儿童感染幽门螺杆菌对克拉霉素耐药情况及耐药机制初步研究

Prevalence and mechanism of Helicobacter pylori resistance to clarithromycin in children

目的 了解儿童幽门螺杆菌 (Helicobacterpylor,Hp)对克拉霉素的耐药情况 ,探讨Hp对克拉霉素耐药性与 2 3SrRNA基因突变的关系。方法 10 8例胃黏膜标本均取自 2 0 0 2年 10月～2 0 0 4年 1月在浙江大学医学院附属儿童医院进行胃镜检查的患儿 ,经分离培养鉴定为Hp菌株后 ,分别采用E test法和琼脂稀释法检测克拉霉素的最低抑菌浓度 (minimuminhibitoryconcentration ,MIC) ,确定Hp菌株对克拉霉素的耐药性。提取所有 10 8株Hp基因组DNA进行PCR扩增 ,用限制性片段长度多态性 (restrictionfragmentlengthpolymorphism ,RFLP)检测克拉霉素耐药菌株的点突变。结果 Hp菌株对克拉霉素耐药率为 14 8% (16 / 10 8)。 16株耐药菌株 2 3SrRNAV功能区PCR扩增片段RFLP分析 ,13株被BsaI酶切 ,提示 2 14 4位点存在A→G点突变 ,3株被BbsI酶切 ,提示2 14 3位点存在A→G点突变 ,所有敏感菌株均不能被BsaI和BbsI酶切 ,提示无 2 3SrRNA基因相应位点的点突变。同时本研究中并未发现突变形式与耐药程度的相关性。结论 儿童Hp感染对克拉霉素耐药率较高 ;2 3SrRNA基因点突变是Hp对克拉霉素耐药的重要因素。耐药菌株存在A2 14 4G和A2 14 3G突变 ,以前者为主。

Objective To investigate th e prevalence of Helicobacter pylori(H. pylori) resistance to clarithromycin ( CLM) in children and to demonstrate the correlation of 23S rRNA gene mutation to clarithromycin resistance of Helicobacter pylori isolates. Meth ods Totally 108 clinical strains of H. pylori were isolated fro m gastric biopsy specimens obtained from children who underwent endoscopy during the period from October 2002 to January 2004 in Children′s Hospital Affiliated to Medical College of Zhejiang University. H. pylori was identified by mo rphology and biochemical tests after culture. Clarithromycin susceptibility of H. pylori isolates was determined by both E-test and two-fold agar diluti on method. A strain was considered resistant when the MIC was defined as ≥1 μ g/ml. Genome DNAs of the 108 isolates were extracted and prepared for PCR to de tect the corresponding gene in the V domain of the 23S rRNA. The amplified frag ments were recognized and analyzed by restriction fragment length polymorphism ( RFLP) when an additional restriction site is created by the mutation. The PCR p roducts of all sensitive and resistant strains were digested with restriction en zyme BbsI and BsaI and were analyzed on a 1.5% agarose gel to discriminate diff erent kinds of mutant genotype. Results Sixteen of 108 i solates of H. pylori were resistant to clarithromycin by the agar dilution method and E-test method in clinical isolates from children, and the CLM resist ance rate was 14.8% (16/108)with MICs ranging from 1 μg/ ml to 128 μg/ ml. Comparison of results of the two methods showed that these two methods were qu ite consistent in determination of susceptibility and resistance. The target fr agment 425 bp in length containing 23S rRNA corresponding gene was successfully amplified. An A2144G mutation digested with BsaI was detected in 13 resistant i solates, but an A2143G mutation digested with BbsI in only 3 among all 16 clarit hromycin resistant strains. None of the sensitive isolates was cleaved by eithe r BsaI or BbsI enzyme, indicating that there was no mutation on them. It was al so found that all the fragments from the resistant strains were not completely d igested, and 425 bp uncut fragments were also visible and showed three bands ind icating that they were heterozygotic strains with a mixture of wild-types and A →G genotypes. In addition, in this study, no statistically significant differe nce between mutations at positions 2143 and 2144 with respect to the MIC was obs erved ( r=0.035, P>0.05). Conclution A high prevalence of clarithromycin-resistant H. pylori strains were detected am ong strains isolated from Chinese children studied. The 23S rRNA gene mutation at positions A2143G and A2144G plays an important role in clarithromycin resista nce of H. pylori and A2144G mutation is the predominant finding among the r esistant strains.