摘要
目的诱导多黏菌素耐药的肺炎克雷伯菌,探究其对多黏菌素的耐药机制。方法药物浓度倍增法诱导多黏菌素耐药株,微量肉汤稀释法测定最低抑菌浓度(minimum inhibitory concentration, MIC)和最低杀菌浓度(minimum bactericidal concentration,MBC);PCR扩增并测序确定突变部分;终点显色法检测内毒素含量;实时荧光定量PCR(qRT-PCR)检测相关基因表达量的变化。比浊法和活菌计数法分别测定生长曲线;结晶紫半定量法分析生物被膜形成能力。结果诱导得到4株耐药株,菌株MIC和MBC均大幅提升且有一株mgrB上游基因突变,内毒素含量升高,mgrB表达量下降,PhoPQ和pmrHFIJKLM相关基因表达均上升。突变株生长状况与野生株基本一致,生物被膜形成能力增强,waaA基因表达上升。结论肺炎克雷伯菌mgrB上游基因突变是多黏菌素耐药原因之一,并可引起内毒素含量升高和生物被膜形成能力增强。
Objective To induce polymyxin-resistant Klebsiella pneumoniae and to investigate the mechanism of polymyxin resistance. Methods Klebsiella pneumoniae was induced by polymyxin in vitro using concentration doubling method. Minimum inhibitory concentration （MIC） and minimum bactericidal concentration （MBC） were determined by the broth microdilution technique. The mutant genes were determined by PCR amplification and sequencing. The endotoxin was detected by limulus amebocyte lysate （LAL） assay. The genes expressions were evaluated by qRT-PCR. Bacterial growth curves were plotted using turbidimetry and plate counting. Biofilm formation was assayed using a modified crystal violet staining method. Results Four resistant strains were obtained and the MIC and MBC of the resistant strains were significantly increased. An insertional inactivation of mgrB upstream gene was found. The mutant strain had increased endotoxin content compared to the wild strain. Transcription of phoP, phoQ, pmrA, pmrD, pmrH and waaA was upregulated. Meanwhile the transcription of mgrB was downregulated. The growth of the mutant strain was basically consistent with that of the wild strain. In addition, the biofilm formation ability was higher than the wild strain. Conclusion Mutation of mgrB upstream gene is one of the causes of polymyxin resistance, and it can increase endotoxin level and biofilm formation ability.
作者
纪乃琪
陈向东
汪辉
任聪
鲍张杰
Ji Nai-qi;Chen Xiang-dong;Wang Hui;Ren Cong;Bao Zhang-jie(School of Life Science and Technology,China Pharmaceutical University,Nanjing 211198)
出处
《中国抗生素杂志》
CAS
CSCD
2018年第11期1443-1448,共6页
Chinese Journal of Antibiotics
基金
江苏高校优势学科建设工程资助项目(No.PPZY2015A057)
