Site-specific mutations in the 23S rRNA gene of Helicobacter pylori confer two types of resistance to macrolide-lincosamide-streptogramin B antibiotics

Abstract

Clarithromycin resistance in Helicobacter pylori is mainly due to A-to-G mutations within the peptidyltransferase region of the 23S rRNA. In the present study, cross-resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics (MLS phenotypes) has been investigated for several clinical isolates of H. pylori. Two major types of MLS resistance were identified and correlated with specific point mutations in the 23S rRNA gene. The A2142G mutation was linked with high-level cross-resistance to all MLS antibiotics (type I), and the A2143G mutation gave rise to an intermediate level of resistance to clarithromycin and clindamycin but no resistance to streptogramin B (type II). In addition, streptogramin A and streptogramin B were demonstrated to have a synergistic effect on both MLS-sensitive and MLS-resistant H. pylori strains. To further understand the mechanism of MLS resistance in H. pylori, we performed in vitro site-directed mutagenesis (substitution of G, C, or T for A at either position 2142 or 2143 of the 23S rRNA gene). The site-directed point mutations were introduced into a clarithromycin-susceptible strain, H. pylori UA802, by natural transformation followed by characterization of their effects on MLS resistance in an isogenic background. Strains with A-to-G and A-to-C mutations at the same position within the 23S rRNA gene had similar levels of clarithromycin resistance, and this level of resistance was higher than that for strains with the A-to-T mutation. Mutations at position 2142 conferred a higher level of clarithromycin resistance than mutations at position 2143. All mutations at position 2142 conferred cross-resistance to all MLS antibiotics, which corresponds to the type I MLS phenotype, whereas mutations at position 2143 were associated with a type II MLS phenotype with no resistance to streptogramin B. To explain that A-to-G transitions were predominantly observed in clarithromycin-resistant clinical isolates, we propose a possible mechanism by which A-to-G mutations are preferentially produced in H. pylori.

FIG. 1

Outline of procedures for construction and characterization of site-directed mutations. The steps are numbered in series. (Step 1) Site-directed mutagenesis. Wild-type H. pylori UA802 chromosomal DNA was used as the template for sequential PCR. The figure shows an example for constructing an A2142G mutation, and the primers used (Table 1) are indicated. (Step 2) Natural transformation. It includes DNA uptake, as illustrated by a heavy arrow, into the cell and subsequent homologous DNA recombination into the chromosome of a recipient cell, as depicted by a double crossover event (X X symbol). (Step 3) Selection for clarithromycin resistance. This step includes 3 to 4 days of incubation for the formation of single colonies and subsequent subculturing to obtain genetically stable Cla^r transformants. (Step 4) Genotypic identification of Cla^r mutants by DNA sequencing. (Step 5) Characterization of MLS phenotypes of Cla^r mutants by the MIC test.

FIG. 2

Nucleotide sequences of the short region in the 23S rRNA gene from Cla^s H. pylori UA802 (wild type) and the constructed Cla^r mutants showing their relevant genotypes. The corresponding sequence for the wild-type strain (see Fig. 3) is indicated on the left, with both adenines at positions 2142 and 2143 highlighted with black dots. The position of a specific base substitution(s) in each particular mutant is marked with an asterisk.

FIG. 3

Secondary structure of the central part of domain V (peptidyltransferase loop) of the H. pylori 23S rRNA gene based on the model of Egebjerg et al. (9)), with indication of the mutations that confer MLS resistance. The mutation sites are numbered according to the newly proposed numbering system (30), and the equivalent positions in E. coli are indicated in parentheses. The base substitutions made by site-directed mutagenesis in this work are indicated by arrows, and the associated MLS phenotypes are indicated. Abbreviations for antibiotics: Cla, clarithromycin; Cln, clindamycin; Qnp, quinupristin.