Retrospective study on outcome of salvage Helicobacter pylori eradication therapies based on molecular genetic susceptibility testing.

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.

Outcome of second- and third-line Helicobacter pylori eradication therapies based on antimicrobial susceptibility testing.

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.

Aegle marmelos fruit extract attenuates Helicobacter pylori Lipopolysaccharide induced oxidative stress in Sprague Dawley rats.

BACKGROUND: Bael (Aegle marmelos (L.) Corr.) has been widely used in indigenous systems of Indian medicine to exploit its medicinal properties including astringent, antidiarrheal, antidysenteric, demulcent, antipyretic, antiulcer, anti-inflammatory and anti cancer activities. The present study aims to evaluate the antioxidative and antiulcer effect of methanolic extract of unripe fruit of Aegle marmelos (MEAM) against Helicobacter pylori-Lipopolysaccharide (HP-LPS) induced gastric ulcer in Sprague Dawley (SD) rats. METHODS: Dose and duration of HP-LPS and MEAM were fixed based on ulcer index of gastric tissue of experimental animals. Various gastric secretory parameters such as volume of gastric juice, free and total acidity, acid output, pepsin concentration were analyzed. The activities of enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione transferase), non-enzymatic antioxidants (reduced glutathione, vitamin C and vitamin E) and the levels of lipid peroxidation products were measured. Histological analysis was performed to evaluate the effect of Aegle marmelos on HP-LPS induced gastric ulcer. RESULTS: Oral administration of HP-LPS (50 µg per animal) for four consecutive days resulted in induction of ulcer with the increase in gastric secretory parameters such as volume of gastric juice, free and total acidity, acid output, pepsin concentration. Oral administration of methanolic extract of Aegle marmelos fruit (MEAM) (25, 50, 100, 250 and 500 mg/kg) reduced the gastric ulcer by 2.8 %, 52.4 %, 73 %, 93 % and 93.98 %, respectively, compared to 89.2 % reduction by sucralfate (100 mg/kg). MEAM treatment significantly (p < 0.05) inhibited the increase in gastric secretory parameters in ulcerated rats, and it also prevented the reduction of enzymatic (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione transferase) and non-enzymatic antioxidants (reduced glutathione, vitamin C and vitamin E) after HP-LPS induction. In addition, lipid peroxidation was inhibited by MEAM in HP-LPS induced rats. Results of histological analysis correlated well with biochemical parameters. CONCLUSION: These observations explored the antioxidant properties of MEAM contributing to the gastroprotective effect in HP-LPS induced gastric ulcer model.

Prospective multicentre study on antimicrobial resistance of Helicobacter pylori in Germany.

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.

Helicobacter pylori resistance to antibiotics in Europe and its relationship to antibiotic consumption.

OBJECTIVE: Resistance to antibiotics is the major cause of treatment failure of Helicobacter pylori infection. A study was conducted to assess prospectively the antibacterial resistance rates of H pylori in Europe and to study the link between outpatient antibiotic use and resistance levels in different countries. DESIGN: Primary antibiotic resistance rates of H pylori were determined from April 2008 to June 2009 in 18 European countries. Data on yearly and cumulative use over several years of systemic antibacterial agents in ambulatory care for the period 2001-8 were expressed in Defined Daily Doses (DDD) per 1000 inhabitants per day. The fit of models and the degree of ecological association between antibiotic use and resistance data were assessed using generalised linear mixed models. RESULTS: Of 2204 patients included, H pylori resistance rates for adults were 17.5% for clarithromycin, 14.1% for levofloxacin and 34.9% for metronidazole, and were significantly higher for clarithromycin and levofloxacin in Western/Central and Southern Europe (>20%) than in Northern European countries (<10%). Model fit improved for each additional year of antibiotic use accumulated, but the best fit was obtained for 2005. A significant association was found between outpatient quinolone use and the proportion of levofloxacin resistance (p=0.0013) and between the use of long-acting macrolides only and clarithromycin resistance (p=0.036). CONCLUSION: In many countries the high rate of clarithromycin resistance no longer allows its empirical use in standard anti-H pylori regimens. The knowledge of outpatient antibiotic consumption may provide a simple tool to predict the susceptibility of H pylori to quinolones and to macrolides and to adapt the treatment strategies.

High secondary resistance to quinolones in German Helicobacter pylori clinical isolates.

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.

Culture of a gastric non-Helicobacter pylori Helicobacter from the stomach of a 14-year-old girl.

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.

[Differences of vacA alleles and cagA gene positivity of Helicobacter pylori strains isolated from two different countries: Turkey and Germany]. / Iki Farkli Ülkeden (Türkiye ve Almanya) Izole Edilen Helicobacter pylori Izolatlarinda VacA Allellerindeki Farklilik ve cagA Geni Pozitifligi.

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.

A novel system of cytoskeletal elements in the human pathogen Helicobacter pylori.

Pathogenicity of the human pathogen Helicobacter pylori relies upon its capacity to adapt to a hostile environment and to escape from the host response. Therefore, cell shape, motility, and pH homeostasis of these bacteria are specifically adapted to the gastric mucus. We have found that the helical shape of H. pylori depends on coiled coil rich proteins (Ccrp), which form extended filamentous structures in vitro and in vivo, and are differentially required for the maintenance of cell morphology. We have developed an in vivo localization system for this pathogen. Consistent with a cytoskeleton-like structure, Ccrp proteins localized in a regular punctuate and static pattern within H. pylori cells. Ccrp genes show a high degree of sequence variation, which could be the reason for the morphological diversity between H. pylori strains. In contrast to other bacteria, the actin-like MreB protein is dispensable for viability in H. pylori, and does not affect cell shape, but cell length and chromosome segregation. In addition, mreB mutant cells displayed significantly reduced urease activity, and thus compromise a major pathogenicity factor of H. pylori. Our findings reveal that Ccrp proteins, but not MreB, affect cell morphology, while both cytoskeletal components affect the development of pathogenicity factors and/or cell cycle progression.

Identification and molecular characterization of triple- and quadruple-resistant Helicobacter pylori clinical isolates in Germany.

OBJECTIVES: The aim of this study was to estimate the frequency of triple- and quadruple-resistant Helicobacter pylori isolated in Germany, to characterize those isolates molecular genetically and to identify risk factors for the development of multiresistance. METHODS: Antimicrobial susceptibility to metronidazole, clarithromycin, amoxicillin, tetracycline, ciprofloxacin/levofloxacin and rifampicin in 1118 clinical isolates obtained between July 2006 and December 2007 was tested by the Etest method. For patients harbouring triple- or quadruple-resistant strains (n = 169), data on prior eradication therapies and underlying diseases were collected and evaluated. A select number of quadruple- and triple-resistant strains were examined for resistance-mediating mutations in their 23S rRNA, 16S rRNA, gyrA and rpoB genes, respectively. RESULTS: From 1118 clinical isolates, 13.4% (n = 150) showed phenotypic resistance to metronidazole, clarithromycin and quinolones and 0.9% (n = 10) to metronidazole, clarithromycin and rifampicin; one isolate exhibited resistance to clarithromycin, quinolones and rifampicin. In eight isolates (0.7%), we detected phenotypic quadruple resistance to metronidazole, clarithromycin, quinolones and rifampicin or tetracycline. Triple- and quadruple-resistant strains harboured resistance-associated mutations in their 23S rRNA, 16S rRNA, gyrA or rpoB genes and were nearly exclusively isolated from patients who had already been unsuccessfully treated on multiple occasions. CONCLUSIONS: We show that more than 15% of H. pylori strains isolated from routine samples in the German National Reference Centre are resistant to three or more antimicrobials and identified prior unsuccessful eradication therapies as a key factor for the development of multiresistance. Our data emphasize the need for further comprehensive surveillance studies monitoring the role of treatment regimens in antimicrobial resistance in H. pylori.

Helicobacter pylori multiplex serology.

BACKGROUND: Helicobacter pylori infection is associated with severe gastrointestinal disease including cancer. It induces complex antibody responses that might vary depending on disease state but currently cannot be assessed adequately. The objective of this work was the development of a sensitive and specific H. pylori multiplex serology assay with high-throughput capability that allows simultaneous detection of antibodies to a protein array. METHODS: Seventeen proteins of up to three H. pylori strains (26695, G27, 151), including CagA, VacA, UreA, Catalase, Omp, and GroEL, were recombinantly expressed as glutathione-S-transferase fusion proteins, affinity-purified, and used as antigens in a fluorescent bead-based antibody-binding assay. Reference sera (n = 317) characterized by commercial assays (screening ELISA with Western blot confirmation) were used for validation. RESULTS: H. pylori seropositivity by multiplex serology defined as reactivity with at least four proteins showed good agreement (kappa: 0.70) with commercial serologic assay classification, and a sensitivity of 89% and specificity of 82%. For individual antigens, agreement with Western blot was good for CagA (kappa: 0.77), moderate for UreA (kappa: 0.53), and weak for VacA (kappa: 0.12). Of the 13 proteins expressed from two strains, only VacA showed serologic strain differences. High antibody reactivity to CagA (Type I infection) was negatively associated with antibodies to GroEL, Cad, CagM, catalase, HcpC, NapA, and UreA, suggesting type-specific differences in protein expression patterns and/or immune response. CONCLUSION: With its high-throughput and simultaneous detection abilities, H. pylori multiplex serology appears suited as tool for large seroepidemiologic studies assessing H. pylori prevalence, antibody patterns, and associations with specific diseases.

Effect of methanolic extract of Terminalia arjuna against Helicobacter pylori 26695 lipopolysaccharide-induced gastric ulcer in rats.

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 mug 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.

Re-analysis of the risks attributed to ciprofloxacin-resistant Campylobacter jejuni infections.

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 11,000 cases. We conclude that fluoroquinolone-resistant Campylobacter infections are not more severe than susceptible infections.

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.

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.

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.

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.

Impact and management of Campylobacter in human medicine--European perspective.

In 2001 in Germany, Campylobacter was second to Salmonella as the most frequent foodborne pathogen isolated from humans. This is also true for the major part of the European population with incidence data available, namely Germany, Hungary, Belgium, Denmark, Austria and Spain, with a total population of 154.5 million, and incidence rates for Salmonella and Campylobacter of 81.8/100 000 and 49.0/100 000 respectively. Countries where Campylobacter is predominant are the UK, Switzerland, Sweden, Finland, Norway, and The Netherlands, with a total population of 94.2 million, and incidence rates for Campylobacter and Salmonella of 79.6/100 000 and 33.3/100 000, respectively. Shigella is rarely isolated in Europe (1.9-4.5/100 000). Epidemiologic risk factors for human campylobacteriosis are poultry, untreated milk, and surface water. Travel plays an important role, especially in Scandinavia and the UK. Isolates acquired outside these countries are much more often resistant to fluoroquinolones than are domestic strains. Fluoroquinolone resistance in human Campylobacter isolates is lowest in the UK and Denmark, at 12%. Resistance quotes of around 20-40% are found in Finland, The Netherlands, Norway, Sweden, Austria, and Germany, and peaks in Spain reaching 70-80%. Usually, uncomplicated Campylobacter enteritis is symptomatically treated. If, in severe cases, antimicrobials are indicated, in all European countries except Switzerland, macrolides are used as first-line drugs, followed by fluoroquinolones or doxycycline.