- 學位論文
凝固酶陰性葡萄球菌之夫西地酸抗藥基因分析
Fusidic acid resistance determinants in clinical isolates of coagulase-negative staphylococci
摘要
臨床上分離之凝固酶陰性葡萄球菌(coagulase-negative staphylococci, CoNS)常被認為是污染而非有意義的病原菌，但近年來發現其在臨床上的重要性，如表皮葡萄球菌(Staphylococcus epidermidis)可以造成菌血症、感染性心內膜炎等；里昴葡萄球菌(Staphylococcus lugdunensis)可以造成感染性心內膜炎、腦膜炎及皮膚與軟組織感染等。因此，準確的鑑定出凝固酶陰性葡萄球菌對疾病診斷及流行病學等均具有重要意義。 夫西地酸(fusidic acid)藉由與EF-G-GTP/GDP-核醣體結合，使EF-G/GDP無法離開核醣體，從而抑制細菌的蛋白蛋合成，臨床上主要用於治療葡萄球菌所引起的皮膚感染或較為嚴重的全身性症狀。細菌可經由表現保護藥物作用標的之蛋白質(FusB-protein family)來對抗夫西地酸的作用，因而限制了夫西地酸在臨床上的使用，瞭解抗藥基因在葡萄球菌的盛行率、抗藥程度與分析抗藥基因結構等具有很重要的意義。 本研究收集了臺大醫院2010年至2012年的凝固酶陰性葡萄球菌，利用分子鑑定方法對收集到的菌株重新鑑定後，共497株，其中242株對夫西地酸為抗性。討論其夫西地酸抗藥基因(fusB、fusC、fusD與fusF)的盛行率及抗藥程度的關聯性，發現在菌種間具有特定的趨勢。在頭部葡萄球菌解脲亞種(Staphylococcus capitis subsp. urealyticus，49/51，96.1%)、表皮葡萄球菌(Staphylococcus epidermidis，89/89，100%)與溶血葡萄球菌(Staphylococcus haemolyticus，65/70，92.9%)裡，主要帶有fusB引起抗藥(MIC範圍為2至32μg/ml)。在10株人葡萄球菌人亞種(Staphylococcus hominis subsp. hominis)中，有9株(90%)因為帶有fusC造成抗藥(MIC範圍為2至16μg/ml)。於8株腐生葡萄球菌(Staphylococcus saprophyticus)中，全都因為帶有fusD導致抗藥(MIC範圍為2至4μg/ml)。而在11株科氏葡萄球菌解脲亞種(Staphylococcus cohnii subsp. urealyticus)中，有10株因為帶有fusF產生抗藥(MIC範圍為2至4μg/ml)。FusB-family protein導致的夫西地酸抗藥程度屬於低抗藥性。根據aj1-LP-fusB片段分為三個型別，在表皮葡萄球菌裡發現，相較於帶有第一型及第三型aj1-LP-fusB片段之菌株(MICs為2至8μg/ml)，帶有第二型之菌株可表現相對較高程度抗藥(MICs為4至16μg/ml)。 對於溶血葡萄球菌帶有fusB基因結構的研究比較少，故以PFGE進行菌株分型，發現65株共可分為6種pulsotypes，以pulsotype E (51/65)所含菌株數最多，再利用LA-PCR與Inverse-PCR分析了一株臨床菌株之fusB基因結構，發現其位在smpB下游，與一自巴西分離出之溶血葡萄球菌Sh29/312/L2結構十分相似。 在208株帶有fusB抗藥基因的菌株中，83株(39.9%)嵌入groEL基因下游之抗藥島嶼(resistance islands)，1株(0.48%)嵌入rpsR基因下游之抗藥島嶼，49株(23.6%)嵌入smpB基因下游之抗藥島嶼，沒有發現嵌在pNVH96質體上，還有75株(36.1%)的fusB抗藥基因嵌在未知的位置。
並列摘要
Coagulase-negative staphylococci (CoNS) isolates from clinical specimens are usually considered as contaminants. However, CoNS have, in recent years, been recognized as emerging opportunistic pathogens in nosocomial infections. Staphylococcus epidermidis is well known as a cause of bacteremia and infective endocarditis (IE), and other CoNS such as Staphylococcus lugdunensis, S. hominis, and S. haemolyticus are also associated with various infections. Thus, accurate identification of CoNS to the species level is essential in investigating clinical signigicance, progress of disease, and epidemiologic characteristics. Fusidic acid is a steroid antibiotic used as topical agent for skin infection and systemic treatment against staphylococcal infection. Fusidic acid inhibits bacterial protein synthesis by preventing release of EF-G/GDP complex from ribosome. Resistance to fusidic acid results from alternation of drug target site or protection of drug target site by FusB-family protein. The emergence of fusidic acid resistance restricts the clinical usage. It is important to clarify the prevalence of the resistance determinants, the resistance level, and genetic organizations of resistance determinants. In this study, clinical isolates of CoNS were recovered from NTUH in 2010 to 2012. A total of 497 CoNS were collected. Among 497 CoNS, 242 were resistant to fusidic acid. Resistance determinants of fusB, fusC, fusD and fusF were determined by PCR. Most Staphylcoccus capitis subsp. urealyticus (49/51, 96.1%), S. epidermidis (89/89, 100%) and S. haemolyticus (65/70. 92.9%) carried fusB (MIC ranges from 2 to 32μg/ml). Among 10 Staphylococcus hominis subsp. hominis, 9 (90%) isolates carried fusC (MIC ranges from 2 to 16μg/ml). All isolates of S. saprophyticus (8) carried fusD (MIC ranges from 2 to 4μg/ml) and 10 isolates of Staphylococcus cohnii subsp. urealyticus carried fusF (MIC ranges from 2 to 4μg/ml). Low-level resistance to fusidic acid (MICs≦32μg/ml) was found in FusB-family protein positive isolate. According to sequence of aj1-LP-fusB region, fusB carrying isolates can be divided in three types. Staphylococcus epidermidis isolates with type II aj1 displayed slightly higher-level of resistance to fusidic acid (MIC ranges from 4 to 16μg/ml) compared with those with type I or type III aj1 (MIC ranges from 2 to 8μg/ml). Only a few reports of S. haemolyticus’fusB were announced. Here we analyze the genetic relatedness using PFGE, 65 isolates can be separated into 6 groups. The pulsotype E was the most predominant (51/65). Using LA-PCR and Inverse-PCR to analyse the fusB structure of a clinical isolate. The results indicated that the fusB was located downstream of smpB, and displaying high similarity with that in S. haemolyticus Sh29/312/L2 isolated from Brazil. Among 208 isolates, 83 (39.9%) fusB resistance islands (RIs) were located downstream of groEL, 1 (0.48%) was located downstream of rpsR, and 49 (23.6%) were located downstream of smpB. 75 (36.1%) remained unidentified.