【作者】 刘远飞；

【导师】 佟恒敏；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2002， 博士

【摘要】 本实验研究了五种氟喹诺酮类抗菌药环丙沙星、氧氟沙星、恩诺沙星、单诺沙星和沙拉沙星对金葡球菌标准菌株ATCC25923和大肠杆菌标准菌株ATCC25922的体外抗菌活性、体外抗菌后效应(post-antibiotic effect，PAE)及抗菌药后亚抑菌浓度效应(post-antibiotic sub-MIC effect，PA-SME)，同时研究了不同抗菌药物浓度、细菌与药物接触时间、细菌接种量对PAE的影响。通过在兔背部两侧皮下埋植组织笼成功建立了兔组织笼感染支架模型，以改进的体内接触药物法研究了单诺沙星对金葡球菌和大肠杆菌的体内PAE。另外，通过透射电镜观察了药物对细菌超微结构的影响，并以新生霉素一琼脂糖CL-6B亲和层析柱分离并纯化了大肠杆菌DNA旋转酶的A、B亚单位，对氟喹诺酮类药物抗菌机制及PAE产生机制进行了研究。 抗菌药物活性实验测得五种氟喹诺酮类抗菌药对金葡球菌和大肠杆菌的MIC值大多低于0.25μg／ml，MBC／MIC值在2～4之间。表明五种氟喹诺酮类抗菌药具有强大的抗菌活性。体外PAE实验表明五种氟喹诺酮类抗菌药的PAE较长，对金葡球菌最长可分别达到3.58、2.22、3.22、2.82和2.55h，对大肠杆菌的最长PAE分别为4.11、2.76、4.24、4.51和2.88h。不同的抗菌药物浓度、接触时间和细菌接种量均可对PAE产生影响，尤其是抗菌药物浓度对PAE的影响最大，在1～4MIC浓度范围内随着药物浓度的升高PAE明显延长，表明氟喹诺酮类药物对金葡球菌和大肠杆菌的PAE呈现明显的浓度依赖性；氟喹诺酮类抗菌药与药物接触时间对PAE也有一定的影响，PAE随接触时间(1h、2h)的延长而延长，但不如药物浓度对PAE的影响明显：细菌接种量(106、10~7和10~8CFU／ml)越大，PAE越短。另外，五种氟喹诺酮类抗菌药对金葡球菌和大肠杆菌具有比PAE更长的PA-SME，表明临床上氟喹诺酮类抗菌药可能具有比体外PAE更长的体内PAE。 通过在兔背部两侧皮下埋植组织笼成功建立了兔组织笼感染支架模型，引进药代动力学、以改进的体内接触药物法研究了单诺沙星对金葡球菌和大肠杆菌的体内PAE。实验结果表明单诺沙星对金葡球菌和大肠杆菌的体内PAE较长，在4MIC浓度作用1h后的PAE分别为3.41h和4.86h，比同时测定的体外PAE(2.86h、3.72h)具有显著性差异(＜0.05)，进一步表明单诺沙星具有良好的体内PAE。临床上可适当延长给药间隔，改进给药方案，即可考虑以单诺沙星血药浓度高于敏感菌MIC的时间加上PAE的时间作为给药间隔。 本实验通过透射电镜观察了单诺沙星在0.5MIC、2MIC和4MIC时作用2h对大肠杆菌和金葡球菌超微结构的影响。结果表明超MIC浓度单诺沙星可导致细菌发生不可逆的损伤，出现细胞膜皱缩，胞质稀疏，细胞膜连续性丧失，甚至细菌解体、细胞破裂死亡。经亚MIC浓度药物作用之后，金葡球菌菌体增大，横隔增宽；大肠杆 东北农业大学 博士学位论文20025菌则表现为菌体显著增长，菌丝形成等。证实单诺沙星为杀菌类抗菌药物，单诺沙星对细菌的抗菌作用PAE的产生是通过抑制DNA旋转酶所致。 以 Epoxyactivated Sepharose 6B和新生霉素为原料，制备新生霉素一琼脂糖CL－6B亲和层析柱，分离了大肠杆菌DNA旋转酶的A、B亚单位，测定了单诺沙星的半抑酶浓度门。。），并研究了单诺沙星对酶活性的抑制及除药后酶活性的恢复情况。结果表明所分离的DNA旋转酶A、B亚单位以2＊的比例重组，全酶活性最强；单诺沙星对 DNA旋转酶的 IC。。为 1．38 H g／IYll，比单诺沙星对大肠杆菌 MIC的高出约10倍；单诺沙星以 2倍C。的浓度与 DNA旋转酶作用 lh，透析去除药物后，酶活性没有完全恢复。表明单诺沙星对DNA旋转酶的抑制是PAE产生的机制之一。 本研究结果表明，氟喳诺酮类抗菌药对金葡球菌和大肠杆菌具有良好的体外PAE和PA－SME，单诺沙星在兔感染组织支架模型内具有比体外更长的PAE。单诺沙星对DNA旋转酶的抑制是其抗菌作用机制之一。单诺沙星对大肠杆菌DNA旋转酶的抑制和对细菌超微结构的影响是产生PAE的重要原因。更多还原

【Abstract】 The antibacterial activity of fluoroquinolones against Staphylococcus aureus 25923and Escherichia coli 25922 in vitro were stodied. At the same time, the post-antibioticeffects (PAE ) and post-antibiotic Sub-MIC effects (PA-SME) offluoroquionlones againsttwo bacteria strains were observed. The infected tissue cage models that had been improvedwere built in rabbits by implanting plastic tissue cages for studying -the PAE ofdanofloxacin against StaPhylococcus aureus and Escherichia coli in vivo. ThemorPhologica1 and ultra-strUctural changes of tWo strains exposed to danofloxacin in highand Sub-inhibitory concentrations were observed with transmission electronic microscope.The DNA gyrase of Escherichia coli were purified by novobiocin-Sepharose CL-6B, andthe activity of enzyme and the 50% inhibitory concentrations (IC,,) of danofloxacin weredetected.The PAE and PA-SME of fluoroquionlones against two bacteria strains weredetermined. DiIuting method was used fOr the drugs removaI and the growth of bacteriawas monitored by Muelle-Hinton (MH ) agar colony counting method in the study. Resultsshowed that the minimal inhibitory concentrations (MIC ) of four in five fluoroquinolones(ciprofloxacin, ofloxacin, enrofloxacin, danofloxacin and saIafloxacin ) were under 0.25u g/ml, the minimal bactercidal concentration(MBC )were under l .0 u g/ml, and the valuesof MBC/MIC were 2~4. It appeared that the fluoroquinolones had not only stronginhibitory activities, but also had quick killing activities.The results of PAE and PA-SME showed that there have long period persistentsuppression of bacterial groWth after a limited exposure to fluoroquinolones. The longestPAE of ciprofloxacin, ofloxacin, enrofloxacin, danofloxacin and salafloxacin againstStaphylococcus aureus are 3.58, 2.22, 3.22, 2.82 and 2.55 hours, respectively. The valuesagainst Escherichia coli are 4. l l, 2.76. 4.24. 4.5l and 2.88 hours, respectively. The va1uesof PAE increased as increasing of drug concentration in l~4MIC, and the period ofexposing to drugs and the quantities of inoculum also affected the PAE. The PA-SMEvalues of fluoroquinplones were longer than that of PAE in the same inducementconcentration.32002.5The infected tissue cage model in rabbits with normal host defending mechanism had been set up successfully using subcutaneously two plastic tissue cages implanted in each animal. The improved model exposing to drugs in vivo were adopted for determining PAE of danofloxacin against Staphylococcus aureus and Escherichia coli after exposed to drug for 1 hour. The PAE of danofloxacin against Staphylococcus and Escherichia were 3.41 and 4.86 hours, respectively. The long period of PAE in vitro and in vivo of danofloxacin showed that the projection of administration in clinic should been improved. It was that the interval of administration should equal the sum of the period of drug concentration in blood over MIC and the period of PAE.The morphological and ultra-structural changes of two strains exposed to both high and Sub-inhibitory concentrations of danofloxacin for 2 hours were observed under scanning electron microscope. Shrinking of the cell membrane, rupture of cell wall, and vacuoles in the cell or even lysis of the cell were seen as the bacteria were hatched with 2MIC and 4MIC danofloxacin. When the exposed concentration was 0.5MIC, the Staphylococcus aureus became larger with irregular shape, decreased dense and a thick wall, and Escherichia coli presented elongation or even filamentation with or without vacuoles in the cell. The results indicated that the danofloxacin inhibited the DNA gyrase mainly.The novobiocin-Sepharose CL-6B affinity chromatography was prepared by Epoxy-activated Sepharose 6B and novobiocin to purifying DNA gyrase which were used for determining the half inhibitory concentration ( ICJO ) of danofloxacin against DNA gyrase. The results showed that the 50% inhibitory concentrations (ICJO) of danofloxacin was 1.38 V- g/ml, and the period更多还原