包氏不動桿菌為一非葡萄糖發酵性之格蘭氏陰性菌，為院內感染的其中一種病原菌。在免疫力低下患者造成的死亡率相當高。造成此菌盛行的其中一個原因，可能來自於其快速產生多重抗藥性的能力。Carbapenem 類乃是目前治療革蘭氏陰性菌感染症最強效的抗生素，然而由於包氏不動桿菌對此類抗生素產生抗藥性的情形日益嚴重，已使得有效治療其感染症的抗生素選擇越來越少。因此尋求新的抗生素治療有其必要性。近年來，學者發現單獨 carbapenem hydrolyzing class D beta-lactamases (CHDLs) 基因過度表現即可造成對 carbapenem 高度的抗藥性，而且幾乎所有 carbapenem 抗藥性包氏不動桿菌都帶有這些 CHDL 基因。因此，對carbapenem抗藥性包氏不動桿菌的策略之一，就是有效抑制CHDL。欲找到可能的抑制 CHDL方式，要先了解其水解carbapenem的機制及活性位置 (active site)。
我們研究團隊發現帶有ISAba1-blaOXA-51-like的質體是造成臺灣地區包氏不動桿菌 carbapenem 抗藥性的主因之一，ISAba1-blaOXA-51-like 是啟動子 (promotor) 可使基因 blaOXA-51-like 大量表現 。然而吾人對於OXA-51-like水解 carbapenem 的特性所知卻十分有限。本研究利用比較兩種OXA-51-like水解酵素，它們僅僅相差一個胺基酸 (Leu167Val) 卻造成不同程度的 carbapenem 抗藥性，帶有 ISAba1-blaOXA-82 的菌株比基因 ISAba1-blaOXA-66對於抗生素美羅培南具較高抗藥性。此研究中，我們探討兩菌株對 carbapenem 產生不同程度抗藥性的機制。抗生素美羅培南對於兩菌株 ATCC15151 (pYMAb2 ISAba1-blaOXA-66) 與 ATCC15151 (pYMAb2 ISAba1-blaOXA-82)有不同的最小抑菌濃度分別為2 與 24 μg/mL，前者對抗生素具敏感性，後者具抗藥性。西方點墨法實驗數據顯示，於短時間反應 (2 – 8 hr) ATCC15151 (pYMAb2 ISAba1-blaOXA-66) 產生oxacillinase 的量約為OXA-82 的 1.5-2 倍；觀測較長的反應時間 (8 – 16 hr)，ATCC15151 (pYMAb2 ISAba1-blaOXA-82) 產生oxacillinase 的量約為 OXA-66 的 1.2 倍。液相層析串聯質譜儀分析、酵素動力學數據顯示，OXA-66 水解效率 (hydrolytic efficiency, kcat/Km = 0.04 μM-1sec-1) 比 OXA-82 ((hydrolytic efficiency, kcat/Km = 0.01 μM-1sec-1)好。改良賀治實驗中，抑菌環的變化了解 OXA-82 水解抗生素美羅培南的量比 OXA-66 多。於 mRNA 即時聚合酶鏈反應實驗中，兩菌在實驗結果中無顯著性差異。觀測三維模擬立體結構，兩酵素蛋白無結構上的差異。從實驗數據中了解具較低 MIC 數值的菌株對於抗生素會表現出較高的水解效率。OXA-82 水解美羅培南的效率較 OXA-66 差，ATCC15151 (pYMAb2 ISAba1-blaOXA-82) 產生 OXA-82 較 ATCC15151 (pYMAb2 ISAba1-blaOXA-66) 慢，但 ATCC15151 (pYMAb2 ISAba1-blaOXA-82) 製造OXA-82 的蛋白表現量多於 OXA-66，對於抗生素有較高的忍受度 (tolerance)。這些實驗結果闡述為何同為 class D OXA-51-like菌株ATCC15151 (pYMAb2 ISAba1-blaOXA-82) MIC 數值高於 ATCC15151 (pYMAb2 ISAba1-blaOXA-66) 的原因。

Acinetobacter baumannii is a non-fermenting Gram negative bacterium and one of the leading pathogens of nosocomial infection worldwide. The carbapenem resistance has been developed in a extremely speed among A. baumannii strains, which has severely compromised the therapeutic options. Therefore, new antimicrobials that are effective against CRAb (carbapenem resistant A. baumannii) are urgently needed. Recently, several researches have shown that overproduction of carbapenem hydrolyzing class D beta-lactamases (CHDLs) by itself can confer a high level of carbapenem resistance in A. baumannii, and these CHDLs are found in nearly all CRAb. Inhibition of CHDLs is among one of the effective strategies to settle CRAb infection. This approach often identifies chemicals that target on the active site of the enzymes.
We have firstly identified the plasmid bearing ISAba1-blaOXA-51-like has become one of the major carbapenem resistance determinants in A. baumannii in Taiwan. Insertion sequence ISAba1 acts as a strong promoter to enhance the expression of blaOXA-51-like. However, the carbapenem hydrolyzing characteristics of OXA-51-like carbapenemase have not been elucidated. We have found that different OXA-51-like enzymes contribute to carbapenem resistance in a different extent. The amino acid sequences are the same between ISAba1-blaOXA-66 and ISAba1-blaOXA-82 except for a Leu167Val amino acid substitution. The transformants carrying the ISAba1-blaOXA-82 would exhibit a much higher level of carbapenem resistance than those carrying ISAba1-blaOXA-66. In this study, we investigated the mechanism of difference in contribution to meropenem resistance between two blaOXA-51-like alleles. The minimum inhibitory concentrations (MIC) of two A.baumannii isolates, ATCC15151 (pYMAb2 ISAba1-blaOXA-66) and ATCC15151 (pYMAb2 ISAba1-blaOXA-82), were 2 and 24 μg/mL for meropenem respectively. The former was susceptible; the latter was resistant to antibiotic. The results of western blotting were shown that oxacillinase produced by ATCC15151 (pYMAb2 ISAba1-blaOXA-66) was approximately 1.5-2 times quantity of the OXA-82 with the short-time reaction (2 - 8 hr). ATCC15151 (pYMAb2 ISAba1-blaOXA-82) produced about 1.2 times quantity of oxacillinase than OXA-66 with the long-time reaction (8 - 16 hr). Hydrolytic efficiency of OXA-66 (kcat/Km = 0.04 μM-1sec-1) was greater than OXA-82 (kcat/Km = 0.01 μM-1sec-1) in Q-TOF LC/MS spectra, enzyme kinetics. The zone of inhibition with modified Hodge test showed OXA-82 hydrolyzed more meropenem than OXA-66. There was no statistically significant difference with mRNA expression between two strains. There was no different structure with molecular modeling between OXA-66 and OXA-82. The strain was possessed the lower MIC value but the oxacillinase was exhibited higher catalytic efficiency for antibiotics. The resistant strain produced more quantity of enzyme to resist and tolerate higher concentration of antibiotic. The results were elaborated the reason why the class D OXA-51-like group strains ATCC15151 (pYMAb2 ISAba1-blaOXA-82) was possessed higher MIC value than ATCC15151 (pYMAb2 ISAba1-blaOXA-66).

1. Abstract.................... 1-4
2. Introduction................ 5-10
3. Materials and Methods....... 11-21
4. Results..................... 22-28
5. Tables and Figures.......... 29-54
6. Discussion.................. 55-57
7. Reference................... 58-65

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