- 學位論文
探討克雷伯氏肺炎桿菌孔蛋白OmpK35和OmpK36與碳氫黴烯抗藥性之關聯
The Role of Klebsiella pneumoniae porin OmpK35 and OmpK36 in Carbapenem Resistance
摘要
臨床目前使用碳氫黴烯作為治療超廣效乙內醯胺酶抗藥株感染的藥物，近年來卻陸續出現抗藥菌株的報導，其中腸桿菌科的克雷伯氏桿菌所占比例甚高，由於此類抗藥菌株限縮用藥選擇，必須仰賴副作用較劇的後線藥物，故尋找可能的抗藥機制，對於公共衛生政策而言極其重要。本篇研究承實驗室先前的研究，分析來自台灣四家教學醫院分離出的克雷伯氏肺炎桿菌之碳氫黴烯抗藥菌株，以聚合酶連鎖反應排除帶有碳氫黴烯酶基因者後，再深入探討革蘭氏陰性菌特有的外膜孔蛋白表現，研究主要孔蛋白OmpK35或OmpK36的缺失或突變是否會影響克雷伯氏肺炎桿菌對於碳氫黴烯類抗生素之感受性。首先，將完整的孔蛋白基因ompK35及ompK36選殖進入質體，並以電穿孔的轉形方式分別補回抗藥性菌株內，測試對於亞胺培南之最低抑菌濃度，發現有34.5%菌株的最低抑菌濃度降低和OmpK35、OmpK36孔蛋白的功能喪失相關，代表菌株孔蛋白的表現改變，或是基因突變或缺失造成功能不彰，進而影響克雷伯氏肺炎桿菌對碳氫黴烯的感受性。故進一步定序分析OmpK35和OmpK36孔蛋白的胺基酸序列，雖有41.2%的菌株胺基酸序列和對碳氫黴烯有感受性的參考菌株一致，但另有41.2%的菌株孔蛋白基因出現插入或單一鹼基剔除，造成移碼突變或提早轉錄終止，對照預測的結構，認為可能影響孔蛋白鑲嵌於外膜。為了解OmpK35和OmpK36孔蛋白實際的表現情形，以基因剔除的方式建構孔蛋白突變株做為對照組，且將外膜蛋白萃取出來觀察其表現量，由於蛋白染色無法依分子量準確區分出特定孔蛋白的位置，還須參照西方墨點法的結果交叉比對，結果顯示21.1%可偵測到OmpK35、52.6%可偵測到OmpK36。因而用反轉錄定量聚合酶連鎖反應分析其核酸表現量，儘管在高滲透壓環境下負責調控孔蛋白的ompR表現增加，但與抗藥性較不具關聯。最後，測定無標記基因置換突變株的抑菌濃度，證實OmpK35近羧端序列對碳氫黴烯抗藥性影響甚鉅。
並列摘要
In recent decades, carbapenem-resistant Klebsiella pneumoniae (CRKPs) emerged rapidly due to the treatment of extended spectrum β-lactamse (ESBLs). To understand whether major outer membrane proteins, OmpK35 and OmpK36, played important roles in carbapenem resistance, we harvested non-carbapenemase-producing and imipenem-resistant K. pneumoniae isolates from four medical centers in Taiwan. In this study, the function of OmpK35 and OmpK36 from 19 of 55 isolates (34.5%) with high MIC (≥ 4μg/mL) was confirmed by using plasmid complementation tests, respectively. Moreover, amino acid sequence analysis revealed that 41.2% (7/19) frame-shift mutation of OmpK35 were caused by one-base pair deletion, whereas 41.2% (7/19) of clinical isolates carried the same sequence of OmpK35 as reference strains. To investigate whether clinical CRKPs were porin-lost or not, the outer membrane proteins of these strains were extracted by Western blots. We found that 21.1% of OmpK35 and 52.6% of OmpK36 were detected. In addition, we used real-time RT-PCR to analyze the transcriptional expression level of ompK35, ompK36 and their response regulator, ompR. The transcriptional expression of ompK35 was downregulated in low osmolality, and the expressions of ompK36 and ompR were upregulated in high osmolality environment. Finally, unmarked replacement OmpK35mutants restored their sensitivity against imipenem. In conclusion, there is no correlation between porins OmpK35 and OmpK36 expression and resistant to imipenem. And this study provides the evidence for importance of carboxyl terminus of OmpK35 in K. pneumoniae’s porin function.