碳青霉烯类耐药肺炎克雷伯菌染色体介导黏菌素耐药机制研究  

The mechanism underlying chromosomally mediated resistance to colistin in carbapenem-resistant Klebsiella pneumoniae

作　　者：张一琦 陈欣 聂志妍[2] 郑永贵 苏佳纯 ZHANG Yiqi;CHEN Xin;NIE Zhiyan;ZHENG Yonggui;SU Jiachun(Institute of Antibiotics,Huashan Hospital,Fudan University,Shanghai 200040,China)

机构地区：[1]复旦大学附属华山医院抗生素研究所,上海200040 [2]上海健康医学院

出　　处：《中国感染与化疗杂志》2022年第3期322-326,共5页Chinese Journal of Infection and Chemotherapy

基　　金：国家自然科学基金青年科学项目(81903673);上海市青年科技英才扬帆计划(19YF1405400)。

摘　　要：目的探究碳青霉烯类耐药肺炎克雷伯菌(CRKP)药物暴露后染色体序列突变,以及突变与黏菌素耐药之间的关系,为临床合理用药、减少耐药发生提供依据。方法黏菌素对临床分离株CRKP 12-130最低抑菌浓度(MIC)为0.25 mg/L,将100μL该菌过夜培养菌液分别涂布于含2倍~32倍MIC黏菌素的MH琼脂平板上,获取耐药突变株;采用细菌基因组提取试剂盒提取基因组DNA并进行二代测序;通过PCR法检测黏菌素耐药相关基因。结果从不同浓度梯度黏菌素的MH琼脂平板中各取50个菌落传代10次后作为受试菌株测其MIC,在2倍MIC和4倍MIC黏菌素的MH琼脂平板上选择出的突变株分别有33株和47株对黏菌素耐药(MIC≥4 mg/L)。8倍MIC、16倍MIC和32倍MIC黏菌素的MH琼脂平板上选择出的突变株均对黏菌素耐药。从中挑选40株MIC值不同的菌株进行二代测序,结果显示有15株菌株mgrB基因完全缺失,2株菌株mgrB基因发生部分缺失,2株菌株的mgrB基因中出现替换(G82T),并导致氨基酸序列改变(G28C)。利用mgrB基因两翼序列进行PCR引物设计,将PCR产物进行电泳后发现,230株耐药突变株中有103株存在mgrB基因的改变。结论CRKP在不同浓度的黏菌素选择压力下,均会选择出自发耐药突变株。mgrB基因的完全缺失是造成该类自发突变株对黏菌素耐药的最主要机制。Objective To explore the chromosomal mutations of carbapenem-resistant Klebsiella pneumoniae(CRKP)after colistin exposure and the relationship between chromosomal mutations and colistin resistance for rational use of colistin and better control of colistin resistance.Methods CRKP 12-130 was a clinical isolate of CRKP.The mimimum inhibitory concentration(MIC)of colistin was 0.25 mg/L against this strain.The resistant mutants were screened by plating 100μL of the overnight suspension culture of CRKP 12-130 on Mueller Hinton(MH)agar containing 2 to 32 times the MIC of colistin.The commercial bacterial genome extraction kit was used to extract bacterial genomic DNA for next generation sequencing(NGS)analysis.The genes related to colistin resistance were detected by PCR.Results Fifty colonies of CRKP 12-130 were harvested from the MH agar containing different colistin concentrations.The mutant strains were cultured for 10 passages on colistin-free MH agar,followed by MIC determination.Overall,33 and 47 strains selected from the MH agar containing 2×MIC and 4×MIC colistin were resistant to colistin(MIC≥4 mg/L),respectively.The strains selected from the MH agar containing 8×,16×,and 32×MIC colistin were all resistant to colistin.Forty strains with different colistin MICs were selected for NGS analysis.The results showed mgrB complete deletion in 15 strains,mgrB partial deletion in 2 strains,and a substitution of G82T(G28C)in mgrB gene in 2 strains.PCR results using primers based on the flank sequences of mgrB gene demonstrated that the mgrB gene mutated in 103 of the 230 colistin-resistant strains obtained by screening. Conclusions Colistin-resistant CRKP mutants can be spontaneously selected under pressure of different concentrations of colistin. The complete deletion of mgrB gene is one of the main mechanisms underlying colistin resistance in the spontaneous resistant mutants.

关 键 词：碳青霉烯类耐药肺炎克雷伯菌 黏菌素 突变 染色体 mgrB基因 

分 类 号：R378.996[医药卫生—病原生物学]