasmid p4821-specific sequences.

Diarrhea↗
PubMed1998

Antibiotic resistance in Salmonella enterica serotype typhimurium.

In order to analyse the development of antibiotic resistance in Salmonella spp., a total of 262 Salmonella strains isolated in 1987 (n = 148) and in 1996 (n = 114) from clinical specimens in Wurzburg, Germany, were tested in parallel by the agar diffusion method. In 1987. most of the strains were Salmonella enterica serotype typhimurium (42.6%), whereas in 1996 most were Salmonella enterica serotype enteritidis (68.4%). The majority of Salmonella enterica serotype enteritidis isolates was fully susceptible in 1987 and 1996. In contrast, the percentage of drug-resistant strains of Salmonella enterica serotype typhimurium increased significantly from 27% in 1987 to 52.4% in 1996. This increase, which might reflect uncontrolled use of antibiotics in the environment, should be of concern to public health authorities.

Anti-Bacterial Agents↗
PubMed1998

Identification of a hotspot for transformation of Neisseria meningitidis by shuttle mutagenesis using signature-tagged transposons.

Shuttle mutagenesis using signature-tagged transposons was employed to generate a library of individually tagged mutants of the Neisseria meningitidis strain B1940, which belongs to serogroup B. The use of tagged transposons allowed us to monitor for enrichment for single mutants during the process of shuttle mutagenesis, by amplification of the tags and subsequent sequence determination. Enrichment of a single clone occurred during the transformation of the meningococci with transposon-containing plasmid DNA. Sequence determination around the site of transposon insertion revealed that the transposon had mutagenized a previously unknown locus, which was designated hrtA (high rate of transformation). hrtA-mediated transformation was independent of TnMax5 and tag sequences, and it most probably involved recombination events. The hrtA locus is restricted to meningococci and gonococci and is present in few apathogenic neisserial species. Chromosomal mapping of hrtA and six further hrt sites revealed a random distribution of highly transforming DNA fragments on the meningococcal chromosome. In conclusion, our data demonstrate that shuttle mutagenesis of naturally competent bacteria using signature-tagged transposons allows the isolation of chromosomal DNA fragments, which exhibit a high transformation efficiency, and which, therefore, are likely to be involved in horizontal gene transfer.

Base Sequence↗
PubMed1998

Neisseria meningitidis producing the Opc adhesin binds epithelial cell proteoglycan receptors.

Neisseria meningitidis possesses a repertoire of surface adhesins that promote bacterial adherence to and entry into mammalian cells. Here, we have identified heparan sulphate proteoglycans as epithelial cell receptors for the meningococcal Opc invasin. Binding studies with radiolabelled heparin and heparin affinity chromatography demonstrated that Opc is a heparin binding protein. Subsequent binding experiments with purified 35SO4-labelled epithelial cell proteoglycan receptors and infection assays with epithelial cells that had been treated with heparitinase to remove glycosaminoglycans confirmed that Opc-expressing meningococci exploit host cell-surface proteoglycans to gain access to the epithelial cell interior. Unexpectedly, Opa28-producing meningococci lacking Opc also bound proteoglycans. These bacteria also bound CEA receptors in contrast to the Opc-expressing phenotype, suggesting that Opa28 may possess domains with specificity for different receptors. Opa/Opc-negative meningococci did not bind either proteoglycan or CEA receptors. Using a set of genetically defined mutants with different lipopolysaccharide (LPS) and capsular phenotype, we were able to demonstrate that surface sialic acids interfere with the Opc-proteoglycan receptor interaction. This effect may provide the molecular basis for the reported modulatory effect of capsule and LPS on meningococcal adherence to and entry into various cell types.

Adhesins, Bacterial↗
PubMed1998

In vitro activities of benzimidazoles against Echinococcus multilocularis metacestodes.

Alveolar echinococcosis, caused by the larval (metacestode) stage of the tapeworm Echinococcus multilocularis, is a lethal parasitosis of the liver prevalent in the Northern Hemisphere. For chemotherapy the benzimidazole derivatives mebendazole and albendazole were introduced, and their use has resulted in a significant improvement in the survival rates. However, data from experiments with animals and clinical observations indicate that these drugs elicit only parasitostatic activity and in most cases are not able to completely eliminate the parasitic metacestode tissue. In the present study, we applied a culture system for the in vitro growth and proliferation of E. multilocularis metacestodes to analyze the parasitostatic and parasitocidal potential of mebendazole. Here, we demonstrate for the first time that at concentrations of >0.1 microM, i.e., at concentrations used for therapy of human alveolar echinococcosis, this antihelminth drug is parasitocidal in vitro. Viability assessment was performed by infection experiments with Meriones unguiculatus and mebendazole-treated metacestode tissue and by reverse transcription-PCR for the detection of E. multilocularis mRNA. The E. multilocularis in vitro model proved to be a valuable tool for the analysis of the potential of antihelminth drugs.

Animals↗
PubMed1998

The central, surface-exposed region of the flagellar hook protein FlgE of Campylobacter jejuni shows hypervariability among strains.

In a previous study, we observed that monoclonal antibodies raised against the hook protein FlgE of Campylobacter jejuni LIO 36, isolate 5226, bound exclusively to this strain. The aim of this study was to elucidate the molecular basis for these binding specificities. The hook protein-encoding gene flgE of C. jejuni was cloned in Escherichia coli and sequenced. The flgE genes of four additional C. jejuni strains were amplified by PCR and also sequenced. Comparison of the deduced amino acid sequences revealed a high degree of variability in the central parts of the FlgE proteins among the strains, including variable and hypervariable domains. These findings may indicate a selective pressure of C. jejuni hosts, forcing the bacteria to generate variations in surface-exposed antigenic determinants.

Amino Acid Sequence↗
PubMed1998

Necessity of molecular techniques to distinguish between Neisseria meningitidis strains isolated from patients with meningococcal disease and from their healthy contacts.

Serogroup C strains of Neisseria meningitidis were isolated from a Germany patient with severe meningococcal disease after a trip to the Czech Republic. These strains (case isolates) were characterized by classical and molecular techniques, as were other strains (carrier isolates) isolated from healthy contacts. Five of 10 carrier isolates had switched off the expression of capsular polysaccharide, as demonstrated by a serogroup-specific PCR. The two case isolates were indistinguishable by multilocus sequence typing and belonged to the ET-37 complex. The carrier isolates belonged to four different sequence types, all unrelated to that of the case strains. Pulsed-field gel electrophoresis showed that the case isolates differed from reference ET-37 complex strains from the Czech Republic and Canada as well as from all the carrier isolates. The isolate from the patient's nasopharynx was indistinguishable from the blood isolate except for a 40,000-bp chromosomal deletion that had occurred during systemic spread.

Adolescent↗
PubMed1997

Monoclonal antibody against species-specific epitope of Pseudomonas aeruginosa Hsp60 protein cross-reacts with Pseudomonas stutzeri and other Pseudomonas species.

In a previous study, we determined the epitope of the Pseudomonas aeruginosa Hsp60 heat shock protein which is recognized by the specific monoclonal antibody (MAb) 2528. Subsequent investigations revealed a weak cross-reactivity of MAb 2528 with P. stutzeri, P. alcaligenes, P. mendocina and P. pseudoalcaligenes. To elucidate the molecular structure for these cross-reactions, we cloned the P. stutzeri hsp60 gene in Escherichia coli and determined the nucleotide sequence of the gene. In addition, the hsp60 gene of further Pseudomonas species was amplified and sequenced and amino acid substitutions within the epitope recognized by MAb 2528 were determined. The decapeptide QADIEARVLQ is unique to the P. aeruginosa Hsp60 protein, and cross-reaction of MAb 2528 reflects the phylogenetic relationship of Pseudomonas species as P. aeruginosa and all four cross-reacting species constitute a DNA homology group within the rRNA group I of the family Pseudomonadaceae, which belong to the gamma-subclass of the Proteobacteria.

Amino Acid Sequence↗
PubMed1997

Induction of human endothelial tissue factor expression by Neisseria meningitidis: the influence of bacterial killing and adherence to the endothelium.

Tissue factor (TF), a small membrane bound high affinity receptor for factor VII, has an important procoagulant role in the haemostatic dysfunction associated with severe sepsis. Using an in vitro model of human endothelial TF expression, defined strains of Neisseria meningitidis were found to upregulate endothelial cell procoagulant activity (PCA) in a dose dependent manner. This TF response was detected with as little as 10(4) cfu/ml and reached similar levels to those seen with high concentrations of purified endotoxin (> 1 ng/ml). Treatment of N. meningitidis with either adult donor immune serum, penicillin or gentamicin failed to enhance this PCA. Limulus amoebocyte lysate assay of lipopolysaccharide in bacterial culture filtrates together with polymyxin B inhibition experiments suggest that endotoxin is largely responsible for endothelial TF induction by N. meningitidis. Incubation of endothelial cells with N. meningitidis B1940 and B1940 siaD- (an eight-fold more adherent unencapsulated isogenic strain), revealed a significantly greater TF response to B1940 siaD- (P < 0.01). In conclusion, bacterial adhesion to the vessel wall and therefore local levels of endotoxin may be important determinants of the endothelial procoagulant response to N. meningitidis and the consequent coagulopathy commonly associated with the disease.

Adult↗
PubMed1997

Molecular divergence of the sia locus in different serogroups of Neisseria meningitidis expressing polysialic acid capsules.

The serogroups B, C, W135 and Y of Neisseria meningitidis express chemically and immunologically distinct capsular polysaccharides containing sialic acid. In the case of serogroup B meningococci sialic acid is synthesized by the gene products of a locus termed sia and forms the homopolymers of the capsule. The organization of the genes required for sialic acid synthesis in serogroups B, C, W135 and Y was elucidated by PCR technology. Cloning, sequencing and the functional expression of the polysialyltransferase (PST) genes of serogroups B and C demonstrated that the difference in capsule composition derives from the presence of related, but distinct siaD genes coding for PSTs. Analysis of meningococci of serogroups W135 and Y expressing sialic acid heteropolymers revealed that the DNA sequences of the corresponding genetic loci in these serogroups were highly homologous, but differed completely from the siaD genes of serogroups B and C. This finding suggests that enzymes unrelated to those of serogroups B and C are required for the formation of sialic acid heteropolymers characteristic of the capsules of serogroups W135 and Y.

Biological Evolution↗
PubMed1997

Functional characterization of an isogenic meningococcal alpha-2,3-sialyltransferase mutant: the role of lipooligosaccharide sialylation for serum resistance in serogroup B meningococci.

The neisserial alpha-2,3-sialyltransferase, which is encoded by the lst gene, terminally links sialic acid to the lacto-N-neotetraose residue of neisserial lipooligosaccharide (LOS). We used the recently published nucleotide sequence of the neisserial lst gene to construct an isogenic serogroup B meningococcal lst mutant by insertion of a kanamycin resistance gene. The resulting lst mutant expressed the unsialylated lacto-N-neotetraose structure. Using bactericidal assays and an infant rat model of meningococcal infection, we were able to demonstrate that lst mutation, in contrast to galE mutation, which results in a truncated LOS, or to siaD mutation, which results in loss of the capsule, neither had an effect on resistance to normal human serum, nor did it impair the ability of meningococci to spread systemically in the non-immune host. The lst mutant was serum resistant despite of the fact that the central factor of complement activation, C3b, was deposited on the lst mutant as efficiently as it was on the galE mutant. Thus, the terminal sialic acid residue linked to the wild-type LOS inhibited C3b deposition on the meningocuccus. However, in contrast to the galE mutant, where C3b deposition is promoted by IgM binding, the lst mutant's surface is not a target for IgM molecules. Thus, the lacto-N-neotetraose residue of neisserial LOS alone, without the presence of terminal sialic acid, is sufficient to block IgM epitopes either on the LOS itself, or on other surface molecules. Our data provide further insight into the complex interplay of capsular and LOS sialic acids in serogroup B meningococci with host effector mechanisms, and suggest that LOS sialylation in meningococci is of a less central importance as it is in gonococci.

Animals↗
PubMed1997

Polymerase chain reaction-based construction of cDNA libraries from minute amounts of third-stage and fourth- and fifth-stage larvae of Dictyocaulus viviparus.

A strategy is described for the amplification and cloning of cDNA from minute amounts of Dictyocaulus viviparus larvae. Initially, third-stage larvae (L3) were used to establish the procedure. Amplification of cDNA synthesized from approximately 400 ng total RNA from 5,000 L3 generated products that were more than 800 bp in length. The unidirectional cloning of amplified cDNA products led to the construction of a UNI ZAP c DNA library with 1 x 10(6) clones. Screening with a homologous oligo(dT)-primed digoxigenin-labeled cDNA probe as well as sequencing of seven randomly picked clones confirmed the successful cloning of lung-worm cDNA. Subsequently, approximately 600 ng total RNA was isolated and polymerase chain reaction (PCR) products of up to 2,400 bp were amplitied from 400 fourth- and fifth-stage larvae (L4/L5). Cloning of these products resulted in a L4/L5 cDNA library of D. viviparus consisting of 5 x 10(5) recombinant clones. In all, 11 clones were randomly picked and sequenced, all revealing typical mRNA/cDNA characteristics. Comparison of the predicted amino acid sequence of the 5' end of clone DvL5/7 revealed 100% homology with the actin gene of several other helminths.

Actins↗
PubMed1997

Non-culture diagnosis and serogroup determination of meningococcal B and C infection by a sialyltransferase (siaD) PCR ELISA.

Rapid, non-culture, serogroup determination of meningococcal infection is important in contact management where vaccination may be possible. The impending availability of polysaccharide-protein conjugate vaccines for serogroup C disease requires maximal case ascertainment, with serogroup determination, at a time when the number of culture confirmed meningococcal infections is decreasing. A polymerase chain reaction assay (PCR), based on a restriction fragment length polymorphism (RFLP) in the meningococcal serogroup B and C sialytransferase (siaD) gene, was developed to combine the non-culture diagnosis of meningococcal infection from CSF, whole blood and serum with serogroup (B and C) identification. The PCR assay was adapted to an ELISA format incorporating hybridization with serogroup-specific B and C oligonucleotide probes. Specificity for CSFs was 100% and sensitivities were respectively 81, 63 and 30% for CSFs, whole blood and sera. The serogroup-specific PCR ELISA is a significant addition to currently available tests for non-culture diagnosis of meningococcal infection and outbreak investigation.

Base Sequence↗
PubMed1997

Complement factor C3 deposition and serum resistance in isogenic capsule and lipooligosaccharide sialic acid mutants of serogroup B Neisseria meningitidis.

Serogroup B meningococci express sialic acids on their surfaces as a modification of the lipooligosaccharide (LOS) and as capsular material consisting of alpha2,8-linked sialic acid homopolymers. The aim of this study was to elucidate the impact of each sialic acid component on the deposition of complement factor C3 and serum resistance. For this purpose, we used isogenic mutants deficient in capsule expression (a polysialyltransferase mutant) or sialylation of the LOS (a galE mutant) or both (a mutant with a deletion of the cps gene locus). Bactericidal assays using 40% normal human serum (NHS) demonstrated that both the capsule and LOS sialic acid are indispensable for serum resistance. By immunoblotting with monoclonal antibody MAb755 that is specific for the C3 alpha-chain, we were able to demonstrate that C3 from 40% NHS was covalently linked to the surface structures of meningococci as C3b and iC3b, irrespective of the surface sialic acid compounds. However, C3b linkage was more pronounced and occurred on a larger number of target molecules in galE mutants with nonsialylated LOS than in meningococci with wild-type LOS, irrespective of the capsule phenotype. C3b deposition was caused by both the classical pathway (CP) and the alternative pathway of complement activation. Use of 10% NHS revealed that at low serum concentrations, C3 deposition occurred via the CP and was detected primarily on nonsialylated-LOS galE mutants, irrespective of the capsular phenotype. Accordingly, immunoglobulin M (IgM) binding to meningococci from heat-inactivated NHS was demonstrated only in both encapsulated and unencapsulated galE mutants. In contrast, inhibition of IgA binding required both encapsulation and LOS sialylation. We conclude that serum resistance in wild-type serogroup B meningococci can only be partly explained by an alteration of the C3b linkage pattern, which seems to depend primarily on the presence of wild-type LOS, since a serum-resistant phenotype also requires capsule expression.

Bacterial Capsules↗
PubMed1997

Use of serum-specific immunoglobulins A and G for detection of Helicobacter pylori infection in patients with chronic gastritis by immunoblot analysis.

Multiple invasive and noninvasive tests for detecting Helicobacter pylori infection are available. The current "gold standard" for the diagnosis of H. pylori infection requires histology and the rapid urease test. Our aim was to test the performance of immunoglobulin A (IgA) and IgG immunoblot assays in comparison with that of the gold standard tests for the diagnosis of H. pylori infection. Ninety patients who underwent gastroscopy were analyzed in a prospective study. Fifty-nine of them were defined to be H. pylori positive by the gold standard tests. The IgA and IgG immunoblot assays correctly identified H. pylori infection in 17 and 58 of these patients, respectively, indicating that determination of IgA antibodies seems to be of low diagnostic value for H. pylori infection. In contrast, the sensitivity and specificity of the IgG immunoblot assay were 98 and 71%, respectively, with positive and negative predictive values of 87 and 96%, respectively. Therefore, the IgG immunoblot assay proved to be a sensitive and useful, noninvasive test for the diagnosis of H. pylori infection.

Adult↗