- 學位論文
臨床凝固酶陰性葡萄球菌菌種之紅黴素抗藥分析
Erythromycin resistance in coagulase-negative staphylococci clinical isolates
摘要
凝固酶陰性葡萄球菌（Coagulase-negative staphylococci, CoNS）為人體皮膚或黏膜的正常菌叢，過去在臨床上曾長期被視為汙染菌株，近年來則成為醫院血液感染最大宗的病原菌，與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，例如紅黴素（Erythromycin）。本研究挑選2015至2016年自臺大醫院細菌室血液檢體中分離，以VITEK 2或MALDI-TOF方法鑑定為凝固酶陰性葡萄球菌的418株臨床菌株，並經由分子方法鑑定後菌種確定的414株凝固酶陰性葡萄球菌作為實驗菌株，分析其對於紅黴素的抗藥表現型與抗藥基因。首先以紙錠擴散試驗區分MLSB抗藥表現型，接著以agar dilution test確認紅黴素對菌株的最小抑制濃度，再以PCR偵測菌株所攜帶的紅黴素抗藥基因。結果顯示414株菌中有263 株（63.5%）具有紅黴素抗藥性，且臨床較常分離的菌種S. epidermidis、S. capitis、S. haemolyticus和S. hominis subsp. hominis的抗藥性都很高，影響到整體凝固酶陰性葡萄球菌的抗藥型態。抗藥表現型主要為cMLSB (54.0%)，最常見的抗藥基因為ermC (38.4 %)和msrA/B (33.5 %)。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），而攜帶msrA/B基因的菌株MIC值主要介於32 μg/ml至64 μg/ml之間。此外，有3株S. epidermidis攜帶罕見基因ermT，以PCR mapping比較他們與團隊先前已知的攜帶ermT的質體結構再進行解序後，發現SE05、SE07與SE01一樣，rep基因被IS431所插入，SE06攜帶ermT的質體除了與SE03相同的前半部分，後半部分結構與S. aureus質體pHSSA1278攜帶fosB基因的結構相同，是全新的基因結構組合。S1 PFGE和southern blot結果顯示SE05和SE06所攜帶的ermT基因不只在一個質體上，且大小皆遠大於5 kb，推測目前已知結構的小質體可能是從較大的質體中分離而出的。將總共7株的S. epidermidis做PFGE分析發現即使他們都是CC5 lineage（過去的CC2 lineage），卻分屬於6個不同的pulsotype，表示並非同一個clone在臺大醫院中持續造成感染。另外，在以分子方法鑑定菌種的過程中，我們發現一株NTUH_8064285在各方法所得到的結果均不同，在與相近菌種比較16S rRNA、dnaJ、gap、groEL、tuf和rpoB的序列並做MLSA分析，還有表現型比較後，目前初步判定NTUH_8064285可能不屬於已知的葡萄球菌，並且在分類上與S. haemolyticus最接近。
並列摘要
Coagulase-negative staphylococci (CoNS) are normal inhabitants of human skin and mucous membranes. They had long been considered as culture contaminants, but have become the major cause of nosocomial blood stream infection, mainly because of catheter-related infections. CoNS are often resistant to many antibiotics, such as erythromycin. In our study, 418 clinical isolates identified as CoNS by VITEK 2 or MALDI-TOF were collected from NTUH from 2015 to 2016 and identified with molecular methods, 414 of which were successfully identified as CoNS. We then analyzed their MLSB phenotype with double disk diffusion test, erythromycin resistance genes with PCR, and MIC values with agar dilution test. 263 of 414 (63.5%) isolates were resistant to erythromycin, and the four species S. epidermidis, S. capitis, S. haemoylticus and S. hominis subsp. hominis more often collected had higher resistant rate then average, resulting in the high resistant rate of CoNS. The major resistant phenotype was cMLSB (54.0%), and the major resistant genes were ermC (38.4%) and msrA/B (33.5 %). High level resistance to erythromycin was found in erm-carrying isolates (>256 μg/ml) whereas low level resistance to erythromycin in msrA/B-carrying isolates (32~64 μg/ml). Furthermore, 3 isolates of S. epidermidis (SE05, SE06 and SE07) carried the rare ermT, and compared with the known ermT-carrying plasmids our team had solved by PCR mapping. Plasmids of SE05 and SE07 resembled that of SE01, featuring the rep gene inserted by IS431. Plasmid of SE06 was composed of two sections: plasmid of SE03 (ermT, aadD and rep gene) and pHSSA1278 of S. aureus containing fosB gene. Results of S1-PFGE and southern blot indicated that there were more than one plasmids carrying ermT gene in SE05 and SE06, and their sizes were far larger then the solved 5kb-plasmids. We speculated that those 5kb plasmids may form from larger plasmids. PFGE analysis of 7 ermT-carrying S. epidermidis isolates showed that even though they were all CC5 lineage, they belonged to 6 different pulsotypes. Furthermore, during species identification we found one isolate, the NTUH_8064285, couldn’t be correctly identified by several methods, and was differentiated from known staphylococcal species on the basis of 16S rRNA, dnaJ, gap, groEL, tuf and rpoB gene sequences and MLSA. These results indicated that NTUH_8064285 may be new staphylococcal species, and that it’s closely related to S. haemolyticus.