Validation of Diffusion Methods for Macrolide Susceptibility Testing ofHelicobacter pylori

Abstract

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.

Quadruplex Real-Time PCR Assay Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to Helicobacter pylori

“…Two phenotypic methods were used to determine the Cla susceptibility of the isolated strains: disk diffusion and Etest. Both methods were used according to a previously described protocol recommended by the French Study Group of Helicobacter (9). A bacterial suspension was prepared to a McFarland 3 turbidity standard (approximately 10 9 CFU/ml of H. pylori), spread with a sterile swab dipped into the inoculum suspension, and swabbed in three directions on a Mueller-Hinton agar plate supplemented with 10% horse blood.…”

“…In routine practice, the detection of Cla resistance is mainly based on phenotypic methods performed after culture: agar diffusion for the disk diffusion and Etest or the agar dilution method, which is considered the reference (9,16). These methods, however, are time-consuming and can take up to 2 weeks to be completed.…”

Fast and Accurate Quantitative Detection of Helicobacter pylori and Identification of Clarithromycin Resistance Mutations in H . pylori Isolates from Gastric Biopsy Specimens by Real-Time PCR

“…Plates were read after 3 days of incubation at 37°C. Strains were considered resistant to clarithromycin if the MIC was Ͼ0.5 g/ml (11).…”

Antimicrobial resistance in Helicobacter pylori: current situation and management strategy in Vietnam

“…On the contrary, the disk diffusion method is the simplest and the most economic for routine susceptibility testing but it is not well standardized for slow-growing bacteria like H. pylori [22]. Interestingly, some studies demonstrated a close relationship between inhibition diameter of disk diffusion method and minimum inhibitory concentration (MIC) by E-test [22][23][24].…”

“…Interestingly, some studies demonstrated a close relationship between inhibition diameter of disk diffusion method and minimum inhibitory concentration (MIC) by E-test [22][23][24]. Molecular techniques able to determine bacterial resistance to some antibiotics within a few days may have advantages [20].…”