Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations

Abstract

In this study, two identical copies of a 23S-5S gene cluster, which are separately situated within the Helicobacter pylori UA802 chromosome, were cloned and sequenced. Comparison of the DNA sequence of the H. pylori 23S rRNA gene with known sequences of other bacterial 23S rRNA genes indicated that the H. pylori UA802 23S rRNA genes are closely related to those of Campylobacter spp. and therefore belong in the proposed Proteobacteria subdivision. The 5'-terminal nucleotide T or A of the 23S rRNA is close to a Pribnow box which could be a -10 region of the transcription promoter for the 23S rRNA gene, suggesting that a posttranscriptional process is likely not involved in the maturation of the H. pylori 23S rRNA. Clinical isolates of H. pylori resistant to clarithromycin were examined by using natural transformation and pulsed-field gel electrophoresis. Cross-resistance to clarithromycin and erythromycin, which was transferred by natural transformation from the Cla(r) Ery(r) donor strain H. pylori E to the Cla(s) Ery(s) recipient strain H. pylori UA802, was associated with an single A-to-G transition mutation at position 2142 of both copies of the 23S rRNA in UA802 Cla(r) Ery(r) mutants. The transformation frequency for Cla(r) and Ery(r) was found to be approximately 2 x 10(-6) transformants per viable cell, and the MICs of both clarithromycin and erythromycin for the Cla(r) Ery(r) mutants were equal to those for the donor isolate. Our results confirmed the previous findings that mutations at positions 2142 and 2143 of the H. pylori 23S rRNA gene are responsible for clarithromycin resistance and suggest that acquisition of clarithromycin resistance in H. pylori could also result from horizontal transfer.

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

“…Versalovic et al [10] have found that clarithromycin resistance in H. pylori is associated with the functioning of three point mutations leading to the substitution of adenine for guanine or cytosine: A2143G, A2143C, and A2144G. Later, new point mutations located at other positions in the 23S rRNA gene were described, which were also associated with resistance of the microbe to clarithromycin: A2115G, G2141A, A2143T, G2224A, C2245T, and T2289C [11][12][13][14][15]. Mutations in the bacterial gene 23S rRNA are dominant or codominant, i.e., the presence of a mutant allele in bacteria carrying two or more ribosome operons is sufficient for the life of bacteria at high macrolide concentrations [16].…”

“…The appearance of H. pylori clarithromycin resistance is connected with a decrease in macrolide affinity for ribosome due to the generation of point mutations in the 23S rRNA gene [10]. There are two copies of a 23S rRNA gene in the H. pylori genome [11]. Several mutations ( A2115C, G2141A, A2143C, A2143T, A2143G, and A2144G ) associated with resistance to clarithromycin have been identified in the peptidyl-transferase region of the V domain of the 23S rRNA gene, but mutations A2143G, A2143C , and A2144G were most frequently encountered [12][13][14].…”

“…Figure 1bpresents MS of minise-MALDI-TOF mass spectra of reaction products obtained after minisequencing the 2140-2144 region of the 23S rRNA gene of H. pylori with primer A2143. (a) Primer A2143 (m/z = 5719); (b) normal allele 2143 of the 23S rRNA gene (DNA samples 2, 4, 5, 7, 13); (c) mutation A2143G (m/z = 7020) in two alleles of the 23S rRNA gene(9,11,12); (d) mutation A2143G (m/z = 7020) in one allele of the 23S rRNA gene (1, 10); (e) mutations A2143G and A2143C in two different alleles of the 23S rRNA gene (8); (f) mutations A2143G and A2144G in one allele of the 23S rRNA gene (DNA samples 6, 10). wt is wild type, mut is mutant type.…”

Characterization of clarithromycin resistance in isolates of Helicobacter pylori from the UAE

“…A mutagenesis study performed by Taylor et al [24] has confirmed that the A2142G and A2143G mutations are associated with clarithromycin resistance. A review by Mêgraud [13] of several studies worldwide demonstrated that 81.5% of the mutations in clarithromycin-resistant isolates were the A2142G or A2143C mutations.…”

Mutations in the 23S rRNA gene of clarithromycin-resistant Helicobacter pylori from Japan

“…tions in the peptidyltransferase-encoding region of 23S rRNA confers resistance to clarithromycin by decreasing the binding of clarithromycin to ribosomes in H. pylori [5]. Most findings show that the major point mutations are the adenine → guanine transition at either position 2142 (A2142G) or 2143 (A2143G), or the adenine → cytosine transition at position 2142 (A2142C) [6].…”