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研究生:江邦興
研究生(外文):kong phang hing
論文名稱:文蛤弧菌對於數種抗菌劑之藥物敏感性實驗研究
論文名稱(外文):The Susceptibilities of Vibrio spp. Isolates from the Hard Clams, Meretrix meretrix, to Several Antibiotics
指導教授:王渭賢
指導教授(外文):Wang Way Shyan
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:89
中文關鍵詞:弧菌抗菌劑藥物敏感性實驗文蛤
外文關鍵詞:Vibrio spp.antibioticdrug susceptibilitieshard clams
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本研究的主要目的在於調查台灣中部地區文蛤養殖場之弧菌分佈及抗葯情形。由文蛤的鰓、肝臟及消化道進行弧菌分離,以了解弧菌菌叢在文蛤體內分佈的情形。並選用一般水生動物常用的抗生素如amoxicillin (AMO)、oxytetracyclin (OTC)、oxolinic acid (OA)、sulfamethoxazole+trimethoprim (5:1) (SXT)、chloramphenicol (CAP)、florfenicol (FLO)、thiamphenicol (TAP)等7種抗生素,進行最小抑菌濃度 (minimal inhibitory concentration;MIC)實驗,以了解細菌對藥物敏感性。實驗中分別收集彰化、雲林縣計六十場次文蛤場之文蛤,計分離出弧菌菌株有205株:Vibrio alginolyticus (103/205)、V. parahaemolyticus (40/205)、V. furnissii (6/205)、V. flurialis (3/205)、V. vulnificus (9/205)、V. mimicus (1/205)、V. cholerae (26/205)、V. cincinnatiensis (15/205)及V. damsela (2/205)。弧菌在文蛤體內分佈位置,為在鰓可分離到V. alginolyticus (51株)、V. parahaemolyticus (20株)、V. furnissii (6株)、V. vulnificus (7株) 、V. flurialis (3株)、V. mimicus (1株)、V. cincinnatiensis (11株)與V. damsela (1株);在肝臟則可分離到V. alginolyticus (21株)、V. parahaemolyticus (5株)、V. vulnificus (1株)、V. cholerae (1株)、V. cincinnatiensis (1株)及 V. damsela (1株);在消化道可分離的弧菌為V. alginolyticus (31株)、V. parahaemolyticus (15株)、V. vulnificus (1株)、V. cholerae (5株)、V. cincinnatiensis (3株)。顯示在文蛤体內弧菌以V. alginolyticus為主,主要分佈之器官為鰓、肝、消化道。
藥物最小抑菌濃度實驗的結果顯示,各抗生素之MIC50分別為OA (0.125 mg/ml);OTC (0.5 mg/ml);FLO (0.5 mg/ml);CAP (0.5 mg/ml);TAP (1 mg/ml);SXT (1 mg/ml);AMO (256 mg/ml)。顯示分離菌株對於各種抗生素之感受性,以OA感受性最高,OTC次之,FLO再次之,CAP再次之,TAP再次之,SXT再次之,以AMO最低。
各種抗生素具有抗藥性之菌株佔總分離菌株之百分比,分別為AMO (63.63% );TAP (19.0%);SXT (16.53%);OTC (4.13%);CAP (4.13%);FLO (1.65%);OA (0.83%)。各抗藥性型態百分比分別為AMO r 50株(60.98%);OTC r 2株(2.44%);SXT r 1株(1.22%);AMO-TAP r 5株(6.08%);AMO-SXT r 6株(7.32%);SXT-TAP r 1株 (2.44%);AMO- SXT-TAP r 8株 (9.76%);AMO- OTC-TAP r 3株 (3.66%);AMO-CAP-TAP r 2株(2.44%);AMO-TAP-SXT-FLO-CAP r 2株(2.44%);AMO-OA-SXT-TAP-CAP r 1株(1.22%)。顯示由文蛤分離出之弧菌菌株其抗藥性,以AMO最為嚴重。因此由分離的弧菌菌株中,對AMO具有抗藥性,其MIC值>25 mg/ml之菌株計有77株,其中經由β-lactamase test檢測,計有65株測檢出具有β-lactamase的產生,有12株沒有測出β-lactamase。再將12株β-lactamase陰性菌株進行amoxicillin+clavunic acid (AMC) disc diffusion test,結果發現其中有9株對AMC敏感,而有3株對AMC具有抗藥性。經由PCR實驗結果顯示弧菌在染色體可測到blaSHV及blaTEM基因,而在質體可測到blaSHV基因。
The aims of this research are to investigate the distribution of Vibrio spp. and the circumstances of their drug resistance in the cultured clam farms of the central part of Taiwan. Through the experiment of Vibrio spp. isolation from gills, liver, and digestive tract, the situation of Vibrio spp. distribution in clam has been found out. Using the antibiotics which are usually used in aquatic animals such as amoxicillin (AMO), oxytetracyclin (OTC), oxolinic acid (OA), sulfamethoxazole+trimethoprim (5:1) (SXT), chloramphenicol (CAP), florfenicol (FLO), thiamphenicol (TAP) to carry out the experiment of minimal inhibitory concentration (MIC), the sensitivities of the isolates to chemical agents have been studied. From sixty times of clam samplings in the county of Changhua and Yunlin, there are 205 strains of Vibrio spp. : Vibrio alginolyticus (103/205), V. parahaemolyticus (40/205), V. furnissii (6/205), V. flurialis (3/205), V. vulnificus (9/205), V. mimicus (1/205), V. cholerae (26/205), V. cincinnatiensis (15/205), V. damsela (2/205) have been isolated. The circumstances of Vibrio spp. distribution in clams are as follows: V. alginolyticus (51strains), V. parahaemolyticus (20 strains), V. furnissii (6 strains), V. vulnificus (7 strains) , V. flurialis (3 strains), V. mimicus (1 strain), V. cincinnatiensis (11 strains), V. damsela (1 strain) in gills, and V. alginolyticus (21 strains), V. parahaemolyticus (5 strains), V. vulnificus (1 strain), V. cholerae (1 strain), V. cincinnatiensis (1 strain), V. damsela (1 strain) in liver, and V. alginolyticus (31 strains), V. parahaemolyticus (15 strains), V. vulnificus (1 strain), V. cholerae (5 strains), V. cincinnatiensis (3 strains) in digestive tract. The results prove that the main Vibrio spp. in clams is V. alginolyticus main distributed in gills, liver, and digestive tract.
The results of the experiment of MIC has shown that each MIC50 are OA (0.125 mg/ml); OTC (0.5 mg/ml); FLO (0.5 mg/ml); CAP (0.5 mg/ml); SXT (1 mg/ml); TAP (1 mg/ml); AMO (256 mg/ml). This has demonstrated that the separated Vibrio spp. are most sensitivity in OA, next in OTC, FLO, CAP, TAP, SXT, and AMO is the least affected antibiotics to the separated Vibrio spp. The percentage of each antibiotics’ drug resistance to Vibrio spp. are AMO (63.63% ); TAP (19.0%); SXT (16.53%); OTC (4.13%); CAP (4.13%); FLO (1.65%); OA (0.83%) respectively.
The numbers and antibiotic resistance patterns are AMO r 50 strains (60.98%);OTC r 2 strains (2.44%);SXT r 1 strain (1.22%);AMO-TAP r 5 strains (6.08%);AMO-SXT r 6 strains (7.32%);SXT-TAP r 2 strains (2.44%);AMO- SXT-TAP r 8 strains (9.76%);AMO- OTC-TAP r 3 strains (3.66%);AMO-CAP-TAP r 2 strains (2.44%);AMO-TAP-SXT-FLO-CAP r 2 strains (2.44%);AMO-OA-SXT-TAP-CAP r 1 strain (1.22%), respectively.
From our results, seventy steven of 121 Vibrio spp are highly resistant (MIC>25 μg/ml ) to amoxicillin (AMO). Amoxicillin resistance are mostly due to the production ofβ-lactamase as shown by 65 of 121 amoxicillin —resistance strains being susceptible to amoxicillin —clavulanic acid and the isolated giving a positive acidimetric method. Of the 12β-lactamase negative stains, 9 strains are resistance to amoxicillin+clavulanic acid (AMC) using the AMC disc diffusion test. Therefore, the amoxicillin resistance strains are investigated for the presence of blaTEM and blaSHV genes coding forβ-lactamase enzymes by PCR. Our data show that the location of SHVβ-lactamase are found in the plasmid and chromosome. However, only TEM gene is found in chromosome.
中文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ I
英文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ III
目錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ VI
表次‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ VII
圖次‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ VIII
壹、緒言‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 1
貳、文獻探討‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 2
參、材料與方法‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 23
肆、結果‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 34
伍、討論‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 45
參考文獻‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 71
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