摘要目的:探讨耐碳青霉烯类肺炎克雷伯菌(CRKP)脂多糖修饰相关的多黏菌素耐药机制。方法:通过接合转移和 mcr基因的检测探究是否存在质粒介导的耐药基因；利用实时荧光定量PCR检测多黏菌素耐药相关基因的相对表达量，并进行全基因组测序分析；采用SDS-PAGE银染试验和MALDI-TOF-MS质谱分析检测脂多糖的改变。 结果:未扩增出 mcr-1~8基因且接合转移试验结果为阴性，未检测到与耐药性相关的可移动元件。7株多黏菌素耐药CRKP中 pmrA、 pmrB、 pmrC基因的相对表达量均增高，其中6株 phoP/ phoQ基因的相对表达量增高；3株 crrA/ crrB基因的相对表达量降低；4株 mgrB基因的相对表达量降低。耐药株发生的错义突变位点主要位于 KPHS_09430、 KPHS_35900、 KPHS_39520、 KPHS_52420等基因上，在CRKP-6菌株中检测到IS Kpn14插入序列。MALDI-TOF-MS观察到耐药菌株脂多糖中天然脂质A有L-Ara4N修饰。 结论:染色体介导的双组分调节系统突变引起脂多糖修饰是本研究CRKP对多黏菌素耐药的主要机制，且双组分调节系统的突变对多黏菌素耐药性的影响在不同菌株背景下也存在差异。

abstractsObjective:To investigate the mechanism of polymyxin resistance related to lipopolysaccharide modification in carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods:Plasmid-mediated drug resistance genes in seven CRKP strains were detected by conjugation assay and mcr gene detection. The expression of polymyxin resistance-related genes was measured using quantitative real-time PCR. The complete genomes of CRKP strains were sequenced. Silver staining and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were performed to analyze the changes in lipopolysaccharide (LPS). Results:The seven CRKP strains were negative for mcr genes and the results of conjugation assay were also negative. Moreover, no mobile genetic elements related to drug resistance were detected. Compared with wild-type strain, all seven CRKP strains that were resistant to polymyxin showed increased expression of pmrA, pmrB and pmrC genes at the transcriptional level; six showed increased expression of phoP/ phoQ genes; three showed decreased expression of crrA/ crrB genes; four showed decreased expression of mgrB gene. The missense mutation sites in drug-resistant strains were mainly in KPHS_09430, KPHS_35900, KPHS_39520 and KPHS_52420. IS Kpn14 insertion sequence was detected in CRKP-6 strain. MALDI-TOF-MS reveals the modification of natural lipid A with L-Ara4N in CRKP LPS. Conclusions:LPS modification induced by chromosome-mediated mutation in the two-component regulatory system was the main molecular mechanism of polymyxin resistance in CRKP isolates in this study. Effects of the mutation in the two-component system on polymyxin resistance varied in different strains.