- 學位論文
臨床抗碳青黴烯類抗生素之綠膿桿菌表現各種不同的oprD基因突變
Diversity of Mutations of oprD in Clinical Isolates of Carbapenem-resistant Pseudomonas aeruginosa
摘要
綠膿桿菌(Pseudomonas aeruginosa)屬於革蘭氏陰性桿菌。臨床上，這個細菌屬於伺機性病源菌，主要感染免疫缺失或重症的病人。這個病原體可以因為本身構造或外來機轉，使其對多種抗生素產生抗藥性。因此，臨床上治療這種細菌所產生的感染症是一大考驗。 碳青黴烯類(Carbapenem)抗生素屬於一種廣效型的乙內醯胺(-lactam)抗生素。這類抗生素對抗綠膿桿菌，有不錯的效果。然而最近幾年出現抗碳青黴烯類抗生素的綠膿桿菌，而且這種抗藥性的綠膿桿菌在臨床上的威脅越來越高。目前研究顯示，這種抗藥的菌株可能是由於細胞膜上的OprD porin出現突變或膜上的Efflux pump激活所導致。 本研究，主要從2010年1月到2010年12月，於南台灣的一個區域教學醫院，收集21株臨床上抗碳青黴烯類抗生素的綠膿桿菌。首先，以PCR得到臨床菌株的16S rDNA片段，加以定序，確定這些臨床的菌株皆為綠膿桿菌。之後從中選取三株臨床抗藥的綠膿桿菌和標準菌株ATCC27853，將其細胞膜蛋白萃取出來，作蛋白質電泳並和標準菌株作比對。蛋白質體分析結果發現這三株選取的抗藥菌株的細胞外膜上，皆缺少OprD porin這個膜蛋白。以PCR得到這21株臨床抗碳青黴烯類抗生素綠膿桿菌的oprD基因片段，轉殖至質體加以定序，結果發現有各式各樣的突變方式，包括點突變導致提早結束轉譯(premature termination of translation)，或嵌入或缺失幾個核苷酸導致轉譯移碼(frameshift)而造成提早結束轉譯或繼續轉譯產生比原來更長的肽鏈，或缺失一大段基因造成提早結束轉譯，或於基因序列中插入一段的嵌入序列(insertion element)。總結來說，在臨床抗碳青黴烯類抗生素的綠膿桿菌中，細胞外膜OprD porin的消失源自於各種不同的oprD基因突變。
並列摘要
Pseudomonas aeruginosa, a Gram-negative bacterium, is an opportunistic pathogen for immune-compromised patients. The organism possesses both an intrinsic and acquired resistance to many antibiotics and treatment of infection by this organism is difficult. Carbapenems have a high potency against a broad spectrum of organisms and are one of the most active groups of -lactam antibiotics against P. aeruginosa. However, the threat of carbapenem-resistant Pseudomonas aeruginosa (CRPA) has been grown in recent years. Recent studies showed the resistance to carbapenem may be attributed to inactivation of OprD porin or overexpression of efflux pumps at the membrane. In this thesis, a total of 21 strains of CRPA were isolated from a regional hospital in south Taiwan from Jan. 2010 to Dec. 2010. The DNA fragments of 16S rDNA of all strains were obtained and sequenced to confirm they indeed are P. aeruginosa. The bacterial membrane fractions of three strains of CRPA and standard strand ATCC27853 were extracted and separated by SDS-PAGE. The proteomic analysis showed they all lacked the outer membrane OprD porins as compared to the standard strain. The DNA fragments of oprD genes of all CRPA strains were obtained by colony PCR, cloned and sequenced. The sequencing data showed diverse mutations in these CRPA strains. These mutations include: 1) premature termination of translation caused by point mutation, insertion of redundant nucleotides, and deletion of nucleotides; 2) insertion of redundant nucleotides to extend the translation at the stop codon; or 3) insertion of a long insertion element. In conclusion, variable mutations of oprD contributed to the disappearance of OprD porin in the outer membrane and resulted in the development of carbapenem resistance in P. aeruginosa.