Fontana Carla, Favaro Marco, Pietroiusti Antonio, Pistoia Enrico Salvatore, Galante Alberto, Favalli Cartesio
Department of Experimental Medicine and Biochemical Sciences, "Tor Vergata" University of Rome, Italy.
J Clin Microbiol. 2003 Aug;41(8):3636-40. doi: 10.1128/JCM.41.8.3636-3640.2003.
The recognition of the role of Helicobacter pylori in gastric diseases has led to the widespread use of antibiotics in the eradication of this pathogen. The most advocated therapy, triple therapy, often includes clarithromycin. It is well known that clarithromycin resistance is one of the major causes of eradication failure. The development of a rapid noninvasive technique that could easily be performed on fecal samples and that could also provide information about the antibiotic resistance of this microorganism is therefore advisable. Previous findings have demonstrated that clarithromycin resistance is due to a single point mutation in the 23S rRNA. All the mutations described have been associated with specific restriction sites, namely BsaI (A2143G), MboII (A2142C/G), and HhaI (T2717C). On this basis we have developed a new method, a seminested PCR, allowing screening for clarithromycin resistance of H. pylori directly on stool samples. This method furnished a 783-bp fragment of the 23S rRNA, which was subsequently digested by MboII, BsaI, and HhaI, in order to identify single point mutations associated with clarithromycin resistance. Of a total of 283 stool samples examined, 125 were H. pylori positive and two of them were shown to contain clarithromycin-resistant strains due to the presence of a mutation at position 2717, whereas no PCR products contained mutations at position 2142 or 2143. In order to evaluate the reliability of the new system, we compared the results of restriction analysis of the PCR products with the MICs shown by the H. pylori isolates by culturing gastric biopsies from the same patients.
对幽门螺杆菌在胃部疾病中作用的认识，促使抗生素在根除这种病原体方面得到广泛应用。最常用的疗法，即三联疗法，通常包括克拉霉素。众所周知，克拉霉素耐药是根除失败的主要原因之一。因此，开发一种能够轻松在粪便样本上进行且能提供该微生物抗生素耐药性信息的快速非侵入性技术是可取的。先前的研究结果表明，克拉霉素耐药是由于23S rRNA中的单点突变所致。所描述的所有突变都与特定的限制性位点相关，即BsaI（A2143G）、MboII（A2142C/G）和HhaI（T2717C）。在此基础上，我们开发了一种新方法，即半巢式PCR，可直接在粪便样本上筛查幽门螺杆菌的克拉霉素耐药性。该方法扩增出23S rRNA的一个783 bp片段，随后用MboII、BsaI和HhaI进行消化，以鉴定与克拉霉素耐药相关的单点突变。在总共检测的283份粪便样本中，125份幽门螺杆菌呈阳性，其中两份因在2717位存在突变而显示含有克拉霉素耐药菌株，而没有PCR产物在2142或2143位含有突变。为了评估新系统的可靠性，我们将PCR产物的限制性分析结果与通过培养同一患者的胃活检样本所显示的幽门螺杆菌分离株的最低抑菌浓度（MIC）进行了比较。
