Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes

Abstract

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.

A2144G Is the Main Mutation in the 23S rRNA Gene of Helicobacter pylori Associated with Clarithromycin Resistance

“…Rapid and reliable methods for detecting point mutations in the 23S rRNA gene of H. pylori have been widely employed in recent years: PCR-RELP, primermismatch PCR, ELISA based on labeled anti-DNA monoclonal antibodies, Inno-Lipa, the use of the resonant mirror biosensors, analysis of annealing duplexes, and direct sequencing of a 23S rRNA gene fragment [17][18][19][20][21][22][23]. Nevertheless, these methods are expensive, require labeling the product, or they do not fulfill the requirements for such parameters as sensitivity/specificity.…”

Antimicrobial resistance of in Poland: a multicentre study

“…Although most resistant isolates contain A2143G, A2142G and A2142C mutations [5,6], other rare mutations have also been identified [7,8] and some isolates, despite high clarithromycin minimal inhibitory concentration (MIC) values, have no detectable mutations in the 23S rRNA gene [7], which could suggest a different resistance mechanism.…”

Resistance to macrolides by ribosomal mutation in clinical isolates of Turicella otitidis

“…This phenotype is similar to that conferred by dimethylation of A2058 encoded by erm genes [14]. A2058C and A2058U mutations have also been reported in Helicobacter pylori where they confer a low level of resistance [15]. By contrast, in Streptococcus pneumoniae mutants the same level of macrolide resistance is conferred regardless of the nature of the substitution [12].…”