- 學位論文
以groESL基因PCR-RFLP鑑定bovis群鏈球菌及分析紅黴素抗藥基因
Identification of bovis group streptococci by PCR-RFLP based on groESL sequence and analysis of erythromycin-resistance determinant
摘要
Streptococcus bovis為生物腸道正常菌叢，與S. equines、S. alactolyticus等菌株歸屬於bovis群，主要會引起老年人的菌血症、心內膜炎等感染，其中因為其與大腸直腸癌具有高度相關，引起科學家對於正確鑑定S. bovis的重視。可根據其不同的生化反應將其區分為三種不同的生化型：生化型Ⅰ、Ⅱ/1、Ⅱ/2)，臨床上所使用的鑑定方法可分為兩大類：傳統的鑑定方法及快速鑑定系統，此兩種鑑定方法除了有耗時的缺點外，更因為其生化反應的多樣性，會導致鑑定錯誤的發生，所以積極發展分子鑑定的方法，目前仍以16S rRNA基因序列為參考準則，但受限於序列在相同specie間具有高度的相似性，因此選擇使用groESL基因，此基因在細菌中普遍存在，具有高度保留性，因此實驗中先利用PCR、基因定序的方法確定其DNA序列後，進一步序列比對與樹狀圖分析，發現不同的生化型可利用序列區分出來，並進一步設計S. bovis特異性的引子，再利用RFLP的方法鑑定三種不同的生化型。 S.bovis對於紅黴素(erythromycin)的抗藥性主要是由erythromycin ribosome methylation(erm)基因引起的，此基因所轉錄出的蛋白會對於紅黴素作用位置(23S rRNA)進行甲基化而使得紅黴素失去作用，而Lincosamide和Streptogramin B這兩種抗生素因為和紅黴素作用位置有重複的地方，因此會同時引起多重抗藥的發生，且因為erm基因表現的不同可分為cMLSB(constituteive MLS)和iMLSB(inducible MLS)，以往在鏈球菌iMLSB抗藥株中所發現的抗藥基因多為ermB，但我們發現S. bovis中iMLS抗藥株卻主要為ermT，因此進一部分析來自於不同患者的菌株是否為單一菌株的傳播或者是各個不同菌株感染。此外，在此段抗藥基因的上游發現有轉錄出ribosomal protein的序列，因此推測此抗藥基因可能位於chromosome上，因此我們進而將包埋在膠中的DNA利用I-ceu I作用，此酵素辨認23S rRNA基因的序列，將DNA切成若干個大片段，利用脈衝場電泳分析及南方墨漬法，得到證明。
並列摘要
Streptococcus bovis group is normal flora of the ruminant and human gut, but will also cause serious infections. Many reports have suggested a potential relationship between underlying infection with this organism and colon cancers. According to biochemical characteristics, S. bovis strains were divided into three biotypes, biotype I, Ⅱ/1 and Ⅱ/2. The clinical identification of bovis group depends on conventional methods or commercial rapid identification systems. But these two methods are time-consuming and not satisfactory. The diversity of biochemical characteristics of bovis group may lead to misidentification. Recently scientists have developed molecular identification based on 16S rRNA gene sequence. Limited to the highly conservation of 16S rRNA gene sequence, it is difficult to differentiate closely related species. Since the taxonomy of the species within bovis group has been proposed, the aim of this part of study was to develop a more rapid and reliable method for identification. In this study, we analyzed groESL sequences, which are ubiquitous and evolutionarily highly conserved, to understand the correlation between the phylogenetic evolution and the biotypes by PCR-direct sequencing method. The results revealed that isolates with the same biotype usually belong to the same genocluster. Then we designed specific PCR for the bovis group, and developed PCR-RFLP method to subsequently differentiate three biotypes. Resistance to erythromycin in S. bovis is mostly due to the target modification, which is mediated by erythromycin ribosome methylation (erm) that methylates 23S rRNA and induces ribosome modification. Because the target site of erythromycin is overlap with Lincosamide and Streptogramin B，ribosome modification can cause three antibiotics resistance known as macrolide-lincosamide-streptogramin B (MLSB) resistance. Expression of MLSB resistance can be either constitutive (cMLSB) or inducible (iMLSB). In previous studies, we found high rate of iMLSB strains in S. bovis. Detection of erythromycin resistance genes by PCR and sequence indicated that some iMLSB strains had erm(T) that is not reported in streptococci before. In this study, we analyzed the structures of erm(T) gene in these iMLSB strains. The SmaI PFGE fragments revealed the heterogeneity and not the expansion of a single clone. Furthermore, we have confirmed that the erm(T) resistance gene is located on chromosome.