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標題: Chloramphenicol、florfenicol及thiamphenicol對水生動物細菌之抗菌效力及殘留檢測方法之開發
The Antibacterial Effect of Some Chemical Agents against Aquatic Animal Bacteria and the Development of Simultaneous Determination Method for Chloramphenicol, Thiamphenicol, and Florfenicol
作者: 許天耀
Hsu, Tain-Yao
關鍵字: 水生動物細菌
antibacterial effect
simultaneous determination method
出版社: 獸醫學系
摘要: 本研究之目的在於檢測鱉、魚類及蝦貝類來源菌株對chloramphenicol (CAP)、thiamphenicol (TAP)及florfenicol (FLO)之感受性,並以amoxicillin (AMO)、oxytetracycline (OTC)及oxolinic acid (OA)進行比較。結果顯示鱉菌株之藥物敏感性順序為OA > FLO, CAP > TAP > AMO, OTC,而抗藥菌株比例,以OA最低 (7.14%),OTC最高 (85.07%);魚類菌株之藥物敏感性順序為OA > FLO > CAP > AMO > OTC > TAP,而抗藥菌株比例,以OA最低 (13.64%),OTC最高 (80.91%);蝦貝類菌株之藥物敏感性順序為OA > CAP, FLO > AMO, OTC, TAP,而抗藥菌株比例,以OA最低 (4.35%),TAP最高 (58.7%)。菌株之抗藥性形式:鱉菌株以AMO-OTCr所佔比例最高 (32.98%);魚類菌株以CAP-TAP-AMO-OTCr所佔比例最高(19.42%);蝦貝類菌株以FLO-CAP-TAP-AMO-OTCr所佔比例最高 (50%)。同時發現測試菌株對CAP、TAP及FLO具有部份交叉抗藥性。 以裝有保護管柱之分離管 (Cosmosil 5C18-MS),移動相0.05 M ammonium acetate-acetonitrile-tetrahydrofuran (76:23:1) (pH7.2),流速1.0 ml/min,並配合220 nm波長之紫外光檢測器可同時精確地分離CAP、TAP及FLO。測試藥物之滯留時間分別為TAP (5.68 min)、FLO (11.95 min)及CAP (16.15 min)。而各藥物之標準濃度曲線相關係數相當好 (r2值可達0.998),且各藥物之最低檢測濃度可達1 ng。利用固相萃取方式進行吳郭魚組織內藥物前處理及萃取,以去離子水清洗數次後,以n-hexane移除脂溶性物質後,再以acetonitrile作為提取液,經濃縮後再以去離子水溶解而後進行分析。結果肌肉組織之回收率為81.5-100.2%,最低檢測濃度為0.01 μg/g,肝臟組織之回收率為81.7-92%,最低檢測濃度為0.025 μg/g,而腎臟組織之回收率為82.4-97.8%,最低檢測濃度為0.05 μg/g。各藥物之回收率曲線相關係數亦相當好 (r2值可達0.99以上)。
The aims of this investigation are to study the antibacterial efficacy of chloramphenicol (CAP), thiamphenicol (TAP), and florfenicol (FLO) against the isolates from soft-shell turtles, fish, and shellfish and to develop the simultaneous detection method for the residues of above chemical agents in the tissue of tilapia. The purpose of the minimum inhibitory concentration test was to evaluate the antibacterial activities of CAP, TAP, and FLO against the bacteria. Amoxicillin (AMO), oxytetracycline (OTC), and oxolinic acid (OA) were used to compare with above agents in this test. The results showed that the order of sensitivities of the isolates from soft-shell turtles for tested chemicals was OA > FLO, CAP > TAP > AMO, OTC. The results of the percentage of the resistant strains indicated that OA was the lowest (7.14%) and OTC was the highest (85.07%). The order of antibacterial activity of tested agents against the isolates from fish was OA > FLO > CAP > AMO > OTC > TAP. The percentage of the resistant strains revealed that OA (13.64%) and OTC (80.91%) was the lowest and highest, respectively. As the similar test, the order of the isolates from shellfish was OA > CAP, FLO > AMO, OTC, TAP. TAP (58.7%) had the greatest percentage among the resistant strains, but OA (4.35%) was the lowest. The most common resistant patterns of the isolates from turtles, fish, and shellfish were AMO-OTCr, CAP-TAP-AMO-OTCr, and FLO-CAP-TAP-AMO-OTCr, respectively. There were partial complete resistance of the resistant isolates among CAP, TAP, and FLO. 0.05 M ammonium acetate (pH7.2)-acetonitrile-tetrahydrofuran (76:23:1) as the mobile phase at a flow rate of 1.0 ml/min, the high performance liquid chromatographic (HPLC) method for the simultaneous determination of CAP, TAP, and FLO was carried out on a C18 column (Cosmosil 5C18-MS, 150 mm X 4.6 mm I. D.) with a guard column. The wavelength of UV detector was set at 220 nm in this investigation. The retention time of CAP, TAP and FLO were 5.68, 11.95, and 16.15 minutes, respectively. The r-square values of standard curve of drug concentration of each drug were up to 0.998. The limited detection concentration of our developed method was 1 ng for each tested agent. The residues of tested drugs in the muscle, liver and kidney of tilapia extracted by solid-phase extraction method were detected with our developed method in this study. The treated tissue was cleaned by following water and n-hexane and eluted by acetonitrile. The results revealed that the recoveries of muscle, liver and kidney from tilapia were 81.5-100.2%, 81.7-92% and 82.4-97.8%, respectively. The detection limit of muscle, liver and kidney from tilapia were 0.01, 0.025 and 0.05 μg/g, respectively. The r-square values of recovery curve of each drug were up to 0.99.
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