金黃色葡萄球菌帶有抗紅黴素基因ermB轉位子Tn6636之新型質體分析

Novel Tn6636-bearing plasmids carrying ermB in erythromycin-resistant Staphylococcus aureus

萬采玟(Tsai-Wen Wan)

指導教授 ： 鄧麗珍

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/博士(2019年)

https://doi.org/10.6342/NTU201901913

摘要

金黃色葡萄球菌 (Staphylococcus aureus) 是臨床上重要的病原菌，其抗藥也一直是嚴重的問題。我們最近發現台灣methicillin-susceptible S. aureus (MSSA)對紅黴素抗藥的菌株，許多是因為帶有ermB基因。而在帶有ermB菌株中，大多為攜帶常見於MRSA sequence type (ST) 59的mobile element structure (MES)-like element。但我們發現少數菌株攜帶源自Enterococcus faecium的新型轉位子”Tn6636”。Tn6636與Tn1546有相同的轉位酶基因，由於vancomycin-resistant S. aureus (VRSA) 的主要抗藥基因結構為轉位子Tn1546攜帶vanA，因此帶有ermB的Tn6636出現在S. aureus非常值得注意。我們分析臺大醫院2000~2016年由菌血症檢體分離之紅黴素抗藥中具ermB的MSSA 112株及methicillin-resistant S. aureus (MRSA) 224株，顯示10株MSSA與10株MRSA具有Tn6636的轉位酶基因。進行multilocus sequence typing (MLST) 分析，發現攜帶Tn6636的轉位酶基因之MSSA有ST7 (n=6)、ST5 (n=3)、ST59 (n=1)，MRSA有ST188 (n=8)、ST965 (n=2)。以PCR mapping分析20株攜帶Tn6636的菌株，顯示其Tn6636結構皆很相似。以S1-PFGE搭配Southern blot分析，顯示10株MSSA及10株MRSA的Tn6636都位於質體上，質體大小約23~48.5 kb間，相同ST的菌株質體大小相似。經由接合作用實驗證實MSSA ST5、ST7、ST59與MRSA ST965的質體可轉移至接受菌株RN2677中，顯示以上質體具傳遞性。進一步以inverse PCR與long and accurate PCR (LA-PCR)解序，發現ST5 MSSA的質體pBS5-3是鑲嵌質體，14-kb的Tn6636插入ST5 MSSA的27-kb質體pWBG744。ST7 MSSA的質體pBS7-2、ST188 MRSA的質體pBR188-6也是鑲嵌質體，Tn6636插入ST93 MRSA的20-kb質體pSaa6159。ST965 MRSA的質體pBR965-1是Tn6636插入ST5 MRSA的25-kb質體pN315。最特別的是ST59 MSSA 47-kb的質體pBS59-1，Tn6636以外的33-kb核苷酸序列在NCBI資料庫內無法比對到相似度大於70%的序列，僅有將編碼區轉成胺基酸序列後才能對到相似度大於99%的序列。此外，本研究並擴大搜尋非血液感染之金黃色葡萄球菌是否帶有Tn6636。結果顯示在兩個收菌期間 (2016年4月至6月、2017年8月至2018年2月)總共230株抗erythromycin的MSSA中有33株具ermB基因，其中11株帶有Tn6636，包括ST7 (n=3)、ST59 (n=2)、ST88 (n=2)、ST188 (n=2)、ST398 (n=1)、ST2592 (n=1)，且S1-PFGE搭配Southern blot結果顯示Tn6636皆位於質體上，3株ST7菌株帶有兩種大小的質體，其他相同ST的菌株帶有相似大小的質體，經由接合作用證實1株ST88的質體可轉移至RN2677菌株中。進一步解序 (pnBS7-1、pnBS59-2、pnBS88-1、pnBS398-1、pnBS2592-1)，發現三種新結構。本研究首度顯示台灣金黃色葡萄球菌出現源自E. faecium並與VRSA抗藥結構有相同轉位酶基因的Tn6636，此結構具二類抗藥基因︰抗紅黴素 (erythromycin) ermB、及抗卡納黴素 (kanamycin)、抗慶大黴素 (gentamicin) 的aacA-aphD。Tn6636位於多種不同結構且具傳遞性的質體上，分佈於不同ST菌株中，此結構的出現非常值得注意及重視。

關鍵字

金黃色葡萄球菌 ； 紅黴素抗藥 ； Tn6636 ； 質體

並列摘要

Staphylococcus aureus is a worldwide pathogen. Antibiotic resistance in S. aureus is a serious problem. Recently, our group reported the most frequent erythromycin resistance genes in blood-isolated methicillin-susceptible S. aureus (MSSA) is ermB in Taiwan. Although most ermB-positive MSSA carried mobile element structure (MES)-like structures, which has been previously reported in sequence type 59 (ST59) methicillin-resistant S. aureus (MRSA), we also identified a novel structure of Enterococcus faecium-originated ermB-positive Tn6636 in MSSA and MRSA. The transposase gene tnp of Tn6636 is identical to Tn1546. Since vancomycin-resistant S. aureus (VRSA) is due to acquisition of the vanA operon, carried by transposon Tn1546, from Enterococcus spp., the emergence of Tn6636 in S. aureus is alarming. In the present study, we examined ermB-carried 112 MSSA and 224 MRSA in blood-isolated during a 17-year period, 2000 to 2016. The results showed 10 MSSA and 10 MRSA carrying tnp gene of Tn6636. There were four ST types, ST7 (n=6), ST5 (n=3) and ST59 (n=1) in MSSA. Two ST types, ST188 (n=8) and ST965 (n=2) were found in Tn6636-carrying MRSA. PCR mapping showed that the above-described 10 MSSA and 10 MRSA carried similar structure of Tn6636. The results of S1-PFGE with Southern blot showed the Tn6636 of above-described 10 MSSA and 10 MRSA located on plasmids with estimated sizes ranging from 23.1 to 48.5 kb. Isolates of the same ST harbored similar size of plasmids. The conjugation test showed plasmids of ST5 MSSA, ST7 MSSA, ST59 MSSA and ST965 MRSA could transfer to recipient strain RN2677. We used inverse PCR and long and accurate (LA) PCR to determine the sequence of the entire plasmids in each ST. The pBS5-3 of ST5 MSSA is a mosaic plasmid with the 14-kb Tn6636 inserted into the 27-kb pWBG744 of ST5 MSSA. The pBS7-2 of ST7 MSSA and pBR188-6 of ST188 MRSA were mosaic plasmids, the Tn6636 inserted into the 20-kb pSaa6159 of ST93 MRSA. The Tn6636 of pBR965-1 of ST965 MRSA inserted into the 25-kb pN315 of ST5 MRSA. The 47-kb pBS59-1 of ST59 MSSA was a unique plasmid. There was no nucleotide sequence in the NCBI database homologous to pBS59-1 except the region of Tn6636. Only amino acids sequence was available in the NCBI database. In addition, we also screened the presence of Tn6636 in 230 erythromycin-resistant MSSA from non-blood specimens. During two collection periods, April to June in 2016 and Aug 2017 to Feb in 2018 of 33 ermB-positive isolates, there were 11 isolates harboring Tn6636. The 11 Tn6636 carrying isolates belonged to six ST types, ST7 (n=3), ST59 (n=2), ST88 (n=2), ST188 (n=2), ST398 (n=1) and ST2592 (n=1). The results of S1-PFGE with Southern blot showed the Tn6636 of 11 MSSA located on plasmids. There were two sizes of plasmid in three ST7 isolates. One plasmid of ST88 isolate could be transferred to recipient strain RN2677. The entire plasmids of pnBS7-1, pnBS59-2, pnBS88-1, pnBS398-1 and pnBS2592-1 were determined by PCR mapping, inverse PCR and LA-PCR. There were three novel Tn6636-encoding plasmids. This is the first report that E. faecium-originated Tn6636 is present in S. aureus. This Tn6636 harboring erythromycin resistance gene ermB and gentamicin and kanamycin resistance gene aacA-aphD. This Tn6636 located on mobile plasmids. Bacteria could improve the drug resistance by horizontal gene transfer. The emergence of the novel Tn6636 needs to pay close attention.

並列關鍵字

Staphylococcus aureus ； erythromycin ； Tn6636 ； ermB ； plasmid

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