Simultaneous colonisation of Helicobacter pylori with and without mutations in the 23S rRNA gene in patients with no history of clarithromycin exposure

Abstract

Background: It was recently reported that A to G transition mutations at positions 2143 and 2144 in the 23S rRNA gene are associated with clarithromycin resistance in Helicobacter pylori.

Aims: To study the incidence and mechanism of development of clarithromycin resistance by analysing these mutations.

Subjects: Eighty two H pylori positive patients who had an endoscopic examination and no history of treatment with macrolide antibiotics.

Methods: Clarithromycin resistance was screened for by polymerase chain reaction-restriction fragment length polymorphism of the 23S rRNA gene coupled with antibiotic susceptibility testing. In clinical isolates with mutations or resistance, mutations in individual colonies were analysed by direct sequencing.

Results: Of the 79 amplicons (DNA fragments amplified by polymerase chain reaction), Alw26I and MboII digestion disclosed the mutation in four (5%) and one (1%) respectively. However, the Alw26I cleavage was incomplete in two of the four amplicons, as was the MboII cleavage. Individual colony analysis of the isolates with incomplete cleavage patterns showed the presence of both wild type and mutated strains in the 23S rRNA genes.

Conclusions: Both clarithromycin sensitive and resistant strains colonised in some patients with no history of exposure to macrolides. The results suggest that resistant strains may not be formed but selected by clarithromycin administration.

Figure 1

Representative polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) pattern of 23S rRNA genes from patients with clarithromycin resistance. Lane M, DNA size marker; lane A, PCR-RFLP pattern obtained with MboII; lane B, PCR-RFLP pattern produced by Alw26I. Case 1-A, Three bands corresponding to the predicted 301, 192, and 109 bp fragments were detected. The amplicon digested by MboII gave an incomplete digestion pattern. Case 1-B, Two bands with predicted sizes (265 and 36 bp) were detected. The amplicon without mutation at 2144G gave this pattern. Case 2-A, One 301 bp band was detected. The amplicon without mutation at 2143G gave this pattern. Case 2-B, The amplicon with a mutation at A2144G was completely digested by Alw26I, producing the predicted 209, 56, and 36 bp fragments. Case 3-A, The pattern was similar to that of case 2-A. Case 3-B, Four bands corresponding in size to 265, 209, 56, and 36 bp were detected, showing an incomplete digestion pattern.