Emergence of a Helicobacter pylori isolate with reduced susceptibility to tetracycline in Germany.
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Aged↗
Biomedical subjects
Manfred Kist
Publications and source records attributed to Manfred Kist.
Helicobacter pylori as a prognostic indicator after curative resection of gastric carcinoma: a prospective study.
BACKGROUND: The effect of Helicobacter-pylori status on survival after curative resection for gastric adenocarcinoma is unknown. We aimed to follow-up patients who were positive or negative for infection with H pylori who had curative (ie, R0) resection for gastric adenocarcinoma to assess differences in relapse-free survival and overall survival. METHODS: Before surgery, we assessed the H pylori status of 166 patients who had R0 resection for gastric adenocarcinoma between 1992 and 2002 with bacterial culture, histological analyses (ie, staining with haematoxylin and eosin and with Warthin-Starry), and serological analyses. FINDINGS: At a median follow-up of 53.0 months (range 1-146), relapse-free survival was 56.7 months (95% CI 4.7-108.7) and overall survival was 61.9 months (13.0-110.9) in patients positive for H pylori, compared with 19.2 months (12.7-25.6) and 19.2 months (7.1-31.3), respectively, in patients negative for H pylori (p=0.0009 for difference in relapse-free survival between groups, and p=0.0017 for difference in overall survival between groups). In multivariate analyses, H pylori was an independent prognostic factor for relapse-free survival (hazard ratio 2.16 [95% CI 1.33-3.49]) and overall survival (2.00 [1.22-3.27]). Depth of tumour invasion (2.60 [1.66-4.08]), lymph-node metastasis (2.11 [1.25-3.57]), and patient age 67.5 years or older (1.75 [1.11-2.75]) were also independent prognostic factors for overall survival. INTERPRETATION: Tumour-specific immune responses might be downregulated in patients who are negative for H pylori, and these patients should be followed up carefully because of a poor outlook.
Adenocarcinoma↗
The novel Helicobacter pylori CznABC metal efflux pump is required for cadmium, zinc, and nickel resistance, urease modulation, and gastric colonization.
Maintaining metal homeostasis is crucial for the adaptation of Helicobacter pylori to the gastric environment. Iron, copper, and nickel homeostasis has recently been demonstrated to be required for the establishment of H. pylori infection in animal models. Here we demonstrate that the HP0969-0971 gene cluster encoding the Czc-type metal export pump homologs HP0969, HP0970, and the H. pylori-specific protein HP0971 forms part of a novel H. pylori metal resistance determinant, which is required for gastric colonization and for the modulation of urease activity. Insertional mutagenesis of the HP0971, HP0970, or HP0969 genes in H. pylori reference strain 26695 resulted in increased sensitivity to cadmium, zinc, and nickel (czn), suggesting that the encoded proteins constitute a metal-specific export pump. Accordingly, the genes were designated cznC (HP0971), cznB (HP0970), and cznA (HP0969). The CznC and CznA proteins play a predominant role in nickel homeostasis, since only the cznC and cznA mutants but not the cznB mutant displayed an 8- to 10-fold increase in urease activity. Nickel-specific affinity chromatography demonstrated that recombinant versions of CznC and CznB can bind to nickel and that the purified CznB protein interacted with cadmium and zinc, since both metals competitively inhibited nickel binding. Finally, single cznA, cznB, and cznC mutants did not colonize the stomach in a Mongolian gerbil-based animal model. This demonstrates that the metal export functions of H. pylori cznABC are essential for gastric colonization and underlines the extraordinary importance of metal ion homeostasis for the survival of H. pylori in the gastric environment.
Animals↗
Novel plasmids for gene expression analysis and for genetic manipulation in the gastric pathogen Helicobacter pylori.
To facilitate gene expression analysis in the human gastric pathogen Helicobacter pylori, we constructed the plasmids pHPLAC-KAN and pHPLAC-CAT containing a promoterless Escherichia coli lacZ gene located upstream from the antibiotic resistance genes aphA-3 or cat, respectively. The suitability of the plasmids for H. pylori mutagenesis and gene expression analysis was evaluated by plasmid integration into the genome of H. pylori strain 1061 by single homologous recombination, using the rpl9 gene encoding ribosomal protein L9 as target. By monitoring beta-galactosidase production from the resulting rpl9::lacZ fusion, it was demonstrated that H. pylori rpl9 displays the classical growth phase-dependent regulation of components of the protein synthesis machinery, as beta-galactosidase production dropped fivefold in the stationary growth phase. The plasmids described in this study extend our methodological repertoire for genetic modification and molecular analysis of H. pylori, and may also be of use for other bacteria, as the resistance cassettes and the lacZ gene are active in the related Campylobacter species.
Bacterial Proteins↗
Mutational analysis of the Helicobacter pylori carbonic anhydrases.
In the gastric microenvironment, Helicobacter pylori is exposed to bicarbonate, urea and acid. Here it is demonstrated that both H. pylori carbonic anhydrases (CAs) are required for maintaining urease activity and therefore influence H. pylori urea resistance at neutral pH. Furthermore, the beta-CA is required for acid resistance as indicated by a growth defect of the corresponding mutant at low pH. The alpha- and beta-CA mutants as well as the double mutant were more resistant to bicarbonate, indicating that both enzymes are involved in bicarbonate metabolism. These phenotypes support important CA-functions in H. pylori urea and bicarbonate metabolism and acid resistance. Thus, both CA enzymes might be required for survival in the gastric niche.
Bicarbonates↗
How effective is moxifloxacin for the first-line treatment of patients with Helicobacter pylori infection?
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Journal Article↗
Transcriptional profiling of Helicobacter pylori Fur- and iron-regulated gene expression.
Intracellular iron homeostasis is a necessity for almost all living organisms, since both iron restriction and iron overload can result in cell death. The ferric uptake regulator protein, Fur, controls iron homeostasis in most Gram-negative bacteria. In the human gastric pathogen Helicobacter pylori, Fur is thought to have acquired extra functions to compensate for the relative paucity of regulatory genes. To identify H. pylori genes regulated by iron and Fur, we used DNA array-based transcriptional profiling with RNA isolated from H. pylori 26695 wild-type and fur mutant cells grown in iron-restricted and iron-replete conditions. Sixteen genes encoding proteins involved in metal metabolism, nitrogen metabolism, motility, cell wall synthesis and cofactor synthesis displayed iron-dependent Fur-repressed expression. Conversely, 16 genes encoding proteins involved in iron storage, respiration, energy metabolism, chemotaxis, and oxygen scavenging displayed iron-induced Fur-dependent expression. Several Fur-regulated genes have been previously shown to be essential for acid resistance or gastric colonization in animal models, such as those encoding the hydrogenase and superoxide dismutase enzymes. Overall, there was a partial overlap between the sets of genes regulated by Fur and those previously identified as growth-phase, iron or acid regulated. Regulatory patterns were confirmed for five selected genes using Northern hybridization. In conclusion, H. pylori Fur is a versatile regulator involved in many pathways essential for gastric colonization. These findings further delineate the central role of Fur in regulating the unique capacity of H. pylori to colonize the human stomach.
Bacterial Proteins↗
Real-time PCR screening for 16S rRNA mutations associated with resistance to tetracycline in Helicobacter pylori.
The effectiveness of recommended first-line therapies for Helicobacter pylori infections is decreasing due to the occurrence of resistance to metronidazole and/or clarithromycin. Quadruple therapies, which include tetracycline and a bismuth salt, are useful alternative regimens. However, resistance to tetracycline, mainly caused by mutations in the 16S rRNA genes (rrnA and rrnB) affecting nucleotides 926 to 928, are already emerging and can impair the efficacies of such second-line regimens. Here, we describe a novel real-time PCR for the detection of 16S rRNA gene mutations associated with tetracycline resistance. Our PCR method was able to distinguish between wild-type strains and resistant strains exhibiting single-, double, or triple-base-pair mutations. The method was applicable both to DNA extracted from pure cultures and to DNA extracted from fresh or frozen H. pylori-infected gastric biopsy samples. We therefore conclude that this real-time PCR is an excellent method for determination of H. pylori tetracycline resistance even when live bacteria are no longer available.
DNA, Bacterial↗
Iron-responsive regulation of the Helicobacter pylori iron-cofactored superoxide dismutase SodB is mediated by Fur.
Maintaining iron homeostasis is a necessity for all living organisms, as free iron augments the generation of reactive oxygen species like superoxide anions, at the risk of subsequent lethal cellular damage. The iron-responsive regulator Fur controls iron metabolism in many bacteria, including the important human pathogen Helicobacter pylori, and thus is directly or indirectly involved in regulation of oxidative stress defense. Here we demonstrate that Fur is a direct regulator of the H. pylori iron-cofactored superoxide dismutase SodB, which is essential for the defense against toxic superoxide radicals. Transcription of the sodB gene was iron induced in H. pylori wild-type strain 26695, resulting in expression of the SodB protein in iron-replete conditions but an absence of expression in iron-restricted conditions. Mutation of the fur gene resulted in constitutive, iron-independent expression of SodB. Recombinant H. pylori Fur protein bound with low affinity to the sodB promoter region, but addition of the iron substitute Mn2+ abolished binding. The operator sequence of the iron-free form of Fur, as identified by DNase I footprinting, was located directly upstream of the sodB gene at positions -5 to -47 from the transcription start site. The direct role of Fur in regulation of the H. pylori sodB gene contrasts with the small-RNA-mediated sodB regulation observed in Escherichia coli. In conclusion, H. pylori Fur is a versatile regulator involved in many pathways essential for gastric colonization, including superoxide stress defense.
Bacterial Proteins↗
The Helicobacter pylori CrdRS two-component regulation system (HP1364/HP1365) is required for copper-mediated induction of the copper resistance determinant CrdA.
Here we describe that the Helicobacter pylori sensor kinase produced by HP1364 and the response regulator produced by HP1365 and designated CrdS and CrdR, respectively, are both required for transcriptional induction of the H. pylori copper resistance determinant CrdA by copper ions. CrdRS-deficient mutants lacked copper induction of crdA expression and were copper sensitive. A direct role of CrdR in transcriptional regulation of crdA was confirmed by in vitro binding of CrdR to the crdA upstream region. A 21-nucleotide sequence located near the crdA promoter was shown to be required for CrdR binding.
Bacterial Proteins↗
Effect of Helicobacter pylori eradication on high-density lipoprotein cholesterol.
We examined the effect of Helicobacter pylori (H. pylori) eradication on lipids and apolipoproteins in 87 patients with duodenal ulcers. A significant increase was observed in high-density lipoprotein (HDL) cholesterol (+24.7%, p <0.001), apolipoprotein AI (+9.0%, p <0.001), and apolipoprotein AII (+11.7%, p <0.001) after eradication. Minor increases occurred in total cholesterol, triglycerides, and apolipoprotein B, whereas low-density lipoprotein cholesterol remained unchanged. Our results suggest that chronic H. pylori infection reduces plasma levels of HDL cholesterol and that eradication improves the lipoprotein pattern.
Apolipoprotein A-I↗
Pathogenesis of Helicobacter pylori infection.
Research in the last year has provided new insights into the function of the the cag-associated type IV secretion system and the vacuolating toxin VacA. A quite new aspect was disclosed by the finding that Helicobacter pylori in Mongolian gerbils colonizes a very distinct topology in the gastric mucous layer, obviously providing optimal conditions for long-term survival. Further research activities focused on H. pylori ammonia and metal metabolism as well as on bacterial stress defence mechanisms. Differential expression of approximately 7% of the bacterial genome was found at low pH suggesting that H. pylori has evolved a multitude of acid-adaptive mechanisms. VacA was shown to interrupt phagosome maturation in macrophage cell lines as well as to modulate and interfere with T lymphocyte immunological functions. Gastric mucosa as well as the H. pylori-infected epithelial cell line AGS strongly express IL-8 receptor A and B, which might contribute to the augmentation of the inflammatory response. Accumulating evidence implicates genetic variation in the inflammatory response to H. pylori in the etiology of the increased risk of gastric cancer after H. pylori infection. The chronic imbalance between apoptosis and cell proliferation is the first step of gastric carcinogenesis. In this regard, it was demonstrated that coexpression of two H. pylori proteins, CagA and HspB, in AGS cells, caused an increase in E2F transcription factor, cyclin D3, and phosphorylated retinoblastoma protein. Taken together, we now have a better understanding of the role of different virulence factors of H. pylori. There is still a lot to be learned, but the promising discoveries summarized here, demonstrate that the investigation of the bacterial survival strategies will give novel insights into pathogenesis and disease development.
Adaptation, Physiological↗
Rapid detection of point mutations in the gyrA gene of Helicobacter pylori conferring resistance to ciprofloxacin by a fluorescence resonance energy transfer-based real-time PCR approach.
As a result of the increasing resistance of Helicobacter pylori against first-line antibiotics, other drugs, such as quinolones, will be needed for eradication therapy in the future. We developed a real-time PCR to detect mutations in the gyrA gene associated with ciprofloxacin resistance of H. pylori, thereby contributing to the selection of patients who could be treated by ciprofloxacin-based therapy.
Anti-Infective Agents↗
Analysis of the rdxA gene in high-level metronidazole-resistant clinical isolates confirms a limited use of rdxA mutations as a marker for prediction of metronidazole resistance in Helicobacter pylori.
Metronidazole (Mtz) resistance in the gastric pathogen Helicobacter pylori is closely associated with inactivation of the nitroreductase gene rdxA. In order to identify respective mutations for diagnostic purposes we analyzed the rdxA gene in a collection of high-level Mtz-resistant clinical H. pylori isolates. Size alterations in the rdxA gene region were found in only two out of 45 and one out of 40 isolates showing lower-level (minimal inhibitory concentrations (MICs) 32-192 microg ml(-1)) and high-level (MIC>/=256 microg ml(-1)) Mtz resistance, respectively. Point mutations that interrupt the rdxA reading frame were detected in two out of eight high-level resistant isolates (MICs>/=256 microg ml(-1)). Most remarkably, the rdxA gene sequence was found to be identical in four out of five high-level Mtz-resistant and -susceptible paired H. pylori isolates from the same patients each. Taken together, these results demonstrate that although some isolates carry classical resistance-associated rdxA mutations, as described earlier, the use of rdxA mutations as a marker for prediction of Mtz resistance is limited.
Anti-Bacterial Agents↗
Identification of beta-subunit of bacterial RNA-polymerase--a non-species-specific bacterial protein--as target of antibodies in primary biliary cirrhosis.
Several observations suggest that bacteria induce autoimmunity in primary biliary cirrhosis (PBC). Since no PBC-specific bacterial species could be identified, it can be speculated that the triggers are non-species-specific bacterial proteins. This hypothesis would imply that several or even all bacterial species can trigger PBC. Therefore, we investigated whether PBC exhibits immune reactions to non-species-specific bacterial antigens. Yersinia enterocolitica O3 was screened for the presence of proteins that were labeled by immunoblotting using PBC sera. We focused our investigations on a 160-kDa protein, which was further enriched and characterized by partial N-terminal amino acid sequencing. The prevalence of antibodies to this protein was determined by immunoblotting in a variety of diseases. The 160-kDa protein was identified as the beta-subunit of bacterial RNA-polymerase, a highly conserved bacterial protein with a very high degree of sequence identity among all bacterial species. Antibodies to the beta-subunit of bacterial RNA polymerase were specific for this protein. Until now no mammalian protein could be found that cross-reacts with these antibodies. The prevalence of antibodies to the beta-subunit of bacterial RNA polymerase (ARPA) using the protein from Yersinia enterocolitica O3 (serum dilution 1:1000) was: healthy controls (HC, N = 101) 7.9%, primary biliary cirrhosis (PBC, N = 61) 32.8%, autoimmune hepatitis type 1 (AIH, N = 46) 26.1%, alcoholic liver cirrhosis (ALC, N = 44) 9.1%, Crohn's disease (CD, N = 38) 7.9%, ulcerative colitis (UC, N = 24) 8.3%, primary sclerosing cholangitis + UC (PSC/UC, N = 11) 0%, acute yersiniosis (Yers, N = 36) 19.4%, acute infection with Campylobacter jejuni (Camp, N = 10) 0%, acute Q-fever (QF, N = 16) 6.25%, chronic hepatitis C (HCV, N = 39) 7.7%, c-ANCA-positive vasculitis (Vasc, N = 40) 15%, systemic lupus erythematosus (SLE, N = 28) 10.7%, and malaria tropica (MT, N = 24) 16.7%. There was no significant difference between PBC and AIH. The group of autoimmune liver diseases (PBC + AIH, N = 107, 29.9%) differed highly significantly from HC, chronic inflammatory bowel diseases (CD + UC + PSC/UC, N = 73, 6.8%), ALC, and HCV and also differed significantly (P = 0.01) from the group with bacterial and parasitic diseases (Yers + Camp + QF + MT, N = 86,13.95%) and from the group with Vasc + SLE (N = 68,13.2%). Testing of ARPA using the protein from E. coli yielded nearly identical results. In conclusion, an increased prevalence of antibodies to the beta-subunit of bacterial RNA polymerase, a highly conserved non-species-specific bacterial protein, can be found in primary biliary cirrhosis, but also in autoimmune hepatitis type I. These findings do not add an argument for a bacterial trigger of PBC. Rather, they suggest that ARPA belong to the pool of natural antibodies that are up-regulated in autoimmune liver diseases.
Antibodies, Bacterial↗
Recent developments in Campylobacter pathogenesis.
PURPOSE OF REVIEW: The Campylobacter species jejuni and coli are leading causes of enteritis and enterocolitis worldwide. Arthritis, Reiter syndrome, and Guillain-Barré syndrome represent post-infectious sequelae. Although the acute and chronic clinical manifestations highlight Campylobacter species as excellent models for the identification of mechanisms involved in pathogenesis, detailed investigations at the molecular level are complicated by the fastidious growth requirements of the bacteria and by the tremendous variability displayed by Campylobacter isolates. Thus, research activities in this field constitute a substantial challenge for scientists of many different disciplines. The genome information has greatly stimulated investigations at the molecular level and the resulting modern research trends lead to a better understanding of Campylobacter-associated diseases providing the basis for new developments in prevention and therapy. RECENT FINDINGS: This review summarizes results from the most recent investigations in the field of Campylobacter pathogenesis. Topics include genome analysis, surface structures and post-infectious complications, adaptation, host cell interaction and cell toxicity. SUMMARY: During its coevolution with human and other vertebrate hosts, Campylobacter species have developed specific survival strategies, which are required for host adaptation and establishment in the intestinal environment. The bacterial factors involved in these processes are the subject of intensive research activities. With a focus on molecular aspects of the most important human pathogen, C. jejuni, this review intends to summarize the recent trends and developments in Campylobacter research by highlighting selected publications in the field of microbial pathogenesis.
Arthritis, Reactive↗
Differential regulation of amidase- and formamidase-mediated ammonia production by the Helicobacter pylori fur repressor.
The production of high levels of ammonia allows the human gastric pathogen Helicobacter pylori to survive the acidic conditions in the human stomach. H. pylori produces ammonia through urease-mediated degradation of urea, but it is also able to convert a range of amide substrates into ammonia via its AmiE amidase and AmiF formamidase enzymes. Here data are provided that demonstrate that the iron-responsive regulatory protein Fur directly and indirectly regulates the activity of the two H. pylori amidases. In contrast to other amidase-positive bacteria, amidase and formamidase enzyme activities were not induced by medium supplementation with their respective substrates, acrylamide and formamide. AmiE protein expression and amidase enzyme activity were iron-repressed in H. pylori 26695 but constitutive in the isogenic fur mutant. This regulation was mediated at the transcriptional level via the binding of Fur to the amiE promoter region. In contrast, formamidase enzyme activity was not iron-repressed but was significantly higher in the fur mutant. This effect was not mediated at the transcriptional level, and Fur did not bind to the amiF promoter region. These roles of Fur in regulation of the H. pylori amidases suggest that the H. pylori Fur regulator may have acquired extra functions to compensate for the absence of other regulatory systems.
Amidohydrolases↗
Molecular microbiology and pathogenesis of Helicobacter and Campylobacter updated: a meeting report of the 11th conference on Campylobacter, Helicobacter and related organisms.
The genome analysis of the gastrointestinal pathogens Helicobacter and Campylobacter has stimulated a wealth of new research activities, which are presented every 2 years at the international conferences on Campylobacter, Helicobacter and Related Organisms (CHRO). Both organisms represent excellent models for the identification of new molecular mechanisms involved in pathogenesis, host response and physiological adaptation in course of acute and chronic infectious diseases. The investigation of their global distribution, pronounced genetic and antigenic diversity as well as the molecular mechanisms allowing long-term persistence in hostile and unusual microbial habitats, is a challenge for scientists of many different disciplines world-wide. With a focus on the molecular microbiology aspects, this review summarizes recent trends in Helicobacter and Campylobacter research by highlighting selected presentations at the 11th CHRO conference. The topics include the discovery of new virulence factors, functional analysis of protein secretion systems, host signalling pathways, adaptation to stress conditions, global gene regulation, and genetic variability.
Animals↗