【摘要】：目的(1)了解長(zhǎng)沙地區(qū)臨床分離金黃色葡萄球菌(以下簡(jiǎn)稱(chēng)金葡菌)對(duì)常用抗菌藥物的耐藥現(xiàn)狀,探討金黃色葡萄球菌對(duì)甲氧西林和萬(wàn)古霉素的耐藥水平。(2)構(gòu)建金葡菌甲氧西林耐藥輔助基因femB的原核表達(dá)載體并在大腸桿菌中表達(dá),為進(jìn)一步研究金葡菌對(duì)甲氧西林的耐藥機(jī)制奠定基礎(chǔ)。 方法(1)收集長(zhǎng)沙地區(qū)11家醫(yī)院2009年11月-2010年11月臨床分離的非重復(fù)金葡菌279株,應(yīng)用Vitek-2全自動(dòng)微生物分析系統(tǒng)進(jìn)行鑒定,K-B法檢測(cè)金葡菌對(duì)24種藥物的敏感性,產(chǎn)色頭孢菌素試驗(yàn)檢測(cè)β-內(nèi)酰胺酶以及D試驗(yàn)檢測(cè)誘導(dǎo)型克林霉素耐藥。(2)應(yīng)用頭孢西丁和苯唑西林紙片擴(kuò)散法篩查耐甲氧西林的金葡菌(MRSA);瓊脂稀釋法檢測(cè)頭孢西丁,苯唑西林和萬(wàn)古霉素的最低抑菌濃度(MIC)。(3)提取金葡菌基因組DNA,并以此為模板進(jìn)行femB基因的PCR擴(kuò)增,構(gòu)建重組質(zhì)粒pGEX-4T-1-femB,并將重組質(zhì)粒導(dǎo)入大腸桿菌BL21中進(jìn)行表達(dá)。采用SDS-PAGE及Western blot分析對(duì)表達(dá)蛋白進(jìn)行驗(yàn)證。 結(jié)果(1)279株分離的金葡菌對(duì)24種藥物的耐藥分析顯示,敏感率50%的藥物為9種,敏感率最高的為萬(wàn)古霉素、替考拉寧和利奈唑胺,均為100%,其次為呋喃妥因(97.1%)、氯霉素(93.5%)和復(fù)方新諾明(87.1%)。耐藥率50%的抗菌藥物有11種,其中以青霉素G和氨芐西林的耐藥率最高,均為97.1%。MRSA的分離率為54.5%,除萬(wàn)古霉素、替考拉寧、利奈唑胺、呋喃妥因均高度敏感外,MRSA對(duì)常用的16種抗生素的耐藥率均顯著高于MSSA。(2) 279株金黃葡菌中,p-內(nèi)酰胺酶陽(yáng)性250株(89.6%),紅霉素耐藥而克林霉素敏感、中介的30株菌中,D試驗(yàn)陽(yáng)性22株(73.3%)。(3)苯唑西林(OXA)和頭孢西丁(FOX) MIC范圍分別為O.125μg/ml～256μg/ml和2μg/ml～256μg/ml,苯唑西林的MIC50和MIC90分別為128μg/ml和256μg/ml,頭孢西丁的MIC50和MIC90分別為64μg/ml和256μg/ml。萬(wàn)古霉素MIC范圍為1～2μg/ml,MIC50和MIC90均為2μg/ml。(4)經(jīng)PCR,雙酶切以及測(cè)序驗(yàn)證,成功的構(gòu)建了重組質(zhì)粒pGEX-4T-1-femB;重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21經(jīng)IPTG誘導(dǎo)后,SDS-PAGE和Western blot分析證實(shí)表達(dá)出49KDa目的蛋白。 結(jié)論(1)長(zhǎng)沙地區(qū)臨床分離的金葡菌p-內(nèi)酰胺酶陽(yáng)性率,克林霉素誘導(dǎo)型耐藥率均較高,且呈多重耐藥特征。(2)長(zhǎng)沙地區(qū)臨床分離的金葡菌不僅MRSA分離率高,而且對(duì)甲氧西林呈高水平耐藥；所有菌株對(duì)萬(wàn)古霉素均敏感,但MIC已接近中介水平,應(yīng)引起高度重視。(3)成功構(gòu)建了重組質(zhì)粒pGEX-4T-1-femB,并在大腸桿菌中高效的表達(dá)。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R346

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