- 學位論文
首次發現台灣腸球菌因IS1216V截入慶大黴素抗藥基因aacA-aphD使其高度抗藥性消失
Novel IS1216V insertion into aacA-aphD leading to low-level resistance of enterococci to gentamicin in Taiwan
摘要
腸球菌 (Enterococcus species)為革蘭氏陽性球菌，是腸胃道中的正常菌叢，但也是造成社區型或醫療照護相關感染的病原菌，其中，糞腸球菌 (Enterococcus faecalis)和屎腸球菌 (Enterococcus faecium)為最常見的腸球菌臨床分離菌種。對於腸球菌相關感染，一般治療方法為複合性療法，由β-內醯胺類抗生素 (β-lactam antibiotics)搭配胺基醣苷類抗生素 (aminoglycosides)，例如: 盤尼西林 (penicillin)和慶大黴素 (gentamicin)，然而當腸球菌獲得aminoglycosides的抗藥基因 (如aacA-aphD)，將對於胺基醣苷類抗生素從原本先天低度抗藥性 (intrinsic low-level resistance)轉為後天高度抗藥性 (acquired high-level resistance)，進而影響臨床治療方針。 本研究中共分析149株臨床腸球菌，菌株皆來自2014年的高雄醫學大學附設醫院。發現其中17株菌株，分別為兩株E. faecalis和15株E. faecium，雖然在聚合酶連鎖反應 (PCR)檢測下具有抗藥基因aacA-aphD，但卻無對於慶大黴素的高度抗藥性表現。且這17株菌株經南方墨點法使用aacA-aphD探針進行核酸雜交實驗，推測菌株可能只具有單一抗藥基因aacA-aphD。 透過桑格定序 (Sanger sequencing)和次世代定序 (Illumina Miseq)來了解抗藥基因aacA-aphD周圍序列變化，發現除了其中一株E. faecalis之外，其餘16株菌株其抗藥基因aacA-aphD皆發生被IS1216V破壞的情形。根據IS1216V截斷aacA-aphD的位置可將16株菌株分成三大群: 第一群為僅有一株的E. faecalis，其aacA-aphD被IS1216V截斷在位置139的核甘酸 (nucleotide)；第二群為八株E. faecium，其aacA-aphD被IS1216V截斷在位置129的核甘酸，並且其中四株菌株的IS1216V被IS3截斷在位置578的核甘酸；第三群為七株E. faecium，其aacA-aphD被IS1216V截斷在位置337的核甘酸，並且其中六株菌株的aacA-aphD有額外被ISEF1截斷在位置764的核甘酸。 透過细菌多位點序列分型 (Multilocus sequence typing)和脈衝場凝膠電泳 (pulsed field gel electrophoresis)分析菌株分型，並發現其結果與上述的基因分群有高度相似性。 最後，透過比較對慶大黴素高度抗藥或低度抗藥兩種腸球菌的生長趨勢，來分析菌種適應性高低，並且利用公式計算對慶大黴素高度抗藥之腸球菌失去慶大黴素高度抗藥性的機率。我們發現雖然慶大黴素高度抗藥或低度抗藥兩種腸球菌的生長趨勢無明顯差異，但慶大黴素高度抗藥之腸球菌在長期於無抗生素環境培養下，因同源重組(homologous recombination)失去慶大黴素高度抗藥性的機率為1.127×10-4。 我們推論台灣近年臨床的胺基醣苷類抗生素使用量下降可能與腸球菌的抗藥基因aacA-aphD被IS1216V破壞有關。
並列摘要
Enterococcus species are Gram-positive commensal organisms in gastrointestinal tract and pathogen causing community- and healthcare-associated infections. Among the enterococcal clinical samples, E. faecalis and E. faecium are the most commonly isolated species. General treatment of enterococcal infections is combined therapy with a cell wall-active agent (e.g. penicillin) and an aminoglycoside (e.g. gentamicin). When enterococci acquire aminoglycosides resistance gene such as aacA-aphD, however, the aminoglycosides resistance would change from low-level (intrinsic low-level resistance) to high-level (acquired high-level resistance), challenging the treatment strategies. In the study, we analysed 149 clinical enterococcal isolates collected from Kaohsiung Medical University Hospital in 2014, but 17 strains (2 E. faecalis and 15 E. faecium) had aacA-aphD confirmed by PCR without high-level gentamicin resistance. The 17 strains had a single copy of aacA-aphD proved by Southern blot hybridization with an aacA-aphD probe. Adjacent sequences of aacA-aphD were determined by Sanger sequencing and Illumina Miseq technique. Except one E. faecalis strain, aacA-aphD of the other 16 strains had been disrupted by IS1216V. Based on the insertion site of IS1216V, 16 strains were divided into three types. Type I (n=1) had IS1216V insertion into the 139 nucleotide (nt) of aacA-aphD in the only one E. faecalis. Type II (n=8) had IS1216V insertion into 129 nt of aacA-aphD; and four strains had a second insertion of IS3 into 578 nt of IS1216V. Type III (n=7) had IS1216V insertion into 337 nt of aacA-aphD; six strains had a second insertion of ISEf1 into 764 nt of aacA-aphD. Molecular typing was achieved by multilocus sequence typing and pulsed-filed gel electrophoresis. The type of IS1216V insertion into aacA-aphD was highly associated with genetic relatedness. Finally, ecological fitness was assessed by the comparison of growth curve between high-level gentamicin-resistant and low-level gentamicin-resistant enterococci, and the probability of loss of high-level gentamicin resistance in enterococci was evaluated. Although no significant difference in growth rate, the high possibility (1.127×10-4) of enterococci to disrupt aacA-aphD by homologous recombination in the non-antibiotic environment was confirmed. We concluded that decreased usage of aminoglycosides in Taiwan in recent years might be related to aacA-aphD disruption by IS1216V in enterococci.