Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/14063
標題: 沙門氏菌及豬放線桿菌胸膜肺炎菌fluoroquinolone抗藥性研究
The study of fluoroquinolone resistance in Salmonella and Actinobacillus pleuropneumoniae
作者: 王裕智
Wang, Yu-Chih
關鍵字: Salmonella;沙門氏菌;Actinobacillus pleuropneumoniae;fluoroquinolone;豬放線桿菌胸膜肺炎菌;氟化奎林酮類抗生素
出版社: 獸醫學系暨研究所
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摘要: 
Salmonella Choleraesuis及Actinobacillus pleuropneumoniae為豬常見的致病菌,由於在台灣其對quinolone抗藥性的增加顯著上昇,為了解此抗藥現象,因此收集中興大學動物疾病診斷中心所收集的菌株加以分析。先以定序的方式確認quinolone resistant-determining region(QRDR)突變。再測量菌株對quinolone藥物之最低抑制濃度,藉由加入efflux抑制劑與否的差異來評估efflux的活化。結果發現S. Choleraesuis及A. pleuropneumoniae皆因QRDR造成fluoroquinolone抗藥性,且抗藥性菌株有efflux pump的過度活化。再以Quantitative real-time RT-PCR分析acrAB-tolC也可得到相同結果。另外測量S. Choleraesuis efflux pump調節基因在mRNA表現量上的差異,結果發現抗藥菌株比具感受性菌株來的多,或是在enrofloxacin處理後增加更高的量。而acrS表現量在acrR(Gln78Stp)突變株的確表現較高,enrofloxacin處理後增加倍數更是遠高於沒突變之菌株,然而在acrAB-tolC的表現量卻未呈現相同的增加,顯示其可能只是其中之一的調節者。而acrAB-tolC的表現量與ramA、marA、soxS、rob則呈正比,特別是rob,幾乎是相同程度的表現量增加。然而這些調控基因的相互作用及對quinolone抗藥性的影響仍需進一步研究。研究結果發現菌株的quinolone抗藥性主要因為目標基因的突變及efflux被活化所致。利用PFGE及MLST分析S. Choleraesuis菌株基因型,發現菌株其在基因上多屬於同一來源ST66(MLST)-1B(PFGE)- S83F, D87N(gyrA)-T57S, S80I(parC)-Q78stop, V213F(acrR),由此可推測這些菌株的出現可能為一抗藥菌株散播的結果。最後利用parC mismatch amplification mutation assay-PCR偵測parC突變來取代藥物敏感性試驗偵測菌株fluoroquinolone抗藥性。實驗結果與定序結果相符,可作為偵測菌株fluoroquinolone抗藥性的方法之一。

This study described fluoroquinolone resistance of Salmonella Choleraesuis and Actinobacillus pleuropneumoniae isolated by animal disease diagnostic center in National Chung Hsing University. Selected strains studied carried several mutations in the quinolone resistance-determining region (QRDR). Among several fluoroquinolones tested in combination with or without efflux pump inhibitor Phe-Arg-β-naphthylamide, the MICs were reduced significantly in fluoroquinolone-resistant isolates than in susceptible isolates. This result suggested that active efflux as additional resistance mechanism. The gene expression of acrAB-tolC was assessed by quantitative real-time PCR. The acrAB-tolC expression of fluoroquinolone-resistant isolates were higher than susceptible isolates. In acrR mutant strains(Gln78Stp), causing up-regulation of acrS, were detected. The result indicated acrAB-tolC expression may be affected slightly. After enrofloxacin treated, the efflux regulatory gene such as ramA, marA, soxS and rob, expression increased, it is parallel to acrAB-tolC does. According to the PFGE, MLST and QRDR sequencing, we found that ST66(MLST)-1B(PFGE)- S83F, D87N(gyrA)- T57S, S80I(parC)- Q78stop, V213F(acrR) was the most common genotype. This major clonal fluoroquinolone-resistant S. Choleraesuis have been disseminated in central Taiwan. A rapid test based on a multiplex allele-specific PCR (MAS-PCR) was designed to detect these parC mutations. The results showed that comparable data were obtained between MAS-PCR and sequencing. A strong association was observed between the presence of mutations, as detected by MAS-PCR, and the resistance of the strains. The MAS-PCR method could be an useful tool for identification of fluoroquinolone-resistant strains of Salmonella.
URI: http://hdl.handle.net/11455/14063
其他識別: U0005-0505201015470400
Appears in Collections:獸醫學系所

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