Helicobacter pylori resistance to macrolides is increasing, and the need for susceptibility testing has become crucial. The only standardized method is agar dilution, which is not adapted to clinical practice. The present work aimed: (1) to optimize the technical conditions and to assess the reproducibility of the E-test and disk diffusion method for macrolides susceptibility testing of H. pylori, and (2) to assess the performances of these two phenotypic methods in detecting strains harboring a resistance mechanism to macrolides. We used 191 isolates collected in nine centers of France and Belgium. Phenotypic tests were performed on Mueller-Hinton agar supplemented with 10% horse blood, inoculated with a 2-day-old H. pylori suspension (10(8) CFU/ml), and incubated for 72 hr at 37 degrees C under microaerophilic conditions. The reproducibility studied on two randomly selected strains was better for disk diffusion than for the E-test for both clarithromycin and erythromycin. For a subset of 10 strains, the MICs of erythromycin and clarithromycin did not differ from more than one two-fold dilution when determined by E-test or agar dilution method. The breakpoints were for MICs: 1 mg/L for both clarithromycin and erythromycin and for inhibition diameters, 22 mm for clarithromycin and 17 mm for erythromycin. There was a 100% concordance between susceptibility to erythromycin and clarithromycin. However, the susceptible and resistant populations were better separated by testing erythromycin. Of 34 resistant strains, two lacked the A2142G and A2143G point mutations in 23S rRNA by PCR-RFLP. None of 15 tested sensitive strains were positive for one of these two point mutations. For clinical practice, we recommend to assess macrolide susceptibility of H. pylori by using one of these two phenotypic methods under the described technical conditions.

Validation of diffusion methods for macrolide susceptibility testing of Helicobacter pylori.

The realization that peptic ulcer disease was caused by an infectious agent has led to the still ongoing search for the most appropriate therapy. Many therapies have been tried, with the conclusion that multiple drug combinations are at present essential to the achievement of acceptable outcomes; cure of infection in >80% of patients is based on intention to treat. One class of drugs, which has been a constant from the early work of Marshall and Warren, is the 5-nitroimidazoles, principally tinidazole and metronidazole (103), agents that had been used in the treatment of anaerobic bacterial and selected protozoan infections (133). Two other drugs were also used with success in the following years: a macrolide compound, clarithromycin, and a β-lactam compound, amoxicillin. The MACH-1 (94) and MACH-2 (110) studies provided data that the combination of a proton pump inhibitor together with clarithromycin and metronidazole or clarithromycin and amoxicillin represented the most effective drug combinations for the treatment of Helicobacter pylori infection. Yet these combinations are subject to failure due to resistance (4). While the mechanisms of macrolide resistance in H. pylori are well understood, the same cannot be said for resistance to 5-nitroimidazoles. Indeed, the issue of resistance to the 5-nitroimidazoles is confused by apparent problems in relation to the accuracy and reproducibility of susceptibility testing, the definition of resistance, and the perceived clinical relevance of resistance. The issues relating to drug selection and antibiotic resistance as well as the prospect of using genomics for drug discovery will be reviewed in this chapter.

Antibiotic susceptibility and resistance

Background

Antibiotic resistance is an important cause of H. pylori treatment failure. This study aimed to examine the change in H. pylori antibiotic resistance profile in Singapore over the course of 15 years.



Materials and methods

The study period was from 2000 to 2014. Gastric mucosal biopsies obtained from H. pylori-positive patients were cultured. Antibiotic susceptibility to metronidazole, clarithromycin, levofloxacin, tetracycline, and amoxicillin was tested. The change in resistance rates over time was analyzed.



Results

A total of 708 H. pylori isolates were cultured. There was a significant increase in resistance rates for metronidazole (2000–2002: 24.8%; 2012–2014: 48.2%; p < .001), clarithromycin (2000–2002: 7.9%; 2012–2014: 17.1%; p = .022), and levofloxacin (2000–2002: 5%; 2012–2014: 14.7%; p = .007). The resistance rates for tetracycline (2000–2002: 5%; 2012–2014: 7.6%) and amoxicillin (2000–2002: 3%; 2012–2014: 4.4%) remained stable. Increase in dual (2000–2002: 6.9%; 2012–2014: 9.4%; p = .479) and triple antibiotic resistance rates (2000–2002: 0; 2012–2014: 7.6%; p < .001) were observed. Overall, the most common dual and triple resistance patterns were metronidazole/clarithromycin (4.4%) and metronidazole/clarithromycin/levofloxacin (1.8%), respectively.



Conclusions

Over 15 years, H. pylori resistance rates to metronidazole, clarithromycin and levofloxacin had increased. There was increased resistance to multiple antibiotics.

The Changing Profile of Helicobacter pylori Antibiotic Resistance in Singapore: A 15-Year Study

Helicobacter pylori is one of the most commonly encountered human pathogens. It has been shown to be closely associated with peptic ulcer disease (PUD), gastric adenocarcinoma, and the gastric mucosa-associated lymphoid tissue (MALT) that may lead to gastric lymphoma. The current diagnostic methods include histology, microbiological culture, classic serology, urease activity detection, polymerase chain reaction (PCR) and stool antigen detection. Its treatment modality options are multiple; however, a triple regimen consisting of a proton pump inhibitor (PPI), and two antibiotics for 10 to 14 days is preferred. Drug resistance is a growing problem in this organism and new therapeutic options are currently limited.

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Helicobacter pylori infection: Epidemiology, pathophysiology, and therapy

Antibiotic susceptibility testing of Helicobacter pylori isolates was performed by broth microdilution method with MegaCellTM RPMI-1640 Medium (SIGMA). Fifty five clinical isolates of H. pylori were tested against metronidazole, tinidazole, amoxicillin, and clarithromycin. The results were compared to those obtained by standard agar dilution method. The microdilution method performed with new medium, showed excellent correlation with agar dilution results, with 100% agreement for metronidazole, 96.3% for amoxicillin, 90.7% for clarithromycin, and 92.8% for tinidazole. MICs determined by proposed method were highly reproducible: replicate results were variable within one-two-fold dilution by using different inocula and different batches of medium.

Antimicrobial Susceptibility Testing of Helicobacter pylori Determined by Microdilution Method Using a New Medium

Metronidazole and tetracycline E tests were compared to an agar dilution method for the antimicrobial susceptibility testing of Helicobacter pylori. Sixteen strains were tested by using tetrazolium egg yolk (TEY) agar. The characteristic E test inhibition ellipse was clearer on TEY agar than on standard blood agar and gave results comparable to those of the agar dilution test. The use of TEY medium is preferable to that of blood agar medium in E test MIC determinations for H. pylori.

Antimicrobial susceptibility of Helicobacter pylori determined by the E test using tetrazolium egg yolk agar.

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Carlos León

Papers

Antimicrobial susceptibility of Helicobacter pylori determined by the E test using tetrazolium egg yolk agar.

Diagnóstico molecular para Leishmaniasis