【作者】 司红彬；

【导师】 胡功政；

【作者基本信息】 河南农业大学 ， 基础兽医学， 2006， 硕士

【摘要】 本文采用试管二倍稀释法对临床分离的鸡志贺氏菌常用抗菌药的MIC值测定，利用双纸片增效法对其所产超广谱β-内酰胺酶(ESBLs)的检测，并用PCR方法确定其基因型。试制出复方头孢噻呋混悬液，并观察了其对人工复制的产β-内酰胺酶志贺氏菌感染治疗效果，以了解鸡志贺氏菌的耐药现状及耐药机制，探讨其产酶耐药的分子机制，为临床治疗提供理论依据，同时为新兽药制剂的开发提供新的思路。 β-内酰胺酶检测及ESBLs的试验结果表明：临床分离的鸡志贺氏菌用特异性的色原头孢菌素法检测呈β-内酰胺酶阳性，AmpC酶为阴性。双纸片增效法检测到超广谱β-内酰胺酶，对临床志贺氏菌所产ESBLs设计引物PCR扩增、克隆、测序，利用Dnastar MegAlign Sequence软件与U48775(TEM-1)对比分析，发现临床株与U48775同源性99.2％，属于TEM型，共有七处碱基发生变化，相应引起三个氨基酸发生改变，33位D(天冬氨酸)变为G(甘氨酸)、186位T(苏氨酸)变为A(丙氨酸)、271位R(精氨酸)变为Q(谷氨酰胺)，与其它序列相比，AY903309(TEM-116)同源性最高，为99.8％，共两处碱基发生变化，其中151位碱基A变为G，引起51位丝氨酸变为甘氨酸，403位碱基T变为C，没有引起氨基酸变化，为沉默突变，因与TEM-116有一个氨基酸差异，故该基因型暂称其为新型TEM-1V，上传至GenBank获序列号DQ211973。 体外抑菌实验结果表明：临床产ESBLs志贺氏菌对大部分单方药物敏感性明显下降，除碳青酶烯类亚胺培南、美罗培南敏感性较高，MIC值分别为0.25μg／ml、0.0625μg／ml；其它单药都相对不敏感，第三代头孢类头孢噻呋、头孢曲松对其MIC值分别为＞128μg／ml、128μg／ml；青霉素类、氟喹诺酮类、氨基糖苷类MIC值均＞128μg／ml，但β-内酰胺酶抑制剂可显著增强β-内酰胺类抗菌药的对产酶菌抗菌活性，头孢噻呋／他唑巴坦(2：1)、头孢曲松／他唑巴坦(2：1)、阿莫西林／棒酸(2：1)对其MIC值分别为8μg／ml、8μg／ml、32μg／ml，提示对产酶菌感染疾病治疗时使用三代头孢类抗生素与抑制剂联用效果较好。 本文还研究了复方头孢噻呋油性混悬注射液处方、物理稳定性和对临床产超广谱β-内酰胺酶(ESBLs)鸡志贺氏菌感染的疗效。根据ESBLs基因型为TEM型及药敏试验确定头孢噻呋和他唑巴坦联用(2：1)对产ESBLs的鸡志贺氏菌较为敏感，MIC值为16μg／ml，是头孢噻呋MIC值(＞128gμg／ml)的1／8；用此为主药处方和辅料羧甲基纤维素钠0.2％、司本-80 1.2％制备的复方头孢噻呋油性混悬液其沉降比为0.997、再分散性良好，经过在温度为60℃、湿度为75％环境下加速试验6个月后观察，颜色无变化，无粘壁现象，无颗粒凝结，表明物理稳定性良好；该制剂按20mg／Kg体重肌肉注射一次，对产ESBLs鸡志贺氏菌感染的有效率90％，治愈率86％。更多还原

【Abstract】 MIC of common antibiotics against shigella isolated from chicken were determined by the ameliorative two fold dilution method ,the ESBLs produced by shigella were detected by double slips synergy and its phenotypes were also made sure with PCR in this paper.The treatment results of compound suspension preparation of ceftiofur which were tentatively made by our laboratory against diseases infected by shigella produced ESBLs were observed, in order to understand the current situation and mechanism of drugs’resistance and investigated molecule mechanism of ita drugs’resistance. theories based on the experiment were offered for clinical trsatments,new thoughts on exploitation of new veterinary drugs were offered at the same time.The results of the detection of BLA and ESBLs showed that shigella clinical strains isolated from chichen had produced BLA by nitrocefin disc test, but none of AmpC . ESBLs was found by double-disc synergy method. The primer was designed for the ESBLs produced by shigella and amplification by PCR,then cloning and sequencing, an analysis was executed by contrasting with the sequence of U48775 with a software called Dnastar MegAlign Sequence, the homology gene with 99.2% was identitied to TEM-1(U48775), the result revealed the 287-amino-acid protein differing by three amino acid substitutions from β-lactamase TEM-1(Asp33 Glu, Thr186 Ala, Arg271Gln),It harbored a TEM -116 type gene with 99.8% identity to AY903309 (TEM-116), Amino acid differences in comparison to the amino acid sequence of ESBLs TEM-116 with Dnastar MegAlign Sequence Distances were found differing only one(Ser 51 Glu),although at the point 403 T changed into C, but it should be silent mutation and not lead to the amino acid changement. The genetype of enzyme was called TEM-1V temporarily because there were an amino acid which was different with sequence of TEM-116 and got serial-number DQ211973 by uploading to Genbank.The results of drug susceptibility in vitro showed that susceptibility of single更多还原