【作者】 肖潇；

【导师】 李英伦；

【作者基本信息】 四川农业大学 ， 基础兽医学， 2009， 硕士

【摘要】 本文是关于“乳炎康”注射液对奶牛乳源金黄色葡萄球菌耐药性消除作用的研究。以临床分离的一株乳源金黄色葡萄球菌,作为本试验的受试菌株。用生化鉴定方法与利用细菌通用引物对细菌的16S rRNA基因进行PCR扩增和测序,再通过测序结果与已知种属的16S rRNA基因序列进行同源性比较确定临床分离菌株的种类。结果显示:临床分离株为金黄色葡萄球菌。选用15种抗生素进行药物敏感性检测,以确定分离株耐药谱,结果显示:金葡菌对氨苄西林(AMP),阿莫西林(AMO),红霉素(EYR)MIC均＞128μg/mL,呈高度耐药,青霉素(PG)对金葡菌MIC为4μg/mL,呈高度耐药,对其他10种抗生素敏感。以“乳炎康”注射液为消除剂,临床分离菌株为靶细菌,用影印培养法,进行体外耐药质粒消除试验,并以SDS作为对照,结果显示:“乳炎康”注射液对耐药金黄色葡萄球菌质粒有消除作用,作用24h其1/2MIC、1/4 MIC、1/8 MIC浓度的消除率分别为2%、2.6%、3.6%,延长作用时间至48h其1/2MIC、1/4 MIC、1/8 MIC浓度的消除率分别为2.8%、3.6%、4.4%。对照组SDS消除率分别为0.4%、0.2%、0%,延长作用时间至48h消除率分别为0.8%、0.6%、0%。将中药和SDS对受试菌株的消除率经X~2检验,“乳炎康”注射液消除结果与SDS对照组比较,均有极显著差异(P＜0.01)。对消除子进行质粒抽提,经琼脂凝胶糖电泳后,结果显示:经“乳炎康”注射液作用后,消除菌株缺失1至2条质粒带,也有消除菌株没有缺失质粒带,但条带明显变淡。消除菌株和原始耐药株药敏试验结果显示:质粒条带消失的消除菌株的抑菌圈明显扩大,特别是阿莫西林的抑菌圈扩大至20.30mm,成为AMO敏感菌株,PG、AMP抑菌圈也有所增大,但仍在耐药范围,ERY的抑菌圈作用前后没有变化;质粒条带未消失的消除菌株抑菌圈明显扩大,AMO抑菌圈扩大至17.96 mm,仍为耐药菌株。PG、AMP、ERY抑菌圈变化与前者相同。结果提示,耐药金葡菌对AMO的耐药可能是由质粒引起。对“乳炎康”注射液作用前后受试菌株的blaⅠ和blaR基因进行PCR扩增,测序和对比分析,结果表明:blaⅠ的基因在“乳炎康”注射液作用的前后碱基没有发生改变。而“乳炎康”注射液作用后,blaR基因在发现1003-1050bp缺失了48bp,在1699处发现AT置换,在1704处缺失碱基C,在1710处缺失碱基A。结果提示,中药“乳炎康”注射液对金葡菌的耐药基因有诱导突变作用,使金葡菌的耐药性随着基因的突变而丧失,并且“乳炎康”注射液的作用位点具有选择性。本次试验运用分子生物学手段对“乳炎康”注射液作用后的金黄色葡萄球菌β-内酰胺耐药基因进行测序,对“乳炎康”注射液具体机制进行了初步探讨,“乳炎康”注射液对金葡菌的耐药基因有诱导突变作用,使金葡菌的耐药性随着基因的突变而丧失。初步揭示了“乳炎康”注射液消除耐药金黄色葡萄球菌耐药性机制,为进一步研究中药消除耐药性提供了实验依据,从而在分子水平上阐述中草药复方制剂的作用机制,同时为中草药在临床生产实践中的应用提供有利的证据。利用基因测序和质粒DNA抽提,细菌总DNA提取,初步了解金黄色葡萄球菌获得耐药性的途径。更多还原

【Abstract】 In this study,the drug resistance eliminate function of "Ru yan kang" injection on staphylococcus aureus which isolated from cow’s milk has been detected.The strain of this study was isolated clinical.And the strain has been detected by biochemistry characterization, sequencing and blast 16S rRNA with the sequences from Genebank.From the result,the strain had been characterized as staphylococcus aureus.Drug sensitivity of this strain had been detected by 15 antibiotics in order to definite its ability of drug resistant.From the result,the strain resistant strongly with AMP,AMO,EYR whose mic＞128ug/ml,while sensitive others.The strain which was cultivated by replica plating had been operate by"Ru yan kang" injection as drug resistant elimination for extraorgan plasmid elimination test.From the result, elimination effect had been detected on"Ru yan kang"injection.Elimination coefficient of 1/2MIC、1/4 MIC、1/8 MIC group is 2.8%、3.6%、4.4%,respectively.While the negative control group is 0.4%、0.2%、0%.Extended time of operation to 48h,Elimination coefficient of 1/2MIC、1/4 MIC、1/8 MIC group is0.8%、0.6%、0%,respectively.While negative control group operated same time is 0.8%、0.6%、0%.The elimination coefficient of group operate by "Ru yan kang" injection is significant deviation(P＜0.01) with negative control group by useing chi-square test.The result of plasmid agar gel electrophoresis demonstrated that one or two plasmid strip of eliminate strain were absence.Plasmid strip of others which strip are not absence is thinning.Susceptibility test between original drug resistant strain and operated strain demonstrated that bacteriostasis collar of the operated strain which strip was absence enlarge obviously.The bacteriostasis collar of amoxicillin had enlarge to 20.30mm and the strain is sensitive with AMO.bacteriostasis collar of PG、AMP enlarge too.Bacteriostasis collar of ERY had not change.Bacteriostasis collar of the strain which strip are not absence has enlarge ERY had not change.Bacteriostasis collar of the strain which strip are not absence has enlarge obviously too.Bacteriostasis collar of AMO enlarge to 17.96 mm which are drug resistant still.The same as PG、AMP、ERY.The result demonstrate that the resistant for AMO is induced by plasmid.PCR amplification bla I and bla R of original strain and strain operated by "Ru yan kang" injection,sequencing and analysis.The result demonstrated that the bla I had not change after operated.The bla R had beed detected that 47bp absenced on 1003-1050 site,AT replacement on 1699bp,basyl absence on 1709.The result demonstrates that"Ru yan kang" injection can induce mutation on drug resistant gene.The function can eliminate drug resistance and can identify effect site.In this study,the function of"Ru yan kang" injection had been investigated by molecular biology method such as sequencing.The result demonstrates that the"Ru yan kang" injection can induce mutation on drug resistant gene that induces drug resistance.Eliminate drug resistance mechanism of "Ru yan kang" injection had revealed initialiy and to provided experiment groundwork for futher study on eliminating drug resistance by the traditional Chinese medicine.Moreover,this study demonstrated mechanism of action and provided positive evidence for using the traditional Chinese medicine on clinical.The way that staphylococcus aureus get drug resistant ability had been revealed by sequencing,extracting plasmid and genomic DNA.更多还原