Molecular Epidemiology and Resistance Mechanisms of Ciprofloxacin-Resistant S. typhimurium and S. choleraesuis

Salmonella enterica為一常見引起腹瀉的致病菌，由於在台灣其對quinolone類藥物抗性的增加顯著上昇，為了解此抗藥現象，因此收集台大醫院分離 (1996~2000)、2001年三月到八月所收集的全省菌株 ( SMART study ) 以及1997~2002年從養豬場的豬隻身上收集到的15株菌株加以分析。利用PFGE分析其基因型，發現同一血清型的菌株其在基因上多屬於同一來源，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。為了探知這些菌株的來源，因此再分析從各地所得的動物菌株，發現有些菌株基因型與人體分離菌株相同，可證實由於食用具抗藥菌株的家畜家禽導致這些菌株傳播到人體的推測。 細菌對quinolone類藥物抗藥機轉目前認為主要是topoisomerase上發生突變以及efflux pump的活化所造成。首先以核酸定序的方式知道topoisomerase上QRDR (quinolone resistant-determining region)的位置的確有發生突變，故此應為造成抗藥的原因之一。再測量菌株對有機溶劑的忍受度以及對ciprofloxacin的累積程度，結果也說明抗藥菌株確實有efflux的活化。進一步以北方墨點法以及西方墨點法分析S. cholerasuis主要efflux pump AcrA在RNA以及蛋白表現上的差異，結果發現抗藥菌株比具感受性菌株來的多，同時在基因序列上也發現它的抑制蛋白基因acrR在Gln78Stp (CAG→TAG)發生點突變可能會導致AcrR無法產生，進而促進efflux的活化。而在S. typhimurium內並沒有發現到相同的結果推測其efflux pump AcrAB活化可能是由於上游基因 (marRAB, soxRS regulons)調控所致，需要再進一步的研究。Salmonella enterica is one of the human pathogens that causes diarrhea and salmonellosis. Fluoroquinolones are often the choice of tratment in the case of salmonellosis. Unfortunately, the quinolone resistant strains significantly increased in recent year. To elevate the dissemination of this strain, clinical isolates from 1996-2001 were collected and analyzed. Pulsed-Field Gel Electrophoresis (PFGE) showed that majority of these clones that with identical serotype of S. enterica sharing similar PFGE pattern. This result suggested that these clinical pathogens were disseminated by similar source. In other word, this is the result of clone spreading effect. In addition, we collected animal infection strains and the result was compared with those clinical isolates. Interestingly, we found that some of the animal infection strains sharing the identical pulsoltype with clinical isolated, suggested that swine served as a reservoir for these resistant strains. Recent study shows that the point mutation of topoisomerase and highly expressed of efflux pumps, such as acrAB operon, causes the bacteria increased the resistant ability to quinolone. Our sequencing results demonstrated the point mutation regions in quinolone resistance determination region (QRDR). Furthermore, organic solvent tolerance assay and ciprofloxacin accumulation assay showed the increasing activity of efflux pump in resistance strains. Northern blotting and Western blotting results confirmed the significantly increased expression level of acrA revealed that the activation of efflux pumps expression leads to the resistance effect in Taiwan. Besides, the stop codon mutation of acrR, an inhibitor of acrA, lost its function to inhibit acrA expression level resulting the over-expression of this efflux pumps. We also could not obtained the similar result in Salmonella typhimurium as S. enterica elevated that, except acrR, an unknown genes which might be involved in acrA regulatory system in resulting acrAB regulon increased its expression levels.總目次 總目次…..……………………………………………………………………....i 圖目次…..……………………………………………………………………...ii 表目次………………………………………………………………………....iii 中文摘要…..…………………………………………………………………..iv Abstract…………………………………………………………………………v 第一章 序論 第一節 前言………………….…………………………………………...1 第二節 沙門氏桿菌之感染………….…………………………………...1 第三節 沙門氏菌屬之分類………………………………………………2 第四節 沙門氏桿菌之治療與抗藥情形…………………………………3 第五節 細菌對於quinolone之抗藥機轉………………………………..5 第六節 研究動機…………………………………………………………6 第二章 材料及方法 第一節 實驗儀器及試藥…….…………………………………………..7 第二節 實驗菌株…………………………………………………….…11 第三節 實驗方法…………………………………………………….…11 第三章 結果 第一節 流行病學的分析…….…………………………………………19 第二節 拓樸酶第二及第四型QRDR之分析…………………………20 第三節 運輸幫浦之篩選……………………………………………….20 第四節 確認運輸幫浦之活化………………………………………….21 第五節 造成AcrAB efflux pump活化的原因…………………………21 第四章 討論………………………………………………………………….22 參考文獻………………………………………………………………...……64 圖目次 圖一: 林口長庚所分離之菌株資料………………………………………………..26 圖二: 參與DNA複製的兩個重要酵素…………………………………………….27 圖三: DNA與Topoisomerase II作用的方式…………….………………..…………28 圖四: 台大醫院細菌室報告………………………...………………………………29 圖五: Salmonella enterica serotype Typhimurium PFGE XbaI分型結果……......…30 圖六: Salmonella enterica serotype Choleraesuis PFGE XbaI分型結果…...…...….31 圖七: Salmonella enterica serotype Typhimurium PFGE BlnI分型結果………..….32 圖八: Salmonella enterica serotype Choleraesuis PFGE BlnI分型結果………...….33 圖九: Salmonella enterica serotype Choleraesuis pulsotype b之pulsosubtype……..34 圖十: 細菌體內ciprofloxacin累積試驗 (accumulation of ciprofloxacin)…………35 圖十一: 北方墨點法分析…………………………………………………………...36 圖十二: 西方墨點法分析…………………………………………………………...37圖十三: marRAB & soxRS regulons………………………………………………....38 圖十四: Ciprofloxacin resistance in Samonella species 671 isolates, Taiwan..……...39 圖十五: Fluroquinolone resistance among non-typhoid salmonella in Taiwan……...40 圖十六: [T-1]gyrA上83及87位置氨基酸改變所造成電性的改變……………...41 圖十七: [T-12]gyrA上83及87位置氨基酸改變所造成電性的改變……………...42 表目次 表一: 民國70年至90年台灣地區食品中毒案件病因物質分類表……………..43 表二: 至1999年為止沙門氏桿菌屬及亞種血清型統計………………………….44 表三: Characteristics of S. enterica serotype Typhimurium and Choleraesuis…..45-53 表四: 菌株QRDR之分析……..……………………….………………………..54-59 表五: Salmonella enterica serotype Choleraesuis QRDR序列分析整理…….…60-61 表六: 有機溶劑容忍度試驗 (Organic solvent tolerance test)……………...…62 表七: Characteristics of Salmonella enterica isolates from human in Taiwan…....…6