建立適用動物分離細菌之抗生素分子檢測平台

Establish an antibiotics molecular detection platform suitable for animal isolated bacteria

盧思彤(Szu-Tung Lu)

指導教授 ： 王祥宇

國立屏東科技大學/獸醫學院/動物疫苗科技研究所/碩士(2021年)

https://doi.org/10.6346/NPUST202100416

摘要

細菌性疾病使動物嚴重死亡並造成經濟上的損失，而現今防治與治療皆以投予抗生素為主。然而，由於其便宜又方便，因此常出現抗生素濫用的情況，不只造成藥物殘留，還會產生抗藥性。抗藥性基因可藉由細菌間互相傳遞，種類多元且複雜。本研究規劃出篩選抗藥性基因之流程,並建立細菌的抗藥性基因檢測平台。利用收集自屏東東海地區水產動物細菌分離株(72株)及哺乳類與家禽類動物之糞便分離細菌(32株)，檢測4種抗生素（amoxicillin, erythromycin, doxycycline及 florfenicol）藥物敏感性。由細菌抗藥性基因庫(CARD)尋找前述4種抗藥性基因，建立基因篩選流程、序列整理與設計評估，分析以聚合酶連鎖反應結果搭配抗生素感受性試驗資料進行檢驗效能。五組抗藥性基因(TEM-1, ermB, tetM, floR, fexA)平均敏感性上，哺乳類及禽類細菌組於不分類細菌結果約34%，數據較低，我們將細菌做革蘭氏陰性及陽性分類後，可看出革蘭氏陽性細菌敏感度上升至47％，而革蘭氏陰性菌則下降為21.2%。而水產細菌於本次實驗中，數值皆較哺乳細菌低。於哺乳類與禽類細菌中，有4個基因檢測敏感度達50%以上（革蘭氏陰性菌的floR、革蘭氏陽性菌的ermB, tetM及fexA）。而本實驗所收集的水產動物細菌經鑑定後，高於50%沒有收錄在CARD資料庫當中。在所有水產細菌檢測當中，只有革蘭氏陰性菌的floR檢測敏感度(sensitivity)超過50%。綜合上述，利用CARD的prevalence database的資料可快速獲得普及的抗藥性基因，但方法使用之對象必須符合資料庫中所收集到的221種細菌屬，且在區分革蘭氏陰陽性可提高方法之可信度。

關鍵字

抗生素 ； 抗藥性基因 ； CARD

並列摘要

Bacterial diseases cause severe death of animals and cause economic losses. Nowadays, prevention and treatment are mainly based on the administration of antibiotics. However, due to its cheapness and convenience, antibiotics are often abused, not only causing drug residues, but also drug resistance. Drug resistance genes can be transmitted between bacteria, and the types are diverse and complex. This research plans to screen the process of drug resistance genes, and establish a bacterial drug resistance gene detection platform. Using bacterial isolates (72 strains) collected from aquatic animals in the East China Sea area of Pingtung and isolated bacteria (32 strains) from the feces of mammals and poultry animals, the drug susceptibility of 4 antibiotics (amoxicillin, erythromycin, doxycycline and florfenicol) was tested. From the bacterial antibiotic resistance gene bank, Comprehensive Antibiotic Resistance Database (CARD) to search for the aforementioned four antibiotic resistance genes, establish a gene screening process, sequence arrangement and design evaluation. Later, the PCR results were combined with the antibiotic susceptibility test data for statistical analysis of the test efficiency. With regard to the average sensitivity of the five groups of drug resistance genes (TEM-1, ermB, tetM, floR, fexA), the results of mammalian and poultry bacteria in the unclassified bacteria group are about 34%, and the data is low. We set the bacteria as Gram-negative and positive classification, it can be seen that the sensitivity of Gram-positive bacteria has increased to 47%, while that of Gram-negative bacteria has dropped to 21.2%. In this experiment, the values of aquatic bacteria are lower than those of mammalian and poultry bacteria. In mammals and poultry bacteria, 4 genes have a sensitivity of more than 50% (floR for gram-negative bacteria, ermB, tetM and fexA for gram-positive bacteria). After the identification of the aquatic animal bacteria collected in this experiment, more than 50% were not included in the CARD database. Among all aquatic bacteria testing, only gram-negative bacteria have a floR sensitivity of more than 50%. In summary, using CARD's prevalence database data can quickly obtain popular antimicrobial resistance genes, but the method used must conform to the 221 bacterial genera collected in the database, and the distinction between gram negative and positive can improve the accuracy of analysis.

並列關鍵字

Antibiotic ； Antibiotic resistant gene ； CARD

參考文獻

[1]黃子鳴*、鄭劭蕙、涂堅. 101 年度魚類鏈球菌藥物感受性調查. 家畜衛試所研報 2013;88:83–8.

[2]Akobeng AK. Understanding diagnostic tests 1: Sensitivity, specificity and predictive values. Acta Paediatr Int J Paediatr 2007;96:338–41.

[3]Chopra I, Roberts M. Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiol Mol Biol Rev 2001;65:232–60.

[4]Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests: Approved standard - Eleventh edition. vol. 32. 2012.

[5]CLSI. M45. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria ; Proposed Guideline. vol. 35. 2015.