【作者】 田勇；

【导师】 乔健；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2003， 硕士

【摘要】 目的和方法 本试验探讨外输泵抑制剂维拉帕米（VP）对耐恩诺沙星（EN）鸡源性大肠杆菌（E．coli）耐药性的影响，为开发拮抗耐药性的兽药制剂打下基础。为此从以下三个方面进行研究。 1．耐恩诺沙星大肠杆菌的分离鉴定和耐药性检测：从河北省某肉鸡集团公司的商品代鸡场分离E．coli，进行血清型的鉴定。随机选取20株E．coli，用Kirby-Bauer法对15种抗菌药物进行药敏试验，筛选出耐EN菌株用于体外试验。 2．体外试验：用试管倍比稀释法分别测定EN、VP对耐药菌株和标准菌株0₂、0₇₈的最小抑菌浓度（MIC）。然后采用试管棋盘法进行联合药敏试验，用部分抑菌浓度指数（FIC）判断EN与VP联用效果，选择有增敏效果的一株用于体内试验。 3．体内试验：30日龄240只商品代肉仔鸡，随机分成4组，即阳性对照组、EN组、EN+VP组（此三组每只鸡腹腔注射1.5ml试验菌液）和阴性对照组（每只鸡腹腔注射1.5ml生理盐水）。出现明显临床症状后，EN组每只鸡每天肌注二次EN 2.5mg／kg B．W；EN+VP组每只鸡每天肌注二次EN 2.5mg／kg B．W和VP6.0mg／kg B．W。观测并记录每组的死亡数、治愈数和体重变化情况。同时分别在2、4、6、8、10天，每组随机抽取6只测定股动脉压，剖检后观察病理变化和菌检率。 结果与结论 1．从206份病料中分离出183株大肠杆菌，平均分离率为89.3％。除23株未能定型外，鉴定出160个分离株分属32个血清型，以0₇₈、0₂、0₁、0₈₈、0₇₆等5个血清型为本地区的优势血清型。对随机选取的20株E．coli，经药敏试验发现：所有菌株全部对2种或多种抗菌药物有耐药性，20株耐药菌中有16种耐药谱型，全都是多重耐药。对恩诺沙星的耐药率为60％。 2．体外联合药敏试验表明，VP和EN联用对8株耐药株中7株菌表现为不同程度的协同作用（MIC值降低2～4倍），2株标准株(0₂、0₇₈)表现为无关作用，拮抗作用的为零。说明VP对耐恩诺沙星的鸡源性大肠杆菌的耐药性具有一定的拮抗作用，但对非耐恩诺沙星的大肠杆菌无明显影响。VP单独对10株菌的MIC值均大于640μg／ml，说明VP本身对大肠杆菌无抑制作用。 3．体内试验表明，VP对耐EN大肠杆菌的耐药性也具有一定的拮抗作用，具体表现为：①EN+VP组的死亡率比EN组有所降低，而有效率、治愈率有所升高；②增重效果分析，EN+VP组显著高于EN组和阳性对照组（P＜0.05）；③EN+VP组病理变化（肝周炎、心包炎、气囊炎）和菌检率均低于同日龄EN组；④VP+EN组鸡的股动脉压在2天和4天分别高于EN组，未表现特别明显的循环阻碍。 综上所述，VP在体内、外对耐EN大肠杆菌的耐药性都具有一定的拮抗作用。更多还原

【Abstract】 Objective and methodThe three following experiments were conducted to investigate the effect of verapamil on enrofloxacin resistance in enrofloxacin-resistant E.coli.1 .The isolation of Enrofloxacin-resistant E.coli strains and sensitivity test: Serotype O of avian E.coli, isolated from broiler farms in HeBei province, were identified.The resistance of 20 strains taken randomly were tested by using Kirby-Bauer disk diffusion test. Eight strains of enrofloxacin-resistant E.coli were selected for the experiment in vitro.2. The experiment in vitro: The minimal inhibitory concentrations(MICs) of enrofloxacin and verapamil on 10 strains E.coli (including standard O2.O78) were tested with the method of broth two-fold dilution. Then the checkboard system was used to determine the fractional inhibitory concentration (FIC) index of enrofloxacin combined with verapamil. The one strain was selected for experiment in vivo.3. The experiment in vivo: 240 commercial broiler chickens were randomly divided into 4 groups on 30 days of age. The positive control and EN, EN+VP groups were inoculated with 1.5ml E.coli suspension by an intraperitoneal injection to induce Avian colibacillosis. The fouth group was negative control. When clinical signs emerged 4 hours postinoculation, the EN group was administrated with EN at a dose of 2.5 mg/kg.b.w twice each day , while the EN+VP group with EN (2.5 mg/kg.b w) and VP (6.0 mg/kg.b.w) twice each day. The mortality, curative ratio, and weight gain were observed every day. At the day 2,4,6,8,10 after inoculation, 6 birds were randomly taken from each group for detecting the mean femoral artery pressure (FAPm), macroscopic leisions and positive rate of E.coli.Result and conclusion1. 183 isolates of E.coli were got from 206 samples, with a mean isolation rate of 83.9%. 160 of 180 isolates were identified as 32 serotypes. The main serotypes were O78.O2.O1.O88.O76 in this district. The sensitivity test showed that 20 strains of E.coli taken randomly from the 160 were all resistant to two or more antibiotics. They could be distinguished to belong to 16 resistance spectrum . All of them showed multiple-resistant. The enrofloxacin resistance rate was 60%.2. The combined sensitivity test in vitro indicated that synergic effect were 87.5 % (7/8) of risistant-EN E.coli, indifferent effect was showed in two standard strains (O78. O2) and no antagonistic effect was found. That means VP can reduce the resistance of E.coli to EN in vitro. The MICs of verapamil against 10 strains revealed efflux pump inhibitor (verapamil ) has little effect itself on the E.coli.3. The experiments in vivo indicated that VP can promote the susceptibility of E.coli to enrofloxacin. The results were as follow: (1?The mortality of EN+VP group was lower than that of EN group, while the protective rate and curative ratio were a bit higher than that of EN group. (2) The weight gain of EN+VP group was significantly higher than that of EN group. (3) The rate of typical pathologic changes (pericarditis, perihepatitis) caused by colibacillosis of EN+VP group lower than that of EN at the same day, the positive rate of E.coli was the same. (4) At 2,4 days postinoculation, the FAPm of EN+VP group was higher than that of EN group. These conclution proved that enrofloxacin combined with verapamil by mascular injection no effect on cardiovascular system of chickens obviously.更多还原