The Association of Age and Antibiotic Resistance of Helicobacter Pylori

The antibiotic resistance of Helicobacter pylori (H pylori) is steadily increasing worldwide, resulting in the low efficiency of the current therapeutic approaches for eradication. In this study, we investigated the relationship between antibiotic resistances, the year of sample collection, and the ages of the infected individuals. A total of 29,034 gastric mucosa biopsy samples were randomly collected from January 1, 2009 to December 9, 2014 in Jiaxing City, Zhejiang Province, China. An antibiotic susceptibility testing was determined using an agar-dilution method. The statistical significance was tested using the chi-squared (x) test. A total of 9687 strains were isolated. The resistance rate to clarithromycin, levofloxacin, and metronidazole were 17.76%, 19.66%, and 95.5%, respectively. Resistance was rare against amoxicillin, gentamicin, and furazolidone. The metronidazole resistance rate stayed at a consistently high level. In contrast, the resistance rates of clarithromycin and levofloxacin increased rapidly from 2009 to 2011, gradually decreased from 2012 to 2013, and then increased again in 2014. Although patients ages 31 to 50 and 71 to 80 years had lower infection rates of H pylori, they also had higher resistance rates to clarithromycin Sc, Miaoying Tu, B ang, BSc, Zhang, MD may thus represent an important category for the future study of H pylori infection. Age plays a key element in H pylori resistance to clarithromycin and levofloxacin. It is therefore necessary to consider individualized therapy for the optimized treatment of H pylori-infected patients. (Medicine 95(8):e2831) Abbreviations: AMX = amoxicillin, CFU = colony forming units, CLR = clarithromycin, FR = furazolidone, LVX = levofloxacin, MTZ = metronidazole, PBS = phosphate-buffered saline, PPI = proton pump inhibitor. INTRODUCTION Helicobacter pylori (H pylori) is a gram-negative and spiralshaped microaerophilic bacterium that colonizes the human gastric antrum and duodenal mucosa. Several treatments for H pylori infection have been developed over the last 30 years. Nevertheless, the treatment success rate does not exceed 80% globally, and has fallen into an unacceptable range (<70%) in some studies. Triple therapy, which consists of 1 proton pump inhibitor (PPI) and 2 antibiotics (clarithromycin and amoxicillin or metronidazole), has been commonly used as the first-line treatment regimen for the planned eradication of H pylori. Unfortunately, the success of the eradication program has been seriously hampered by increasing antibiotic resistance of H pylori, especially against clarithromycin. Traditional quadruple therapy consists of a bismuth, tetracycline hydrochloride, metronidazole, and PPI, which is not a good choice in areas where bismuth is not available or high metronidazole resistance is observed, although metronidazole resistance could be partially overcome by increased doses and treatment durations. Poor success rates have even made nonbismuthbased quadruple (concomitant) therapy unacceptable as an empiric therapy option. Sequential therapy consisted of PPI and amoxicillin for the first 5 days, followed by triple therapy to complete the 10-day therapy. However, the cure rate is also insufficiently improved by the presence of infections with multidrug resistances, such as both clarithromycin and metronidazole resistance. A major cause of treatment failure is the excessive and indiscriminate use of antibiotics. Antibiotic resistance rates of H pylori vary in different countries, and between developed and developing countries. In most countries, clarithromycin resistance has exceeded the minimum value (15%) of the Maastricht IV consensus recommendations. It is reported to be 28% in Japan and 38.5% in Korea. Metronidazole resistance to H pylori is now considered to be ubiquitous, ce rate ranges from 14.4% to 93.2%. sing rate of levofloxacin resistance in the s has drawn global attention. www.md-journal.com | 1 The development of antibiotic resistance in H pylori is an evolving process and varies across different countries and even between the age groups of affected patients. To obtain comprehensive epidemiologic surveillances of H pylori resistance, it is necessary to perform a prevalence survey in a country or city. In this study, we performed the latest survey of H pylori antibiotic resistance from 2009 to 2014 in Jiaxing City, Zhejiang Province, China, and analyzed the pattern of H pylori resistance to currently recommended therapies over a 6-year period and further investigated the relationship between antibiotic resistance and different age groups. The results provide valuable insights into the choice of available treatment strategies for H pylori infections. MATERIALS AND METHODS Patient and Tissue Samples Gastric mucosa biopsy samples were collected from 29,034 patients (14,003 males and 15,031 females) who were diagnosed at the First Hospital of Jiaxing City, Zhejiang Province, China, from January 1, 2009 to December 9, 2014. The average age of these patients was 48.18 13.73, and they were subdivided into 7 groups (<20, 21–30, 31–40, 41–50, 51–60, 61–70, and 71–80 years of age). Subsequently, the gastric mucosa biopsy specimens were collected by gastrointestinal endoscopy and were stored immediately in a brain–heart infusion broth (Oxoid, Dardilly, France) with 5% glycerin. They were then sent to the laboratory at Hangzhou Zhiyuan Medical Inspection Institute for antibiotics susceptibility testing. This study was approved by the Ethics Committee of the National Institute for Communicable Disease Control and Prevention. Additionally, each patient wrote his or her informed consent and agreed to H pylori isolation prior to gastrointestinal endoscopy. Inclusion Criteria and Exclusion Criteria The inclusion criteria were as follows. Firstly, patients had symptoms of abdominal pain, bloating, acid reflux, belching, nausea, vomiting, heartburn, chest pain, vomiting, melena, etc. Secondly, patients were unused antibiotics, bismuth, H2 receptor antagonists, or PPI in the last 2 weeks before gastrointestinal endoscopy. Thirdly, patients agreed to H pylori culture and sensitivity testing taken by endoscopy gastric biopsy specimens. The exclusion criteria were as follows. Firstly, patients with severe heart, liver, kidney dysfunction, pregnant, or lactating women were not allowed in this study. Secondly, patients with complications of bleeding, perforation, pyloric obstruction, cancer or esophageal, and gastrointestinal surgery history were not allowed in this study. Thirdly, patients whose could not properly express their complaints, such as psychosis, severe neurosis were not allowed in this study. Fourthly, patients with allergic to penicillin or either drugs of the 6 antibiotic tested by susceptibility testing were not allowed in this study. Isolation of H pylori Strains The isolation of H pylori was based on Gram staining and enzyme activity testing as described previously. Briefly, a gastric mucosa biopsy sample was ground and inoculated directly onto a Columbia Agar (Oxoid) plate containing 5% defibrinated sheep blood. The plate was then incubated under microaerophilic conditions (5% O and 10% CO ) for 3 days Ji et al 2 2 at 378C. Translucent colonies were identified by colony morphology with Gram staining and urease, catalase, and oxidase activity testing. Spiral gram-negative strains that were 2 | www.md-journal.com positive for all 3 enzyme activities were identified as H pylori. Sequentially, these strains were collected in phosphate-buffered saline (PBS, pH 7.4) at a concentration of 4 108 CFU/mL for antibiotic susceptibility testing. Antibiotic Susceptibility Testing The susceptibility of the isolated H pylori strains to 6 antibiotics (clarithromycin, levofloxacin, metronidazole, amoxicillin, gentamicin, and furazolidone) was determined by the agar dilution method. The resistance breakpoints of the 6 antibiotics were defined for clarithromycin 1, levofloxacin 2, metronidazole 8, amoxicillin 2, gentamicin 16, and furazolidone 2mg/mL. Two microliter suspensions of H pylori were transferred onto Mueller–Hintonagar (Oxoid) supplemented with 5% sheep blood and a single antibiotic. They were then grown in a microaerobic humidified atmosphere at 378C for 3 days. As a control, a standard H pylori strain (ATCC43504/NCTC11637) was used. All tests were repeated and conducted at the Hangzhou Zhiyuan Medical Inspection Institute. Statistical Analysis The statistical significances in the resistance rates among the different collection years, to the different antibiotics and in the different patient ages were analyzed by the chi-squared (x) test using the SPSS statistical software package version 19.0 (SPSS Inc., Chicago, IL). Probability (P) values< 0.05 were considered significant.