以PCR方法鑑定臨床分離之腸球菌和毒性因子分析

PCR assay for species identification and virulence genes among clinical isolates of Enterococcus

張慧貞(Hui-Jen Chang)

指導教授 ： 鄧麗珍

國立臺灣大學/醫學院/醫事技術學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01378

摘要

腸球菌主要引起的疾病包括菌血症，尿道感染，心內膜炎，傷口感染等，近年來，腸球菌在世界各地引起的感染症日益增加，甚至成為院內感染的主要病菌之一，所以為了有效的治療及監控腸球菌的感染，快速且正確的菌種鑑定是非常重要的。而以傳統的生化方式鑑定腸球菌是非常耗時的，而市售的快速鑑定系統，對於腸球菌少見菌種的鑑定，正確率並不高。因此本研究希望利用分子生物學的方法，以groESL基因為標的，發展對腸球菌菌種鑑定有效的方法。
由於本實驗室之前已得到Enterococcus faecalis ATCC 29212之參考菌株的groESL基因全長序列及其他八株腸球菌參考菌株近乎全長的groESL基因序列。在本實驗中，欲利用已得到的groESL基因序列，發展multiplex PCR的分析方法，正確的區分腸球菌不同的菌種(species)。將腸球菌不同菌種的groESL基因序列排列之後，選擇序列變異性大的位置設計對不同species有專一性的引子，藉由所增幅出的PCR片段大小的不同，期望能用簡單的multiplex PCR的方式，正確的鑑定不同種(species)的腸球菌。所增幅出的PCR產物大小為141 bp至745 bp不等，其中除了E. avium和E. raffinosus產物大小無法區分，E. hirae和E. mundtii無法區分之外，其他菌種皆可增幅出專一的PCR產物。如欲再區分以上菌種，可再利用PCR-RFLP的方式，以Hind III限制酵素處理PCR產物，藉由產生不同的RFLP pattern，區分multiplex PCR片段大小相同的菌種。首先，先以標準菌株測試，可行之後，再測試由台大醫院收集的114株腸球菌的臨床菌株，所用的臨床菌株經過16S rDNA定序確認，測試後發現我們所發展出的利用multiplex PCR鑑定腸球菌的方法，具有100%的專一性，可提供另一正確鑑定腸球菌菌種的方法，另外，也與自動化鑑定系統Phoenix的鑑定結果作比較。此外，也結合了臨床上常見的革蘭氏陽性球菌(GPC)，包括Staphylococcus aureus, Streptococcus species,和Enterococcus species，以不同的基因為標的，利用multiplex PCR的方式，區分此三類的革蘭氏陽性球菌，未來希望能直接從檢體中區分出常見的GPC。
另一部分，由於以往認為E. faecium致病性較低，但近年來發現E. faecium與E. faecalis一樣，具有esp這個毒力基因，且具有hyl這個有潛力的毒力基因，加上本實驗室之前發現，以groESL基因為標的，利用PCR-RFLP的方式可以將E. faecium區分為兩種type，因此欲探討E. faecium不同的PCR-RFLP types與毒力基因之間的關係。實驗中，共測試了86株臨床菌株，發現esp，hyl這兩個毒力基因只有在PCR-RFLP type I中出現，type II並沒有發現，不過這可能是因為type II的臨床菌株數目較少的關係。另外，若分為VRE和non-VRE來比較的話，發現在VRE中，esp基因所佔的比例高達67.4 %，而hyl基因高達60.46 %，相對於non-VRE (13.95 %, 13.95 %) 來說高出釵h。且esp和hyl基因常伴隨存在。

關鍵字

毒力基因 ； 腸球菌 ； 熱刺激蛋白基因

並列摘要

Identification of Enterococcus to the species level by conventional methods is time-consuming and complicated. Accurate identification of bacteria is important for effective treatment. In this study, we developed a multiplex PCR assay targeting groEL gene to identify Enterococcus to the species level. Species-specific primers from 9 species were designed based on the variable regions of each groEL gene. Each species generated different size (141 to 745 bp) except E. avium/ E. raffinosus and E. hirae/ E. mundtii. Further digestion of PCR products with Hind III can distinguish E. avium from E. raffinosus and E. hirae from E. mundtii. A total of 114 clinical isolates were tested and showed that the assay had 100% agreement with identification by 16S rDNA sequence. This multiplex PCR assay is easy to perform and accurate for identification of Enterococcus species including E. faecalis/ faecium and non- E. faecalis/ faecium species. In another part, three pairs of primers were combined to differentiate common gram positive cocci, including Staphylococcus aureus, Streptococcus species, and Enterococcus species.
Furthermore, the prevalence of esp and hyl of E. faecium was also investigated. E. faecium is generally considered to be a species of limited virulence. Recently, some data suggest that E. faecium strains may have become more and more virulent. esp and hyl are potential virulence genes of E. faecium strains. Besides, in our previous study, we found that E. faecium clinical isolates could group into two PCR-RFLP types based on groESL genes. The aim of the study was to find the correlation between different PCR-RFLP types of E. faecium and esp, hyl virulence genes. Of 86 strains tested, both determinants were found predominately in groESL PCR-RFLP type I strains. All PCR-RFLP type II strains have neither esp nor hyl virulence genes. However, comparing VRE with non-VRE, both esp and hyl virulence genes were mainly present in VRE.

並列關鍵字

virulence gene ； Enterococcus ； groESL

參考文獻

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