2016-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/660036摘要：金黃色葡萄球菌 (Staphylococcus aureus)是人與動物常見的重要病原菌，可引起院內感染或社區感染。臨床上嚴重的金黃色葡萄球菌感染極不易治療，近來 methicillin抗藥金黃色葡萄球菌(MRSA)菌株的增加，更增加治療的困難。而凝固酶陰性葡萄球菌(coagulase-negative staphylococci, CoNS)則主要是共生菌，但也會引起機會性感染。夫西地酸(Fusidic acid)為一種類固醇類(steroid-like)的抗生素，臨床上主要用於皮膚感染，及少數的全身性感染，例如骨頭關節葡萄球菌的感染。夫西地酸抗藥菌株雖不是很多，但近年來已有增加趨勢，因此了解抗藥機轉為重要課題。夫西地酸主要作用於細菌在轉譯 (translation)合成蛋白質的過程當中，與 elongation factor G (EF-G)結合而抑制細菌生長。若細菌 EF-G (fusA)有變異或得到外來因子(FusB-family proteins, 主要是 FusB, FusC,或 FusD)保護 EF-G 的作用，則細菌會對夫西地酸產生抗藥性。自 2002 年起，本實驗室團隊開始探討葡萄球菌之夫西地酸抗藥基因分析。一開始(2002-2006 年)先針對菌血症分離之金黃色葡萄球菌及幾種常見凝固酶陰性葡萄球菌菌株，分析夫西地酸抗藥基因的分佈，發現在 MRSA 與 methicillin-susceptible S. aureus (MSSA)即有很大不同 (Antimicrobial Agents and Chemotherapy 2010)。在 MRSA中，大多是因為 fusA 點突變導致夫西地酸高抗藥性，而 MSSA則多為 fusB或 fusC，且表現的是比較低抗藥。不過近期研究發現夫西地酸抗藥之 MRSA(主要是 ST239/SCCmecIII)攜帶 fusC 有增加趨勢，並發現其 fusC 是位於一特殊結構 SCCfusC，位於 SCCmec上游。由於 fusB 或 fusC 可能會經由菌株間或菌種間傳遞，特別是與 CoNS 間傳遞，因此我們也分析了自菌血症分離 Staphylococcus epidermidis的抗夫西地酸基因結構，發現以 fusB為主。S. epidermidis之 fusB 基因位於外來之抗藥島嶼(resistance island)上，基因構造極具多樣性並位於不同染色體位置上，包括 rpsR、groEL、smpB等(Antimicrobial Agents and Chemotherapy 2011與 2013)。由於夫西地酸目前主要用於皮膚感染治療，為了解人體皮膚常在菌叢是否已對夫西地酸產生抗藥，本實驗室已自健康受試者分離帶有抗夫西地酸之凝固酶陰性葡萄球菌菌株。因此將於第一年計畫分析已收集之人體手部皮膚常在菌叢之凝固酶陰性葡萄球菌菌株抗夫西地酸基因結構。此外，血液分離菌株的部份，之前只分析 S. aureus及 S. epidermidis，尚未分析其他 CoNS 菌種。因此擬於第二年測試近年來臨床血液分離凝固酶陰性葡萄球菌之夫西地酸抗藥基因結構，並與皮膚常在菌叢菌株比較菌株特性及基因分型。第三年︰由於我們初步試驗發現在 S. cohnii subsp. urealyticus 有一新抗藥基因型 fusF，因此將針對此新型 fusF，進行完整基因序列分析、探討基因結構及對夫西地酸抗藥功能分析探討。此外，為更全面了解 S. cohnii subsp. urealyticus 的特性，也將進行其全基因體定序。<br> Abstract: Staphylococcus aureus is an important pathogen causing nosocomial or community-acquired infections. Resistance to antimicrobial agents is an increasing problem. Coagulase-negative staphylococci (CoNS) are mainly commensal organisms, but some of them may be associated with opportunistic infections. Fusidic acid (FA), a steroid-like antibiotic, has been used as a topical agent for skin infection and for some systemic infections caused by S. aureus. FA inhibits bacteria protein synthesis by binding to a complex of elongation factor G (EF-G)-GTP/GDP and the ribosome, and then inhibiting the release of EF-G-GDP complex. Resistance to FA may result from alternation of drug target site (fusA or rplF point mutation) or protection of drug target site by FusB-family proteins encoded by fusB, fusC, or fusD. The FusB protein resulted in resistance to fusidic acid by directly binding to EF-G on the ribosome, following promoting the dissociation of ribosome/EF-G/GDP/fusidic acid complexes, and them allowing ribosome to resume translation. For S. aureus, we have previously found that the distribution of fusA mutations, fusB, and fusC was significantly different between the methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) groups. Recently, we found fusC-positive MRSA has been increasing and the increasing prevalence of fusC in fusidic acid-resistant MRSA isolates was due to a novel SCC structure, SCCfusC, which was integrated into orfX (rlmH) and located upstream of SCCmec. The SCCfusC element was found in MRSA with SCCmec type III and contained speG, which also contribute to the polyamine resistance. For S. epidermidis, we identified the fusB genetic structures and found that it was located in phage-related pathogenicity islands (resistance islands) downstream of groEL, rpsR or smpB. Since FA is used in skin infection. Is it possible that the commensal organisms, especially CoNS will develop resistance to FA and serve as reservoir of resistance determinants? Thus in the first year of this project, we will focus on analysis of genetic structures of FA resistance elements from staphylococci isolated from skin flora. In the second year, we will analyse the FA resistance elements from FA-resistant CoNS isolated from blood. The reason is that we have previously analysed resistance structure from S. aureus and S. epidermidis, but not yet tested other CoNS isolated from blood. In addition, our preliminary data revealed that a novel type of FA resistance determinant, fusF, was found in S. cohnii subsp. urealyticus. Thus in the third year, we will perform the sequencing of fusF and flanking regions and cloning of fusF with plasmids to test the role of the gene. Furthermore, to gain more insight into S. cohnii subsp. urealyticus, we will perform whole genome sequencing.葡萄球菌夫西地酸抗藥基因抗藥島嶼staphylococcifusidic acid resistance determinantsfusBfusCfusFresistance islands