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M Kist

Publications and source records attributed to M Kist.

At least 37 records · Page 2Linked to original sources
PubMed2000

Identification and molecular analysis of superoxide dismutase isoforms in Helicobacter pylori.

Three electromorphs of iron superoxide dismutase (FeSOD) were identified among 29 Helicobacter pylori isolates by native gel electrophoresis and activity staining. The electromorphs designated isoforms A, B, and C are characterized by slow, intermediate and fast electrophoretic migration, respectively, which was not observed under denaturing conditions. The isoforms were not associated with virulence determinants and with the outcome of disease. Sequence analysis of the sodB gene in strains producing different FeSOD isoforms and comparison of deduced protein sequences revealed that differences in the electric migration behavior are associated with exchange of charged amino acids, suggesting that faster migration is caused by a more negative total charge of the proteins. Electrophoretic migration of native FeSOD was not influenced by changes in the iron cofactor concentration, oxidative stress, and different media, indicating that FeSOD isoforms represent stable strain-specific markers.

Amino Acid Sequence↗
PubMed2000

New approaches for genotyping of Helicobacter pylori based on amplification of polymorphisms in intergenic DNA regions and at the insertion site of the cag pathogenicity island.

The population of the gastric pathogen Helicobacter pylori shows a high degree of genetic diversity. It is well established that heterogeneity at the isolate level is caused by nucleotide transitions within genes, differences in the gene order, and by genetic instability of single genes as well as of a large virulence-associated genomic DNA region, the cag pathogenicity island (PAI). Analysis of intergenic regions with specific PCR-assays developed in this study, revealed that DNA polymorphisms in the noncoding DNA localized in front of the genes ribA and vacA and at the insertion site of the cag PAI contribute to the genetic diversity of H. pylori and are useful for differentiation of individual isolates. Thirteen individual genotypes were identified by PCR analysis of these polymorphic loci in 487, 241, and 182 clinical H. pylori isolates. Sequence analysis revealed that genetic variability in front of genes ribA and vacA, and in the intergenic region at the PAI insertion site is caused by insertion and deletions of so-far-unknown DNA sequences as well as by parts of the H. pylori IS elements IS605 and IS606, respectively. The new genotypes identified could be used to differentiate antrum and corpus isolates from the same patients. Their combination with vacA allele subtypes and with the cagA status allowed to differentiate 140 isolates in 51 subtypes. In 36 cases the corresponding genotype patterns were isolate specific. In summary, the results confirm that DNA polymorphisms in intergenic regions contribute to the genetic diversity of H. pylori. Although individual H. pylori genotypes were not associated with peptic ulcer disease, the PCR-based approaches for their detection developed here should be of use for further investigation of genetic diversity in H. pylori and for epidemiological purposes.

Adult↗
PubMed2000

Secondary resistance among 554 isolates of Helicobacter pylori after failure of therapy.

In a study to determine secondary resistance among Helicobacter pylori isolates, gastroenterologists from several German cities submitted over a 3-year period to centre A (Regensburg) or centre B (Freiburg) gastric biopsies from patients in whom one or more therapies to eradicate Helicobacter pylori had failed. Rates of resistance among the collections of 302 (centre A) and 252 (centre B) isolates were, respectively, as follows: to metronidazole, 75% and 66%; to clarithromycin, 58% and 49%; to amoxicillin, 0%; to ciprofloxacin, 9%; to doxycycline, 0%; and to rifampin, 0%. Resistance to clarithromycin was associated with metronidazole resistance in 89% and 85% of the isolates in centre A and centre B, respectively.

Amoxicillin↗
PubMed2000

Diagnosis of small intestinal bacterial overgrowth in patients with cirrhosis of the liver: poor performance of the glucose breath hydrogen test.

BACKGROUND/AIMS: Small intestinal bacterial overgrowth is known to occur in association with cirrhosis of the liver and studies are needed to assess its pathophysiological role. The glucose breath hydrogen test as an indirect test for small intestinal bacterial overgrowth has been applied to patients with cirrhosis but has not yet been validated against quantitative culture of jejunal secretion in this particular patient population. METHODS: Forty patients with cirrhosis underwent glucose breath hydrogen test and jejunoscopy. Jejunal secretions were cultivated quantitatively for aerobe and anaerobe microorganisms. RESULTS: Small intestinal bacterial overgrowth was detected by culture of jejunal aspirates in 73% of patients, being associated with age and the administration of acid-suppressive therapy. The glucose breath hydrogen test correlated poorly with culture results, sensitivity and specificity ranging from 27%-52% and 36%-80%, respectively. CONCLUSIONS: In patients with cirrhosis, the glucose breath hydrogen test correlates poorly with the diagnostic gold standard for small intestinal bacterial overgrowth. Until other non-invasive tests have been validated, studies addressing the role of small intestinal bacterial overgrowth in patients with cirrhosis should resort to microbiological culture of jejunal secretions.

Adult↗
PubMed2000

Eradication of H. pylori with pantoprazole, clarithromycin, and metronidazole in duodenal ulcer patients: a head-to-head comparison between two regimens of different duration.

BACKGROUND: The study was conducted to compare the efficacy and tolerability of two pantoprazole-based triple therapies of different length in the eradication of H. pylori. METHODS: In this double-blind, multicenter parallel group comparison, H. pylori-positive patients were randomly assigned to either the PCM-7 group (7 days of pantoprazole 40 mg bid, clarithromycin 500 mg bid, metronidazole 500 mg bid) or the PCM-14 m group (modified 14 day therapy of the same regimen with metronidazole only given for 10 days due to labeling reasons). H. pylori status was determined by urease test, histology, culture, and 13C-urea breath test. Treatment outcome was assessed 6 weeks after intake of the last study medication. RESULTS: The following eradication rates were achieved: for PCM-7 in the MITT population 83% (89/107), in the PP population 84% (81/97); for PCM-14 m in MITT 87% (92/106), in PP 88% (91/104). Ulcer healing rates were: for PCM-7 in MITT population 99% (106/107), in the PP population 99% (96/97); for PCM-14 m in MITT 99% (105/106), in PP 99% (103/104). Gastrointestinal symptoms and gastritis scores decreased in both treatment groups. Equivalence of treatment regimens could be proven for all populations. In total, 64 patients reported adverse events. Five serious adverse events occurred, all unrelated to the study medication. CONCLUSION: The two pantoprazole-based triple therapies tested in this study are equally effective in H. pylori eradication, ulcer healing and relief from ulcer pain. It is concluded that 7 days of triple therapy are generally sufficient.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed2000

Regulation of ferritin-mediated cytoplasmic iron storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori.

Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen Helicobacter pylori contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in H. pylori. Analysis of an isogenic fur-negative mutant revealed that H. pylori Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the H. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.

Bacterial Proteins↗
PubMed2000

Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric tissue by fluorescent in situ hybridisation.

BACKGROUND: The development of macrolide resistance in Helicobacter pylori is considered an essential reason for failure of antibiotic eradication therapies. The predominant mechanism of resistance to macrolides, particularly clarithromycin, is based on three defined mutations within 23S rRNA, resulting in decreased binding of the antibiotic to the bacterial ribosome. AIM: To develop an rRNA based whole cell hybridisation method to detect Helicobacter species in situ within gastric tissue, simultaneously with its clarithromycin resistance genotype. METHODS: A set of fluorescent labelled oligonucleotide probes was developed, binding either to H pylori 16S rRNA or 23S rRNA sequences containing specific point mutations responsible for clarithromycin resistance. After hybridisation and stringent washing procedures, labelling of intact single bacteria was monitored by fluorescence microscopy. The new approach was compared with PCR based assays, histology, and microbiological culture. RESULTS: In comparison with the phenotypic resistance measurement by E test, the genotypic clarithromycin resistance correlated perfectly (100%) for 35 H pylori isolates analysed. In a set of gastric biopsy specimens (27) H pylori infection was confirmed by histology (17/27) and correctly detected by whole cell hybridisation. Five clarithromycin resistant strains were identified in gastric tissue specimens directly. Furthermore, non-cultivable coccoid forms of H pylori were easily detectable by whole cell hybridisation. CONCLUSIONS: Whole cell hybridisation of rRNA holds great promise for cultivation independent, reliable, and rapid (three hours) genotypic determination of clarithromycin resistance in H pylori. Compared with PCR techniques it is independent of nucleic acid preparations, not prone to inhibition, and allows semiquantitative visualisation of the bacteria within intact tissue samples.

Anti-Bacterial Agents↗
PubMed1999

Routine identification of Campylobacter jejuni and Campylobacter coli from human stool samples.

Correct identification of Campylobacter jejuni and Campylobacter coli isolates to the species or subspecies level is a cumbersome but nevertheless important task for a routine diagnostic laboratory. The widely used biochemical tests might be often misleading while more sophisticated phenotypic or genotypic methods are not generally available. This investigation was performed to assess the performance of common biochemical identification in comparison with species-specific PCR and gas liquid chromatography of whole cell fatty acid extracts (GLC). A total of 150 consecutive isolates from human stool samples were investigated (134 C. jejuni ssp. jejuni, 14 C. coli, two Helicobacter pullorum). From these 144, 145 and 149 isolates were correctly identified by biochemistry, GLC and PCR, respectively. Biochemical identification of all C. jejuni isolates was confirmed by PCR. GLC detected both H. pullorum strains but misidentified two C. coli strains as C. jejuni and one C. jejuni strain as C. coli. No single method can be defined as 'gold standard' for identification of C. jejuni and C. coli but a combination of techniques is needed. Therefore a stepwise identification scheme starting with biochemical reactions is suggested. All results other than C. jejuni should be confirmed by further methods. For indoxyl acetate-positive isolates species-specific PCR is recommended while GLC seems to be advantageous in indoxyl acetate-negative isolates.

Campylobacter coli↗
PubMed1999

The ferric uptake regulator (Fur) homologue of Helicobacter pylori: functional analysis of the coding gene and controlled production of the recombinant protein in Escherichia coli.

A homologue of the ferric uptake regulator protein Fur has recently been identified within the Helicobacter pylori genome. The promoterless gene on a plasmid did partially complement a fur-negative mutant of Escherichia coli, and was strongly positive in the Fur titration assay (FURTA). The genetic and functional characterization of the complete fur homologue performed in this study revealed that the gene is conserved among H. pylori strains ( > 95% identity), and does not carry nucleotide transitions in iron-resistant mutants of H. pylori. The fur homologue on a plasmid mediated full iron-dependent ferric uptake regulator activity in the fur-deficient mutant strains H1681 and H1780 of E. coli. Immunoblot analysis revealed that Fur from H. pylori cross-reacts with antibodies raised against Fur from E. coli. The fact that inactivation of the fur gene abolished the FURTA-positive phenotype in the E. coli indicator strain H1717, indicated that this phenotype is rather caused by the encoded protein than by real Fur titration. Subcloning of the fur gene into an expression vector allowed controlled production in E. coli, and purification of a recombinant version of the H. pylori Fur protein. In summary, the results confirm the function of the H. pylori Fur homologue as iron-dependent transcriptional repressor by its ability to interact with the Fur-regulated promoters of the genes fiu and fhuF in E. coli.

Amino Acid Sequence↗
PubMed1999

Detection of antibodies against Campylobacter jejuni serogroup PEN O:19 purified flagellar protein in a patient with Guillain-Barré syndrome.

C. jejuni serogroup PEN O:19 was isolated from a stool specimen from a patient with Guillain-Barré syndrome (GBS). Flagellar protein was isolated and purified from reference strain C. jejuni PEN O:19, ATCC 43,446, as well as from a homologous patient strain. Antibodies against flagellar protein were detected by means of immunoblotting, enzyme-linked immunosorbent assay (ELISA) and tube agglutination test. The antibody titres were found to be directly correlated at the beginning and in the recovery phase of GBS. Antibodies of IgG and IgA classes were present from the very onset of the disease as well as 5 months later, but with a lower titre population. However, antibodies of the IgM class were persistent only at the onset of the infection and disappeared during the following 5 months. Our results strongly support the hypothesis that in GBS patients, antiflagellar antibodies are induced during C. jejuni infection and can be used in the diagnosis of C. jejuni-associated GBS.

Adult↗
PubMed1999

High prevalence of antibodies to calreticulin of the IgA class in primary biliary cirrhosis: a possible role of gut-derived bacterial antigens in its aetiology?

BACKGROUND: In a preliminary study we showed that antibodies to the endoplasmic reticulum protein calreticulin (CR) occur in primary biliary cirrhosis (PBC) and autoimmune hepatitis type 1 (AIH). Since anti-CR antibodies have also been found in patients with infectious diseases, we investigated their prevalence and immunoglobulin classes in patients with various hepatic and intestinal diseases, hoping to get some information on a possible relationship between an infectious trigger and the induction of a certain class of anti-CR antibodies. METHODS: Sera were tested for anti-CR antibodies of the IgA, IgG, and IgM class by Western blotting, using CR isolated from human liver: in autoimmune liver diseases (primary biliary cirrhosis (PBC) (n = 86) and autoimmune hepatitis (AIH) type 1 (n = 57)), alcoholic liver cirrhosis (ALC) (n = 32), viral liver infections (acute hepatitis A (n = 8), acute hepatitis B (n = 20), and chronic hepatitis C (n = 28)), and intestinal diseases (Crohn disease (CD) (n = 30), acute yersiniosis (n = 26)). Sera from 100 healthy individuals served as negative controls. RESULTS: The most prominent finding was the high prevalence of anti-CR antibodies of the IgA class and the similarity in the anti-CR antibody class pattern in PBC (IgA, 62%; IgG, 43%; IgM, 55%) and yersiniosis (IgA, 62%; IgG, 39%; IgM, 42%). Class IgA anti-CR antibodies also occurred frequently in ALC (IgA, 44%; IgG, 41%; IgM, 19%). In contrast, in AIH anti-CR antibodies were predominantly of class IgG (IgA, 28%; IgG, 60%; IgM, 33%). In hepatitis A anti-CR antibodies were absent. In the other diseases they had a low prevalence and were mostly of class IgG (acute hepatitis B: IgA, 0%; IgG, 15%; IgM, 0%; chronic hepatitis C: IgA, 7%; IgG, 21%; IgM, 0%; CD: IgA, 13%; IgG, 20%; IgM, 13%). Of the healthy individuals 7% had anti-CR antibodies exclusively of class IgG. CONCLUSIONS: The high prevalence of anti-CR antibodies of class IgA in patients with PBC and yersiniosis as well as in alcoholic liver disease reflects a reactivity of the gut-associated immune system and could imply that a still undefined gut-derived bacterial (?) agent may trigger PBC.

Antigens, Bacterial↗
PubMed1999

Susceptibility in vitro of Helicobacter pylori to cetylpyridinium chloride.

The antimicrobial agent cetylpyridinium chloride (CPC) which is used in therapy of oro-pharyngeal infections and for antiseptic treatment of the oral cavity is active against different bacterial species. Determination of the minimal inhibitory concentration (MIC) using the agar dilution technique revealed that the gastric pathogen Helicobacter pylori in vitro is highly susceptible to CPC as indicated by an MIC of 10 microM (3.4 microg ml(-1)) which was significantly lower than the MIC of CPC against other bacterial species, which were analyzed in comparison to H. pylori. Bacteria of the genus Campylobacter, various Streptococcus spp., Staphylococcus aureus and Escherichia coli showed higher MICs ranging from 100 microM to 2 mM. In summary, this finding renders CPC-containing drugs candidates possibly useful for eradication or for the prevention of transmission of the gastric pathogen.

Agar↗
PubMed1999

Impact of ELISA and immunoblot as diagnostic tools one year after eradication of Helicobacter pylori in a multicentre treatment study.

The performance of serological tests for Helicobacter pylori infections is hampered by the persistence of antibodies after eradication therapy or spontaneous healing. Detection of different antigens or immunoglobulin classes might have an impact on the validity of serodiagnosis. The aim of this study was to assess the decrease in IgA and IgG antibody levels after eradication of H. pylori. Serum samples of 242 patients with active duodenal ulcer were tested with the ELISA and the immunoblot (IB) techniques for H. pylori-specific IgA and IgG antibodies before therapy and 1 year after successful eradication. From a total of 81 patients paired sera were available. At the end of the follow-up period ELISA antibody titres from the IgA class had decreased from a mean value of 6.69 to 4.26 units (P = 0.0001), and IgG class antibody titres from a mean value of 21.9 to 12.1 units (P = 0.0001). Regarding seroreversion, from 34 initially IgA positive sera 16 (47%), and from 74 IgG positive sera 18 (24%), had definitively reverted to 'negative'. One year after eradication, when tested with the immunoblot, the antibody responses against specific antigens of 37% IgA-positive sera (23/62) and 8% IgG-positive sera (6/78) reverted to 'negative', compared to a seroreversion rate of 27% of the anti-CagA IgA-positive sera (18/67) and of 9% of the anti-CagA IgG-positive sera (7/79). In conclusion, despite an overall significant decrease of H. pylori antibodies, both tests cannot be recommended for monitoring treatment success.

Adult↗
PubMed1999

Molecular analysis of riboflavin synthesis genes in Bartonella henselae and use of the ribC gene for differentiation of Bartonella species by PCR.

The biosynthesis pathway for riboflavin (vitamin B(2)), the precursor of the essential cofactors flavin mononucleotide and flavin adenine dinucleotide, is present in bacteria and plants but is absent in vertebrates. Due to their conservation in bacterial species and their absence in humans, the riboflavin synthesis genes should be well suited either for detection of bacterial DNA in human specimens or for the differentiation of pathogenic bacteria by molecular techniques. A DNA fragment carrying the genes ribD, ribC, and ribE, which encode homologues of riboflavin deaminase (RibD) and subunits of riboflavin synthetase (RibC and RibE), respectively, was isolated from a plasmid-based DNA library of the human pathogen Bartonella henselae by complementation of a ribC mutation in Escherichia coli. Sequence analysis of the ribC gene region in strains of B. henselae, which were previously shown to be genetically different, revealed that the ribC gene is highly conserved at the species level. PCR amplification with primers derived from the ribC locus of B. henselae was used to isolate the corresponding DNA regions in B. bacilliformis, B. clarridgeiae, and B. quintana. Sequence analysis indicated that the riboflavin synthesis genes are conserved and show the same operon-like genetic organization in all four Bartonella species. Primer oligonucleotides designed on the basis of localized differences within the ribC DNA region were successfully used to develop species-specific PCR assays for the differentiation of B. henselae, B. clarridgeiae, B. quintana, and B. bacilliformis. The results obtained indicate that the riboflavin synthesis genes are excellent targets for PCR-directed differentiation of these emerging pathogens. The PCR assays developed should increase our diagnostic potential to differentiate Bartonella species, especially B. henselae and the newly recognized species B. clarridgeiae.

Amino Acid Sequence↗
PubMed1998

Discrimination of Helicobacter pullorum and Campylobacter lari by analysis of whole cell fatty acid extracts.

Helicobacter pullorum and Campylobacter lari are rarely isolated from humans with acute enteritis. Hitherto the two species could only be identified by genotypic techniques. Gas liquid chromatography of whole cell fatty acid extracts is described as the first phenotypic method for discrimination of the two species. Cholesteryl glucoside, a characteristic feature of the genus Helicobacter, but seldom found in other bacteria, could not be detected in Helicobacter pullorum. Therefore, rapid determination of this glycolipid may serve as a discrimination marker for Helicobacter pullorum from most other Helicobacter species.

Animals↗
PubMed1998

Cloning and characterization of the fur gene from Helicobacter pylori.

The fur homologue of Helicobacter pylori was isolated by screening a plasmid-based, genomic DNA library using the Fur titration assay (FURTA). The analysis of the DNA sequence revealed significant homology with Fur proteins from various other bacterial species. The highest degree of homology was observed for the Fur protein from Campylobacter jejuni. The H. pylori fur gene on a plasmid could partially complement the fur mutation in Escherichia coli strain H1681. The repressor activity depended on addition of iron to the medium indicating that iron acts as a co-repressor for the H. pylori protein similar to Fur from other bacteria. Comparison of Fur from H. pylori strain NCTC11638 with the recently published genomic DNA sequence of another strain (26695) confirmed the identity of the fur homologue and revealed that the fur locus is highly conserved in both strains.

Amino Acid Sequence↗