长沙地区临床分离金黄色葡萄球菌的耐药特征及femB基因的克隆和原核表达

发布时间：2018-09-01 06:13

【摘要】：目的(1)了解长沙地区临床分离金黄色葡萄球菌(以下简称金葡菌)对常用抗菌药物的耐药现状,探讨金黄色葡萄球菌对甲氧西林和万古霉素的耐药水平。(2)构建金葡菌甲氧西林耐药辅助基因femB的原核表达载体并在大肠杆菌中表达,为进一步研究金葡菌对甲氧西林的耐药机制奠定基础。方法(1)收集长沙地区11家医院2009年11月-2010年11月临床分离的非重复金葡菌279株,应用Vitek-2全自动微生物分析系统进行鉴定,K-B法检测金葡菌对24种药物的敏感性,产色头孢菌素试验检测β-内酰胺酶以及D试验检测诱导型克林霉素耐药。(2)应用头孢西丁和苯唑西林纸片扩散法筛查耐甲氧西林的金葡菌(MRSA);琼脂稀释法检测头孢西丁,苯唑西林和万古霉素的最低抑菌浓度(MIC)。(3)提取金葡菌基因组DNA,并以此为模板进行femB基因的PCR扩增,构建重组质粒pGEX-4T-1-femB,并将重组质粒导入大肠杆菌BL21中进行表达。采用SDS-PAGE及Western blot分析对表达蛋白进行验证。结果(1)279株分离的金葡菌对24种药物的耐药分析显示,敏感率50%的药物为9种,敏感率最高的为万古霉素、替考拉宁和利奈唑胺,均为100%,其次为呋喃妥因(97.1%)、氯霉素(93.5%)和复方新诺明(87.1%)。耐药率50%的抗菌药物有11种,其中以青霉素G和氨苄西林的耐药率最高,均为97.1%。MRSA的分离率为54.5%,除万古霉素、替考拉宁、利奈唑胺、呋喃妥因均高度敏感外,MRSA对常用的16种抗生素的耐药率均显著高于MSSA。(2) 279株金黄葡菌中,p-内酰胺酶阳性250株(89.6%),红霉素耐药而克林霉素敏感、中介的30株菌中,D试验阳性22株(73.3%)。(3)苯唑西林(OXA)和头孢西丁(FOX) MIC范围分别为O.125μg/ml～256μg/ml和2μg/ml～256μg/ml,苯唑西林的MIC50和MIC90分别为128μg/ml和256μg/ml,头孢西丁的MIC50和MIC90分别为64μg/ml和256μg/ml。万古霉素MIC范围为1～2μg/ml,MIC50和MIC90均为2μg/ml。(4)经PCR,双酶切以及测序验证,成功的构建了重组质粒pGEX-4T-1-femB;重组质粒转化大肠杆菌BL21经IPTG诱导后,SDS-PAGE和Western blot分析证实表达出49KDa目的蛋白。结论(1)长沙地区临床分离的金葡菌p-内酰胺酶阳性率,克林霉素诱导型耐药率均较高,且呈多重耐药特征。(2)长沙地区临床分离的金葡菌不仅MRSA分离率高,而且对甲氧西林呈高水平耐药；所有菌株对万古霉素均敏感,但MIC已接近中介水平,应引起高度重视。(3)成功构建了重组质粒pGEX-4T-1-femB,并在大肠杆菌中高效的表达。
[Abstract]:Objective (1) To investigate the resistance of clinical isolates of Staphylococcus aureus (S. aureus) to common antibiotics in Changsha, and to investigate the resistance level of S. aureus to methicillin and vancomycin. (2) To construct the prokaryotic expression vector of S. aureus methicillin resistance auxiliary gene femB and express it in E. coli. To further study the mechanism of Staphylococcus aureus resistance to methicillin.
Methods (1) 279 strains of non-repetitive Staphylococcus aureus were collected from 11 hospitals in Changsha from November 2009 to November 2010. The susceptibility of Staphylococcus aureus to 24 kinds of drugs was detected by Vitek-2 automatic microbial analysis system. The sensitivity of Staphylococcus aureus to 24 kinds of drugs was detected by K-B method. (2) Methicillin-resistant Staphylococcus aureus (MRSA) was screened by cefoxitin and oxacillin disk diffusion method, and the minimal inhibitory concentration (MIC) of cefoxitin, oxacillin and vancomycin was detected by agar dilution method. (3) Genomic DNA of Staphylococcus aureus was extracted and used as a template for PCR amplification of femB gene. The recombinant plasmid pGEX-4T-1-femB was constructed and its weight was determined. The expressed protein was confirmed by SDS-PAGE and Western blot analysis.
Results (1) Drug resistance of 279 strains of Staphylococcus aureus to 24 kinds of drugs showed that there were 9 kinds of drugs with 50% susceptibility rate, the highest susceptibility rate was vancomycin, teicoplanin and linezolid, all of them were 100%, followed by furantoin (97.1%), chloramphenicol (93.5%) and cotrimoxazole (87.1%). The isolation rate of MRSA was 54.5%. Except for vancomycin, teicoplanin, linezolid and furantoin, the resistance rate of MRSA to 16 commonly used antibiotics was significantly higher than that of MSSA. (2) Among 279 strains of Staphylococcus aureus, 250 strains (89.6%) were positive for p-lactamase, and 250 strains (89.6%) were resistant to erythromycin but sensitive to clindamycin. Among the 30 strains, 22 (73.3%) were positive for D test. (3) The MIC ranges of oxacillin (OXA) and cefoxitin (FOX) were 0.125 ug/ml to 256 ug/ml, 2 ug/ml to 256 ug/ml, oxacillin MIC50 and MIC90 were 128 ug/ml and 256 ug/ml, cefoxitin MIC50 and MIC90 were 64 ug/ml and 256 ug/ml, respectively. (4) The recombinant plasmid pGEX-4T-1-femB was successfully constructed by PCR, double enzyme digestion and sequencing. The recombinant plasmid was transformed into E.coli BL21 and induced by IPTG. SDS-PAGE and Western blot analysis confirmed the expression of 49KDa protein.
Conclusion (1) The P-lactamase positive rate of clinical isolates of Staphylococcus aureus in Changsha is high, and the induced resistance rate of clindamycin is high, and the characteristics of multiple resistance are shown. (2) The clinical isolates of Staphylococcus aureus in Changsha are not only high isolation rate of MRSA, but also high resistance to methicillin. The recombinant plasmid pGEX-4T-1-femB was successfully constructed and expressed in E.coli.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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