Resistance rate to antibiotics of Helicobacter pylori isolates in eastern Taiwan

BACKGROUND AND AIM: Prevalence of Helicobacter pylori (H. pylori) strains resistant to metronidazole, clarithromycin and amoxicillin is increasing worldwide. The aim of this study was to determine the antibiotic susceptibility patterns in H. pylori strains isolated from eastern Taiwan. METHODS: One strain each of H. pylori was isolated from 133 symptomatic patients and subjected to determination of the minimal inhibitory concentration (MIC) by the Epsilometer test (E-test) for four antibiotics commonly used in the treatment of H. pylori infections. RESULTS: None of the strains were resistant to tetracycline. Resistance to metronidazole (8 microg/mL), clarithromycin (1 microg/mL) and amoxicillin (8 microg/mL) was found in 51.9%, 13.5% and 36.1% of the isolates, respectively. Metronidazole-resistant strains were isolated more frequently from women (49/78; 62.8%) than from men (20/55; 36.4%). Resistance to at least two antimicrobial agents was detected in 33.8% of the isolates. There was a high rate of resistance to both metronidazole and amoxicillin (18.1%). CONCLUSIONS: Clarithromycin and tetracycline may provide useful components of treatment regimens in eastern Taiwan. In addition, pretreatment microbial susceptibility testing rather than empiric therapy is highly recommended for eradication of H. pylori infection.     

Prevalences of metronidazole‐ and clarithromycin‐resistant Helicobacter pylori isolates in Shanghai and molecular mechanism of resistance to clarithromycin

OBJECTIVE : To investigate the prevalences of metronidazole‐ and clarithromycin‐resistant Helicobacter pylori over the period from 1995 to 1999 in Shanghai, and the molecular mechanism of resistance to clarithromycin. METHODS : A total of 150 H. pylori strains were randomly selected from the isolates collected in 1995, 1997 and 1999, and tested for sensitivity against metronidazole and clarithromycin by using the E‐test. The mechanism of resistance was studied by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP). RESULTS : It was found that 42% (21/50), 57% (27/50) and 70% (35/50) of the tested strains were resistant to metronidazole among the isolates collected in 1995, 1997 and 1999, respectively. In 1995, there was no strain (0/50) resistant to clarithromycin, of which the prevalence rose to 2% (1/50) in 1997, and to 10% (5/50) in 1999. The prevalences of metronidazole‐ and clarithromycin‐resistant H. pylori in 1999 were significantly higher than those in 1995 (P < 0.05). Of nine clarithromycin‐resistant H. pylori strains, eight were found to have an A→G mutation at position 2144 of domain V of the 23S rRNA. CONCLUSIONS : These results suggest a significant increase in the prevalences of metronidazole‐ and clarithromycin‐resistant H. pylori in Shanghai during the 1995–1999 period. The majority (88.8%) of clarithromycin‐resistant H. pylori isolates have an A2144G mutation in domain V of the 23S rRNA.     

A randomized controlled comparison of three quadruple therapy regimens in a population with low Helicobacter pylori eradication rates

BACKGROUND AND AIM: We sought to compare the efficacy and tolerability of an omeprazole/clarithromycin/bismuth/tetracycline-based quadruple therapy to that of a ranitidine/metronidazole/bismuth/tetracycline-based quadruple therapy of 2 or 3 weeks duration in a population with a high prevalence of metronidazole-resistant Helicobacter pylori and low triple therapy eradication rates. METHODS: Two hundred and twenty-one patients who presented endoscopically proven duodenal ulcers and a positive rapid urease test were randomized to receive either: (i) omeprazole 20 mg b.i.d., clarithromycin 250 mg b.i.d., bismuth subcitrate 240 mg b.i.d. and tetracycline 500 mg b.i.d (OCBT) for 2 weeks; (ii) ranitidine 300 mg b.i.d., metronidazole 500 mg b.i.d, bismuth subcitrate 240 mg b.i.d. and tetracycline 500 mg b.i.d. (RMBT2) for 2 weeks; or (iii) ranitidine 300 mg b.i.d., metronidazole 500 mg b.i.d, bismuth subcitrate 240 mg b.i.d. and tetracycline 500 mg b.i.d. (RMBT3) for 3 weeks. Patients were interviewed 2 weeks after the completion of therapy to review compliance and side-effects. Eradication of H. pylori was assessed 8 weeks after the completion of therapy with the use of a 14C-urea breath test. RESULTS: The per-protocol eradication rate was significantly higher with OCBT (88%) than RMBT2 (73%) or RMBT3 (71%) (P<0.05). The intent-to-treat eradication rate was numerically higher with OCBT (80%) than RMBT2 (68%) or RMBT3 (68%), although this difference did not reach statistical significance (P=0.09). Per-protocol or intent-to-treat eradication rates were similar with RMBT2 and RMBT3. There were significantly greater side-effects with the RMBT2 regimen. CONCLUSIONS: The omeprazole/clarithromycin/bismuth/tetracycline-based quadruple therapy provides higher H. pylori eradication rates than the ranitidine/metronidazole/bismuth/tetracycline-based quadruple therapy when administered per protocol. The prolongation of the latter regimen from 2 to 3 weeks did not increase eradication rates.     

Mechanism of metronidazole resistance in Helicobacter pylori

OBJECTIVE : Drug resistance to Helicobacter pylori is one of the most important reasons for the failure of anti‐H. pylori treatment. Metronidazole is a preferred drug for the elimination of H. pylori. However, using this drug alone can easily lead to resistance. The aim of the present study was to investigate the mechanism of metronidazole resistance in H. pylori. METHODS : International standard strain NCTC11639 was used. For the nitrate reduction test, four strains were used as positive controls, two strains of Bacillus coli and two strains of Vibrio cholerae. Metronidazole was used as tablets (0.2 g per tablet). The following techniques were used: (i) metronidazole used to apply selective pressure; (ii) sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS‐PAGE); (iii) nitrate reduction test; and (iv) test to detect the enzyme activities associated with 95 different substrates for Gram‐negative bacteria. RESULTS : As a result of the selective pressure of metronidazole, 20 H. pylori colonies survived; the bacteriostatic zones of these colonies were 0 mm in diameter, whereas the original NCTC11639 bacteriostatic zones were 25 mm in diameter. There was no significant difference seen among the 18 strains of drug‐resistant H. pylori and the original NCTC11639 strain protein electrophoresis strips. In the nitrate reduction test, both sensitive and resistant H. pylori all tested negative and the control strains of Bacillus coli and Vibrio cholerae tested positive. After the mutation of H. pylori from sensitive to resistant, the activities of enzymes associated with mono‐methyl succinate, succinic acid and D ‐alanine metabolism were decreased, and those associated with L ‐fucose and 6‐phosphate glucose metabolism were increased. CONCLUSIONS : The resistance of H. pylori to metronidazole is not related to nitroreductase. There may be no obvious changes in membrane protein in the drug‐resistant strain. The metronidazole resistance of H. pylori is associated with its metabolism and a change in enzyme activities.     

Assessing heterogeneity in meta‐analyses of Helicobacter pylori infection‐related clinical studies: a critical appraisal

OBJECTIVE: To critically assess the meta‐analyses of Helicobacter pylori infection‐related clinical studies, particularly the handling of between‐study heterogeneity. METHODS: A qualitative, all‐language, systematic literature search was performed in Medline, PubMed, BioMed Central and Embase up to February 2003, supplemented by a manual search of major relevant journals. Assessment was according to modified criteria for literature searching, eligibility criteria, validity assessment, data extraction and presentation. Five parameters were used to assess the quality of the meta‐analyses in handling between‐study heterogeneity. RESULTS: Of 84 potentially relevant citations, 47 were systematic reviews and of them 38 were meta‐analyses. Of these 38 studies, 15 (39.5%) had conducted a literature search of multiple databases and 34 (89.5%) had conducted a supplementary manual search. The eligibility criteria were clearly presented in 81.6% of studies, but the quality of the primary studies was assessed in only 26.3%. The process and strategy for data extraction was reported in 57.9% of all studies; 19 (50%) studies planned statistical tests of between‐study homogeneity and the results were reported in 18, but the level of statistical significance was reported in only 11 (57.9%). The selection of and justification for a statistical model was presented in 39.5% and 26.3% of studies, respectively. Among the 11 meta‐analyses in which statistical between‐study heterogeneity was reported, 54.5% ignored the statistical findings and proceeded to pool the study results. The implications of between‐study heterogeneity were discussed in only 8 studies. CONCLUSIONS: Many methodological flaws were identified in the meta‐analyses of H. pylori‐related clinical studies, particularly for assessing, reporting and interpreting between‐study heterogeneity. This warrants consistent and urgent adherence by reviewers and journal editors to the methodological guidelines for meta‐analyses.     

Effect of Helicobacter pylori infection on cyclooxygenase‐2 expression in gastric antral mucosa

OBJECTIVE: Helicobacter pylori infection is a major etiological cause of chronic gastritis. Inducible cyclooxygenase (COX‐2) is an important regulator of mucosal inflammation. Recent studies indicate that expression of COX‐2 may contribute to gastro­intestinal carcinogenesis. The aim of this study was to investigate the effects of H. pylori infection and eradication therapy on COX‐2 expression in gastric antral mucosa. METHODS: Antral biopsies were taken from 46 H. pylori‐infected patients, who also had chronic gastritis, both before and after anti‐H. pylori treatment. The COX‐2 protein was stained by using immunohistochemical methods and COX‐2 expression was quantified as the percentage of epithelial cells expressing COX‐2. Gastritis and H. pylori infection status were graded according to the Sydney system. RESULTS: Cyclooxygenase‐2 expression was detected in the cytoplasm of gastric antral epithelial cells both before and after the eradication of H. pylori. Cyclooxygenase‐2 expression in mucosa with H. pylori infection was compared with the corresponding mucosa after successful H. pylori eradication (20.1 ± 13.1%vs 13.8 ± 5.9%; P < 0.05). At the same time, COX‐2 expression in H. pylori‐infected mucosa was com­pared with the normal controls (18.0 ± 14.1%vs 12.3 ± 4.6%, P < 0.05). Expression of COX‐2 was correlated with the degree of chronic inflammation (r= 0.78, P < 0.05). CONCLUSIONS: Our results showed that H. pylori infection leads to gastric mucosal overexpression of COX‐2 protein, suggesting that the enzyme is involved in H. pylori‐related gastric pathology in humans.     

幽门螺杆菌体外诱导耐药试验和耐药率监测

目的 抗生素耐药越来越被公认为是根除幽门螺杆菌（Hp)治疗失败的主要原因。比较根除Hp常用抗生素耐药性发生倾向和监测耐药率。方法 选用7株敏感菌株（其中2株为标准菌株）进行阿莫西林，四环素，呋喃唑酮，甲硝唑和克拉霉素5种抗生素的体外诱导耐药试验。随机选取2000-2001年间保存的165株菌株，用琼脂稀释法测定最低抑菌浓度（MIC），对上述5种抗生素进行耐药监测。结果 体外诱导试验显示，7株Hp中5株诱导出甲硝唑耐药，且可诱导倍数最高。5株诱导出四环素耐药。虽未诱导出克拉霉素耐药，但有1株可诱导倍数较高。未诱导出阿莫西林或呋喃唑酮耐药，呋喃唑酮可诱导倍数最低。耐药监测甲硝唑耐药率为49.7%(82/165)，克拉霉素为7.3%(12/165)，阿莫西林为1.2%(2/165)，四环素为2.4%(4/165)，呋喃唑酮为1.2%(2/165)。结论 Hp对甲硝唑很容易产生耐药，对克拉霉素可产生耐药，对呋喃唑酮和阿莫西林不易产生耐药。除四环素外，Hp对其他4种抗生素发生耐药的难易程度与实际监测的耐药率高低相关，这有助于预测感染Hp后抗生素耐药率变迁的倾向。     

The Loeffler's methylene blue stain: An inexpensive and rapid method for detection of Helicobacter pylori

Abstract Loeffler's methylene blue, commonly used as a counter-stain for acid fast bacilli, was used to detect Helicobacter pylori in paraffin sections and touch smears of gastric mucosal biopsies from 15 patients with duodenal ulcer and 35 with non-ulcer dyspepsia, and the results were compared with the modified Giemsa stain. The time taken to stain smears by Loeffler's methylene blue was approximately 10 min and the results correlated well with those stained by the modified Giemsa stain. However, the Loeffler's methylene blue method was found to be simpler, quicker and cheaper than the modified Giemsa stain.     

Relevance of antibiotic sensitivities in predicting failure of omeprazole, clarithromycin, and tinidazole to eradicate Helicobacter pylori

Omeprazole 20 mg once (o.d.) or twice daily (b.d.), clarithromycin 250 mg b.d., and tinidazole 500 mg b.d. for 7 days (OCT) is an effective regimen against Helicobacter pylori, but the effect of 5-nitroimidazole resistance is unclear. We aimed to evaluate this using the disc diffusion technique (Mast Diagnostics, Bootle, UK) and E-test (Cambridge Diagnostics Services, Cambridge, UK) to assess 5-nitroimidazole resistance. H. pylori was cultured from antral biopsies of infected patients, as determined by ¹³C-urea breath test (¹³C-UBT), histology, and/or rapid urease test. Patients were prescribed OCT and H. pylori eradication was assessed by ¹³C-UBT at least 4 weeks after completion of therapy. Antibiotic sensitivities to metronidazole and clarithromycin were evaluated by the disc diffusion method and by the measurement of minimum inhibitory concentration (MIC) using the E-test. One hundred and forty-one H. pylori-infected patients were enrolled in the study and the organism was successfully cultured from 119 patients (84%). The overall eradication rate was 125/141 (89%). OCT was successful in 62/69 (90%) patients harboring fully sensitive strains of H. pylori, compared with 42/45 (93%) of patients with strains that were resistant to metronidazole alone (P = 0.74, Fisher's exact test). MIC was assessed in 22 samples. Using a cut-off point of >32 μg/ml to define metronidazole resistance eradication rates were higher against sensitive (9/12; 75%) than resistant (3/10; 30%) strains (P = 0.08, Fisher's exact test). 5-Nitroimidazole resistance assessed by the disc diffusion technique is not helpful in predicting OCT failure, but the E-test may be of value. (Received June 9, 1997; accepted Aug. 22, 1997)     

嗜酸乳杆菌对球形幽门螺杆菌的细胞黏附和增殖作用的影响

目的研究球形幽门螺杆菌(Hp)对AGS细胞的黏附和增殖作用及热灭活状态和活菌状态嗜酸乳杆菌对其产生的影响。方法制备AGS细胞爬片,按球形Hp对照组、球形Hp与活/灭活嗜酸乳杆菌实验组加入细菌,电镜、革兰染色后光镜观察Hp对AGS细胞黏附指数,同时用荧光抗体染色法评价黏附于AGS细胞的Hp密度。运用CCK-8比色法检测球形Hp对AGS细胞的增殖影响及比较加入嗜酸乳杆菌产生的变化。结果实验组与对照组比较,球形Hp对细胞的黏附指数及黏附密度均明显下降(P0.05)。低浓度的球形Hp促进细胞增殖,高浓度的球形Hp抑制细胞增殖。两种不同状态的嗜酸乳杆菌均能降低Hp对AGS细胞增殖的影响。结论球形Hp对AGS细胞具有黏附能力并随浓度不同对其产生不同增殖影响,嗜酸乳杆菌能降低其作用。     

幽门螺杆菌提取液诱导AGS细胞内活性氧类物质水平的变化

目的 探讨幽门螺杆菌(Hp)感染与AGS细胞内活性氧类物质(ROS)的关系.方法 将Hp11638(CagA~+,VacA~+)菌株及其突变株Hp11638M(CagA~+,VacA~-)提取液与AGS细胞共同孵育,收集细胞及培养上清液.流式细胞术检测细胞内ROS水平,分光光度计550 nm处检测培养上清液中细胞色素C含量.结果 AGS细胞内ROS水平与2株Hp提取液呈浓度及时间依赖关系,经Hp11638提取液刺激的AGS细胞内ROS水平高于Hp11638M提取液.细胞色素C减少证实了这一结果 .结论 AGS细胞内ROS升高是Hp多种蛋白共同作用的结果 ,VacA蛋白参与了这一过程.     

Cholestenone functions as an antibiotic against Helicobacter pylori by inhibiting biosynthesis of the cell wall component CGL

Helicobacter pylori, a pathogen responsible for gastric cancer, contains a unique glycolipid, cholesteryl-α-D-glucopyranoside (CGL), in its cell wall. Moreover, O-glycans having α1,4-linked N-acetylglucosamine residues (αGlcNAc) are secreted from gland mucous cells of gastric mucosa. Previously, we demonstrated that CGL is critical for H. pylori survival and that αGlcNAc serves as antibiotic against H. pylori by inhibiting CGL biosynthesis. In this study, we tested whether a cholesterol analog, cholest-4-en 3-one (cholestenone), exhibits antibacterial activity against H. pylori in vitro and in vivo. When the H. pylori standard strain ATCC 43504 was cultured in the presence of cholestenone, microbial growth was significantly suppressed dose-dependently relative to microbes cultured with cholesterol, and cholestenone inhibitory effects were not altered by the presence of cholesterol. Morphologically, cholestenone-treated H. pylori exhibited coccoid forms. We obtained comparable results when we examined the clarithromycin-resistant H. pylori strain “2460.” We also show that biosynthesis of CGL and its derivatives cholesteryl-6-O-tetradecanoyl-α-D-glucopyranoside and cholesteryl-6-O-phosphatidyl-α-D-glucopyranoside in H. pylori is remarkably inhibited in cultures containing cholestenone. Lastly, we asked whether orally administered cholestenone eradicated H. pylori strain SS1 in C57BL/6 mice. Strikingly, mice fed a cholestenone-containing diet showed significant eradication of H. pylori from the gastric mucosa compared with mice fed a control diet. These results overall strongly suggest that cholestenone could serve as an oral medicine to treat patients infected with H. pylori, including antimicrobial-resistant strains.

Helicobacter pylori is a gram-negative microaerophilic pathogen that colonizes the human stomach in approximately half the world’s population. It is well established that H. pylori infection is closely associated with pathogenesis of chronic active gastritis, peptic ulcer, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma (1–4). Thus, in 1994, H. pylori was categorized as a Group I carcinogen by the World Health Organization’s International Agency for Research on Cancer. Accordingly, eradication therapy for H. pylori is expected to decrease the incidence of gastric cancer (5–7). In fact, eradication of the bacterium has been successfully achieved in ∼90% of infected patients using a combination of three drugs, namely, a proton pump inhibitor (PPI), clarithromycin, and amoxicillin (8, 9). However, successful eradication has been challenged by emergence of drug-resistant strains, in particular, clarithromycin-resistant H. pylori (10). Thus, development of new strategies as eradication therapy for H. pylori including drug-resistant strains is needed.The cell wall of Helicobacter species, including H. pylori, characteristically contains unique glycolipid α-cholesteryl glucosides (αCGs), of which the major components are cholesteryl-α-D-glucopyranoside (CGL), cholesteryl-6-O-tetradecanoyl-α-D-glucopyranoside (CAG), and cholesteryl-6-O-phosphatidyl-α-D-glucopyranoside (CPG) (11). αCGs are synthesized by cholesterol α-glucosyltransferase (αCgT), which transfers glucose from UDP-glucose to a carbon atom at the third position of cholesterol with an α1,3-linkage (SI Appendix, Fig. S1A). On the other hand, gastric gland mucous cells secrete unique O-glycans having terminal α1,4-linked N-acetylglucosamine (αGlcNAc) attached to the scaffold protein MUC6. Previously, we revealed that αGlcNAc suppresses H. pylori growth by inhibiting αCgT activity, which forms CGL (12, 13). Because the H. pylori genome does not encode enzymes required for cholesterol biosynthesis, microbes require exogenous cholesterol to synthesize αCGs (14, 15).Cholestenone is a cholesterol analog catabolized from cholesterol by intestinal bacteria, including human-derived Escherichia coli, Eubacterium, and Bacteroides sp. that replace the steroid 3β-hydroxyl group with a keto group (16–20). Because the hydroxyl group at the cholesterol third position is critical to form CGL, we hypothesized that cholestenone cannot serve as an αCgT substrate (SI Appendix, Fig. S1B) and thus that cholestenone treatment could inhibit H. pylori growth due to defective CGL biosynthesis.In the present study, we examined growth capacity of H. pylori in vitro in the presence of cholesterol and analogs including cholestenone, β-sitosterol, and cholestanol (SI Appendix, Fig. S2). Our results clearly indicate that growth of H. pylori, including that of a clarithromycin-resistant strain, was significantly suppressed by cholestenone through inhibition of CGL biosynthesis. When cholestenone was orally administered to H. pylori-infected C57BL/6 mice, mice showed successful eradication of the microbe. Because cholestenone is safe, therapy using cholestenone could be a promising approach to eliminate H. pylori infection in humans, including infection with antibiotic-resistant strains.     

Prevalence of Helicobacter pylori resistance to antibiotics in Northeast Italy: a multicentre study. GISU. Interdisciplinary Group for the Study of Ulcer.

Aims. To evaluate prevalence of primary Helicobacter pylori antibiotic resistances in Northeast Italy and to identify risk factors associated with this resistance.

Materials and methods. A total of 248 patients undergoing upper gastrointestinal endoscopy were enrolled from 19 Endoscopy Units over a 6-month period. From each patient, 4 gastric biopsies were taken for histology and 2 were sent to the Central Referral Microbiological Laboratory for culture and determination of antibiotic activity against Helicobacter pylori by means of E-test. Strains were considered resistant when minimum inhibitory concentration was >8 μg/ml for metronidazole and > 1 μg/ml for clarithromycin. No cut-off value was predefined for amoxycillin.

Results. Culture of Helicobacter pylori was successfully performed in 167 patients. Primary resistance to metronidazole, clarithromycin or amoxycillin was 14.9%, 1.8% and 0.%, respectively. Patients infected with Helicobacter pylori strains resistant to antibiotics were more frequently females than males (70.3% vs 41.4%), had a significantly lower coffee intake (66.6% vs 86.6%) and lower body mass index (23.7±2.6 vs 25.3±3.6) than patients with susceptible Helicobacter pylori strains. Age, smoking, alcohol use, family history of Helicobacter pylori infection, concomitant diseases and treatments, endoscopic diagnoses, Helicobacter pylori density and histological activity of chronic gastritis were not associated with antibiotic resistance. Multivariate analysis confirmed that female gender (odds RATIO = 2.74, 95% confidence INTERVAL = 1.03–7.27) was the only significant risk factor associated with antibiotic resistance.

Conclusions. In this population, primary Helicobacter pylori resistance to metronidazole was higher than resistance to clarithromycin, and female gender was significantly associated with this resistance. The low prevalence of resistance to metronidazole, clarithromycin and amoxycillin identified in this geographical area suggests that proton pump inhibitor-based triple regimens including these antibiotics may still be used as first line therapies against Helicobacter pylori infection.