Sakinc Türkan, Baars Barbara, Wüppenhorst Nicole, Kist Manfred, Huebner Johannes, Opferkuch Wolfgang
Division of Infectious Diseases, University Hospital Freiburg, Hugstetter Strasse 55, Freiburg, D-79106, Germany.
BMC Res Notes. 2012 Oct 30;5:603. doi: 10.1186/1756-0500-5-603.
Clarithromycin (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.
Twenty-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.
Twenty-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).
Here 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.
克拉霉素（CLR）是幽门螺杆菌根除方案中最常推荐使用的抗生素，但对CLR耐药的幽门螺杆菌患病率正在上升。CLR耐药与23S rRNA基因突变有关。然而，三联疗法仍可实现幽门螺杆菌的根除，多种抗生素可能会产生相加作用。
检测了26株CLR耐药菌株。如其他文献所述，通过在哥伦比亚琼脂上进行琼脂稀释试验来测定克拉霉素的最低抑菌浓度（MIC）。扩增幽门螺杆菌23S rRNA基因在核苷酸位置1445和2846之间的保守区域[GenBank：U27270]。对1402 bp的PCR产物进行限制性片段长度多态性（RFLP）分析和序列分析。使用琼脂稀释棋盘法评估克拉霉素和阿莫西林之间的协同作用。为了证实A2143G位点突变与亚抑菌浓度的阿莫西林协同效应之间的相关性，对4株CLR敏感的幽门螺杆菌分离株进行了定点诱变。
检测了26株克拉霉素耐药菌株。扩增了幽门螺杆菌23S rRNA基因的保守区域，并通过限制性片段长度多态性（RFLP）分析和测序检查纯化的PCR产物是否存在突变。在12株携带A2142G突变的幽门螺杆菌菌株中，仅3株（25%）发现有协同效应；在10株携带A2143G突变的幽门螺杆菌菌株中，5株（50%）发现有协同效应（部分抑菌浓度：FIC<0.5，最低抑菌浓度：MIC<2 mg/L）。对4株CLR敏感的幽门螺杆菌分离株进行了定点诱变。这4株分离株中有3株在A2143G位点发生突变，在CLR选择下生长（MIC>16 mg/L），并且所有3株菌株均显示出协同效应（FIC<0.5）。相比之下，用在A2142G位点发生突变的DNA片段转化的相同4株菌株中的3株对CLR耐药（MIC>16 mg/L），并且与阿莫西林无协同作用（FIC>2）。
我们在此证明，在100%的体外转化菌株中，A2143G位点的突变导致克拉霉素和阿莫西林之间产生协同效应，而A2142G位点的突变则没有明显作用。
