Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22109
標題: Mycobacterium abscessus 臨床抗藥菌株抗藥機制之研究
Study of the drug-resistance of Mycobacterium abscessus clinical isolates
作者: 劉韋宏
Liu, Wei-Hung
關鍵字: mycobacterium abscessus
臨床抗藥菌株
drug resistance
clinical isolates
抗藥機制
抗藥性
出版社: 分子生物學研究所
引用: 林佳融. 2008. 結合分枝桿菌 ESX-5 基因組與 Rv1793 枝分泌與膿腫分枝桿菌表現型變異之研究. 國立陽明大學微生物及免疫學研究所碩士論文. 張慈庭. 2009. 台灣重要 9 種分枝桿菌之分子鑑定. 國立中興大學生命科學院碩士論文. American Thoracic Society (1997). Diagnosis and treatment of disease caused by nontuberculous mycobacteria. This official statement of the American Thoracic Society was approved by the Board of Directors, March 1997. Medical Section of the American Lung Association. Am J Respir Crit Care Med 156, S1-25. Brennan, P. J., and Nikaido, H. (1995). The envelope of mycobacteria. Annu Rev Biochem 64, 29-63. Covert, T. C., Rodgers, M. R., Reyes, A. L., and Stelma, G. N., Jr. (1999). Occurrence of nontuberculous mycobacteria in environmental samples. Appl Environ Microbiol 65, 2492-2496. Hobbie, S. N., Bruell, C., Kalapala, S., Akshay, S., Schmidt, S., Pfister, P., and Bottger, E. C. (2006). A genetic model to investigate drug-target interactions at the ribosomal decoding site. Biochimie 88, 1033-1043. Hobbie, S. N., Pfister, P., Brull, C., Westhof, E., and Bottger, E. C. (2005). Analysis of the contribution of individual substituents in 4,6-aminoglycoside-ribosome interaction. Antimicrob Agents Chemother 49, 5112-5118. Lewin, B. (2008). Genes IX, 9th edn (Sudbury, Mass.: Jones and Bartlett Publishers). Lin, D., and Lehmann, P. F. (1995). Random amplified polymorphic DNA for strain delineation within Candida tropicalis. J Med Vet Mycol 33, 241-246. Murray, C. K., and Hospenthal, D. R. (2005). Treatment of multidrug resistant Acinetobacter. Curr Opin Infect Dis 18, 502-506. Recht, M. I., Douthwaite, S., Dahlquist, K. D., and Puglisi, J. D. (1999). Effect of mutations in the A site of 16 S rRNA on aminoglycoside antibiotic-ribosome interaction. J Mol Biol 286, 33-43. Riley, J. G., Menggad, M., Montoya-Peleaz, P. J., Szarek, W. A., Marolda, C. L., Valvano, M. A., Schutzbach, J. S., and Brockhausen, I. (2005). The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide. Glycobiology 15, 605-613. Ripoll, F., Deshayes, C., Pasek, S., Laval, F., Beretti, J. L., Biet, F., Risler, J. L., Daffe, M., Etienne, G., Gaillard, J. L., and Reyrat, J. M. (2007). Genomics of glycopeptidolipid biosynthesis in Mycobacterium abscessus and M. chelonae. BMC Genomics 8, 114. Sander, P., Prammananan, T., Meier, A., Frischkorn, K., and Bottger, E. C. (1997). The role of ribosomal RNAs in macrolide resistance. Mol Microbiol 26, 469-480. Shen, G. H., Wu, B. D., Wu, K. M., and Chen, J. H. (2007). In Vitro activities of isepamicin, other aminoglycosides, and capreomycin against clinical isolates of rapidly growing mycobacteria in Taiwan. Antimicrob Agents Chemother 51, 1849-1851. Stahl, C., Kubetzko, S., Kaps, I., Seeber, S., Engelhardt, H., and Niederweis, M. (2001). MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis. Mol Microbiol 40, 451-464. Stephan, J., Bender, J., Wolschendorf, F., Hoffmann, C., Roth, E., Mailander, C., Engelhardt, H., and Niederweis, M. (2005). The growth rate of Mycobacterium smegmatis depends on sufficient porin-mediated influx of nutrients. Mol Microbiol 58, 714-730. Stephan, J., Mailaender, C., Etienne, G., Daffe, M., and Niederweis, M. (2004). Multidrug resistance of a porin deletion mutant of Mycobacterium smegmatis. Antimicrob Agents Chemother 48, 4163-4170. Trias, J., Jarlier, V., and Benz, R. (1992). Porins in the cell wall of mycobacteria. Science 258, 1479-1481. Wang, T. W., and Tseng, Y. H. (1992). Electrotransformation of Xanthomonas campestris by RF DNA of filamentous phage phi Lf. Lett Appl Microbiol 14, 65-68.
摘要: 快速生長型分枝桿菌會引起大範圍的散播及局部性的疾病,Mycobacterium abscessus 是其中一株致病性菌株,亞洲地區主要以 aminoglycoside 抗生素中的 Amikacin 及 Isepamicin 進行臨床治療。為了檢測 M. abscessus 的抗藥機制是否由16S rRNA 突變所至,本研究首先檢測 M. abscessus 臨床多重抗藥性菌株 R31 與 R39 的 16S rRNA 基因 rrs 的 DNA 序列,發現 R31 在其 16S rRNA 的 bp 976 與 bp 1375 皆有 A to G 的點突變,且在 rrs 基因下游 31 bp 處有 T to C 點突變,並依序以 m1、m2、及 m3 命名之。其中, m2 即是已知可使 E. coli 及同屬快速生長型分枝桿菌的 M. smegmatis 獲得對 Amikacin 至少 1024 ug/ml 抗藥性的 16S rRNA A1408G 點突變。接著以 Amikacin 對 m1、 m2 及 m3點突變在單一、兩兩及三者皆存在的轉型株進行最低抑菌濃度試驗,發現只要有 m2 存在,轉型株皆可獲得至少 32000 ug/ml 的抗藥性,而 m1 單獨存在時結果亦相同,顯示 16S rRNA 的突變是 R31對 Amikacin 等藥物產生抗藥性的原因之一。 m3 單一點突變與野生株並無差異,但若是 m3 與 m1 同時存在,最低抑菌濃度試驗結果則與野生株相同。根據 RNA 二級結構預測結果,推論 16S rRNA 會因為 m1 的存在而產生二級結構變異,導致 Amikacin 無法結合,轉型株得以進行轉譯作用而存活。因此, m3 可能是藉由改變或抑制 m1 所引起的結構變異,使得 Amikacin 可與 16S rRNA 結合而抑制轉譯作用,進而抑制菌株的生長。 此外,M. abscessus 共有兩段可表現 porin 蛋白的基因 MAB_1080 與 MAB_1081,為了檢測 M. abscessus 的臨床抗藥性是否源自於其細胞膜上通道蛋白的缺失所致,首先分析 M. abscessus 感藥性菌株 ATCC19977 與 CS1C.S,及抗藥性菌株 R31 與 R39 的通道蛋白基因 porin 的 DNA 序列及轉譯後蛋白序列。發現抗藥性菌株 R31 與感藥性菌株 CS1C.S 的MAB_1080 有嚴重缺失而MAB_1080 有點突變;抗藥性菌株 R39 與 感藥性菌株ATCC19977 的 MAB_1080 與 MAB_1081 的 DNA序列則完全相同,顯示 porin 基因的缺失與R31 及 R39 對 Amikaicn 等抗生素產生抗藥性無直接的相關性。
RGM ( rapid growing mycobacterium ) can cause large-scale and localized spread of the disease. Mycobacterium abscessus is one of the pathogenic strain in Asia. In the clinical treatment, aminoglycoside antibiotics have the potential to be extremely active against RGM, especially Amikacin and Isepamicin. To test the resistance mechanism of M. abscessus whether caused by the 16S rRNA mutations, this study first analyze the DNA sequence of 16S rRNA gene, rrs, of the multiple-drug-resistance M. abscessus, R31 and R39. We find out that rrs of R31 has A to G point mutation at bp 976 and bp 1375 in rrs, named m1 and m2, and T to C point mutation at bp 31 after rrs, named m3. By testing the MIC ( minimum inhibitory concentration ) for Amikacin of the different combination with m1, m2, or m3, we find out that as long as m2 exist, the transformants will obtain at least 32000 ug/ml of drug resistance to Amikacin, and the same as m1 exist alone. It means the mutation of 16S rRNA is one of the reasons for R31 to resist Amikacin. On the other hand, when m3 exist alone, the result of MIC test has no different with wild type strains, but when m3 combined with m1, the high resistance to Amikaicn obtained from m1 will be inhibit and restored to the same concentration as wild type strain. According to the predicted secondary structure of RNA, we suggest that m1 can cause the secondary structure change of 16S rRNA, resulting that Amikacin can not combine with 16S rRNA and transformants can survive by the functional translation. For this reason, m3 might modify or inhibit the conformational change caused by m1 to make 16S rRNA to resume the Amikacin binding site, simultaneously suppressing the transformanst to grow up by inhibiting the translation. In addition, M. abscessus has two porin expression genes, MAB_1080 and MAB_1081. To test the resistance mechanism of M. abscessus whether caused by the defect of membrane channel protein, we first analyze the porin expression genes of the strains sensitive to Amikacin, M. abscessus ATCC19977 and CS1C.S, and the strains resistant to Amikacin, R31 and R39. R31 has serious defect of MAB_1080 and have point mutation of MAB_1081 but CS1C.S has the similar result, and we can't complement the normal porin expression to R31 to make sure whether the porin defect is relative to Amikacin reietance or not. On the other hand, MAB_1080 and MAB_1081 of R39 are totally identical to ATCC19977. It means that drug resistant of R39 has no relationship with porin mutation.
URI: http://hdl.handle.net/11455/22109
其他識別: U0005-1108201014065600
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1108201014065600
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