Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes.
Resistance of Helicobacter pylori to macrolides is a major cause of failure of eradication therapies. Single base substitutions in the H. pylori 23S rRNA genes have been associated with macrolide resistance in the United States. Our goal was to extend this work to European strains, to determine the consequence of this mutation on erythromycin binding to H. pylori ribosomes, and to find a quick method to detect the mutation. Seven pairs of H. pylori strains were used, the parent strain being naturally susceptible to macrolides and the second strain having acquired an in vivo resistance during a treatment regimen that included clarithromycin. The identity of the strains was confirmed by random amplified polymorphic DNA testing with two different primers, indicating that resistance was the result of the selection of variants of the infecting strain. All resistant strains were found to have point mutations at position 2143 (three cases) or 2144 (four cases) but never on the opposite DNA fragment of domain V of the 23S rRNA gene. The mutation was A-->G in all cases except one (A-->C) at position 2143. Using BsaI and BbsI restriction enzymes on the amplified products, we confirmed the mutations of A-->G at positions 2144 and 2143, respectively. Macrolide binding was tested on purified ribosomes isolated from four pairs of strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the resistant one. In conclusion we suggest that the limited disruption of the peptidyltransferase loop conformation, caused by a point mutation, reduces drug binding and consequently confers resistance to macrolides. Finally, the macrolide resistance could be detected without sequencing by performing restriction fragment length polymorphism with appropriate restriction enzymes.
Occhialini
A
A
Laboratoire de Bactériologie, Hôpital Pellegrin, and Université de Bordeaux 2, France.
Urdaci
M
M
Doucet-Populaire
F
F
Bébéar
C M
CM
Lamouliatte
H
H
Mégraud
F
F
D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology*; D002250:Carbon Radioisotopes; D016172:DNA Fingerprinting; D004352:Drug Resistance, Microbial / Q000235:genetics; D004917:Erythromycin / Q000378:metabolism; D016480:Helicobacter pylori / Q000187:drug effects* / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D017354:Point Mutation; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D002250:Carbon Radioisotopes; D012338:RNA, Ribosomal, 23S; D004917:Erythromycin