氨苄西林诱导志贺菌耐受与hipA和hipB基因突变的关系

The relationship between Shigellatolerance induced with ampicillin and mutation of the hipA and hipB genes

目的探讨氨苄西林诱导志贺菌(Shigella)耐受与hipA和hipB基因突变的关系,为细菌耐药的预防提供理论基础。方法对2014年分离于河南省睢县腹泻患者粪便样本中的志贺菌进行生化和血清学鉴定,采用Kirby-Bauer(K-B)纸片扩散法筛选氨苄西林敏感株。用10MIC氨苄西林间隔诱导志贺菌,测定其MDK99。PCR扩增志贺菌原代株和耐受株hipA和hipB基因,分析诱导前后两基因碱基序列的变化。结果成功诱导出两株志贺菌耐受株,分别命名为mel-ss2014011/YD和mel-ss2014021/YD。诱导前后志贺菌对氨苄西林的MIC保持不变,mel-ss2014011/YD和melss2014021/YD的MDK99分别是诱导前的4倍和6倍。mel-ss2014021/YD株hipA基因在226位点处有单碱基G的插入,插入区域位于编码区,hipB基因第217位点(C→T Pro→Leu)出现错义突变。mel-ss2014011/YD株未见hipA和hipB;因突变。结论 hipA和hipB基因突变可能会提高耐受水平,志贺菌在发生耐药前先进入细菌耐受状态。

Objective To examine the association between Shigellatolerance and mutation of the hipA and hipB genes in order to determine a principle for the prevention of bacterial resistance. Methods Biochemical and serological indicators of Shigellabacteria isolated from fecal samples from patients with diarrhea in Sui County(Henan Province)in 2014 were identified.Shigella strains were screened for sensitivity to ampicillin using Kirby-Bauer(K-B)disc diffusion and were then exposed to 10 MIC ampicillin at certain intervals.MDK99 was subsequently measured.The hipA and hipB genes from original strains and tolerant strains were amplified using PCR,and the base sequences of hipA and hipB were analyzed after tolerance was induced. Results Tolerance was successfully induced in 2strains of Shigella,named melss2014011/YD and mel-ss2014021/YD.The MIC of Shigellaremained unchanged after tolerance was induced.After tolerance was induced,the MDK99 of mel-ss2014011/YD and mel-ss2014021/YD increased 4-fold and 6-fold,respectively.Sequencing indicated that the mel-ss2014021/YD inducer hipA had a single base G insertion at site 226,and the insertion region was located in the coding region.There was a missense mutation in the 217 th loci of the hipB gene(C to T Pro to Leu),and hipA and hipB were not mutated in mel-ss2014011/YD strain. Conclusion Mutation of hipA and hipB may increase the tolerance of Shigellaso that Shigella becomes tolerant before becoming drug-resistant.