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Journal of Digestive Diseases 2015; 16; 464–470
doi: 10.1111/1751-2980.12271
Original article
Susceptibility of Helicobacter pylori to antibiotics in Chinese patients Peng BAI,* Li Ya ZHOU,* Xiu Mei XIAO,† Yang LUO* & Yu DING* *Department of Gastroenterology and †Department of Laboratory Medicine, Peking University Third Hospital, Beijing, China
OBJECTIVE: Antibiotic resistance to Helicobacter pylori (H. pylori) has been increasing worldwide. The study aimed to evaluate in vitro susceptibility and resistance patterns to antibiotics in empirical H. pylori eradication regimens, and to determine the optimal antibiotics for treatment. METHODS: H. pylori strains (n =181) were obtained from gastric biopsies of patients with upper gastrointestinal symptoms who underwent esophagogastroduodenoscopy from March to December 2013. The susceptibility of H. pylori strains to amoxicillin (AMX), metronidazole (MTZ), clarithromycin (CLR), amoxicillin-clavulanate (AMC), cephalothin (CEP), cefuroxime (CXM), cefixime (CFM), moxifloxacin (MFX) and minocycline (MNO) was determined. RESULTS: Dual resistance to MTZ + CLR was detected in 48 (26.5%) isolates, MTZ + MFX in 94 KEY WORDS: moxifloxacin.
CONCLUSIONS: AMX, AMC, MNO and cephalosporins, but not MTZ, CLR and MFX, showed good in vitro anti-H. pylori activity. Among cephalosporins, CXM was the most active. H. pylori resistance is higher in patients with previous H. pylori eradication.
antibiotic resistance, susceptibility, clarithromycin, Helicobacter pylori, metronidazole,
INTRODUCTION Helicobacter pylori (H. pylori) was first identified by Warren and Marshall in 1983, since then numerous Correspondence to: Li Ya ZHOU, Department of Gastroenterology, Peking University Third Hospital, 49 North Garden Road,Haidian District, Beijing 100191, China. Email: [email protected] Conflict of interest: None. © 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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(51.9%), and CLR + MFX in 49 (27.1%). Overall, 41 (22.7%) were resistant to MTZ + CLR + MFX. MTZ and CLR resistance rates were significantly associated with the history of H. pylori eradication but there was no significant difference in MFX resistance rates between treated and untreated patients (P = 0.674). No significant relationship was found between antibiotic resistance and patient’s gender, age, endoscopic findings, inflammatory severity or gastric atrophy.
studies have demonstrated that H. pylori infection is closely associated with the development of chronic gastritis, peptic ulcers and gastric adenocarcinoma. The World Health Organization classified H. pylori as a type I carcinogen in 1994. H. pylori eradication could effectively alleviate or prevent disease development and progression.1 In recent years the eradication rate of H. pylori with standard triple therapy, including a proton pump inhibitor (PPI) and two of the three antibiotics (amoxicillin [AMX], clarithromycin [CLR] and metronidazole [MTZ]), has reduced to below 80% in
Journal of Digestive Diseases 2015; 16; 464–470 many regions of the world.2–4 This unacceptably low eradication rate is mainly caused by the resistance of H. pylori to antibiotics. Therefore, identifying other antibiotics that are safe and sensitive has been the focus in H. pylori eradication. Investigating the resistance to antibiotics among different regions using bacterial culture and susceptibility tests could be valuable in identifying sensitive antibiotics, thereby improving the eradication rate. Few studies to date have investigated the susceptibility of H. pylori to cephalosporins and minocyclines, especially in China. In the present study, we aimed to investigate the susceptibility of H. pylori to frequently used classical antibiotics (AMX, CLR and MTZ) and some alternate antibiotics, including amoxicillinclavulanate (AMC), moxifloxacin (MFX), minocycline (MNO) and cephalosporins. We also aimed to investigate the risk factors involved in H. pylori resistance to antibiotics. PATIENTS AND METHODS Patients Patients who underwent esophagogastroduodenoscopy (EGD) for upper gastrointestinal (GI) symptoms and were confirmed with H. pylori infection by rapid urease test (RUT) at the Peking University Third Hospital between March and December 2013 were enrolled in the study. Exclusion criteria were: (i) aged 70 years; (ii) patients who had received drugs such as antibiotics, antacids or bismuth that may influence H. pylori survival within the last 4 weeks before their enrollment in the study; (iii) GI malignancy; (iv) previous gastric or esophageal surgery; (v) severe concomitant diseases; (vi) pregnant or lactating; and (vii) alcohol abuse. During the endoscopic examination gastric biopsy specimens (two for each from the corpus and antrum) were collected for further analysis. The patients’ characteristics including age, gender, previous treatments for H. pylori infection and endoscopic findings were recorded. This study was approved by the Institutional Ethics Committee of Peking University Third Hospital, and written informed consent was obtained from each participant. Histological assessment The biopsy specimens (one from the corpus and one from the antrum) were evaluated using hematoxylin
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and eosin (HE) and Warthin–Starry stain. Histological assessment was performed according to the updated Sydney system.5 A visual analogue score was used to categorize the histological findings: H. pylori density, intensity of acute (polymorphonuclear) infiltrates, intensity of chronic (lymphoplasmacytic) infiltration, lymphoid aggregates, atrophic gastritis and intestinal metaplasia. Histological scores for inflammation severity were graded as: 0, none; 1, mild; 2, moderate; and 3, severe. Isolation, culture and identification of H. pylori The biopsy tissues (one from the corpus and one from the antrum) were stored in the same vial of Brucella broth with 10% glycerol until analysis. After the specimens were ground in sterilized tissue grinders, the tissue suspension was transferred using sterilized pipettes to Karmali Agar Base (CM0935, Basingstoke, Oxoid, UK) containing the H. pylori-selective supplement (SR0147, Oxoid Limited, Hampshire, UK) and 7% (v/v) horse blood. Segment separation of the suspension was performed using a sterilized inoculating loop. The plates were incubated under a microaerophilic environment (AnaeroPack, Oxoid, UK) at 37°C for 3 days. Pinpoint-sized, round, translucent bacterial colonies with a smooth wet surface were selected for the identification of H. pylori. Gram-negative spiral or seagullshaped bacteria with positive oxidase and urease test results were identified as the H. pylori strain. Antibiotic susceptibility test The Epsilome test was used to determine the susceptibility of H. pylori to AMX, AMC, CLR, MTZ, MFX, cephalothin (CEP), cefuroxime (CXM), cefixime (CFM) and MNO. The susceptibility to antibiotics was determined according to the European Committee on Antimicrobial Susceptibility Testing (AMX >0.12 mg/L, CLR >0.5 mg/L, and MTZ >8 mg/L) and previous studies (AMC >2 mg/L,6 MFX >1 mg/L,7,8 CEP >8 mg/L, CXM >8 mg/L, CFM >2 mg/L9 and MNO >8 mg/L10). Statistical analysis Statistical analyses were performed using SPSS 17.0 (IBM, Armonk, NY, USA). Qualitative data among different groups were compared using χ2 test and Fisher’s exact test; non-conditional logistic regression was used to investigate the factors affecting antibiotic resistance,
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Journal of Digestive Diseases 2015; 16; 464–470
and odds ratio (OR) and corresponding 95% confidence interval (CI) were used to assess the strength of this association. A two-sided P value of 0.12 >2 >8 >0.5 >1 >8 >8 >8 >2
7 (3.9) 0 (0) 111 (61.3) 56 (30.9) 134 (74.0) 12 (6.6) 2 (1.1) 0 (0) 1 (0.55)
0.016 0.016 128 0.38 3 2 0.016 0.016 0.032
0.032 0.023 >256 256 32 8 0.094 0.047 0.125
AMC, amoxicillin–clavulanate; AMX, amoxicillin; CEP, cephalothin; CFM, cefixime; CLR, clarithromycin; CXM, cefuroxime; MFX, moxifloxacin; MIC, minimum inhibitory concentration; MIC50, 50% minimum inhibitory concentration; MIC90, 90% minimum inhibitory concentration; MNO, minocycline; MTZ, metronidazole.
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
Journal of Digestive Diseases 2015; 16; 464–470 Table 2.
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Association between patients’ characteristics and their antibiotic resistance
Characteristics, n (%) Gender Male (n = 108) Female (n = 73) P value Age (years) ≤35 (n = 55) 35–60 (n = 98) ≥60 (n = 28) P value Previous treatment No (n = 144) Yes (n = 37) P value Endoscopic finding Gastritis (n = 114) Peptic ulcer (n = 67) P value Gastric atrophy No (n = 139) Yes (n = 42) P value Inflammatory severity* Mild (n = 36) Moderate to severe (n = 128) P value
MTZ (n = 111)
CLR (n = 56)
MFX (n = 134)
66 (61.1) 45 (61.6) 1.000
32 (29.6) 24 (32.9) 0.743
83 (76.9) 51 (69.9) 0.305
31 (56.4) 62 (63.3) 18 (64.3) 0.660
19 (34.5) 30 (30.6) 7 (25.0) 0.670
40 (72.7) 72 (73.5) 22 (78.6) 0.833
80 (55.6) 31 (83.8) 0.002
37 (25.7) 19 (51.4) 0.005
105 (72.9) 29 (78.4) 0.674
71 (62.3) 40 (59.7) 0.638
42 (36.8) 14 (20.9) 0.021
85 (74.6) 49 (73.1) 0.727
84 (60.4) 27 (64.3) 0.720
40 (28.8) 16 (38.1) 0.259
100 (71.9) 34 (81.0) 1.000
25 (69.4) 72 (56.3) 0.054
13 (36.1) 39 (30.5) 0.547
30 (83.3) 94 (73.4) 0.276
P < 0.05 indicated in bold (χ2 test).*Seventeen patients are excluded from this analysis because their biopsy samples are not adequate for the evaluation of inflammatory severity. CLR, clarithromycin; MFX, moxifloxacin; MTZ, metronidazole.
Multidrug resistance was found in the H. pylori strains. Briefly, 26.5% (n = 48) of the strains were resistant to both MTZ and CLR, 51.9% (n = 94) were resistant to both MTZ and MFX, 27.1% (n = 49) were resistant to both CLR and MFX, and 22.7% (n = 41) strains were resistant to MTZ, MFX and CLR. Risk factors for antibiotic resistance The resistance rates to AMX, AMC, MNO and cephalosporins were relatively low; therefore, statistical analysis could not be performed due to the small sample sizes. CLR, MTZ and MFX, which had high resistance rates, were selected for potential influence factor analysis. The resistance to antibiotics was compared in terms of patient’s gender, age, previous treatment, endoscopic findings, atrophy and inflammatory severity (Table 2), showing that resistance rates to MTZ and CLR were significantly higher in the treated group than in the untreated group (P < 0.05). The resistance rate to CLR was significantly higher in patients with chronic gastritis than in those with peptic ulcers (P = 0.021).
Univariate and multivariate logistic regression analysis for the risk factors of antibiotic resistance Univariate logistic regression was performed using the abovementioned factors as independent variables, and MTZ, CLR and MFX resistance as the dependent variables to explore the risk factors influencing antibiotic resistance. The results showed that the resistance to MTZ was associated with a previous eradication of H. pylori and the severity of mucosal inflammation. The patients with previous H. pylori eradication had a higher risk of developing resistance to MTZ than untreated patients (OR 4.133, 95% CI 1.624–10.517, P = 0.003), while patients with moderate to severe inflammation had a lower risk of developing resistance to MTZ than those with mild inflammation (OR 0.429, 95% CI 0.187–0.948, P = 0.046). Resistance to CLR was associated with previous eradication of H. pylori and endoscopic findings. Treated patients also had a higher risk of developing resistance to CLR than untreated patients (OR 3.053, 95% CI 1.449– 6.431, P = 0.003), and patients with chronic gastritis had a higher risk of developing resistance to CLR than those with peptic ulcers (OR 2.282, 95% CI
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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1.132–4.598, P = 0.021). No significant association between each of the risk factors and the resistance to MFX was found (P > 0.05). Multivariate logistic regression analysis showed that patients with a previous H. pylori eradication had a higher risk of developing resistance to MTZ than those without (OR 3.836, 95% CI 1.456–10.109, P = 0.007) after adjusting for age and gender; however, there was no significant association between inflammatory severity and the resistance to MTZ after the adjustment (P = 0.055). For CLR resistance, there was still an association between previous treatment and the resistance to CLR, the treated patients had a higher risk of developing resistance than the untreated patients (OR 3.354, 95% CI 1.514–7.429, P = 0.003); however, the association between the disease type and antibiotic resistance disappeared after the adjustment (OR 2.101, 95% CI 0.969–4.169, P = 0.061). DISCUSSION H. pylori resistance to antibiotics has been sharply increasing in recent years. A multicenter clinical study showed that triple therapy using standard doses of PPI and two antibiotics for H. pylori infection resulted in an eradication rate below 80%.12 Such a low eradication rate is mainly attributed to the resistance to commonly used antibiotics, especially CLR and MTZ. Antibiotic resistance is a serious issue in China, and thus the eradication of H. pylori is, to some extent, difficult in China. Researchers have already proposed several strategies to meet this challenge: (i) change the drug combination; and (ii) identify other safe, effective and sensitive antibiotics, or choose antibiotics based on antibiotic susceptibility test results. CLR is frequently used in clinical practice for H. pylori eradication. Our previous study showed that CLR resistance, which decreased the eradication rate of triple therapy by about 50%, could be used as an independent predictor for treatment failure.12 Therefore, the Maastricht IV consensus suggested that CLR should not be used as the first choice when the CLR resistance rate in the region is more than 15–20%.1 The present study also demonstrated that CLR should not be used as the first-line antibiotic for triple therapy. In addition, the MIC50 of CLR was relatively low, and the CLR bacteriostatic activity was still high for sensitive strains; therefore, if possible, tailored therapy could be applied based on the results of antibiotic susceptibility test. H. pylori resistance to MTZ has been regarded as a global challenge. Our study demonstrated that the
Journal of Digestive Diseases 2015; 16; 464–470 MTZ resistance rate was 61.3% in vitro. The Maastricht IV consensus has suggested that the therapeutic regimen containing MTZ should be abandoned when MTZ resistance rate is more than 40%.1 In addition, our findings also found that 26.5% of the strains were resistant to both MTZ and CLR; therefore, the combination of these two antibiotics should be abandoned in regions with such high resistance rates. Because H. pylori resistance to AMX is associated with the mutations in penicillin-binding proteins but not β-lactamase production,13 AMX resistance rate is very low worldwide.14 AMX has thus been used as the choice of treatment for H. pylori eradication. The in vitro bacteriostatic activity of AMX was very high and the resistance rate was only 3.9% in this study, suggesting that AMX is an effective antibiotic for H. pylori eradication. Several other antibiotics have recently been used to eradicate H. pylori. Some studies have demonstrated that MFX, a fourth generation of quinolone drugs, could achieve a better efficacy of H. pylori eradication than CLR-containing triple therapy and bismuthcontaining quadruple therapy when used as a first-line and second-line drugs, respectively.15 The eradication rate is closely associated with MFX resistance.16 Because there is cross-resistance of H. pylori to quinolone drugs, a single mutation of gyrA, the quinolone resistance-determining region, could result in the resistance to quinolones including MFX.17 The levofloxacin resistance rate was found to increase over time, with an increased MFX resistance rate accordingly. Our findings demonstrated that the MFX resistance rate was as high as 74.0% in vitro. Currently, a resistance rate of 10% has been regarded as the threshold for an acceptable resistance rate to quinolone drugs.18,19 Therefore, MFX should not be used as a first-line therapy for the treatment of H.pylori in China, which has a very high quinolone drug resistance rate; however, MFX could be used as a rescue therapy based on in vitro antibiotic susceptibility test in patients with multiple previous eradication failures. The low resistance rate of tetracycline has allowed it to be a classical drug used for H. pylori eradication. However, tetracycline could cause stained teeth and bone marrow suppression and thus is rarely used in clinical practice. Nevertheless, we can hypothesize that another tetracycline-type antibiotic can also achieve a comparable antibacterial effect but without such side effects. This study revealed that MNO could effectively eradicate H. pylori in vitro, and the resistance rate was only 6.6%. Currently, MNO is used in a few countries
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
Journal of Digestive Diseases 2015; 16; 464–470 for H. pylori eradication. The combined use of MNO, MTZ and PPI has been recommended only in Japan as a second-line treatment for H. pylori eradication.20 Further large-scale clinical studies and in vitro drug sensitivity tests are needed to investigate the efficacy and side effects of MNO in H. pylori eradication. AMX has a high bacteriostatic activity and a low resistance rate; however, its resistance rate is still increasing in several countries, especially in developing countries. In addition, AMX cannot be used for some patients because of their allergy to penicillin or AMX-related GI side effects, and only limited kinds of antibiotics can be used in such patients, especially for those in China, a country with a high antibiotic resistance rate. The mechanisms of action for cephalosporins are similar to those of AMX; however, cephalosporins have several advantages including good tolerance to the penicillin enzyme, good absorption after oral administration as well as mild side effects. Pichichero et al.21 demonstrated that the cross-allergy between cephalosporins and penicillin was mainly found in the first-generation cephalosporins but not in the second-generation or third-generation cephalosporins, which could be a result of the chemical structure. In the present study, antibiotic susceptibility tests were performed for the representatives of first-, second- and third-generation cephalosporins, showing that all these cephalosporins had high bacteriostatic activity against H. pylori in vitro. This was especially true for CXM, a second-generation cephalosporin that was found to have the lowest resistance rate and MIC values (MIC50 0.016 mg/L, MIC90 0.047 mg/L). In Westblom et al.’ study,22 CXM concentrations in blood and gastric mucosa were examined in animal experiments, and two peaks in the mucosal concentration were found after 1 h and 8 h; the corresponding mucosa/serum ratio was 36.75, suggesting that using a standard dose of CXM could result in a mucosal concentration over MIC90. The results indicated that cephalosporins could be used for H. pylori eradication. However, most studies on H. pylori treatment with cephalosporins are in vitro experiments, and further studies are needed to investigate its pharmacokinetics and clinical profiles. Our findings also found that the resistance rates and multidrug resistance (MDR) rate for AMX and cephalosporins were relatively low, suggesting these drugs could be good choices for combination therapy; however, in vivo studies are needed to validate the interactions among these drugs. Previous studies showed that the MTZ and CLR resistance rates differed in terms of patient’s age, gender and disease type. A meta-analysis from the USA showed that
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the MTZ and CLR resistance rates were significantly higher in women than in men, and aged patients also had a higher risk of developing CLR resistance.23 In the present study, three antibiotics including MTZ, CLR and MFX, which had relatively high antibiotic resistance rates, were further investigated. Multivariate analyses showed that previous treatment was a risk factor for developing resistance to both MTZ and CLR, while age, gender, disease type and pathological type was not significantly associated with antibiotic resistance. Because treatment failures could significantly increase the antibiotic resistance rate, we should try to choose the best antibiotics regimen to achieve successful treatment for the first time. For the patients who have had several treatment failures, antibiotics should be chosen based on antibiotic susceptibility results. In conclusion, H. pylori resistance to antibiotics is becoming a serious problem that could directly influence its eradication rate, especially in China. Therefore, special attention should be paid to antibiotic susceptibility test results and tailored therapy should be administered according to these results. Studies have suggested that among the commonly used antibiotics, AMX is still a good choice of treatment, while the combined use of CLR and MTZ should not be used as a first-line treatment strategy. MFX is also not a good choice as a first-line drug due to its high resistance rate. AMC, MNO and cephalosporins could be used for H. pylori eradication because they have a low resistance rate and good bacteriostatic activity against H. pylori in vitro. The findings also suggest that the antibiotic resistance rate could be higher in patients who had been treated for H. pylori, and for those who have experienced several treatment failures, antibiotics should be chosen based on antibiotic susceptibility results, which could help to increase the eradication rate and prevent the development of MDR. ACKNOWLEDGEMENT This study was funded by the Projects from the National Science Technology Pillar Program in the Twelfth Five-Year Plan Period, China (No.2012BAI06B02) and was performed in Key Laborotory for Helicobacter pylori Infection and Upper Gastrointestinal Diseases (BZ0371). REFERENCES 1 Malfertheiner P, Megraud F, O’Morain CA et al.; European Helicobacter Study Group. Management of Helicobacter pylori infection – the Maastricht IV/ Florence consensus report. Gut 2012; 61: 646–64.
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2 Fujimura K, Kuwana M, Kurata Y et al. Is eradication therapy useful as the first line of treatment in Helicobacter pylori-positive idiopathic thrombocytopenic purpura? Analysis of 207 eradicated chronic ITP cases in Japan. Int J Hematol 2005; 81: 162–8. 3 Chuah SK, Tsay FW, Hsu PI, Wu DC. A new look at anti-Helicobacter pylori therapy. World J Gastroenterol 2011; 17: 3971–5. 4 Xie T, Cui X, Zheng H, Chen D, He L, Jiang B. Meta-analysis: eradication of Helicobacter pylori infection is associated with the development of endoscopic gastroesophageal reflux disease. Eur J Gastroenterol Hepatol 2013; 25: 1195–205. 5 Genta RM, Dixon MF. The Sydney system revisited: the Houston International Gastritis Workshop. Am J Gastroenterol 1995; 90: 1039–41. 6 Yang JC, Lee PI, Hsueh PR. In vitro activity of nemonoxacin, tigecycline, and other antimicrobial agents against Helicobacter pylori isolates in Taiwan, 1998–2007. Eur J Clin Microbiol Infect Dis 2010; 29: 1369–75. 7 Kim JM, Kim JS, Kim N, Kim SG, Jung HC, Song IS. Comparison of primary and secondary antimicrobial minimum inhibitory concentrations for Helicobacter pylori isolated from Korean patients. Int J Antimicrob Agents 2006; 28: 6–13. 8 Cheng A, Sheng WH, Liou JM et al. Comparative in vitro antimicrobial susceptibility and synergistic activity of antimicrobial combinations against Helicobacter pylori isolates in Taiwan. J Microbiol Immunol Infect 2015; 48: 72–9. 9 Horiki N, Omata F, Uemura M et al. Annual change of primary resistance to clarithromycin among Helicobacter pylori isolates from 1996 through 2008 in Japan. Helicobacter 2009; 14: 86–90. 10 Nakajima S, Inoue H, Inoue T, Maruoka Y. Multiple-antibiotic-resistant Helicobacter pylori infection eradicated with a tailor-made quadruple therapy. J Gastroenterol Hepatol 2012; 27 Suppl 3: 108–11. 11 Zhou L, Lin S, Ding S et al. Relationship of Helicobacter pylori eradication with gastric cancer and gastric mucosal histological changes: a 10-year follow-up study. Chin Med J (Engl) 2014; 127: 1454–8. 12 Zhou L, Zhang J, Chen M et al. A comparative study of sequential therapy and standard triple therapy for Helicobacter pylori infection: a randomized multicenter trial. Am J Gastroenterol 2014; 109: 535–41.
Journal of Digestive Diseases 2015; 16; 464–470 13 Rimbara E, Noguchi N, Kawai T, Sasatsu M. Mutations in penicillin-binding proteins 1, 2 and 3 are responsible for amoxicillin resistance in Helicobacter pylori. J Antimicrob Chemother 2008; 61: 995–8. 14 Vakil N, Megraud F. Eradication therapy for Helicobacter pylori. Gastroenterology 2007; 133: 985–1001. 15 Zhang G, Zou J, Liu F et al. The efficacy of moxifloxacin-based triple therapy in treatment of Helicobacter pylori infection: a systematic review and meta-analysis of randomized clinical trials. Braz J Med Biol Res 2013; 46: 607–13. 16 Bago J, Majstorovic´ K, Belosic´-Halle Z et al. Antimicrobial resistance of H. pylori to the outcome of 10-days vs. 7-days moxifloxacin based therapy for the eradication: a randomized controlled trial. Ann Clin Microbiol Antimicrob 2010; 9: 13. 17 Tankovic J, Lascols C, Sculo Q, Petit JC, Soussy CJ. Single and double mutations in gyrA but not in gyrB are associated with low- and high-level fluoroquinolone resistance in Helicobacter pylori. Antimicrob Agents Chemother 2003; 47: 3942–4. 18 Marzio L, Coraggio D, Capodicasa S, Grossi L, Cappello G. Role of the preliminary susceptibility testing for initial and after failed therapy of Helicobacter pylori infection with levofloxacin, amoxicillin, and esomeprazole. Helicobacter 2006; 11: 237–42. 19 Gisbert JP, Fernández-Bermejo M, Molina-Infante J et al. First-line triple therapy with levofloxacin for Helicobacter pylori eradication. Aliment Pharmacol Ther 2007; 26: 495–500. 20 Murakami K, Sato R, Okimoto T et al. Effectiveness of minocycline-based triple therapy for eradication of Helicobacter pylori infection. J Gastroenterol Hepatol 2006; 21: 262–7. 21 Pichichero ME. Use of selected cephalosporins in penicillin-allergic patients: a paradigm shift. Diagn Microbiol Infect Dis 2007; 57: 13S–18S. 22 Westblom TU, Duriex DE. Pharmacokinetics of cefuroxime and ciprofloxacin in gastric mucosa: comparison to in vitro inhibitory concentrations against Helicobacter pylori. Microbiologica 1991; 14: 37–43. 23 Meyer JM, Silliman NP, Wang W et al. Risk factors for Helicobacter pylori resistance in the United States: the surveillance of H. pylori antimicrobial resistance partnership (SHARP) study, 1993–1999. Ann Intern Med 2002; 136: 13–24.
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
Journal of Digestive Diseases 2015; 16; 464–470
doi: 10.1111/1751-2980.12271
Original article
Susceptibility of Helicobacter pylori to antibiotics in Chinese patients Peng BAI,* Li Ya ZHOU,* Xiu Mei XIAO,† Yang LUO* & Yu DING* *Department of Gastroenterology and †Department of Laboratory Medicine, Peking University Third Hospital, Beijing, China
OBJECTIVE: Antibiotic resistance to Helicobacter pylori (H. pylori) has been increasing worldwide. The study aimed to evaluate in vitro susceptibility and resistance patterns to antibiotics in empirical H. pylori eradication regimens, and to determine the optimal antibiotics for treatment. METHODS: H. pylori strains (n =181) were obtained from gastric biopsies of patients with upper gastrointestinal symptoms who underwent esophagogastroduodenoscopy from March to December 2013. The susceptibility of H. pylori strains to amoxicillin (AMX), metronidazole (MTZ), clarithromycin (CLR), amoxicillin-clavulanate (AMC), cephalothin (CEP), cefuroxime (CXM), cefixime (CFM), moxifloxacin (MFX) and minocycline (MNO) was determined. RESULTS: Dual resistance to MTZ + CLR was detected in 48 (26.5%) isolates, MTZ + MFX in 94 KEY WORDS: moxifloxacin.
CONCLUSIONS: AMX, AMC, MNO and cephalosporins, but not MTZ, CLR and MFX, showed good in vitro anti-H. pylori activity. Among cephalosporins, CXM was the most active. H. pylori resistance is higher in patients with previous H. pylori eradication.
antibiotic resistance, susceptibility, clarithromycin, Helicobacter pylori, metronidazole,
INTRODUCTION Helicobacter pylori (H. pylori) was first identified by Warren and Marshall in 1983, since then numerous Correspondence to: Li Ya ZHOU, Department of Gastroenterology, Peking University Third Hospital, 49 North Garden Road,Haidian District, Beijing 100191, China. Email: [email protected] Conflict of interest: None. © 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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(51.9%), and CLR + MFX in 49 (27.1%). Overall, 41 (22.7%) were resistant to MTZ + CLR + MFX. MTZ and CLR resistance rates were significantly associated with the history of H. pylori eradication but there was no significant difference in MFX resistance rates between treated and untreated patients (P = 0.674). No significant relationship was found between antibiotic resistance and patient’s gender, age, endoscopic findings, inflammatory severity or gastric atrophy.
studies have demonstrated that H. pylori infection is closely associated with the development of chronic gastritis, peptic ulcers and gastric adenocarcinoma. The World Health Organization classified H. pylori as a type I carcinogen in 1994. H. pylori eradication could effectively alleviate or prevent disease development and progression.1 In recent years the eradication rate of H. pylori with standard triple therapy, including a proton pump inhibitor (PPI) and two of the three antibiotics (amoxicillin [AMX], clarithromycin [CLR] and metronidazole [MTZ]), has reduced to below 80% in
Journal of Digestive Diseases 2015; 16; 464–470 many regions of the world.2–4 This unacceptably low eradication rate is mainly caused by the resistance of H. pylori to antibiotics. Therefore, identifying other antibiotics that are safe and sensitive has been the focus in H. pylori eradication. Investigating the resistance to antibiotics among different regions using bacterial culture and susceptibility tests could be valuable in identifying sensitive antibiotics, thereby improving the eradication rate. Few studies to date have investigated the susceptibility of H. pylori to cephalosporins and minocyclines, especially in China. In the present study, we aimed to investigate the susceptibility of H. pylori to frequently used classical antibiotics (AMX, CLR and MTZ) and some alternate antibiotics, including amoxicillinclavulanate (AMC), moxifloxacin (MFX), minocycline (MNO) and cephalosporins. We also aimed to investigate the risk factors involved in H. pylori resistance to antibiotics. PATIENTS AND METHODS Patients Patients who underwent esophagogastroduodenoscopy (EGD) for upper gastrointestinal (GI) symptoms and were confirmed with H. pylori infection by rapid urease test (RUT) at the Peking University Third Hospital between March and December 2013 were enrolled in the study. Exclusion criteria were: (i) aged 70 years; (ii) patients who had received drugs such as antibiotics, antacids or bismuth that may influence H. pylori survival within the last 4 weeks before their enrollment in the study; (iii) GI malignancy; (iv) previous gastric or esophageal surgery; (v) severe concomitant diseases; (vi) pregnant or lactating; and (vii) alcohol abuse. During the endoscopic examination gastric biopsy specimens (two for each from the corpus and antrum) were collected for further analysis. The patients’ characteristics including age, gender, previous treatments for H. pylori infection and endoscopic findings were recorded. This study was approved by the Institutional Ethics Committee of Peking University Third Hospital, and written informed consent was obtained from each participant. Histological assessment The biopsy specimens (one from the corpus and one from the antrum) were evaluated using hematoxylin
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and eosin (HE) and Warthin–Starry stain. Histological assessment was performed according to the updated Sydney system.5 A visual analogue score was used to categorize the histological findings: H. pylori density, intensity of acute (polymorphonuclear) infiltrates, intensity of chronic (lymphoplasmacytic) infiltration, lymphoid aggregates, atrophic gastritis and intestinal metaplasia. Histological scores for inflammation severity were graded as: 0, none; 1, mild; 2, moderate; and 3, severe. Isolation, culture and identification of H. pylori The biopsy tissues (one from the corpus and one from the antrum) were stored in the same vial of Brucella broth with 10% glycerol until analysis. After the specimens were ground in sterilized tissue grinders, the tissue suspension was transferred using sterilized pipettes to Karmali Agar Base (CM0935, Basingstoke, Oxoid, UK) containing the H. pylori-selective supplement (SR0147, Oxoid Limited, Hampshire, UK) and 7% (v/v) horse blood. Segment separation of the suspension was performed using a sterilized inoculating loop. The plates were incubated under a microaerophilic environment (AnaeroPack, Oxoid, UK) at 37°C for 3 days. Pinpoint-sized, round, translucent bacterial colonies with a smooth wet surface were selected for the identification of H. pylori. Gram-negative spiral or seagullshaped bacteria with positive oxidase and urease test results were identified as the H. pylori strain. Antibiotic susceptibility test The Epsilome test was used to determine the susceptibility of H. pylori to AMX, AMC, CLR, MTZ, MFX, cephalothin (CEP), cefuroxime (CXM), cefixime (CFM) and MNO. The susceptibility to antibiotics was determined according to the European Committee on Antimicrobial Susceptibility Testing (AMX >0.12 mg/L, CLR >0.5 mg/L, and MTZ >8 mg/L) and previous studies (AMC >2 mg/L,6 MFX >1 mg/L,7,8 CEP >8 mg/L, CXM >8 mg/L, CFM >2 mg/L9 and MNO >8 mg/L10). Statistical analysis Statistical analyses were performed using SPSS 17.0 (IBM, Armonk, NY, USA). Qualitative data among different groups were compared using χ2 test and Fisher’s exact test; non-conditional logistic regression was used to investigate the factors affecting antibiotic resistance,
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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and odds ratio (OR) and corresponding 95% confidence interval (CI) were used to assess the strength of this association. A two-sided P value of 0.12 >2 >8 >0.5 >1 >8 >8 >8 >2
7 (3.9) 0 (0) 111 (61.3) 56 (30.9) 134 (74.0) 12 (6.6) 2 (1.1) 0 (0) 1 (0.55)
0.016 0.016 128 0.38 3 2 0.016 0.016 0.032
0.032 0.023 >256 256 32 8 0.094 0.047 0.125
AMC, amoxicillin–clavulanate; AMX, amoxicillin; CEP, cephalothin; CFM, cefixime; CLR, clarithromycin; CXM, cefuroxime; MFX, moxifloxacin; MIC, minimum inhibitory concentration; MIC50, 50% minimum inhibitory concentration; MIC90, 90% minimum inhibitory concentration; MNO, minocycline; MTZ, metronidazole.
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Association between patients’ characteristics and their antibiotic resistance
Characteristics, n (%) Gender Male (n = 108) Female (n = 73) P value Age (years) ≤35 (n = 55) 35–60 (n = 98) ≥60 (n = 28) P value Previous treatment No (n = 144) Yes (n = 37) P value Endoscopic finding Gastritis (n = 114) Peptic ulcer (n = 67) P value Gastric atrophy No (n = 139) Yes (n = 42) P value Inflammatory severity* Mild (n = 36) Moderate to severe (n = 128) P value
MTZ (n = 111)
CLR (n = 56)
MFX (n = 134)
66 (61.1) 45 (61.6) 1.000
32 (29.6) 24 (32.9) 0.743
83 (76.9) 51 (69.9) 0.305
31 (56.4) 62 (63.3) 18 (64.3) 0.660
19 (34.5) 30 (30.6) 7 (25.0) 0.670
40 (72.7) 72 (73.5) 22 (78.6) 0.833
80 (55.6) 31 (83.8) 0.002
37 (25.7) 19 (51.4) 0.005
105 (72.9) 29 (78.4) 0.674
71 (62.3) 40 (59.7) 0.638
42 (36.8) 14 (20.9) 0.021
85 (74.6) 49 (73.1) 0.727
84 (60.4) 27 (64.3) 0.720
40 (28.8) 16 (38.1) 0.259
100 (71.9) 34 (81.0) 1.000
25 (69.4) 72 (56.3) 0.054
13 (36.1) 39 (30.5) 0.547
30 (83.3) 94 (73.4) 0.276
P < 0.05 indicated in bold (χ2 test).*Seventeen patients are excluded from this analysis because their biopsy samples are not adequate for the evaluation of inflammatory severity. CLR, clarithromycin; MFX, moxifloxacin; MTZ, metronidazole.
Multidrug resistance was found in the H. pylori strains. Briefly, 26.5% (n = 48) of the strains were resistant to both MTZ and CLR, 51.9% (n = 94) were resistant to both MTZ and MFX, 27.1% (n = 49) were resistant to both CLR and MFX, and 22.7% (n = 41) strains were resistant to MTZ, MFX and CLR. Risk factors for antibiotic resistance The resistance rates to AMX, AMC, MNO and cephalosporins were relatively low; therefore, statistical analysis could not be performed due to the small sample sizes. CLR, MTZ and MFX, which had high resistance rates, were selected for potential influence factor analysis. The resistance to antibiotics was compared in terms of patient’s gender, age, previous treatment, endoscopic findings, atrophy and inflammatory severity (Table 2), showing that resistance rates to MTZ and CLR were significantly higher in the treated group than in the untreated group (P < 0.05). The resistance rate to CLR was significantly higher in patients with chronic gastritis than in those with peptic ulcers (P = 0.021).
Univariate and multivariate logistic regression analysis for the risk factors of antibiotic resistance Univariate logistic regression was performed using the abovementioned factors as independent variables, and MTZ, CLR and MFX resistance as the dependent variables to explore the risk factors influencing antibiotic resistance. The results showed that the resistance to MTZ was associated with a previous eradication of H. pylori and the severity of mucosal inflammation. The patients with previous H. pylori eradication had a higher risk of developing resistance to MTZ than untreated patients (OR 4.133, 95% CI 1.624–10.517, P = 0.003), while patients with moderate to severe inflammation had a lower risk of developing resistance to MTZ than those with mild inflammation (OR 0.429, 95% CI 0.187–0.948, P = 0.046). Resistance to CLR was associated with previous eradication of H. pylori and endoscopic findings. Treated patients also had a higher risk of developing resistance to CLR than untreated patients (OR 3.053, 95% CI 1.449– 6.431, P = 0.003), and patients with chronic gastritis had a higher risk of developing resistance to CLR than those with peptic ulcers (OR 2.282, 95% CI
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1.132–4.598, P = 0.021). No significant association between each of the risk factors and the resistance to MFX was found (P > 0.05). Multivariate logistic regression analysis showed that patients with a previous H. pylori eradication had a higher risk of developing resistance to MTZ than those without (OR 3.836, 95% CI 1.456–10.109, P = 0.007) after adjusting for age and gender; however, there was no significant association between inflammatory severity and the resistance to MTZ after the adjustment (P = 0.055). For CLR resistance, there was still an association between previous treatment and the resistance to CLR, the treated patients had a higher risk of developing resistance than the untreated patients (OR 3.354, 95% CI 1.514–7.429, P = 0.003); however, the association between the disease type and antibiotic resistance disappeared after the adjustment (OR 2.101, 95% CI 0.969–4.169, P = 0.061). DISCUSSION H. pylori resistance to antibiotics has been sharply increasing in recent years. A multicenter clinical study showed that triple therapy using standard doses of PPI and two antibiotics for H. pylori infection resulted in an eradication rate below 80%.12 Such a low eradication rate is mainly attributed to the resistance to commonly used antibiotics, especially CLR and MTZ. Antibiotic resistance is a serious issue in China, and thus the eradication of H. pylori is, to some extent, difficult in China. Researchers have already proposed several strategies to meet this challenge: (i) change the drug combination; and (ii) identify other safe, effective and sensitive antibiotics, or choose antibiotics based on antibiotic susceptibility test results. CLR is frequently used in clinical practice for H. pylori eradication. Our previous study showed that CLR resistance, which decreased the eradication rate of triple therapy by about 50%, could be used as an independent predictor for treatment failure.12 Therefore, the Maastricht IV consensus suggested that CLR should not be used as the first choice when the CLR resistance rate in the region is more than 15–20%.1 The present study also demonstrated that CLR should not be used as the first-line antibiotic for triple therapy. In addition, the MIC50 of CLR was relatively low, and the CLR bacteriostatic activity was still high for sensitive strains; therefore, if possible, tailored therapy could be applied based on the results of antibiotic susceptibility test. H. pylori resistance to MTZ has been regarded as a global challenge. Our study demonstrated that the
Journal of Digestive Diseases 2015; 16; 464–470 MTZ resistance rate was 61.3% in vitro. The Maastricht IV consensus has suggested that the therapeutic regimen containing MTZ should be abandoned when MTZ resistance rate is more than 40%.1 In addition, our findings also found that 26.5% of the strains were resistant to both MTZ and CLR; therefore, the combination of these two antibiotics should be abandoned in regions with such high resistance rates. Because H. pylori resistance to AMX is associated with the mutations in penicillin-binding proteins but not β-lactamase production,13 AMX resistance rate is very low worldwide.14 AMX has thus been used as the choice of treatment for H. pylori eradication. The in vitro bacteriostatic activity of AMX was very high and the resistance rate was only 3.9% in this study, suggesting that AMX is an effective antibiotic for H. pylori eradication. Several other antibiotics have recently been used to eradicate H. pylori. Some studies have demonstrated that MFX, a fourth generation of quinolone drugs, could achieve a better efficacy of H. pylori eradication than CLR-containing triple therapy and bismuthcontaining quadruple therapy when used as a first-line and second-line drugs, respectively.15 The eradication rate is closely associated with MFX resistance.16 Because there is cross-resistance of H. pylori to quinolone drugs, a single mutation of gyrA, the quinolone resistance-determining region, could result in the resistance to quinolones including MFX.17 The levofloxacin resistance rate was found to increase over time, with an increased MFX resistance rate accordingly. Our findings demonstrated that the MFX resistance rate was as high as 74.0% in vitro. Currently, a resistance rate of 10% has been regarded as the threshold for an acceptable resistance rate to quinolone drugs.18,19 Therefore, MFX should not be used as a first-line therapy for the treatment of H.pylori in China, which has a very high quinolone drug resistance rate; however, MFX could be used as a rescue therapy based on in vitro antibiotic susceptibility test in patients with multiple previous eradication failures. The low resistance rate of tetracycline has allowed it to be a classical drug used for H. pylori eradication. However, tetracycline could cause stained teeth and bone marrow suppression and thus is rarely used in clinical practice. Nevertheless, we can hypothesize that another tetracycline-type antibiotic can also achieve a comparable antibacterial effect but without such side effects. This study revealed that MNO could effectively eradicate H. pylori in vitro, and the resistance rate was only 6.6%. Currently, MNO is used in a few countries
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
Journal of Digestive Diseases 2015; 16; 464–470 for H. pylori eradication. The combined use of MNO, MTZ and PPI has been recommended only in Japan as a second-line treatment for H. pylori eradication.20 Further large-scale clinical studies and in vitro drug sensitivity tests are needed to investigate the efficacy and side effects of MNO in H. pylori eradication. AMX has a high bacteriostatic activity and a low resistance rate; however, its resistance rate is still increasing in several countries, especially in developing countries. In addition, AMX cannot be used for some patients because of their allergy to penicillin or AMX-related GI side effects, and only limited kinds of antibiotics can be used in such patients, especially for those in China, a country with a high antibiotic resistance rate. The mechanisms of action for cephalosporins are similar to those of AMX; however, cephalosporins have several advantages including good tolerance to the penicillin enzyme, good absorption after oral administration as well as mild side effects. Pichichero et al.21 demonstrated that the cross-allergy between cephalosporins and penicillin was mainly found in the first-generation cephalosporins but not in the second-generation or third-generation cephalosporins, which could be a result of the chemical structure. In the present study, antibiotic susceptibility tests were performed for the representatives of first-, second- and third-generation cephalosporins, showing that all these cephalosporins had high bacteriostatic activity against H. pylori in vitro. This was especially true for CXM, a second-generation cephalosporin that was found to have the lowest resistance rate and MIC values (MIC50 0.016 mg/L, MIC90 0.047 mg/L). In Westblom et al.’ study,22 CXM concentrations in blood and gastric mucosa were examined in animal experiments, and two peaks in the mucosal concentration were found after 1 h and 8 h; the corresponding mucosa/serum ratio was 36.75, suggesting that using a standard dose of CXM could result in a mucosal concentration over MIC90. The results indicated that cephalosporins could be used for H. pylori eradication. However, most studies on H. pylori treatment with cephalosporins are in vitro experiments, and further studies are needed to investigate its pharmacokinetics and clinical profiles. Our findings also found that the resistance rates and multidrug resistance (MDR) rate for AMX and cephalosporins were relatively low, suggesting these drugs could be good choices for combination therapy; however, in vivo studies are needed to validate the interactions among these drugs. Previous studies showed that the MTZ and CLR resistance rates differed in terms of patient’s age, gender and disease type. A meta-analysis from the USA showed that
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the MTZ and CLR resistance rates were significantly higher in women than in men, and aged patients also had a higher risk of developing CLR resistance.23 In the present study, three antibiotics including MTZ, CLR and MFX, which had relatively high antibiotic resistance rates, were further investigated. Multivariate analyses showed that previous treatment was a risk factor for developing resistance to both MTZ and CLR, while age, gender, disease type and pathological type was not significantly associated with antibiotic resistance. Because treatment failures could significantly increase the antibiotic resistance rate, we should try to choose the best antibiotics regimen to achieve successful treatment for the first time. For the patients who have had several treatment failures, antibiotics should be chosen based on antibiotic susceptibility results. In conclusion, H. pylori resistance to antibiotics is becoming a serious problem that could directly influence its eradication rate, especially in China. Therefore, special attention should be paid to antibiotic susceptibility test results and tailored therapy should be administered according to these results. Studies have suggested that among the commonly used antibiotics, AMX is still a good choice of treatment, while the combined use of CLR and MTZ should not be used as a first-line treatment strategy. MFX is also not a good choice as a first-line drug due to its high resistance rate. AMC, MNO and cephalosporins could be used for H. pylori eradication because they have a low resistance rate and good bacteriostatic activity against H. pylori in vitro. The findings also suggest that the antibiotic resistance rate could be higher in patients who had been treated for H. pylori, and for those who have experienced several treatment failures, antibiotics should be chosen based on antibiotic susceptibility results, which could help to increase the eradication rate and prevent the development of MDR. ACKNOWLEDGEMENT This study was funded by the Projects from the National Science Technology Pillar Program in the Twelfth Five-Year Plan Period, China (No.2012BAI06B02) and was performed in Key Laborotory for Helicobacter pylori Infection and Upper Gastrointestinal Diseases (BZ0371). REFERENCES 1 Malfertheiner P, Megraud F, O’Morain CA et al.; European Helicobacter Study Group. Management of Helicobacter pylori infection – the Maastricht IV/ Florence consensus report. Gut 2012; 61: 646–64.
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2 Fujimura K, Kuwana M, Kurata Y et al. Is eradication therapy useful as the first line of treatment in Helicobacter pylori-positive idiopathic thrombocytopenic purpura? Analysis of 207 eradicated chronic ITP cases in Japan. Int J Hematol 2005; 81: 162–8. 3 Chuah SK, Tsay FW, Hsu PI, Wu DC. A new look at anti-Helicobacter pylori therapy. World J Gastroenterol 2011; 17: 3971–5. 4 Xie T, Cui X, Zheng H, Chen D, He L, Jiang B. Meta-analysis: eradication of Helicobacter pylori infection is associated with the development of endoscopic gastroesophageal reflux disease. Eur J Gastroenterol Hepatol 2013; 25: 1195–205. 5 Genta RM, Dixon MF. The Sydney system revisited: the Houston International Gastritis Workshop. Am J Gastroenterol 1995; 90: 1039–41. 6 Yang JC, Lee PI, Hsueh PR. In vitro activity of nemonoxacin, tigecycline, and other antimicrobial agents against Helicobacter pylori isolates in Taiwan, 1998–2007. Eur J Clin Microbiol Infect Dis 2010; 29: 1369–75. 7 Kim JM, Kim JS, Kim N, Kim SG, Jung HC, Song IS. Comparison of primary and secondary antimicrobial minimum inhibitory concentrations for Helicobacter pylori isolated from Korean patients. Int J Antimicrob Agents 2006; 28: 6–13. 8 Cheng A, Sheng WH, Liou JM et al. Comparative in vitro antimicrobial susceptibility and synergistic activity of antimicrobial combinations against Helicobacter pylori isolates in Taiwan. J Microbiol Immunol Infect 2015; 48: 72–9. 9 Horiki N, Omata F, Uemura M et al. Annual change of primary resistance to clarithromycin among Helicobacter pylori isolates from 1996 through 2008 in Japan. Helicobacter 2009; 14: 86–90. 10 Nakajima S, Inoue H, Inoue T, Maruoka Y. Multiple-antibiotic-resistant Helicobacter pylori infection eradicated with a tailor-made quadruple therapy. J Gastroenterol Hepatol 2012; 27 Suppl 3: 108–11. 11 Zhou L, Lin S, Ding S et al. Relationship of Helicobacter pylori eradication with gastric cancer and gastric mucosal histological changes: a 10-year follow-up study. Chin Med J (Engl) 2014; 127: 1454–8. 12 Zhou L, Zhang J, Chen M et al. A comparative study of sequential therapy and standard triple therapy for Helicobacter pylori infection: a randomized multicenter trial. Am J Gastroenterol 2014; 109: 535–41.
Journal of Digestive Diseases 2015; 16; 464–470 13 Rimbara E, Noguchi N, Kawai T, Sasatsu M. Mutations in penicillin-binding proteins 1, 2 and 3 are responsible for amoxicillin resistance in Helicobacter pylori. J Antimicrob Chemother 2008; 61: 995–8. 14 Vakil N, Megraud F. Eradication therapy for Helicobacter pylori. Gastroenterology 2007; 133: 985–1001. 15 Zhang G, Zou J, Liu F et al. The efficacy of moxifloxacin-based triple therapy in treatment of Helicobacter pylori infection: a systematic review and meta-analysis of randomized clinical trials. Braz J Med Biol Res 2013; 46: 607–13. 16 Bago J, Majstorovic´ K, Belosic´-Halle Z et al. Antimicrobial resistance of H. pylori to the outcome of 10-days vs. 7-days moxifloxacin based therapy for the eradication: a randomized controlled trial. Ann Clin Microbiol Antimicrob 2010; 9: 13. 17 Tankovic J, Lascols C, Sculo Q, Petit JC, Soussy CJ. Single and double mutations in gyrA but not in gyrB are associated with low- and high-level fluoroquinolone resistance in Helicobacter pylori. Antimicrob Agents Chemother 2003; 47: 3942–4. 18 Marzio L, Coraggio D, Capodicasa S, Grossi L, Cappello G. Role of the preliminary susceptibility testing for initial and after failed therapy of Helicobacter pylori infection with levofloxacin, amoxicillin, and esomeprazole. Helicobacter 2006; 11: 237–42. 19 Gisbert JP, Fernández-Bermejo M, Molina-Infante J et al. First-line triple therapy with levofloxacin for Helicobacter pylori eradication. Aliment Pharmacol Ther 2007; 26: 495–500. 20 Murakami K, Sato R, Okimoto T et al. Effectiveness of minocycline-based triple therapy for eradication of Helicobacter pylori infection. J Gastroenterol Hepatol 2006; 21: 262–7. 21 Pichichero ME. Use of selected cephalosporins in penicillin-allergic patients: a paradigm shift. Diagn Microbiol Infect Dis 2007; 57: 13S–18S. 22 Westblom TU, Duriex DE. Pharmacokinetics of cefuroxime and ciprofloxacin in gastric mucosa: comparison to in vitro inhibitory concentrations against Helicobacter pylori. Microbiologica 1991; 14: 37–43. 23 Meyer JM, Silliman NP, Wang W et al. Risk factors for Helicobacter pylori resistance in the United States: the surveillance of H. pylori antimicrobial resistance partnership (SHARP) study, 1993–1999. Ann Intern Med 2002; 136: 13–24.
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