O157:H7型大腸桿菌之分子檢驗分型與逆境反應之研究

Abstract

微生物的檢驗包含分離鑑定與分型兩大部分。O157:H7型大腸桿菌之分離純化、生化試驗與血清型別檢驗及鑑定約需5-7天，因疫情或臨床治療上需要必須進行並尋找縮短檢驗及鑑定時間之方法。分子分型技術近年來不只應用在動、植物或微生物之分類上，目前應用於流行病學調查研究工作，更有長足的進步。本論文共分為四部分，第一部分配合疾病管制局進行國內首件腸道出血性大腸桿菌感染症之檢驗與分型研究；第二部分針對脈場膠體電泳 (pulsed-filed gel electrophoresis, PFGE) 及放大片段長度多型性分析法 (amplified-fragment length polymorphism, AFLP) 等分子分型方法進行標準化及基因資料庫之建構，並應用於分子流行病學之研究，探討本土及國外分離菌株基因型之差異；第三部分應用反轉錄即時聚合酶鏈反應 (reverse transcription real-time polymerase chain reaction) 針對O157:H7 型大腸桿菌進行偵測，發展具良好專一性及靈敏度之檢驗及鑑定方法，並應用此方法分辨樣品中細菌是否存活，減少傳統聚合酶鏈反應 (polymerase chain reaction, PCR) 所造成之偽陽性；第四部份已先前所發展具有良好定性及定量效果之反轉錄即時聚合酶鏈反應，進行經逆境處理後之O157:H7型大腸桿菌菌株第二型類志賀氏毒素及黏附因子基因表現之定量。臺灣首次發生腸道出血性大腸桿菌感染症之病原菌檢測鑑定部分，根據生化試驗、快速檢驗試劑及聚合酶鏈反應結果顯示，由病患、病患家屬及環境檢體所分離之49個檢體中，由病患腹瀉檢體中分離出O157:H7型大腸桿菌，但48個由病患親屬及環境所分離之檢體皆呈現O157:H7型大腸桿菌陰性。在分子分型研究部分，收集O157:H7型大腸桿菌標準株及參考株共37株，分別來自美國、加拿大、日本及台灣地區，將此些菌株進行脈場膠體電泳與放大片段多型性試驗分析，再與國內首次病歷所分離之O157:H7型大腸桿菌比對，研究結果顯示，分離之O157:H7型大腸桿菌基因型別與台灣地區之分離菌株並無相關性，證實與台灣地區無直接關係。與各國之收集菌株比對，除與美國地區分離株基因型較為相似外亦無相同之基因型，證實此為一獨立發生於臺灣地區之O157:H7型大腸桿菌感染事件。第三部分，本研究以 multiplex real-time PCR with TaqMan® system 和 RT multiplex real-time PCR with TaqMan® system 偵測E. coli O157的目標基因和辨認活細胞，並利用前處理方式提高了RT multiplex real-time PCR with TaqMan® system分辨活細胞的偵測極限；結果顯示本方法具有很好的專一性、偵測極限與效率，節省製備樣本的時間，並可辨別活細胞及定量目標基因。第四部份，菌株經過化學方式逆境處理將促進菌株之毒素與黏附因子基因之表現，造成感染之風險亦會增加；溫度逆境處理部份，菌株在低溫保存過後並不會誘導其毒素基因或黏附因子基因表現之增加，其所造成之風險較以化學藥劑處理為低。檢驗與分型是流行病學調查與防治重要的一環，本研究之結果將可作為流行病學研究參考的佐證。

The detection of microorganisms includes identification and subtyping generally. For the control of disease, rapid and automatic detection methods have to be developed. The molecular subtyping is the best evidence for epidemiology. It is also an important key point for the procedure of detection methods. Enterohaemorrhagic Escherichia coli O157:H7 is an important pathogen these days. Outbreaks of its infection have been reported all over the world, in Australia, Canada, Japan, the United States, and in various countries in Europe and South Africa. In the summer of 2001, the first clinical infection case by E. coli O157:H7 was identified in Taiwan. In this study, we describe the results of the isolation and identification of this strain and molecular typing for comparison with previously reported strains. Biochemical and molecular biological tests were used to confirm that this patient, who developed bloody diarrhea and kidney failure as a result of the infection, was indeed infected with E. coli O157:H7. None of the patients’ close contacts were affected. The two molecular subtyping methods we used are pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). The isolates from the USA, Canada, Japan, and Taiwan each showed a unique molecular fingerprinting pattern. The strains isolated from cattle in Taiwan showed closer relationships with each other, and their similarity was in the range of 75-85%. The first human clinical strain isolated in Taiwan in 2001 was similar to the strains from North America but not closely related to the strains isolated from cattle in Taiwan. The establishment of a database of our own and joining the global network database is an important task if we want to control such agricultural and food-borne pathogens, and reduce the number of victims and sufferings, as well as the economic losses due to the infection. Three combinations of primers and probes were designed to detect and identify E. coli O157 on the TaqMan® detection system which focus on the specific genes eae, rfbO157, and stxII. Reverse transcription (RT) multiplex TaqMan® PCR were carried out to detect viable target cells correctly. Furthermore, the acidic pretreatment and immunomagnetic separation (IMS) of food and stool samples also improved the specificity and accuracy of the RT multiplex TaqMan® PCR. The developed multiplex TaqMan® PCR was effective in differentiating E. coli O157, enterovirulent E. coli, and non-E. coli pathogens from 100 strains which were isolated from human clinical patients and the cattle in Taiwan. Viable and non-viable cells were also distinguished by this assay. The pretreatment protocol, which included IMS to concentrate and purify the E. coli O157, was developed and the sensitivity of the assay was improved to 100 CFU/mL in pure culture, food and stool samples. On the other hand, the RT multiplex TaqMan® PCR with these combinations primers and probes were used to quantify the genes expression of stxII and eaeA of E. coli O157:H7 TWC01 which was treated with 7 different conditions. As the results, the chemical reagents promote the gene expression of stxII and eaeA in E. coli O157:H7 TWC01. On the treatment with storage temperature, the treated strain were not improved the gene expression of stxII and eaeA. It shortens the processing time and increases the specificity of the pathogens detection. This is critical for improving the safety and sanitation of our food supply.