中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
Chinese Journal of Microbiology and Immunology
2015年
9期
666-671
,共6页
张少妮%孙颜红%楼永良%严杰%孙爱华
張少妮%孫顏紅%樓永良%嚴傑%孫愛華
장소니%손안홍%루영량%엄걸%손애화
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
肺炎鏈毬菌%二元信號傳導繫統%CiaH蛋白%β-內酰胺類抗生素%耐藥性
폐염련구균%이원신호전도계통%CiaH단백%β-내선알류항생소%내약성
Streptococcus pneumoniae%Two-component signaling system%CiaH protein%β-lactam antibiotics%Drug resistance
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。
目的：構建肺炎鏈毬菌ciaH基因敲除（ΔciaH）突變株，瞭解肺炎鏈毬菌二元信號繫統CiaH／CiaR中ciaH基因與β-內酰胺類抗生素耐藥的相關性。方法採用PCR 擴增肺炎鏈毬菌ATCC6306株ciaH基因片段，T-A剋隆後測序。構建用于敲除ciaH基因的自殺質粒pEVP3ciaH ，CaCl2法將其轉化入肺炎鏈毬菌ATCC6306株，通過同源重組、插入失活及氯黴素篩選穫得肺炎鏈毬菌ATCC6306株ciaH基因敲除突變株ΔciaH。採用PCR、測序及激光共聚焦顯微鏡法對ΔciaH突變株進行鑒定。採用二倍瓊脂稀釋法測定併比較青黴素G（ PCN）或頭孢噻肟（ CTX）對ΔciaH突變株和野生株最低抑菌濃度（ MIC）及其差異。採用實時熒光定量RT-PCR（ qRT-PCR）檢測1／4 MIC PCN或CTX作用前後ciaH-mRNA水平變化。結果 PCR和測序結果顯示，所構建的ΔciaH突變株染色體DNA中ciaH基因被插入失活；免疫熒光法檢測結果顯示，ΔciaH突變株不錶達CiaH蛋白。 PCN和CTX對ΔciaH突變株MIC分彆為32μg／ml 和64μg／ml，明顯高于野生株的0．06μg／ml 和1μg／ml （ P＜0．01）。1／4 MIC PCN或CTX作用後，肺炎鏈毬菌ciaH-mRNA水平顯著升高（P＜0．01）。結論肺炎鏈毬菌CiaH／CiaR二元信號繫統中CiaH與其對β-內酰胺類抗生素耐藥性有關。
목적：구건폐염련구균ciaH기인고제（ΔciaH）돌변주，료해폐염련구균이원신호계통CiaH／CiaR중ciaH기인여β-내선알류항생소내약적상관성。방법채용PCR 확증폐염련구균ATCC6306주ciaH기인편단，T-A극륭후측서。구건용우고제ciaH기인적자살질립pEVP3ciaH ，CaCl2법장기전화입폐염련구균ATCC6306주，통과동원중조、삽입실활급록매소사선획득폐염련구균ATCC6306주ciaH기인고제돌변주ΔciaH。채용PCR、측서급격광공취초현미경법대ΔciaH돌변주진행감정。채용이배경지희석법측정병비교청매소G（ PCN）혹두포새우（ CTX）대ΔciaH돌변주화야생주최저억균농도（ MIC）급기차이。채용실시형광정량RT-PCR（ qRT-PCR）검측1／4 MIC PCN혹CTX작용전후ciaH-mRNA수평변화。결과 PCR화측서결과현시，소구건적ΔciaH돌변주염색체DNA중ciaH기인피삽입실활；면역형광법검측결과현시，ΔciaH돌변주불표체CiaH단백。 PCN화CTX대ΔciaH돌변주MIC분별위32μg／ml 화64μg／ml，명현고우야생주적0．06μg／ml 화1μg／ml （ P＜0．01）。1／4 MIC PCN혹CTX작용후，폐염련구균ciaH-mRNA수평현저승고（P＜0．01）。결론폐염련구균CiaH／CiaR이원신호계통중CiaH여기대β-내선알류항생소내약성유관。
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.
