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OBJECTIVES The objective of this study was to assess the outcome of antimicrobial susceptibility-guided therapies in Helicobacter pylori-infected individuals who had undergone unsuccessful prior eradication treatments. METHODS From October 2004 to December 2013, 481 H. pylori-positive patients with prior unsuccessful eradication treatments were administered susceptibility-guided salvage eradication treatments. Six months on, treatment outcome was assessed by urea breath test, stool antigen ELISA, Helicobacter urease test or microbiology and/or histopathology. RESULTS Resistance to metronidazole and clarithromycin was high in patients with prior unsuccessful eradication treatments and was dependent on the number of treatment failures. Susceptibility-guided salvage eradication treatments achieved eradication rates of nearly 70% in these patients. No particular regimen was significantly better than another. CONCLUSIONS Antimicrobial susceptibility testing prevents prescription of inefficient antimicrobials and enables individualized and promising salvage treatments in patients with prior unsuccessful eradication treatments.

Objectives: The aim of this study was to update data on levofloxacin/ciprofloxacin and triple resistance (resistance to metronidazole, clarithromycin and levofloxacin/ciprofloxacin) in Helicobacter pylori clinical isolates and to identify the impact of prior eradication therapies on their development. Methods: We tested the antimicrobial susceptibility to amoxicillin, metronidazole, clarithromycin, levofloxacin/ ciprofloxacin, tetracycline and rifampicin of 5296 clinical H. pylori strains isolated between 2006 and 2011. Information on prior eradication therapies was gathered and their impact on the development of antimicrobial resistance, in particular to levofloxacin/ciprofloxacin and triple resistance, was analysed. Results: From 2006 onwards, both levofloxacin/ciprofloxacin and triple resistance have steadily increased and peaked in 2011 with 29.1% and 18.6%, respectively. Unsuccessful prior eradication attempts proved a major risk factor for resistance development. Patients who had undergone unsuccessful eradication attempts harboured levofloxacin/ciprofloxacin- and triple-resistant isolates significantly more often than untreated individuals (26.7% and 18.1% versus 10.6% and 1.6%). Levofloxacin/ciprofloxacin and triple resistance occurred significantly more often in patients who had received quinolones when compared with patients who had not (44.5% versus 23.1% and 28.7% versus 15.6%). We did not observe any significant differences in resistance rates in the different German federal states. Conclusions: Resistance to levofloxacin/ciprofloxacin and triple resistance have continuously risen and reached worrying numbers. Hence we strongly advise against the use of quinolones in empirical second-line therapies for H. pylori without prior susceptibility testing and/or a carefully taken patient medical history.

Helicobacter felis belongs to the fastidious gastric non-Helicobacter pylori helicobacter species that are typically found in the stomach of cats and dogs. These bacteria have the potential to colonize the human stomach and are then associated with gastritis, gastroduodenal ulcers, and MALT lymphoma. Strains cultured from the human stomach are rare. Here, we present the first isolation of H. felis from a gastric biopsy specimen of a 14-year-old girl who presented with persistent epigastric pain. The strain was cultured using our routine protocol for H. pylori and identified by phylogenetic analyses of partial urease AB and gyrB gene sequences.

Vom Robert Koch-Institut werden in Abstimmung mit dem Bundesministerium fur Gesundheit seit dem Jahr 1995 in Deutschland verstarkt Nationale Referenzzentren und Konsiliarlaboratorien zur laborgestutzten Uberwachung ausgewahlter Infektionserreger und Syndrome berufen. Sie sollen zu einem effizienten Infektionsschutz beitragen und die infektionsepidemiologische Surveillance durch die mikrobiologische Uberwachung bestimmter Erreger erganzen. Gegenwartig bestehen 19 Nationale Referenzzentren und 48 Konsiliarlaboratorien, die jeweils fur eine Dauer von drei Jahren berufen werden. Um die bisherige erfolgreiche Arbeit der Nationalen Referenzzentren und Konsiliarlaboratorien weiter zu optimieren und Synergien mehr als bisher zu nutzen, wurden im Jahr 2009 insgesamt zehn Referenznetzwerke etabliert, in denen Nationale Referenzzentren mit assoziierten Konsiliarlaboratorien im Rahmen wissenschaftlicher Projekte zusammenarbeiten. Das Ziel der Netzwerke besteht insbesondere darin, den Erfahrungsaustausch zu diagnostischen Methoden und Praventionsansatzen zu fordern und die Reprasentativitat in der Flache zu verbessern. Durch die Netzwerke besteht zudem die Moglichkeit, erregerubergreifende Fragestellungen noch effizienter zu bearbeiten. In Zukunft sollen auch externe nationale und internationale Spezialisten verstarkt einbezogen werden. Die Arbeit der Netzwerke wird durch die Kommission Infektionsepidemiologie evaluiert. Sie arbeitet die Modalitaten der Forderung aus, um die Zusammenarbeit zwischen Nationalen Referenzzentren und Konsiliarlaboratorien zu unterstutzen und die Strukturen den Erfordernissen auf nationaler und internationaler Ebene anzupassen.

The world-wide increase in Clostridium difficile-associated infections (CDI) and the more severe course of infection being observed have recently raised the level of attention given to this problem. In particular, CDI should be included in the differential diagnosis of watery diarrhoea. Laboratory detection methods should include microbiological culture for increased sensitivity. Antibiotic therapy is still a primary risk factor for the development of CDI and it appears that the increasing use of fluoroquinolones plays a particular role with regard to the hypervirulent strains of C. difficile (ribotype 027 and 078). Rational use of antibiotics and compliance with infection control measures are essential to prevent CDI. Since C. difficile produces highly resistant spores, special attention must be devoted to disinfection and hand hygiene. Furthermore, surveillance of cases in hospital is required to implement the necessary infection control measures.

Introduction: In September 2007 an increase of severe Clostridium difficile-associated infections (CDI) was noticed in a hospital in the city of Trier, Germany. It was assumed that a new, possibly hypervirulent strain (PCR ribotype 027) was related to these events. An outbreak investigation was initiated by the local health authorities and the Robert Koch Institute to describe the epidemiology of the possible outbreak and to identify and control the possible sources. Methods: In addition to retrospective case-finding of severe CDI and ribotype 027 infections by analysis of patient documents and certificates of death, an active surveillance system for severe CDI and ribotype 027 infections was established in the 6 hospitals of the affected region. In all suspected cases, a test for toxin A/B and a stool culture for C. difficile were conducted simultaneously. Bacterial isolates were further characterised by PCR ribotyping. Data on the course of disease, case fatality, and possible risk factors for CDI-related deaths were assessed using a standardised questionnaire. Environmental investigations were done. Results: By 31 January 2008, 27 cases of severe CDI and 21 cases with C. difficile ribotype 027 infections were found in the area under investigation. Active surveillance found 76 of 399 (19%) patients positive for C. difficile. In 20 patients, PCR ribotyp 027 could be proven. In total, 9 deaths occurred (19%). An existing immunosupressive therapy (OR 35.8; 95% CI2.8-464.5) was related to case fatality in the multivariate analysis. Severe cases of CDI were also observed in non-ribotype 027 infections. In the screening of hospital personnel (n = 161), 6% were found positive for toxin A/B. Discussion: This investigation demonstrated the endemicity of C. difficile PCR ribotyp 027 in Germany for the first time. As a consequence from this study, severe CDI became a reportable disease in Germany at the end of 2007. In addition to hygienic measures, the critical use of antibiotics is an important measure to prevent a further increase of CDI.

BACKGROUND Antimicrobial susceptibility of Helicobacter (H.) pylori is usually determined by phenotypic methods. When H. pylori cannot be grown owing to contaminations or delay in transport of gastric tissue samples to the microbiological laboratory, molecular genetic testing is a reasonable alternative. The aim of this retrospective study was to assess the outcome of salvage eradication treatments based on molecular genetic susceptibility testing. METHODS Data on 144 H. pylori PCR-positive gastric tissue samples of patients primarily with prior unsuccessful eradication treatments were retrospectively analyzed. Eradication treatments were recommended based on genotypic clarithromycin and/or levofloxacin susceptibility as tested by real-time PCR or reverse hybridization. Treatment success was assessed by attending physicians using urea breath test; stool-antigen ELISA; and microbiology/histopathology. RESULTS Overall success rate of molecular genetic testing-guided salvage treatments was low (68%); none of the regimens chosen was significantly better than another. Multivariable logistic regression analysis did not reveal any factors that may predict treatment failure. CONCLUSIONS Eradication success was poor despite susceptibility testing. Gastroenterologists are advised to prescribe recommended salvage treatments, considering recommended dosages and prolonged treatment duration.

The German Federal Ministry of Health has funded National Reference Centres (NRC) for laboratory-based surveillance of selected infection pathogens and infections disease syndromes. This selection is based on the epidemiologic relevance of the pathogens, specific diagnostic requirements, antimicrobial resistance and need for public health measures. Currently there are 18 NRC, nominated for a duration of 3 years. Toward the end of a nomination period, each NRC is evaluated by an expert committee, based on the catalogue of core tasks. In order to expand the spectrum of competencies 47 consiliary laboratories on additional pathogens of special epidemiologic importance have been named. Their main function is to provide information and consultation on special diagnostic issues. In order to further improve the effectiveness and cooperation of the system Networks have been created. The aim of the Networks is to facilitate exchange of diagnostic methods and prevention concepts and to improve the geographic coverage of the services.

This guideline updates a prior concensus recommendation of the German Society for Digestive and Metabolic Diseases (DGVS) from 1996. It was developed by an interdisciplinary cooperation with representatives of the German Society for Microbiology, the Society for Pediatric Gastroenterology and Nutrition (GPGE) and the German Society for Rheumatology. The guideline is methodologically based on recommendations of the Association of the Scientific Medical Societies in Germany (AWMF) for providing a systematic evidence-based consensus guideline of S 3 level and has also implemented grading criteria according to GRADE (Grading of Recommendations Assessment, Development and Evaluation). Clinical applicability of study results as well as specifics for Germany in terms of epidemiology, antibiotic resistance status, diagnostics and therapy were taken into account.

Helicobacter pylori lipopolysaccharide (HP-LPS) is a potent virulence factor in the causation of gastric ulcer and gastritis. H. pylori-induced gastric pathology is prevalent throughout the world. Herbal medicines are attracting attention because of their traditional values, popularity and belief, as well as for their advantages such as less toxicity, affordability and medicinal value. The present study aimed to evaluate the anti-ulcer effect of a methanolic extract of Terminalia arjuna (TA) against HP-LPS-induced gastric damage in rats. Ulcers were induced with HP-LPS (50 μg per animal) administered orally daily for 3 days. The efficacy of TA on gastric secretory parameters such as volume of gastric juice, pH, free and total acidity, pepsin concentration, and the cytoprotective parameters such as protein-bound carbohydrate complexes in gastric juice and gastric mucosa was assessed. The protective effect of TA was also confirmed by histopathological examination of gastric mucosa. HP-LPS-induced alterations in gastric secretory parameters were altered favourably in rats treated with TA, suggesting that TA has an anti-secretory role. Furthermore, HP-LPS-induced impairments in gastric defence factors were also prevented by treatment with TA. These results suggest that the severe cellular damage and pathological changes caused by HP-LPS are mitigated by TA; these effects are comparable with those of sucralfate. The anti-ulcer effect of TA may reflect its ability to combat factors that damage the gastric mucosa, and to protect the mucosal defensive factors.

Recent publications were compared and analysed in addition to novel surveillance data to assess the hypothesis that fluoroquinolone-resistant Campylobacter infections are causing more severe disease than susceptible infections. The available data did not support this hypothesis. There was no significant difference in duration of disease between susceptible and resistant infections. However, both for resistant and susceptible infections, disease symptoms were prolonged by on average 1–2 days for Campylobacter cases acquired during foreign travel. Nevertheless, the likelihood and duration of hospitalisation were not increased for cases related to foreign travel. These observations were confirmed by a new analysis of almost 11000 cases. We conclude that fluoroquinolone-resistant Campylobacter infections are not more severe than susceptible infections.

The aim of this study was to assess the rate of rifampicin resistance in Helicobacter pylori isolated from patients in Germany, to detect rifampicin resistance-associated mutations and to identify non-resistance-associated genetic variants in the rpoB gene.Susceptibility to rifampicin in a total of 1585 clinical isolates obtained between January 2003 and July 2006 was tested by disc diffusion and/or by the Etest method. The rpoB genes of a selection of both resistant (n=17) and susceptible (n=100) clinical isolates were sequenced in order to distinguish between resistance- and non-resistance-associated genetic alterations. In vitro mutagenesis experiments such as site-directed mutagenesis were carried out to demonstrate the pivotal role of rpoB mutations in rifampicin resistance.From 1585 clinical isolates examined, 22 (1.4%) showed phenotypic resistance to rifampicin (MIC4 mg/L). The majority of the resistant strains harboured point mutations in their rpoB genes at codons 530, 540 and 545 and showed cross-resistance to rifabutin. Four clinical isolates with moderate rifampicin resistance (8 mg/L) showed a rifabutin-susceptible phenotype and did not harbour any mutation in the sequenced rpoB fragments. Sequence analysis of 100 rifampicin-susceptible isolates revealed numerous novel silent mutations in the rpoB genes resulting in amino acid exchanges, but not in resistance.Resistance to rifampicin/rifabutin in H. pylori strains isolated in Germany is still low and is associated with mutations in the rpoB gene. Further surveillance studies analysing the use of rifabutin in H. pylori eradication and its association with the occurrence of rifabutin-resistant strains are required.

We show that quinolone resistance in Helicobacter pylori has reached an alarming level in Germany. Our data suggest that the use of quinolones requires prior antimicrobial susceptibility testing, especially for isolates from patients who have already undergone previous unsuccessful eradication treatments, and also underline the further need for surveillance studies to monitor antibiotic resistance in H. pylori .

Summary 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.

More than one-half of the world population is infected with Helicobacter pylori. Of those, approx. 500,000 die from gastric carcinoma every year. Ulcer disease, gastricatrophy and the rare MALT lymphoma are other sequelae of H. pylori infection. H. pylori possesses an array of virulence factors that include urease, flagellar motility, adhesins, the vacuolating cytotoxin VacA and the protein CagA. The gene encoding CagA is located on the cag pathogenicity island, comprising 29 genes the majority of which encodes components of a type IV secretion system capable of translocating CagA into epithelial cells where it interferes with cellular signal transduction processes. A number of diagnostic tests for H. pylori infection require gastroendoscopy. These include the biopsy urease test, histology, culture with susceptibility testing, and molecular detection methods such as fluorescent in situ hybridization. Non-invasive tests that do not require endoscopy include the (13)C urea breath test, H. pylori stool antigen ELISA and serology. The latter is unsuitable for treatment follow-up, since antibody titres persist up to a year after successful treatment. When patients have never been treated for H. pylori infection, biopsy urease test and histology are usually sufficient for diagnosis. In patients where endoscopy is not required, H. pylori infection can be reliably detected by (13)C urea breath test, stool antigen ELISA or serology. Patients who have under gone one or more unsuccessful cycles of eradication therapy in most cases harbour H. pylori resistant to one or several antibiotics. In these patients, culture and antibiotic susceptibility testing are indicated. Patients who have never been treated for H. pylori infection usually harbour susceptible strains. In such patients, classic "Italian" or "French" triple therapies may achieve eradication in >90% of cases. In the case of treatment failure, second-line antibiotic treatment regiments (rescue therapy) are used, optimally guided by susceptibility data.

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.

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 Mn 2+ 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.

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.

The important human pathogen Helicobacter pylori requires the abundant expression and activity of its urease enzyme for colonization of the gastric mucosa. The transcription, expression, and activity of H. pylori urease were previously demonstrated to be induced by nickel supplementation of growth media. Here it is demonstrated that the HP1338 protein, an ortholog of the Escherichia coli nickel regulatory protein NikR, mediates nickel-responsive induction of urease expression in H. pylori . Mutation of the HP1338 gene ( nikR ) of H. pylori strain 26695 resulted in significant growth inhibition of the nikR mutant in the presence of supplementation with NiCl 2 at ≥100 μM, whereas the wild-type strain tolerated more than 10-fold-higher levels of NiCl 2 . Mutation of nikR did not affect urease subunit expression or urease enzyme activity in unsupplemented growth media. However, the nickel-induced increase in urease subunit expression and urease enzyme activity observed in wild-type H. pylori was absent in the H. pylori nikR mutant. A similar lack of nickel responsiveness was observed upon removal of a 19-bp palindromic sequence in the ureA promoter, as demonstrated by using a genomic ureA :: lacZ reporter gene fusion. In conclusion, the H. pylori NikR protein and a 19-bp operator sequence in the ureA promoter are both essential for nickel-responsive induction of urease expression in H. pylori .

Die akute Campylobacterinfektion verlauft als selbstlimitierende Enteritis. Bakteriamie, Endokarditis, Meningitis, Pankreatitis, septischer Abort und neonatale Sepsis sind selten. Die wichtigste postinfektose Komplikation ist das Guillain-Barre-Syndrom (GBS). Die unkomplizierte Enteritis wird symptomatisch behandelt. Patienten mit schweren Verlaufen oder Immunsuppression erhalten Antibiotika. Resistenzentwicklung gegen Chinolone, seltener gegen Makrolide, ist zu berucksichtigen. Ende 2001 war Campylobacter in Deutschland mit 52.256 Meldungen hinter den Salmonellosen (74.671) und vor den Rotaviren (45.759) der zweithaufigste darmpathogene Erreger. Die fehlende Vermehrung in der Umwelt kompensiert Campylobacter durch eine weite Verbreitung in der Natur und den massiven Befall zahlreicher Wirte. Geflugelfleisch, Rohmilch und Oberflachenwasser sind wichtige alimentare Risikofaktoren fur den Menschen. Die Kontrolle der Campylobacterinfektionen wird erschwert durch eine schnelle Ausbreitung in Schlachtgeflugel wahrend Haltung, Transport und Schlachtprozess. Pasteurisierung der Milch ist eine effektive Intervention, wahrend die Kuhlung von Lebensmitteln das Uberleben von Campylobacter verlangert. Ansatze zur Pravention einer humanen Infektion sind die Quarantanehaltung des Schlachtgeflugels, Verzicht auf das so genannte “Ausdunnen” der Bestande, Dekontamination der Transportkafige, das Vorziehen der Schlachtung nicht infizierter Bestande, hochstmogliche Bruhtemperaturen der Schlachtkorper, Abkuhlung der Korper im Kuhlbad, Zusatz antimikrobieller Substanzen zum Prozesswasser und moglicherweise zukunftig eine dekontaminierende Schlussbehandlung. Forschungsbedarf besteht bei der Aufklarung der Pathogenese von Enteritis und GBS. Die zunehmende Resistenzentwicklung gegen antimikrobielle Substanzen erfordert ein interdisziplinares Uberwachungs- und Interventionsprogramm.

Background. Availability of the essential nutrient iron is thought to vary greatly in the gastric mucosa, and thus the human gastric pathogen Helicobacter pylori requires regulatory responses to these environmental changes. Bacterial iron-responsive regulation is often mediated by Ferric Uptake Regulator (Fur) homologs, and in this study we have determined the role of H. pylori Fur in regulation of H. pylori iron uptake. Methods. Wild-type H. pylori and fur mutant derivatives were compared after growth in iron-restricted and iron-replete conditions. Iron-uptake was measured using 55Fe-labeled iron, whereas gene expression was monitored at the transcriptional level using Northern hybridization and lacZ reporter gene fusions. Results. Iron-uptake and total cellular iron content were approximately five-fold increased in the fur mutant compared with the wild-type strain, which indicated that in the fur mutant iron-uptake is not repressed by excess iron. A comprehensive screening of all H. pylori genes encoding putative iron-uptake proteins indicated that some of these H. pylori genes are constitutively expressed, while others are iron- and Fur-regulated. Conclusions. Iron uptake in H. pylori is in part differently regulated compared with other bacteria, since in H. pylori some iron-uptake systems are constitutively expressed. However, other iron uptake systems of H. pylori display the iron- and Fur-mediated repression that is common in bacteria. Taken together, this Fur-mediated modulation of iron-uptake capacity may be a specific adaptation to the conditions in the human stomach, where iron starvation and iron overload can be encountered in relatively short time intervals.

The only known niche of the human pathogen Helicobacter pylori is the gastric mucosa, where large fluctuations of pH occur, indicating that the bacterial response and resistance to acid are important for successful colonization. One of the few regulatory proteins in the H. pylori genome is a homologue of the ferric uptake regulator (Fur). In most bacteria, the main function of Fur is the regulation of iron homeostasis. However, in Salmonella enterica serovar Typhimurium, Fur also plays an important role in acid resistance. In this study, we determined the role of the H. pylori Fur homologue in acid resistance. Isogenic fur mutants were generated in three H. pylori strains (1061, 26695, and NCTC 11638). At pH 7 there was no difference between the growth rates of mutants and the parent strains. Under acidic conditions, growth of the fur mutants was severely impaired. No differences were observed between the survival of the fur mutant and parent strain 1061 after acid shock. Addition of extra iron or removal of iron from the growth medium did not improve the growth of the fur mutant at acidic pH. This indicates that the phenotype of the fur mutant at low pH was not due to increased iron sensitivity. Transcription of fur was repressed in response to low pH. From this we conclude that Fur is involved in the growth at acidic pH of H. pylori ; as such, it is the first regulatory protein implicated in the acid resistance of this important human pathogen.

OBJECTIVES Antimicrobial resistance of Helicobacter pylori endangers the successful eradication of the bacteria. The aim of this prospective surveillance study (ResiNet) is to continuously keep antimicrobial resistance of H. pylori in Germany under surveillance and to identify risk factors for its development. METHODS From July 2001 until December 2012, we tested the antimicrobial susceptibility of H. pylori strains isolated from 1651 prospectively enrolled patients. We analysed clinical and epidemiological data and identified risk factors for the development of resistance. RESULTS Average primary resistances were 29.4% for metronidazole, 6.7% for clarithromycin and 3.1% for both antimicrobials. Prior unsuccessful eradication treatments, female sex and country or continent of origin were identified as independent risk factors for development of resistance. CONCLUSIONS H. pylori-positive patients without prior eradication therapy can be treated empirically; antimicrobial susceptibility testing is recommended in previously unsuccessfully treated patients and in patients who have received antimicrobial chemotherapies due to unrelated bacterial infections.

The rate of human intestinal infections with more than a single Campylobacter strain was determined and the genetic variabilities of Campylobacter strains throughout an infection episode were investigated by means of pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). For 48 and 49 of 50 patients, all isolates from one sample showed identical patterns by PFGE and ERIC-PCR, respectively. Throughout an infection episode in 47 of 52 patients, the PFGE fingerprints of the isolates remained stable, while in 1 patient two different species were observed and in 4 patients different patterns were observed. Therefore, ERIC-PCR proved less discriminative than PFGE. These findings suggest that human infection with more than one Campylobacter strain is rare and should not significantly impair epidemiologic analyses. However, changes in the genetic fingerprint throughout an infection should be considered in the assessment of epidemiologic studies of Campylobacter spp.

Background. Helicobacter pylori persists in the human stomach for decades. This requires an efficient adaptation of H. pylori to the gastric niche and involves the regulation of bacterial genes in response to environmental stress. Efficient molecular tools to identify regulated H. pylori genes are scarce, therefore we developed a genomic lacZ reporter gene fusion system in H. pylori to screen for stress-regulated genes. Materials and methods. The integration vector pBW was constructed and used to generate random genomic lacZ fusions in H. pylori. Two-hundred-and-fifty H. pylori transformants were selected from this library, replica-plated and screened for differential lacZ expression after exposure to two environmental stress conditions: increased temperature (42°C), and iron-limitation. Results. From a library of H. pylori transformants with random genomic transcriptional lacZ fusions, two stress-regulated H. pylori loci were identified. The transcription of a gene of unknown function (designated hsp12) was increased by incubation at 42°C. The transcription of a locus, consisting of the three fumarate reductase subunit genes (frdCAB) and the HP0190 gene from H. pylori strain 26695, was decreased under iron-limitation. Conclusions. This is the first time that a genomic transcriptional lacZ reporter gene H. pylori library has been used as a tool for the fast and efficient identification of environmental stress-regulated H. pylori genes.

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.

The functions of the riboflavin synthesis gene homologues ribA, ribBA, ribC, and ribD from Helicobacter pylori strain P1 were confirmed by complementation of defined Escherichia coli mutant strains. The H. pylori ribBA gene, which is similar to bifunctional ribBA genes of Gram-positive bacteria, fully complemented the ribB mutation and partially restored growth in a ribC mutant. However, ribBA did not complement the ribA mutation in E. coli, thus explaining the presence of the additional separate copy of the ribA gene in the H. pylori chromosome. In E. coli exclusively ribA conferred hemolytic activity and gave rise to production of molecules with fluorescence characteristics similar to flavins, as observed earlier. The E. coli hemolysin ClyA was not involved in causing the hemolytic phenotype. No riboflavin synthesis genes on plasmids conferred iron uptake functions to a siderophore-deficient mutant of E. coli. Marker exchange mutagenesis of the genes in H. pylori was not successful indicating that riboflavin synthesis is essential for basic metabolic functions of the gastric pathogen.

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. Keywords: Helicobacter pylori; macrolide resistance; clarithromycin; in situ hybridisation; genotypic resistance

The Escherichia coli-based Fur titration assay (FURTA), although a powerful tool for identification of genes regulated by the ferric uptake regulator (Fur), was unsuccessful for the gastric pathogen Helicobacter pylori. The FURTA was modified by construction of an E. coli indicator strain producing H. pylori Fur only. The promoter regions of the ferric citrate receptor homolog fecA2 and the riboflavin synthesis gene ribBA were both positive in the modified FURTA, but negative in the original FURTA. Transcription of fecA2 and ribBA was demonstrated to be iron-repressed in H. pylori. This type of modification should allow FURTA analysis for bacteria with Fur binding sequences poorly recognized by E. coli Fur.

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.

C. jejuni serogroup PEN O:19 was isolated from a stool specimen from a patient with Guillain-Barre 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.

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.

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.

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.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.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.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.

The vacuolating cytotoxin of Helicobacter pylori (VacA) is known to cause cell damage to mammalian cells and is suspected to give rise to gastric epithelial lesions that might lead to peptic ulcer disease. As shown recently, the gene encoding VacA exhibits genetic variation, with three different families of signal sequences (s1a, s1b, and s2) and two families of midregion sequences (m1 and m2). In order to investigate the relationship between the presence of specific vacA genotypes and peptic ulceration, the vacA genotypes of 158 clinical isolates of H. pylori were determined. The study group consisted of 106 patients with duodenal ulceration; 52 patients with nonulcer dyspepsia (NUD) were used as controls. H. pylori of genotype s1 was isolated from 96% of the patients with ulcerations, whereas genotype s2 was only present in 4%, indicating a strong correlation between the vacA genotype and peptic ulceration ( P < 0.001). In contrast, 31% of the patients from the NUD control group were infected with strains of vacA genotype s2. Particular midregion genotypes (m1 and m2) were not associated with clinical manifestations. The midregions from 18% of the isolates could not be classified by the proposed scheme. DNA sequencing revealed high homology between the untypeable midregions and that of genotype m1, with multiple base pair exchanges, some affecting the primer annealing site. Compared to those of m1 and m2 alleles, the divergent midregions from untypeable strains showed clustering, indicating the presence of a further subfamily of sequences in the midregion of vacA in German isolates, for which we propose the term “m1a.” A new specific primer that we designed for typing m1a isolates might be useful in other studies.

Various strains of Helicobacter pylori were able to lyse erythrocytes from sheep, horse, and human when grown on blood agar. The hemolysis did not depend on the production of the vacuolating cytotoxin VacA as demonstrated by the hemolytic behavior of an isogenic vacA-negative mutant strain. The hemolytic activity could be detected in cell-free supernatants and was not regulated by iron. To isolate genes coding for proteins involved in the destruction of erythrocytes, a plasmid-based DNA library was screened for expression of lytic activity on blood agar. This approach revealed that the H. pylori ribA gene confers hemolytic properties to Escherichia coli. The ribA gene encodes the enzyme GTP-cyclohydrolase II [EC 3.5.4.25] that catalyzes the initial step in the synthesis of riboflavin. The predicted amino acid sequence of the H. pylori RibA protein showed a high degree of similarity to equivalent enzymes from microorganisms and from plants. The single gene on a plasmid restored riboflavin synthesis in a ribA mutant of E. coli and induced hemolytic activity. Furthermore, ribA overexpression was associated with the production of a fluorescent yellow molecule that was not identical with riboflavin. Hemolysis was also seen for the ribA gene from E. coli, indicating that this feature was not specific for the H. pylori gene. The presence of ribA in various H. pylori strains was confirmed by Southern blot hybridization and by polymerase chain reaction with specific primers. This analysis revealed that microdiversity exists within the DNA region upstream from ribA, which was further confirmed by nucleotide sequence analysis.

Helicobacter pullorum, recently described as sp. nov., is commonly isolated from asymptomatic poultry. Two cases of human enteritis associated with H. pullorum, one of them in an immunocompromised patient, are reported. Problems in the correct species identification by means of phenotypic and genotypic methods are discussed and for the first time a fatty acid pattern of Helicobacter pullorum is presented.

The stereoisomers of amidinomycin 7 and their intermediates 1-6, which are produced from homochiral 3-oxocyclopentanecarboxylic acids by asymmetric synthesis, are tested for their antimicrobial effects by agar diffusion test and by Bouillon serial dilution assay. Their antibiotic activities against Bacillus subtilis, Staphylococcus aureus, and Micrococcus Iuteus, respectively, are reported. Structure-activity relationships depend on the type and combination of functional groups, on only the relative stereochemistry as well as on the grade of lipophilia of the tested compounds.

Helicobacter pylori colonizes the gastric mucosa of humans and can cause chronic gastritis, peptic ulcer disease, gastric cancer or mucosa-associated-lymphoid tissue (MALT) lymphoma. Here, we report the case of a 61-year-old male patient who presented with tickle of the throat, globus sensation and heartburn. In an esophagogastroduodenoscopy subpharyngeal localized heterotopic gastric mucosa (HGM), reflux esophagitis and a chronic gastritis were diagnosed. HGM and stomach were H. pylori positive as proven by culture and histopathological examination. After eradication therapy with a proton pump inhibitor (PPI), amoxicillin and clarithromycin followed by PPI treatment, the patient reported clinical improvement and the histopathological changes in the HGM due to H. pylori infection improved, too. This case report demonstrates that culture and susceptibility testing of H. pylori using established protocols succeeds not only from tissue samples of the stomach but also from heterotopic gastric mucosa. Eradication therapy may not only improve typical H. pylori associated discomforts of the stomach but also extragastric signs and symptoms of H. pylori infection.

Helicobacter pylori strains isolated from different regions of the world or human ethnic groups exhibit some differences at certain loci. The aim of this study was to determine allelic differences of H.pylori strains isolated from two countries, Turkey and Germany. A total of 72 H.pylori isolates, of which 37 strains were from Kocaeli province of Turkey and 35 strains from Hamburg, Germany were included in this study. H.pylori strains had been isolated from gastric biopsies of the patients. Genomic DNA was extracted with phenol-chloroform-isoamyl alcohol procedure and vacA alleles and cagA regions were identified by polymerase chain reaction (PCR) using specific primer sets. The rates of cagA positivity in Kocaeli and Hamburg strains were found as 75.7% (28/37) and 71.4% (25/35), respectively. VacA s1a allele was predominant both in Turkey and Germany isolates. There were five vacA mosaicisms in Hamburg strains, including 14 for s1a/m1a (40%), nine for s1a/m2 (25.7%), eight for s2/m2 (17.1%), three for s1a/m1 (8.5%) and three for s1b/m2 (8.5%). There were four vacA mosaicisms in Kocaeli strains, including 22 for s1a/m2 (59.4%), eight for s2/m2 (21.6%), four for s1a/m1a (10.8%), and three for s1a/m1 (8.1%). In this study, Hamburg and Kocaeli strains did not reveal significant differences regarding cagA status and vacA s1 allele. Whereas vacA s1a/m1a, cagA(+) type was the most frequent in Hamburg strains and s1a/m2, cagA (+) type in Kocaeli strains.

Helicobacter pylori colonizes the stomachs of at least half of the world's human population. The role of the oral cavity in this colonization is not clear and there are, to date, no comprehensive data that clearly demonstrate the isolation of this bacterium from the oral cavity. The aim of this study was to evaluate the prevalence of H. pylori in the oral cavity of 15 patients who tested positive for H. pylori. A comprehensive dental examination of all patients was conducted. Samples were taken from supragingival and subgingival plaque, saliva, periapical exudates and tongue swabs. All samples were taken before the application of antibiotics. A total of 163 oral samples were investigated by PCR using two different H. pylori-specific primer pairs. A PCR inhibition control using a modified plasmid was always included for the most specific primer pair. In addition, a culture technique was used to confirm PCR results. Despite a PCR detection limit of 10(2) bacteria ml(-1), out of 14 patients, H. pylori could not be detected in any of the samples taken. In one patient, H. pylori-positive PCR signals were obtained in two samples using only one primer pair. H. pylori could not be cultivated from these two PCR-positive samples; therefore, no correlation to oral colonization status could be established. This study challenges the misleading preconception that H. pylori resides in the human oral cavity and suggests that this bacterium should be considered transient and independent of the oral status. To date, positive PCR results for H. pylori in the oral cavity have been overestimated and not critically interpreted in literature.

SUMMARYBetween 2000 and 2009, the total number of patients withClostridium difficileinfections increased considerably in Southeastern Germany. A clear seasonality was observed with a higher number of affected patients occurring in the winter months (January–March). Moxifloxacin and erythromycin-resistantC. difficilePCR ribotypes 001 (72%) and 027 (4·6%) were the most commonly isolated strains.

INTRODUCTION: To establish strategic priorities for the German national public health institute (RKI) and guide the institute's mid-term strategic decisions, we prioritized infectious pathogens in accordance with their importance for national surveillance and epidemiological research. METHODS: We used the Delphi process with internal (RKI) and external experts and a metric-consensus approach to score pathogens according to ten three-tiered criteria. Additional experts were invited to weight each criterion, leading to the calculation of a median weight by which each score was multiplied. We ranked the pathogens according to the total weighted score and divided them into four priority groups. RESULTS: 127 pathogens were scored. Eighty-six experts participated in the weighting; "Case fatality rate" was rated as the most important criterion. Twenty-six pathogens were ranked in the highest priority group; among those were pathogens with internationally recognised importance (e.g., Human Immunodeficiency Virus, Mycobacterium tuberculosis, Influenza virus, Hepatitis C virus, Neisseria meningitides), pathogens frequently causing large outbreaks (e.g., Campylobacter spp.), and nosocomial pathogens associated with antimicrobial resistance. Other pathogens in the highest priority group included Helicobacter pylori, Respiratory Syncytial Virus, Varicella zoster virus and Hantavirus. DISCUSSION: While several pathogens from the highest priority group already have a high profile in national and international health policy documents, high scores for other pathogens (e.g., Helicobacter pylori, Respiratory syncytial virus or Hantavirus) indicate a possible under-recognised importance within the current German public health framework. A process to strengthen respective surveillance systems and research has been started. The prioritization methodology has worked well; its modular structure makes it potentially useful for other settings. peerReviewed