臺灣博碩士論文加值系統

凝固酶陰性葡萄球菌為人體皮膚黏膜正常菌叢。近年來凝固酶陰性葡萄球菌已成為血液檢體常見的病原菌，其重要性與菌血症、血管內裝置或導管造成的院內感染有關。凝固酶陰性葡萄球菌經常對許多抗生素具有抗藥性，如紅黴素，在台灣很少有關於臨床凝固酶陰性葡萄球菌之紅黴素的研究，很有可能解釋為何金黃色葡萄球菌 (Staphylococcus aureus)有高紅黴素抗藥的原因，因此選擇紅黴素來研究凝固酶陰性葡萄球菌抗藥基因的分布。本研究挑選自2012至2014年臺大醫院細菌室415株由VITEK2鑑定為凝固酶陰性葡萄球菌的臨床菌株，經由分子方法鑑定後共有401株凝固酶陰性葡萄球菌作為實驗菌株。首先分析凝固酶陰性葡萄球菌對於紅黴素之抗藥表現型與抗藥基因之盛行率，以紙錠擴散試驗區分MLSB抗藥表現型，利用agar dilution確認紅黴素對菌株的最小抑制濃度，使用PCR分析紅黴素之抗藥基因。結果顯示三年的凝固酶陰性葡萄球菌中有267株具有紅黴素抗藥性，常見的抗藥基因為ermC (37.1 %)和msrA/B (34.8 %)，抗藥表現型主要為cMLSB (55.4 %)，也發現抗藥基因盛行率在各菌種之間有特定分布，S. captis主要以攜帶ermA基因型 (25/35, 71.4 %)、S. epidermidis較多攜帶ermC基因型 (79/157, 50.3 %)、S. haemolyticus攜帶msrAB (30/53, 56.6 %)。接下來探討抗藥基因與MIC關聯，大多數攜帶erm系列基因的菌株對於紅黴素的MIC值都很高 (大於256 μg/ml)，攜帶msrA/B基因型MIC範圍介於16 μg/ml至128 μg/ml。此外在S. capitis和S. epidermidis共有6株菌株發現攜帶罕見基因ermT，目前關於ermT基因在凝固酶陰性葡萄球菌的文獻非常少，且近年報導多發現在金黃色葡萄球菌。為了深入探討此基因，將6株攜帶ermT基因的菌株進行southern Blot和S1-PFGE，發現ermT基因均被質體DNA攜帶且為單一copy數目，挑選菌種不同的兩株NTUH-4483和NTUH-8031399透過self-ligation與定序分析攜帶ermT質體結構，發現兩者質體相似且為約5kb大小的質體結構，經資料庫比對後發現與MSSA ST398-t571攜帶質體pU3912 (GenBank: HE805623)的結構不同，可能為尚未發表的結構。

Coagulase-negative staphylococci (CoNS) are the main species of normal skin flora. Recently, CoNS have been recognized as typical opportunists in nosocomial infection. They are particularly associated with the use of indwelling or implanted foreign bodies. CoNS often have multiple antibiotic resistances, like erythromycin. The study about clinical erythromycin resistance of CoNS in Taiwan is rare. It might explain the higher frequency of erythromycin resistance in Staphylococcus aureus. We analyzed the erythromycin resistance genes of CoNS to investigate the prevalence. In our study, 401 clinical isolates identified as CoNS were collected from NTUH from 2012 to 2014. We analyzed the MLSB resistance phenotype by double disk diffusion test and erythromycin resistance genes by PCR. 267 (66.6 %) isolates were resistant to erythromycin. Among the erythromycin resistance genes, both ermC and msrA/B were the predominant, and the cMLSB was the major MLSB phenotype. We also found that some specific determinants in species. Most Staphylococcus capitis carried ermA Staphylococcus epidermidis carried ermC and Staphylococcus haemolyticus carried msrA/B. To investigate the relationship with resistance genes, we determined the MIC by agar dilution. High level resistance to erythromycin was found in erm-carried isolates (>256 μg/ml) whereas low level resistance to erythromycin in msrA/B-carried isolates. Furthermore, a total of six clinical isolates in S. capitis and S. epidermidis carried the unusual ermT genes. The ermT gens was seldom reported in CoNS but associated with S. aureus recently. To investigate this gene, using southern blot and S1-nuclease PFGE to probe the location of ermT gene. We found the ermT genes were carried by plasmid and only one copy number. Then, select two isolates NTUH-4483 and NTUH-8031399 to analysis the structure of ermT-carried plasmid by self-ligation and Sanger sequence. Both the structure of plasmid were similar 5 kb size. Comparing with NCBI database, the structure of ermT-carreid plasmid was different to pUR3912 carreied by MSSA ST398-t571 (GenBank: HE805623). The 5kb ermT-carried plasmid might be unpublished plasmid structure.

致謝 I
英文摘要 II
中文摘要 IV
目錄 V
圖目錄 VIII
表目錄 IX
第一章、前言 1
1.1凝固酶陰性葡萄球菌簡介 (Coagulase-negative staphylococci, CoNS) 2
1.2紅黴素 (Erythromycin) 2
1.3 Macrolide–Lincosamide-Streptogramin B抗藥機制 2
1.4常見紅黴素抗藥基因erm攜帶方式 3
1.5 ermT基因的攜帶方式 3
1.6凝固酶陰性葡萄球菌的大環內酯 (Macrolide)與Methicillin抗藥之關聯 5
第二章、材料與方法 6
2.1本實驗用菌株與參考菌株 6
2.2細菌菌株培養與保存 6
2.3染色體核酸萃取 (Chromosomal DNA extraction) 6
2.4聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 8
2.5細菌菌種鑑定 9
2.6抗生素藥敏性試驗–紙錠擴散試驗 (Double Disk diffusion Test) 10
2.7抗生素藥敏性試驗–最小抑菌濃度 (Minimum inhibitory concentration) 11
2.8 PCR產物基因純化 (PCR product gene clean) 12
2.9 16S核醣體核酸鑑定 (16S rRNA) 13
2.10質體核酸萃取 (plasmid DNA extraction) 13
2.11南方墨點法 (Southern Blot) 14
2.12 Inverse PCR 17
2.13 S1 nuclease-pulsed field gel electrophoresis (S1 nuclease-PFGE) 19
第三章、結果 22
3.1篩選臨床菌株和鑑定菌種之比較 22
3.2凝固酶陰性葡萄球菌的紅黴素抗藥比例 22
3.3凝固酶陰性葡萄球菌的紅黴素抗藥基因分布 23
3.4 Methicillin抗藥與紅黴素抗藥之間的關聯 23
3.5凝固酶陰性葡萄球菌的SCCmec與紅黴素抗藥基因關聯 24
3.6紅黴素抗藥基因與最小抑菌濃度MIC值關聯 24
3.7 MLST分析攜帶ermT菌株的Sequence type 25
3.8利用Southern Blot確認菌株攜帶ermT基因 25
3.9利用S1-PFGE探討菌株攜帶ermT的方式 26
3.10分析攜帶ermT的質體DNA基因結構 26
3.11限制酶評估NTUH-4483與 NTUH-8031399攜帶ermT的質體DNA大小 26
3.12 PCR評估NTUH-4483與NTUH-8031399攜帶ermT的質體DNA大小 27
3.13以PCR mapping分析攜帶ermT的質體DNA基因結構 27
3.14利用不同限制酶與Southern blot估計NTUH-2831-1和NTUH-1044958攜帶ermT的質體DNA大小 27
3.15 ermT基因攜帶菌株之PFGE結果 28
第四章、討論 29
4.1 凝固酶陰性葡萄球之菌種鑑定 29
4.2 凝固酶陰性葡萄球的紅黴素抗藥情形 29
4.3 凝固酶陰性葡萄球菌之紅黴素抗藥基因分析 30
4.4 紅黴素抗藥基因與MIC值關聯性 31
4.5 凝固酶陰性葡萄球菌之ermT基因分布 31
4.6 分子分型分析4株ermT攜帶之S. epidermidis演化關係之比較 31
4.7 臨床凝固酶陰性葡萄球菌主要為cMLSB表現型 32
4.8 Methicillin抗藥與紅黴素抗藥之間的關聯 32
4.9 攜帶ermT基因的質體DNA結構 33
4.10 兩種葡萄球菌攜帶類似的ermT的質體DNA結構 33
4.11 凝固酶陰性葡萄球菌之抗紅黴素基因結果與2000-2012年台大醫院MSSA的紅黴素抗藥基因之比較 34
第五章、附圖 35
第六章、附表 44
參考文獻 57

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