儿童感染幽门螺杆菌体外诱导抗生素耐药的研究

Preliminary study on in-vitro induction of antibiotic resistance in Helicobacter priori strains isolated from children

目的比较幽门螺杆菌(Hp)对甲硝唑(MTZ)、克拉霉素(CLA)、阿莫西林(AMX)体外获得耐药性的难易程度,初步探索 Hp 耐药的发生规律,指导临床合理用药。方法所有 Hp 均为来源于2004年12月至2005年7月于浙江大学附属儿童医院进行胃镜检查患儿的临床分离株。采用Etest 法测定最低抑菌浓度(MIC 值),筛选出对3种抗生素均敏感的菌株,并进行浓度递增法体外诱导。琼脂稀释法测定各菌株诱导前后的 MIC 值,以比较 Hp 对3种抗生素诱导的难易程度。结果共筛选出7株对3种抗生素均敏感的 Hp,经过6～17代体外诱导,所有7株均获得 MTZ 耐药,3株获得 CLA 耐药,未获得 AMX 耐药。Hp 对3种抗生素的可诱导倍数不同,MTZ 多数(6/7)达32倍以上,CLA 为1～256倍,AMX 为2～16倍。诱导后菌株经不含抗生素的培养基传代3次,对 MTZ 与 CLA的 MIC 值较传代前变化差异无统计学意义(P>0.05),而对 AMX 的 MIC 值较传代前降低(P<0.05)。结论儿童感染的 Hp 经体外诱导很容易获得 MTZ 耐药,可以获得 CLA 耐药,但很难获得AMX 耐药;体内外实验的一致性提示 Hp 对抗生素耐药主要为获得性耐药;体外诱导耐药发生的难易程度有助于预测实际耐药率的变迁,为根除治疗中选择抗生素提供依据。

Objective Many clinical studies indicated that Helicobacter pylori （Hp） strains rarely acquired resistance to amoxicillin but easily to clarithromycin and metronidazole. However, it was unclear whether the antibiotic resistance of Hp strains was induced or passively selected during long-term or frequent treatment with metronidazole, clarithromycin and amoxicillin. To compare the propensity of acquired resistance to antibiotics, Hp strains were exposed to amoxicillin, clarithromycin and metronidazole in vitro in this study. Methods All Hp strains were clinical isolates, derived from biopsy specimens of patients taken during endoscopy in the Affiliated Children＇s Hospital, Zhejiang University School of Medicine from December 2004 to July 2005. To seek susceptible strains, the minimum inhibitory concentrations （MICs） of the three antibiotics were determined by using Epsilometer test （E-test） method. In vitro induction was carried out on serially doubling concentrations of antibiotics incorporated into agar. Isolates were also transferred at least three times on antimicrobial agent-free medium, followed by a redetermination of the final MICs to assess the stability of the selected resistance. Results 7 strains were exposed to antibiotics in vitro. After 6-17 passages on antibiotic plates, 7 and 3 strains respectively acquired resistance to metronidazole and clarithromycin, while none of the strains were resistant to amoxicillin. The inductive folds were different among three groups： 8-128 folds in metronidazole group; 1-256 folds in clarithromycin group; 2-16 folds in amoxicillin group. After three transfers on antimicrobial agent-free medium, the MICs decreased significantly in amoxicillin group （ P 〈 0. 05 ） but had no change in metronidazole group and clarithromycin group （P 〉 0.05）. Conclusions The metronidazole resistance in Hp was easily selected. Strains resistant to clarithromycin could be selected, but the amoxicillin resistance could not be selected after in vitro induction for Hp isolated from children. The correlation between in vitro and in vivo outcomes suggests that acquired resistance was the main cause for the resistance in Hp strains. The laboratory results of in vitro antibiotic induction could help predict the actual rate of resistance and select appropriate antibiotics for treatment.