Antibacterial Activities of Liposomal Linolenic Acids against Antibiotic-Resistant Helicobacter pylori

Helicobacter pylori (H. pylori) infection with its vast prevalence is responsible for various gastric diseases including gastritis, peptic ulcers, and gastric malignancy. While effective, current treatment regimens are challenged by a fast-declining eradication rate due to the increasing emergence of H. pylori strains resistant to existing antibiotics. Therefore, there is an urgent need to develop novel antibacterial strategies against H. pylori. In this study, we developed a liposomal nanoformulation of linolenic acid (LipoLLA) and evaluated its bactericidal activity against resistant strains of H. pylori. Using a laboratory strain of H. pylori, we found that LipoLLA was effective in killing both spiral and coccoid forms of the bacteria via disrupting bacterial membranes. Using a metronidazole-resistant strain of H. pylori and seven clinically isolated strains, we further demonstrated that LipoLLA eradicated all strains of the bacteria regardless of their antibiotic resistance status. Furthermore, under our experimental conditions, the bacteria did not develop drug resistance when cultured with LipoLLA at various sub-bactericidal concentrations, whereas they rapidly acquired resistance to both metronidazole and free linolenic acid (LLA). Our findings suggest that LipoLLA is a promising antibacterial nanotherapeutic to treat antibiotic-resistant H. pylori infection.     

Reacetylated chitosan microspheres for controlled delivery of anti-microbial agents to the gastric mucosa

The high aqueous solubility of chitosan restricts the utility of chitosan microspheres for gastric drug delivery. This paper describes the preparation of reacetylated chitosan microspheres with suitable properties for the controlled release of active anti-microbial agents, such as amoxycillin and metronidazole, in the gastric cavity. Two different microencapsulation approaches were developed and optimized in order to encapsulate hydrophilic (amoxycillin) and hydrophobic (metronidazole) compounds efficiently. The reacetylated chitosan microspheres exhibited a controlled water swelling capacity and gelified at acidic pH, resulting in prolonged release of the encapsulated antibiotics. The reacetylation time was found to be a key factor that affects not only drug release, but also encapsulation efficiency and anti-microbial activity of the encapsulated compound. The last two parameters were also dependent on drug solubility in the reacetylating agent. Using short reacetylation time periods, it was possible to efficiently control the release of both hydrophilic and lipophilic antibiotics while maintaining their activity against different bacteria. Consequently, reacetylated chitosan microspheres are promising vehicles for the controlled delivery of anti-microbial agents to the gastric cavity and, hence, for the eradication of Helicobacter pylori, a pathogen strongly associated with gastric ulcers and possibly gastric carcinoma.     

Reacetylated chitosan microspheres for controlled delivery of anti-microbial agents to the gastric mucosa

The high aqueous solubility of chitosan restricts the utility of chitosan microspheres for gastric drug delivery. This paper describes the preparation of reacetylated chitosan microspheres with suitable properties for the controlled release of active anti-microbial agents, such as amoxycillin and metronidazole, in the gastric cavity. Two different microencapsulation approaches were developed and optimized in order to encapsulate hydrophilic (amoxycillin) and hydrophobic (metronidazole) compounds efficiently. The reacetylated chitosan microspheres exhibited a controlled water swelling capacity and gelified at acidic pH, resulting in prolonged release of the encapsulated antibiotics. The reacetylation time was found to be a key factor that affects not only drug release, but also encapsulation efficiency and anti-microbial activity of the encapsulated compound. The last two parameters were also dependent on drug solubility in the reacetylating agent. Using short reacetylation time periods, it was possible to efficiently control the release of both hydrophilic and lipophilic antibiotics while maintaining their activity against different bacteria. Consequently, reacetylated chitosan microspheres are promising vehicles for the controlled delivery of anti-microbial agents to the gastric cavity and, hence, for the eradication of Helicobacter pylori, a pathogen strongly associated with gastric ulcers and possibly gastric carcinoma.     

Synthesis and in-vitro antibacterial activity of 5-substituted 1-methyl-4-nitro-1H-imidazoles

A series of 5-substituted 1-methyl-4-nitro-1H-imidazole derivatives were synthesized and evaluated for in-vitro antibacterial activity against a panel of microorganisms including Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Enterobacter aerogenes, and Helicobacter pylori using conventional agar dilution method. Among the test compounds, 1-methyl-4-nitro-5-(phenylsulfonyl)-1H-imidazole was the most potent against Gram-positive bacteria, with a MIC value of < or =8 microg/mL. All compounds showed no significant activity against Gram-negative bacteria at concentrations < or =64 microg/mL. The MIC values against 15 clinical isolates of H. pylori indicated that compounds 10 and 11 were the most active compounds in this series in terms of inhibiting the growth of H. pylori (MIC = 2 microg/mL). It was also demonstrated that their corresponding activities were four times larger than that of metronidazole.     

DNA single strand breaks in peripheral blood lymphocytes induced by three nitroimidazole derivatives

Tinidazole (TNZ), ornidazole (ONZ) and metronidazole (MTZ) are antiparasitic drugs (nitroimidazole derivatives) that have proven to be effective against Trichomonas vaginalis, Entoamoeba histolytica, Giardia lamblia and Helicobacter pylori. The reduction of the nitro group and the generation of short-lived reactive intermediates are the basis of its parasiticidal activity. This reduction is associated with its mutagenic activity in bacteria, although in mammalian cells DNA damage seems to be related to the production of reactive oxygen species (ROS). Using alkaline single cell electrophoresis, a significant increase in single strand breaks and alkali labile sites in human peripheral blood lymphocytes (PBL) exposed to MTZ, ONZ and TNZ at 10, 100 and 500 microg/ml is observed. MTZ causes less damage, especially at higher concentrations, when compared with TNZ, the most harmful of the drugs tested. These findings suggest that primary damage is induced under aerobic conditions and confirms that these nitroimidazoles are DNA damaging agents.     

Anti-Helicobacter pylori activity of Chinese tea: in vitro study

BACKGROUND: Chinese tea has an antibacterial activity against a wide range of bacteria. However, its activity against Helicobacter pylori has not been reported. METHOD: In this study the anti-Helicobacter pylori effects of a Chinese tea (Lung Chen tea), and two tea catechins, epigallocatechin gallate and epicatechin and their minimum inhibitory concentrations (MICs) were examined. The effect of Lung Chen on metronidazole resistance was also studied using the E-test. RESULTS: Lung Chen, epigallocatechin gallate and epicatechin all inhibited the growth of H. pylori. The MIC90 for Lung Chen was 0. 25-0.5% (w/w) and that of epigallocatechin gallate and epicatechin were 50-100 and 800-1600 microg/mL, respectively. Epigallocatechin gallate is probably the active ingredient responsible for most of the anti-H. pylori activity of Chinese tea. Lung Chen did not reverse metronidazole resistance. CONCLUSIONS: Chinese tea has anti-H. pylori activity in a daily consumed concentration, and epigallocatechin gallate is probably the active ingredient responsible for the action.     

Effect of cyclo-oxygenase inhibitors on Helicobacter pylori susceptibility to metronidazole and clarithromycin

BACKGROUND: We previously reported that aspirin inhibited Helicobacter pylori growth and suppressed the mutagenic effect of metronidazole. AIM: To determine the effects of a cyclo-oxygenase (COX)-2-specific inhibitor, SC-236, and a non-selective COX inhibitor, indometacin, on the growth, urease activity and antimicrobial susceptibility of H. pylori. METHODS: Three H. pylori reference strains, and 18 clinical isolates were treated with SC-236 or indometacin for 24 and 48 h. Growth, urease activity and susceptibility to clarithromycin and metronidazole of the bacteria were assessed by viable colony counting, spectrophotometry and E-test respectively. RESULTS: SC-236 and indometacin inhibited H. pylori growth in a dose-dependent manner with the lowest inhibitory concentrations of 0.03 and 0.1 mm, and the lethal concentrations of 0.09 and 0.3 mm, respectively. The numbers of CFU/mL in Brucella broth containing 0.09 mm SC-236 were 2 log lower at 24 h, and even 3 log lower at 48 h than that at 0 h (P = 0.035, compared with the vehicle control). Treatment of 0.3 mm indometacin reduced the number of CFU/mL by 1 log at 24 h compared with that at 0 h (P = 0.037 compared with the vehicle control). Helicobacter pylori urease activity began to decrease with 0.06 mm SC-236 at 24 h (P = 0.016), and 0.3 mm indometacin at 48 h (P = 0.025). MICs of metronidazole and clarithromycin against H. pylori were decreased significantly in the presence of 0.03 mm SC-236 or 0.1 mm indometacin (all P < 0.001). CONCLUSION: Both SC-236 and indometacin suppressed the growth and urease activity of H. pylori in a dose-dependent manner, and increased its susceptibility to the antibiotics.     

A new quadruple therapy for Helicobacter pylori: influence of resistant strains on treatment outcome

BACKGROUND: There have been no reports concerning the efficacy and safety of a 1-week quadruple therapy regimen of omeprazole, amoxycillin, roxithromycin and metronidazole for Helicobacter pylori infections and the impact of primary resistance on the eradication rate. METHODS: One hundred and sixty-nine consecutive patients with peptic ulcer disease as well as gastritis with biopsy-proven H. pylori infection were entered into an open study of omeprazole 20 mg o.m., amoxycillin 500 mg t.d.s., roxithromycin 150 mg b.d., and metronidazole 250 mg t.d.s. Helicobacter pylori status was determined by urease test, histology and culture. Susceptibility to amoxycillin, metronidazole and roxithromycin was determined by the E-test. RESULTS: H. pylori was eradicated in 155 out of 169 (92%; 95% CI 88-96%) by intention-to-treat analysis, and in 155 out of 163 (95%; 95% CI 92-98%) by per protocol analysis. The prevalence of primary resistance against amoxycillin, roxithromycin and metronidazole was 2 out of 166 (1%), 16 out of 166 (10%) and 27 out of 166 (16%), respectively. H. pylori was eradicated in 25 out of 27 (93%) patients with metronidazole-resistant strains compared with 130 out of 136 (96%) in patients with metronidazole-sensitive strains of H. pylori. It was eradicated in 15 out of 16 (94%) patients with roxithromycin-resistant strains while in 140 out of 147 (95%) patients with roxithromycin-sensitive strains of H. pylori, and in two out of two (100%) patients with amoxycillin-resistant stains compared with 153 out of 161 (95%) in patients with amoxycillin-sensitive strains. H. pylori was eradicated in three out of four (75%) patients with double resistance against metronidazole and roxithromycin compared with 152 out of 159 (96%) patients with sensitive strains to metronidazole and or roxithromycin. None of these differences were statistically significant. Severe side-effects were found in only one out of 169 patients-anaphylaxis due to penicillin. CONCLUSIONS: The 1-week quadruple therapy with omeprazole, amoxycillin, metronidazole and roxithromycin was found to eradicate H. pylori in over 90% of all patients. This regimen was also found to be beneficial for patients with pre-treatment resistant strains to metronidazole, roxithromycin or amoxycillin, and was observed to be safe and well-tolerated.     

High prevalence and level of resistance to metronidazole, but lack of resistance to other antimicrobials in Helicobacter pylori, isolated from a multiracial population in Kuwait

BACKGROUND: The primary treatment regimen for Helicobacter pylori infection for Kuwaitis does not contain metronidazole, but that for expatriates does. There is also increasing failure of antimicrobial therapy. AIM: To determine the susceptibility of H. pylori from upper gastrointestinal biopsies of Kuwaitis and non-Kuwaitis to find out if differences existed in the susceptibilities of the isolates from the two different populations. METHODS: The susceptibilities of 96 H. pylori isolates were tested against metronidazole, amoxicillin, clarithromycin and tetracycline by the E test. The rdxA gene was analysed from selected metronidazole-susceptible and metronidazole-resistant strains to find out polymorphism and the basis of metronidazole resistance. RESULTS: Approximately, 70% of isolates from both populations were metronidazole resistant with 65% isolates showing high minimum inhibitory concentration values of >256 mug/mL. No resistance to the other three antimicrobials was found. There were novel nonsense and missense mutations with no deletion in the rdxA gene by insertion of mini-IS605. CONCLUSIONS: The prevalence and level of metronidazole resistance in H. pylori in the two populations was high with no difference, in spite of different treatment regimens. Metronidazole resistance in this transitional country appeared to be independent of prior metronidazole use for treatment of H. pylori infection.     

Nizatidine and omeprazole enhance the effect of metronidazole on Helicobacter pylori in vitro

Treatment failures are common in patients infected with metronidazole-resistant Helicobacter pylori in the gastric mucosa when triple therapy including metronidazole is used. In patients with treatment failure and metronidazole-resistant H. pylori, a higher eradication rate for H. pylori was found after secondary treatment with bismuth/ranitidine in combination with antibiotics including metronidazole, compared with the same antibiotics combined with a standard dose of omeprazole. This agrees with our previous finding that bismuth was able to reduce the susceptibility of H. pylori to metronidazole. In this study, we have found that nizatidine, an H(2)-receptor antagonist, is also able to reduce the susceptibility of H. pylori to metronidazole in vitro, despite having no direct inhibitory effect on the growth of H. pylori. This agrees with earlier findings that compounds having the ability to reverse antibiotic resistance do not necessarily have an antibiotic or chemotherapeutic effect in the sense of growth inhibition. Therefore, it was decided to investigate the effect of nizatidine and omeprazole on the oxidative respiratory chain, as it is known that metronidazole is able to inhibit the activity of fumarate reductase of H. pylori. This enzyme is a key enzyme in the alternative respiratory chain under anaerobic conditions. Nizatidine was, in these preliminary experiments, found to inhibit fumarate reductase in a dose-dependent way, like metronidazole, whereas omeprazole had almost no effect on fumarate reductase. No other significant effects on the enzymes of the respiratory chain were found. The synergistic effect of nizatidine on metronidazole resistant H. pylori strains could be explained by the effect on fumarate reductase, whereas the effect of omeprazole is different and could be an inhibition of a proton pump in H. pylori. Reversal of antimicrobial resistance with the help of different non-antibiotics seems to be possible by using quite different compounds, and is therefore to be explained by different molecular mechanisms.     

幽门螺杆菌相关性消化性溃疡的中西医结合治疗

目的观察丹参饮联合奥美拉唑、阿莫西林、甲硝唑治疗幽门螺杆菌（HP）相关性消化性溃疡的临床疗效。方法将150例HP相关性消化性溃疡随机分为治疗组75例,对照组75例。治疗组采用丹参饮联合奥美拉唑、阿莫西林、甲硝唑治疗;对照组单用奥美拉唑、阿莫西林和甲硝唑治疗;观察二组治疗前后胃镜、HP的变化。结果治疗组在溃疡愈合、HP根除,与对照组相比差异有显著性意义（P〈0.05）。结论中西医结合治疗能显著提高幽门螺杆菌感染的根除率,并且患者临床症状有明显改善。     

Enhancement of the antibacterial activity of ampicillin by liposome encapsulation

This study showed that encapsulation of ampicillin (Amp) in liposomes prepared with synthetic lecithins enhanced its antibiotic activity against both Amp-sensitive and Amp-resistant Escherichia coli. This was demonstrated by growth inhibition of E. coli in the presence of the liposomal preparations containing Amp. Growth inhibition was also seen in the presence of exogenous β-lactamase. Adsorption of Amp onto the surface of the liposomes did not result in enhanced activity of the drug against E. coli. The encapsulation efficiency of Amp in liposomes prepared by the Bangham method (BM) was greatly affected by the phospholipids used. The efficiency increased with a rise in the phase transition temperature (T_c) of the phospholipid, the maximum encapsulation of Amp being obtained with distearoylphosphatidylcholine (DSPC). The entrapment efficiency of Amp with the reverse phase evaporation method (REV) was largely superior to that with the BM method. Kinetic studies using DSPC liposomes prepared by the REV and BM protocols demonstrated that Amp-loaded liposomes contained 60 and 72% of drug, respectively, after 24 h at 37°C.     

A novel colorimetric broth microdilution method to determine the minimum inhibitory concentration (MIC) of antibiotics and essential oils against Helicobacter pylori

Helicobacter pylori infections have been associated with the pathogenesis of a number of stomach and gastroduodenal diseases. In order to find alternative drugs for their treatment the search is increasingly focused on new antimicrobial products. However, no standardized methods are available to test the anti-Helicobacter pylori activity in particular of natural substances. Therefore we developed a broth microdilution assay to investigate the susceptibility of this fastidious slow growing bacterium against 15 essential oils widely used to treat disorders of the gastrointestinal tract. The MIC values were determined colorimetrically using p-iodonitrophenyltetrazolium violet (INT) as an indicator for bacterial cell viability. The test sytem was evaluated with three common antibiotics: amoxicillin, ampicillin and levofloxacin. The antibiotic MICs were controlled by Etest. The Helicobacter reference strain was remarkably susceptible to both the antibiotics (amoxicillin MIC: 0.02 microg/ml, ampicillin MIC: 0.064 microg/ml, levofloxacin MIC: 0.39 microg/ml) and the essential oils. Most of their MICs ranged from 0.015 to 0.064% (v/v) and about 140.0 to 280.0 microg/ml, respectively. Interestingly, chamomile oil, orange flower oil and ginger oil inhibited the bacterial growth in extraordinarily low concentrations of 0.0075% (v/v) and about 65 microg/ml, respectively. The bactericidal concentrations were generally one to two dilution steps higher. In conclusion, we could develop an innovative assay for the MIC determination of essential oils and antibiotics against Helicobacter pylori, which is simple to handle, accurate, reproducible and not as time- and material-consuming as traditional agar dilution techniques.     

Amoxicillin and ampicillin are not transferred to gastric juice irrespective of Helicobacter pylori status or acid blockade by omeprazole

BACKGROUND: The effects of proton pump inhibitors and Helicobacter pylori infection on the distribution of drugs used for the eradication of the bacteria are poorly understood. AIM: The aim of this study was to investigate the effects of a 7-day administration of 20 mg of omeprazole on the pharmacokinetics of amoxicillin and ampicillin in the plasma, saliva and gastric juice of individuals with and without H. pylori infection. METHODS: Fifty-four healthy volunteers without endoscopic lesions were enrolled. Twenty-six volunteers were included in the amoxicillin study and 28 individuals in the ampicillin study. Each study had an open randomized two-period crossover design and a 21-day washout period between phases. Plasma, saliva and gastric juice concentrations of amoxicillin and ampicillin in subjects with and without omeprazole pre-treatment were measured by reversed-phase HPLC using UV detection. RESULTS: Neither pre-treatment with omeprazole nor H. pylori infection interfered with the plasma bioavailability of amoxicillin or ampicillin, as assessed by the AUC0-2 h. Neither ampicillin nor amoxicillin were detected in saliva or gastric juice in any study phase. CONCLUSION: Short-term treatment with omeprazole does not interfere with the pharmacokinetics of amoxicillin or ampicillin. Our results also exclude the presence of a transfer mechanism for amoxicillin or ampicillin from the plasma to the gastric lumen.     

Anti-infectious effect of S-benzylisothiourea compound A22, which inhibits the actin-like protein, MreB, in Shigella flexneri

S-Benzylisothiourea compound A22 induces coccoid forms in Escherichia coli by inhibiting the function of the actin-like cytoskeletal protein, MreB. The minimum inhibitory concentration of A22 and the minimum concentration to induce coccoid forms for various pathogenic bacteria were determined. At 10 microg/ml, A22 induced coccoid forms in Shigella flexneri but did not inhibit the growth. No alteration of coccoid forms in the Gram-positive bacteria and anaerobic bacteria tested were observed following treatment with A22. To study the relationship between pathogenicity and alterations in bacterial shape, the infectious capacity of A22-induced coccoid S. flexneri was examined using CHO-K1 cells. Invasion of the coccoid cells was significantly reduced, however, no changes in adherence were observed. Using a mutant defective in the type III secretion apparatus, which delivers effectors to the host, we examined the secretion of effectors by A22-induced coccoid S. flexneri. The amount of secreted effectors in the coccoid cells was clearly decreased compared to rod-shaped cells. These results showed that the maintenance of rod-shaped cells by MreB in bacteria was essential for the secretion of effectors via the type III secretion system. Therefore, our results suggest that A22 is a useful lead compound for a novel anti-infectious agent without bactericidal activity and MreB is a candidate target site for development of new anti-infectious agents.     

雷贝拉唑治疗幽门螺杆菌阳性消化性溃疡的疗效观察

目的:观察雷贝拉唑三联疗法对幽门螺杆菌(HP)阳性的消化性溃疡的疗效.方法:70例符合条件的患者随机分为两组,治疗组35例,应用雷贝拉唑+甲硝唑+克拉霉素三联1周,之后雷贝拉唑3周疗法;对照组35例,应用奥美拉唑+甲硝唑+克拉霉素三联1周,之后奥美拉唑3周疗法.治疗前、治疗4周后均进行胃镜和HP检查,评价疗效.结果:治疗组与对照组溃疡愈合率分别为97.06%和93.75%,两组间差异无统计学意义(P＞0.05);HP根除率分别为91.18%和68.75%,两组间差异有统计学意义(P＜0.05).结论:雷贝拉唑三联疗法可作为HP感染的消化性溃疡的首选疗法.     

Enhancement of the antibacterial activity of ampicillin by liposome encapsulation

Abstract

This study showed that encapsulation of ampicillin (Amp) in liposomes prepared with synthetic lecithins enhanced its antibiotic activity against both Amp-sensitive and Amp-resistant Escherichia coli. This was demonstrated by growth inhibition of E. coli in the presence of the liposomal preparations containing Amp. Growth inhibition was also seen in the presence of exogenous β-lactamase. Adsorption of Amp onto the surface of the liposomes did not result in enhanced activity of the drug against E. coli. The encapsulation efficiency of Amp in liposomes prepared by the Bangham method (BM) was greatly affected by the phospholipids used. The efficiency increased with a rise in the phase transition temperature (T_c) of the phospholipid, the maximum encapsulation of Amp being obtained with distearoylphosphatidylcholine (DSPC). The entrapment efficiency of Amp with the reverse phase evaporation method (REV) was largely superior to that with the BM method. Kinetic studies using DSPC liposomes prepared by the REV and BM protocols demonstrated that Amp-loaded liposomes contained 60 and 72% of drug, respectively, after 24 h at 37°C.     

Helicobacter pylori: cultivability and antibiotic susceptibility of coccoid forms

Helicobacter pylori is an actively dividing helical bacterium that changes to coccoid morphology as the culture ages. It has been suggested that the coccoid forms may be involved in transmission of infection and in relapses following antimicrobial therapy. The aim of this investigation was to determine the survival and susceptibility of the coccoid forms to amoxycillin, erythromycin, gentamicin and metronidazole. Colony counts and microscopic examination were performed after 1–4 weeks of culture. At 2 and 4 weeks, identical cultures were treated with the antibiotics for 24 h. Our results showed that 4-week cultures of coccoid forms were cultivable after antibiotic treatment.     

Potentiation of the action of metronidazole on Helicobacter pylori by omeprazole and bismuth subcitrate

Treatment failures using triple therapy that include metronidazole, are common in patients infected with metronidazole-resistant Helicobacter pylori in the gastric mucosa. Higher eradication rates in such patients have been described when treatment regimens include bismuth salts compared to regimens that include proton pump inhibitors. In the present study, the synergistic effect of subinhibitory concentrations (0.25-0.5 MIC) of either bismuth subcitrate or omeprazole with metronidazole on the susceptibility of 42 H. pylori strains was investigated by agar dilution method and the Epsilometer test (Etest). With 0.5 MIC of either of the two drugs, the susceptibility of all H. pylori4 mg/l) reverted to being metronidazole sensitive. These results suggested that either bismuth salts or proton pump inhibitors may be effective in the treatment of some infections with metronidazole-resistant H. pylori strains when used in sufficiently high doses.     

A practical approach to patients with refractory Helicobacter pylori infection, or who are re-infected after standard therapy.

The vast majority of recurrences of Helicobacter pylori infection after apparent eradication are observed during the first year. Almost all of these early recurrences are due to recrudescence rather than reinfection by a new strain. After the first year, the recurrence rates approximate to the rate of natural acquisition of H. pylori infection. By contrast, in developing countries, higher rates of recurrence suggest a major role of real reinfection. Important predictive factors of H. pylori treatment success are compliance and bacterial susceptibility to antibiotics. The new 1-week triple therapies, based on a proton pump inhibitor (PPI) and 2 antibiotics, lead to treatment discontinuation but rarely. If containing a nitroimidazole, their efficacy is reduced to 60 to 80% by pretreatment in vitro resistance. The prevalence of nitroimidazole resistance varies dependent on the geographical area, with rates over 50% in tropical regions. Resistance against macrolides hinders treatment success in 50 to 80% of patients. In the US, south-western Europe and Japan the prevalence of macrolide resistance amounts to about 10%, in other countries about 3%. After failed treatment, acquired resistance is frequent. Testing for resistance is recommended to facilitate the decision for an alternative triple therapy or for quadruple therapy comprising bismuth, metronidazole, tetracycline and a PPI. It seems reasonable to increase the dose of PPI in a retreatment regimen containing amoxicillin. Post-treatment double resistance against nitroimidazoles and macrolides reduces the success of most of the currently evaluated retreatment regimens. To overcome double resistance, high dose PPI plus amoxicillin is one approach, beside other experimental multidrug treatments.