多重抗藥性包氏不動桿菌Integrase聚合酶反應之分子研究

A Molecular Study of the Integrase Polymerase Chain Reaction in Multidrug Resistant Acinetobacter Baumannii

目的：包氏不動桿菌（Acinetobacter baumannii）為喜好潮濕的革蘭氏陰性球桿菌，普遍存在於人體皮膚、黏膜以及環境中。能廣泛的利用各種碳源，常在加護病房以及燒燙傷病房內免疫力較弱的病人身上引起各種症狀，包括敗血症、心內膜炎、腦膜炎、尿道炎等，但是最主要的，還是在院內感染的肺炎。包氏不動桿菌廣泛帶有對多種抗生素的抗藥性，甚至陸續出現對多種抗生素具抗藥性的菌株（Multidrug-resistant Acinetobacter baumannii;MDR-Ab），使得我們在臨床上治療它們所引發的感染疾病更加困難。因此，我們針對MDR-Ab進行integrase聚合酶反應之分子研究，分析可能的抗藥性基因之水平傳播方式。方法：本研究以中部某區域醫院中所收集的MDR-Ab臨床菌株之流行病學資料為基礎，探討其抗藥性基因是否經水平傳播的方式散佈於各菌株間之可能性。使用的臨床菌株是民國九十一年六月一日到民國九十二年五月三十一日止，在中部某區域醫院分離出的12株Multidrug-resistant包氏不動桿菌為材料，分別再以Disk diffusion experiment與VITEK 2 Advance Expert System等抗生素藥物敏感性測試來測定這12株菌株的抗生素抗藥性表現型。此外，以聚合酶連鎖反應（PCR）方法進行水平傳播平台Integron內含的integrase基因來分析。最後，以南方墨點法來確認這12株MDR-Ab臨床菌株的Integron序列是否相同。結果：PCR結果顯示這12株MDR-Ab菌株均帶有一個約160-bp fragment的第一類integrase gene。南方墨點法結果發現這12株M D R-Ab菌株均帶有一個約2.5Kb的第一類integron，其中的gene cassette排列方式也完全相同，這結果顯示出抗藥基因有水平傳播的可能。結論：這12株MDR- Ab 菌株均帶有一個約160- bp fragment的第一類integrase gene，所以都具有第一類integron，integron是一種基因水平傳播單位，能幫助抗藥基因在不同細菌中散播，這結果顯示出這12株M D R-Ab菌株的抗藥基因有水平傳播的可能性。從integron的定序結果發現，這12株菌株的gene cassettes排列完全相同，推測可能原因有二，一為這12株菌株剛好都得到相同的gene cassettes，另一個可能為這12株菌株的integron位在一個更大的水平移動單位，如轉位子上。

Objectives: Acinetobacter baumannii, a type of gram-negative coccobacilli which prefers a a moist environment, is commonly found on the skin and mucosa of human beings. Capable of utilizing various carbon sources, Acinetobacter baumannii frequently induces septicemia, endocarditis, meningitis and urethritis in intensive care or severely burned patients because of their weaker immunity. Among all hospital-acquired infections, pneumonia caused by Acinetobacter baumannii is primary. Acinetobacter baumannii i is resistant to numerous antibiotics, and several strains of multidrug-resistant Acinetobacter baumannii (MDR-Ab) have been discovered. This makes Acinetobacter baumannii induced infections more difficult to treat. The aim of this study was to analyze the horizontal transfer of possible drug-resistant genes in the integrase polymerase chain reaction of MDR-Ab. Methods: This study utilized epidemiologic data about 12 strains of MDR-Ab collected by a regional hospital in central Taiwan between June 1, 2002 and May 31, 2003. Two drug sensitivity tests, a disk diffusion experiment and the VITEK 2 Advanced Expert System, were used to determine the antibiotic-resistant phenotype of the 12 strains. In addition, a polymerase chain reaction (PCR) was conducted to analyze integrase genes in the integron, a horizontal transfer platform. A southern blot was also used to verify whether these 12 clinical strains of MDR-Ab had an identical integron sequence. Results: PCR results revealed that a class 1 integrase gene about 160-bp fragment was common among these 12 strains of MDR-Ab. The southern blot also showed the existence of a class 1 integron about 2.5Kb in all 12 strains of MDR-Ab; moreover, their gene cassette sequences were identical. Conclusions: These 12 strains of MDR-Ab all had a fragment of class 1 integrase gene about 160-bp, and therefore, they all had a class 1 integron, a gene horizontal transfer unit enabling the spread of drug-resistant genes among bacteria. This finding demonstrated the possibility of a horizontal transfer of the drug resistant genes of these 12 strains of MDRAb. Results from integron sequencing showed that the gene cassettes of these 12 strains of MDR-Ab were completely identical. This may have been because these 12 strains of bacteria happened to have identical gene cassettes, or the integron of these 12 strains of bacteria was located at a larger horizontal transfer unit, e.g.,the transposon.