肺炎链球菌 ciaH 基因与β-内酰胺类抗生素耐药的相关性研究  

Correlation between the ciaH gene of Streptococcus pneumoniae and the resistance toβ-lactam antibi-otics

作　　者：张少妮[1,2] 孙颜红[1,2] 楼永良[1] 严杰[3] 孙爱华[1,2] 

机构地区：[1]温州医科大学检验医学院和生命科学学院,325035 [2]浙江医学高等专科学校基础医学部,310053 [3]浙江大学医学院病原生物学系

出　　处：《中华微生物学和免疫学杂志》2015年第9期666-671,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（81271893）;浙江省卫生高层次创新人才工程项目（[2012]241）;浙江省医药卫生科技计划项目（2011KYA005）

摘　　要：目的：构建肺炎链球菌ciaH基因敲除（ΔciaH）突变株，了解肺炎链球菌二元信号系统CiaH／CiaR中ciaH基因与β-内酰胺类抗生素耐药的相关性。方法采用PCR 扩增肺炎链球菌ATCC6306株ciaH基因片段，T-A克隆后测序。构建用于敲除ciaH基因的自杀质粒pEVP3ciaH ，CaCl2法将其转化入肺炎链球菌ATCC6306株，通过同源重组、插入失活及氯霉素筛选获得肺炎链球菌ATCC6306株ciaH基因敲除突变株ΔciaH。采用PCR、测序及激光共聚焦显微镜法对ΔciaH突变株进行鉴定。采用二倍琼脂稀释法测定并比较青霉素G（ PCN）或头孢噻肟（ CTX）对ΔciaH突变株和野生株最低抑菌浓度（ MIC）及其差异。采用实时荧光定量RT-PCR（ qRT-PCR）检测1／4 MIC PCN或CTX作用前后ciaH-mRNA水平变化。结果 PCR和测序结果显示，所构建的ΔciaH突变株染色体DNA中ciaH基因被插入失活；免疫荧光法检测结果显示，ΔciaH突变株不表达CiaH蛋白。 PCN和CTX对ΔciaH突变株MIC分别为32μg／ml 和64μg／ml，明显高于野生株的0．06μg／ml 和1μg／ml （ P＜0．01）。1／4 MIC PCN或CTX作用后，肺炎链球菌ciaH-mRNA水平显著升高（P＜0．01）。结论肺炎链球菌CiaH／CiaR二元信号系统中CiaH与其对β-内酰胺类抗生素耐药性有关。Objective To construct a mutant strain of Streptococcus pneumoniae （ S.pneumoniae） with ciaH gene-knockout （ΔciaH） and to analyze the correlation between the ciaH gene and the bacterial re-sistance against β-lactam antibiotics.Methods The ciaH gene segament of S.pneumoniae strain ATCC6306 was amplified by PCR.The PCR product was sequenced after T-A cloning.A suicide plasmid pEVP3ciaH was constructed for the deletion of ciaH gene and then transformed into the ATCC 6306 strain by using the CaCl2 method .The mutant strain of S.pneumonia strain ATCC6306 with ciaH gene-knockout （ΔciaH） was genera-ted through homologous recombination , insertion inactivation and amphemycin screening , which was further identified by PCR , sequencing analysis and laser confocal microscopy .Double agar dilution method was used to detect the minimal inhibitory concentrations （ MICs ） of penicillin G （ PCN ） and cefotaxime （ CTX ） against theΔciaH mutant strain and the wild type strain .The differences between the MICs were further ana-lyzed.The changes of ciaH gene expression at mRNA level after treatment with 1/4 MIC of PCN or CTX were detected by real-time fluorescent quantitative RT-PCR （ qRT-PCR ） .Results The ciaH gene in the genomic DNA of the generated ΔciaH mutant strain was inactivated by insertion as indicated by PCR and se-quencing analysis .Results of the immunofluorescence assay showed that the ΔciaH mutant strain did not ex-press the CiaH protein .The MICs of PCN and CTX against the ΔciaH mutant strain were 32 μg/ml and 64μg/ml, respectively, which were significantly higher than that of the wild type strain （0.06 μg/ml and 1μg/ml） （P〈0.01）.The expression of ciaH gene at mRNA level was significantly elevated after treatment S.pneumoniae ATCC6306 strain with 1/4 MIC PCN or CTX （P〈0.01）.Conclusion The CiaH protein in the CiaH/CiaR two-component signaling system is involved in the resistance of S.pneumoniae against β-lac-tam antibiotics.

关 键 词：肺炎链球菌 二元信号传导系统 CiaH蛋白 Β-内酰胺类抗生素 耐药性 

分 类 号：R378[医药卫生—病原生物学]