Influence of a 23S ribosomal RNA mutation in Helicobacter pylori strains on the in vitro synergistic effect of clarithromycin and amoxicillin

Abstract

BackgroundClarithromycin (CLR) is the most commonly recommended antibiotic in Helicobacter pylori eradication regimens, but the prevalence of CLR-resistant H. pylori is increasing. CLR resistance is associated with mutations in the 23S rRNA gene. However, H. pylori eradication can still be achieved with triple therapy, and an additive effect may occur with multiple antibiotics.MethodsTwenty-six CLR-resistant strains were examined. The MIC of clarithromycin was determined by agar-dilution-testing on Columbia agar, as described elsewhere. The conserved region of the H. pylori 23S rRNA gene between nucleotide positions 1445 and 2846 [GenBank: U27270] was amplified. RFLP and sequence analysis were performed with the 1402-bp PCR product. Synergy between clarithromycin and amoxicillin was assessed using the agar dilution checkerboard technique. To confirm the correlation between mutation and synergistic effect with subinhibitory concentrations of AMX, site-directed mutagenesis was performed in four CLR-susceptible H. pylori isolates.ResultsTwenty-six clarithromycin-resistant strains were examined. The conserved region of the H. pylori 23S rRNA gene was amplified, and the purified PCR product was checked for mutations by restriction fragment length polymorphism (RFLP) analysis and sequencing. A synergistic effect was found in only three of the 12 H. pylori strains (25%) with the A2142G mutation and five of the 10 H. pylori strains (50%) with the A2143G mutation (fractional inhibitory concentration: FIC < 0.5, minimal inhibitory concentration: MIC<2 mg/L) was found. Site-directed mutagenesis was performed in four CLR-susceptible H. pylori isolates.Three of these isolates harboring a mutation in position A2143G grew under selection with CLR (MIC >16 mg/L), and all three strains showed the synergistic effect (FIC<0.5). In contrast, three of the same four strains transformed with DNA fragments with a mutation in position A2142G were resistant to CLR (MIC>16 mg/L) and showed no synergism with amoxicillin (FIC>2).ConclusionsHere we demonstrate that in 100% of the in vitro transformed strains, a mutation at position A2143G leads to a synergistic effect between clarithromycin and amoxicillin, whereas a mutation at position at A2142G had no discernible effect.

Low Helicobacter pylori primary resistance to clarithromycin in gastric biopsy specimens from dyspeptic patients of a city in the interior of São Paulo, Brazil

“…We found a higher prevalence of A2143G compared to A2142G mutation in our samples which is in agreement with the majority of Brazilian studies including the States of Minas Gerais, São Paulo and Recife [ 20 , 34 , 36 ]. A2143G but not A2142G point mutation shows a synergistic effect of clarithromycin and amoxicillin, which have been used together in the first-line H. pylori regimen [ 49 ], reinforcing the necessity to investigate this clarithromycin 23S rDNA point mutation before treatment. One sample harbored both A2142G and A2143G mutations at domain V of the H. pylori 23S rDNA that was also found in three H.pylori isolates obtained from patients of the Brazilian State of Minas Gerais [ 20 ].…”

Systematic review and meta-analysis: full- vs. half-dose anti-microbials in clarithromycin-based regimens forHelicobacter pylorieradication

“…demonstrated that the combination of clarithromycin and metronidazole has synergistic activity against 50% of tested H. pylori strains . In one study, synergism between clarithromycin and amoxicillin was only observed for clarithromycin‐susceptible isolates while in another, synergism was noted in the presence of a mutation at position A2143G, but not in the presence of one at position at A2142G . Indirect evidence for this synergy between amoxicillin and clarithromycin was suggested in a small study from Japan where triple regimens containing amoxicillin prevented the selection of secondary clarithromycin resistance compared to non‐amoxicillin‐containing regimens .…”

Resistance to clarithromycin and genotypes in Helicobacter pylori strains isolated in Sicily

“…The presence of the A2143G point of mutation has been shown to significantly affect the eradication rate of H. pylori. Consequently, investigating the distribution of each mutated genotype for resistance to clarithromycin is relevant for managing H. pylori in clinical practice (Sakinc et al, 2012). Thus, various studies have been performed in order to identify the relationship of the mutated genotypes with the bacterial genetic factors involved in virulence and clarithromycin sensitivity (Vannarath et al, 2014;Agudo et al, 2010;Garcia et al, 2010).…”