Search Results (16)

Helicobacter pylori (H. pylori) is regarded as a significant risk factor for gastritis, peptic ulcer disease, and gastric cancer. However, the increasing resistance of H. pylori strains has resulted in low eradication rates and ineffective treatments. Herein, we report on identification of a new quipazine derivative—compound 9c (N-(3-chlorobenzyl)-2-(piperazin-1-yl)quinolin-4-amine), which displayed antibacterial properties (MIC range 2–4 µg/mL) against H. pylori CagA-positive reference strains associated with an increased risk of gastric cancer, including metronidazole-resistant ATCC 43504, clarithromycin-resistant ATCC 700684 and susceptible J99 strain, as well as clinical, multidrug-resistant isolate (3CML, resistant to clarithromycin, metronidazole and levofloxacin). Compound 9c showed bacteriostatic activity (MBC/MIC ratio > 4), demonstrated antibiofilm-forming properties and prevented auto-aggregation of microbial cells. It also displayed an additive effect in ½ MIC (2 µg/mL) when administered with clarithromycin and/or metronidazole. Compound 9c had no impact on gut microbiota reference strains of S. aureus, E. coli, E. faecalis and L. paracasei as well as no hemolytic activity against sheep erythrocytes. Finally, by reducing the viability of the SNU-1 human gastric cancer cell line (IC₅₀ = 3.28 μg/mL), compound 9c might offer important implications regarding the oncogenic characteristics of cagA+ H. pylori strains. Full article

Helicobacter pylori is a pathogenic bacterium that causes gastric and extragastric diseases. We have previously demonstrated that one of the mechanisms of H. pylori-associated chronic immune thrombocytopenia involves immune complexes of platelets, a H. pylori protein Lpp20 and an anti-Lpp20 antibody. However, it remains unclear how Lpp20 enters the body. We hypothesize that bacterial extracellular vesicles (bEVs) transport Lpp20. Thus, this study assessed Lpp20 in the bEVs released from seven clinical H. pylori isolates, using immunoprecipitation (IP), immunoblotting (IB), and surface plasmon resonance imaging (SPRi), with anti-GroEL (a marker of bEVs) and anti-Lpp20 antibodies. Lpp20 and bEVs were each detected in lysates of all seven strains. IP–IB experiments demonstrated that bEVs containing Lpp20 were produced by five of the strains (J99, SS1, HPK5, JSHR3, and JSHR31). SPRi using an anti-Lpp20 antibody demonstrated significantly higher reflectance from the strain HPK5 than from its lpp20-disrupted strains (p < 0.01), indicating localization of Lpp20 on the bEVs’ surface; Lpp20 may also be contained within bEVs. The bEVs containing Lpp20 were not detected from two clinical H. pylori strains (26695 and JSHR6) or from two lpp20-disrupted strains (26695ΔLpp20 and HPK5ΔLpp20). Differences in Lpp20 detection in bEVs are likely due to variations in bEV production resulting from strain diversity. Full article

Peptidoglycan is the basic structural polymer of the bacterial cell wall and maintains the shape and integrity of single cells. Despite years of research conducted on peptidoglycan’s chemical composition, the microscopic elucidation of its nanoscopic architecture still needs to be addressed more thoroughly to advance knowledge on bacterial physiology. Apart from the model organism Escherichia coli, ultrastructural imaging data on the murein architecture of Gram-negative bacteria is mostly missing today. This study therefore intended to further our understanding of bacterial physiology by the isolation of peptidoglycan sacculi from the Gram-negative bacterium Helicobacter pylori J99 and the subsequent nanoscopic imaging of the murein network by Atomic Force Microscopy (AFM). With the ability to purify peptidoglycan sacculi from H. pylori J99 for AFM by a modified peptidoglycan isolation protocol, nanoscopic imaging of the murein network by intermittent-contact AFM in air and under liquid yielded ultrastructural insights into the H. pylori J99 cell wall architecture. High-resolution data acquisition on isolated peptidoglycan from H. pylori J99 by AFM under liquid was performed and revealed a molecular network similar to available data from E. coli. Subsequent enzymatic digestion of the isolated H. pylori J99 sacculi and analysis of the resulting fragments by +ESI-LCMS confirmed the presence of N-acetylglucosamine as an additional marker for successful peptidoglycan isolation. By comparison of the nanoscopic sacculus dimensions of H. pylori J99 to E. coli NU14, this study also identified specific differences in the sacculus morphology of both Gram-negative pathogenic bacteria. Full article

Background/objectives: Bismuth is commonly used in Helicobacter pylori (H. pylori) eradication therapy. However, few studies have examined the in vitro susceptibility of H. pylori to bismuth. Moreover, the exact mechanism of action of bismuth on H. pylori remains unclear. The aim of this study was to identify the anti-bacterial effect of bismuth as well as to evaluate potential synergistic effects between bismuth and various antibiotics. Methods: The minimum inhibitory concentrations (MICs) of three bismuth preparations, bismuth subsalicylate, bismuth potassium citrate, and colloidal bismuth subcitrate (CBS, De-Nol) were determined for H. pylori strains using the agar dilution technique. Agar plates of varying pH values from 5.0 to 8.0 were used to investigate whether acidity influences the anti-bacterial effect of bismuth. A checkerboard assay was performed to assess the synergism between CBS and antibiotics (amoxicillin, clarithromycin, and metronidazole). Results: Twelve H. pylori strains, including three reference strains (H. pylori 26695, J99, and ATCC 43504), and nine clinically isolated strains were tested. The MICs for bismuth subsalicylate, bismuth potassium citrate, and CBS ranged from 4 to 32 μg/mL, 2 to 16 μg/mL, and 1 to 8 μg/mL, respectively. The bismuth MICs for the reference strains were similar at pH 5–8. In the checkerboard assay, no interactions between CBS and any of the antibiotics were observed in the reference H. pylori strains. Conclusions: Bismuth showed in vitro susceptibility against H. pylori. The enhanced eradication efficacy of bismuth-containing regimens appears to be due to mechanisms other than direct synergy with antibiotics. Full article

This is the first registered intervention study for vonoprazan, high-dose amoxicillin, clarithromycin, and metronidazole 14-day concomitant therapy based on a susceptibility test of Helicobacter pylori. We conducted this study as a fourth-line rescue regimen in Japan. Methods: Twenty patients who underwent three rounds of eradication therapies (first- or second-line 7-day triple therapy consisting of amoxicillin and clarithromycin, or metronidazole- and sitafloxacin-based third-line therapy) and had failed eradication based on a urea breath test or fecal antigen test were recruited. All patients underwent endoscopic examination and culture tests before starting eradication therapy. The intervention was concomitant therapy consisting of vonoprazan 20 mg bid, amoxicillin 500 mg qid, clarithromycin 400 mg bid, and metronidazole 250 mg bid for 14 days, which were modified based on the susceptibility test, and the resistant drugs were removed from the regimen. Patients with negative culture results were treated with quadruple therapy. The primary outcome was the eradication rate (UMIN000025765, jRCTs 031180208). Results: The eradication rate of susceptibility-testing-based fourth-line eradication therapy was 63.2% (95%CI: 38.4–83.7%) in intent-to-treat analysis and 70.6% (95%CI: 44.0–89.7%) in per-protocol analysis. Thirteen patients received quadruple therapy, with eradication rates of 61.5% and 75.0%, respectively. No serious adverse events were reported. Conclusions: This vonoprazan-based concomitant therapy modified by the susceptibility test is a potential option as fourth-line eradication after first-line clarithromycin-based 7-day triple, second-line metronidazole-based 7-day triple, and third-line sitafloxacin-based 7-day triple therapy failure. Full article

Research into novel anti-Helicobacter pylori agents represents an important approach for the identification of new treatments for chronic gastritis and peptic ulcers, which are associated with a high risk of developing gastric carcinoma. In this respect, two series of azobenzenesulfonamides were designed, synthesized, and tested against a large panel of human and bacterial CAs to evaluate their inhibitory activity. In addition, computational studies of the novel primary benzenesulfonamides (4a–j) were performed to predict the putative binding mode to both HpCAs. Then, the antimicrobial activity versus H. pylori of the two series was also studied. The best-in-class compounds were found to be 4c and 4e among the primary azobenzenesulfonamides and 5c and 5f belonging to the secondary azobenzenesulfonamides series, showing themselves to exert a promising anti-H. pylori activity, with MIC values of 4–8 μg/mL and MBCs between 4 and 16 μg/mL. Moreover, the evaluation of their toxicity on a G. mellonella larva in vivo model indicated a safe profile for 4c,e and 5c,f. The collected results warrant considering these azobenzenesulfonamides as an interesting starting point for the development of a new class of anti-H. pylori agents. Full article

Background and Objective: Rescue Helicobacter pylori eradication can be challenging. Rifabutin (RBT) demonstrates high activity against Helicobacter pylori and is incorporated into various rescue eradication regimens. This exploratory study was performed to evaluate the efficacy and safety of a rescue regimen comprising RBT, metronidazole (MNZ), and vonoprazan (VPZ). Methods: This prospective, single-center, single-arm, interventional study was performed in Japan. Eligible patients were those who underwent failed primary eradication treatment (7-day treatment with three drugs: VPZ or a proton pump inhibitor [PPI], amoxicillin [AMPC], and clarithromycin) and secondary eradication treatment (7-day treatment with three drugs: VPZ or a PPI, AMPC, and MNZ) and those who were unable to receive first- and second-line therapy because of penicillin allergy. Twenty Helicobacter pylori-positive patients were treated with RBT (150 mg twice daily), MNZ (250 mg twice daily), and VPZ (20 mg twice daily) for 10 days (RBT-MNZ-VPZ therapy). Eradication success was evaluated using the urea breath test. Drug susceptibility test results were available in 16 patients. This study is registered in the Japan Registry of Clinical Trials (jRCT031220504). Results: The intention-to-treat (ITT) and per-protocol (PP) eradication rates of RBT-MNZ-VPZ therapy were 70% (90% confidence interval [CI]: 49.2%–86.0%) and 72.2% (95% CI: 50.2%–88.4%), respectively. In the MNZ-susceptible subgroup, the ITT (n = 8) and PP (n = 7) eradication rates were 100% (90% CI: 68.8%–100%) and 100% (90% CI: 65.2%–100%). In the MNZ-resistant subgroup, the ITT (n = 8) and PP (n = 7) eradication rates were both 62.5% (90% CI: 28.9%–88.9%). All infections were RBT-susceptible. Conclusions: These findings suggest that RBT-MNZ-VPZ therapy may be a promising rescue regimen, especially in MNZ- and RBT-susceptible infections or patients with penicillin allergy. Full article

Helicobacter pylori protects itself from stressful environments by forming biofilms, changing its morphology, or invading eukaryotic cells, including yeast cells. There is little knowledge about the environmental factors that influence the endosymbiotic relationship between bacterium and yeasts. Here, we studied if oxygen availability stimulated the growth of H. pylori within Candida and if this was a bacterial- or yeast strain-dependent relationship. Four H. pylori strains and four Candida strains were co-cultured in Brucella broth plus 5% fetal bovine serum, and incubated under microaerobic, anaerobic, or aerobic conditions. Bacteria-like bodies (BLBs) within yeast cells (Y-BLBs) were detected by microscopy. H. pylori was identified by FISH and by PCR amplification of the 16S rRNA gene of H. pylori from total DNA extracted from Y-BLBs from H. pylori and Candida co-cultures. BLBs viability was confirmed by SYTO-9 fluorescence. Higher Y-BLB percentages were obtained under anaerobic conditions and using H. pylori J99 and C. glabrata combinations. Thus, the H. pylori–Candida endosymbiotic relationship is strain dependent. The FISH and PCR results identified BLBs as intracellular H. pylori. Conclusion: Stressful conditions such as an anaerobic environment significantly increased H. pylori growth within yeast cells, where it remained viable, and the bacterium–yeast endosymbiotic relationship was bacterial strain dependent with a preference for C. glabrata. Full article

DNA methylomes of Helicobacter pylori strains are complex due to the large number of DNA methyltransferases (MTases) they possess. H. pylori J99 M.Hpy99III is a 5-methylcytosine (^m5C) MTase that converts GCGC motifs to G^m5CGC. Homologs of M.Hpy99III are found in essentially all H. pylori strains. Most of these homologs are orphan MTases that lack a cognate restriction endonuclease, and their retention in H. pylori strains suggest they have roles in gene regulation. To address this hypothesis, green fluorescent protein (GFP) reporter genes were constructed with six putative promoters that had a GCGC motif in the extended −10 region, and the expression of the reporter genes was compared in wild-type H. pylori G27 and a mutant lacking the M.Hpy99III homolog (M.HpyGIII). The expression of three of the GFP reporter genes was decreased significantly in the mutant lacking M.HpyGIII. In addition, the growth rate of the H. pylori G27 mutant lacking M.HpyGIII was reduced markedly compared to that of the wild type. These findings suggest that the methylation of the GCGC motif in many H. pylori GCGC-containing promoters is required for the robust expression of genes controlled by these promoters, which may account for the universal retention of M.Hpy99III homologs in H. pylori strains. Full article

Untargeted metabolomic profiling provides the opportunity to comprehensively explore metabolites of interest. Herein, we investigated the metabolic pathways associated with Jhp0106, a glycosyltransferase enzyme in Helicobacter pylori. Through untargeted exometabolomic and metabolomic profiling, we identified 9 and 10 features with significant differences in the culture media and pellets of the wild-type (WT) J99 and jhp0106 mutant (Δjhp0106). After tentative identification, several phosphatidylethanolamines (PEs) were identified in the culture medium, the levels of which were significantly higher in WT J99 than in Δjhp0106. Moreover, the reduced lysophosphatidic acid absorption from the culture medium and the reduced intrinsic diacylglycerol levels observed in Δjhp0106 indicate the possibility of reduced PE synthesis in Δjhp0106. The results suggest an association of the PE synthesis pathway with flagellar formation in H. pylori. Further investigations should be conducted to confirm this finding and the roles of the PE synthesis pathway in flagellar formation. This study successfully demonstrates the feasibility of the proposed extraction procedure and untargeted exometabolomic and metabolomic profiling strategies for microbial metabolomics. They may also extend our understanding of metabolic pathways associated with flagellar formation in H. pylori. Full article

The detection of chemical substances excreted from the human body offers an attractive approach for non-invasive, early diagnostics of certain diseases. In this preliminary study, we proposed a susceptible optical sensor capable of quantitatively detecting ammonia from exhaled breath. The proposed sensor consists of nanoassembled ultrathin films composed of tetrakis(4-sulfophenyl)porphine (TSPP) and poly(diallyldimethylammonium chloride) (PDDA) deposited on quartz substrates using a layer-by-layer method. Measurement principles are based on the ammonia-induced absorbance changes at 489 (Soret band) and 702 nm (Q band), associated with the deprotonation of the J-aggregated TSPPs inside the film. Before exposure to breath, the PDDA/TSPP thin film was calibrated using known concentrations of ammonia gases with a projected detection limit of 102 ± 12 parts per billion (ppb). Calibrated sensor films were then exposed to human breath and urine samples to determine the ammonia concentration. Concentrations of exhaled ammonia are influenced significantly by the consumption of food or the amount of urea. Sensor response and maximum sensitivity, obtained from the absorbance changes induced by ammonia, were achieved by initial sensor exposure to HCl vapor. Previously reported procedures for the Helicobacter pylori (HELIC Ammonia Breath) test based on urea reaction with urease were reproduced using the proposed sensor. The observed behavior corresponded very well with the kinetics of the interactions between urea and urease, i.e., ammonia reached a maximum concentration approximately 5 min after the start of the reaction. A large-scale study involving 41 healthy volunteers in their 20s to 60s was successfully conducted to test the capabilities of the sensor to determine the concentration of exhaled ammonia. The concentration of ammonia for the healthy volunteers ranged between 0.3 and 1.5 ppm, with a mean value of ca. 520 ppb in the morning (before eating) and ca. 420 ppb in the afternoon (immediately after eating). These real-test mean values are meaningful when considered against the projected LOD. Full article

First-line treatment for Helicobacter pylori includes amoxicillin and clarithromycin or metronidazole plus a proton pump inhibitor. Treatment failure is associated with antibiotic resistance and possibly also with internalization of H. pylori into eukaryotic cells, such as yeasts. Factors triggering the entry of H. pylori into yeast are poorly understood. Therefore, the aim of this study was to evaluate whether clarithromycin or amoxicillin trigger the entry of H. pylori into C. albicans cells. Methods: H. pylori J99 and C. albicans ATCC 10231 were co-cultured in the presence of subinhibitory concentrations of amoxicillin and clarithromycin as stressors. Bacterial-bearing yeasts were observed by fresh examination. The viability of bacteria within yeasts was evaluated, confirming the entry of bacteria into Candida, amplifying, by PCR, the H. pylori16S rRNA gene in total yeast DNA. Results: Amoxicillin significantly increased the entry of H. pylori into C. albicans compared to the control. Conclusion: the internalization of H. pylori into C. albicans in the presence of antibiotics is dependent on the type of antibiotic used, and it suggests that a therapy including amoxicillin may stimulate the entry of the bacterium into Candida, thus negatively affecting the success of the treatment. Full article

Helicobacter pylori colonizes the human stomach of half of the world’s population. The infection if not treated, persists through life, leading to chronic gastric inflammation, that may progress to severe diseases as peptic ulcer, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. The first line of treatment, based on 7 to 21 days of two antibiotics associated with a proton pump inhibitor, is, however, already failing most due to patient non-compliance that leads to antibiotic resistance. It is, therefore, urgent to screen for new and more efficient antimicrobials against this bacterium. In this work, Fourier Transform Infrared (FTIR) spectroscopy was evaluated to screen new drugs against H. pylori, in rapid (between 1 to 6 h), and high-throughput mode and based on a microliter volume processes in relation to the agar dilution method. The reference H. pylori strains 26,695 and J99, were evaluated against a peptide-based antimicrobial and the clinical antibiotic clarithromycin, respectively. After optimization of the assay conditions, as the composition of the incubation mixture, the time of incubation, and spectral pre-processing, it was possible to reproducibly observe the effect of the drug on the bacterial molecular fingerprint as pointed by the spectra principal component analysis. The spectra, obtained from both reference strains, after its incubation with drugs concentrations lower than the MIC, presented peak ratios statistically different (p < 0.05) in relation to the bacteria incubated with drugs concentrations equal or higher to the MIC. It was possible to develop a partial least square regression model, enabling to predict from spectra of both bacteria strains, the drug concentration on the assay, with a high correlation coefficient between predicted and experimental data (0.91) and root square error of 40% of the minimum inhibitory concentration. All this points to the high potential of the technique for drug screening against this fastidious growth bacterium. Full article

Yeasts can adapt to a wide range of pH fluctuations (2 to 10), while Helicobacter pylori, a facultative intracellular bacterium, can adapt to a range from pH 6 to 8. This work analyzed if H. pylori J99 can protect itself from acidic pH by entering into Candida albicans ATCC 90028. Growth curves were determined for H. pylori and C. albicans at pH 3, 4, and 7. Both microorganisms were co-incubated at the same pH values, and the presence of intra-yeast bacteria was evaluated. Intra-yeast bacteria-like bodies were detected using wet mounting, and intra-yeast binding of anti-H. pylori antibodies was detected using immunofluorescence. The presence of the H. pylori rDNA 16S gene in total DNA from yeasts was demonstrated after PCR amplification. H. pylori showed larger death percentages at pH 3 and 4 than at pH 7. On the contrary, the viability of the yeast was not affected by any of the pHs evaluated. H. pylori entered into C. albicans at all the pH values assayed but to a greater extent at unfavorable pH values (pH 3 or 4, p = 0.014 and p = 0.001, respectively). In conclusion, it is possible to suggest that H. pylori can shelter itself within C. albicans under unfavorable pH conditions. Full article

Polysaccharide containing extracts from immature fruits of okra (Abelmoschus esculentus) are known to exhibit antiadhesive effects against bacterial adhesion of Helicobacter pylori (H. pylori) to stomach tissue. The present study investigates structural and functional features of polymers responsible for this inhibition of bacterial attachment to host cells. Ammonium sulfate precipitation of an aqueous extract yielded two fractions at 60% and 90% saturation with significant antiadhesive effects against H. pylori, strain J99, (FE_60% 68% ± 15%; FE_90% 75% ± 11% inhibition rates) after preincubation of the bacteria at 1 mg/mL. Sequential extraction of okra fruits yielded hot buffer soluble solids (HBSS) with dose dependent antiadhesive effects against strain J99 and three clinical isolates. Preincubation of H. pylori with HBSS (1 mg/mL) led to reduced binding to 3ʹ-sialyl lactose, sialylated Le^a and Le^x. A reduction of bacterial binding to ligands complementary to BabA and SabA was observed when bacteria were pretreated with FE_90%. Structural analysis of the antiadhesive polysaccharides (molecular weight, monomer composition, linkage analysis, stereochemistry, and acetylation) indicated the presence of acetylated rhamnogalacturonan-I polymers, decorated with short galactose side chains. Deacetylation of HBSS and FE_90% resulted in loss of the antiadhesive activity, indicating esterification being a prerequisite for antiadhesive activity. Full article