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標題: | 北台灣銀色葡萄球菌臨床菌株之分子異質性 Molecular Heterogeneity of Staphylococcus argenteus Clinical Isolates in Northern Taiwan |
作者: | Jia-Chuan Hsu 許家銓 |
指導教授: | 鄧麗珍(Lee-Jene Teng) |
關鍵字: | 銀色葡萄球菌,基因分型,表現型分型,全基因定序, Staphylococcus argenteus,genotyping,phenotyping,whole genome sequencing, |
出版年 : | 2021 |
學位: | 博士 |
摘要: | 銀色葡萄球菌(Staphylococcus argenteus),原是來自無色素之金黃色葡萄球菌(Staphylococcus aureus),其特點為無色素基因(crtOPQMN)而呈現白色菌落,2015年被重新正式命名。由於近年S. argenteus臨床分離菌株明顯增加,但大家對此菌的特性仍不是很了解,因此本研究進行北台灣某醫學中心自臨床血液檢體分離出之S. argenteus 菌株之分子分型及表現型分析,並選取20株進行全基因定序,以探討菌株特性及適應力可能機制。 依MLST分型,有ST2250 (72株)、ST2793 (12株)、ST1223 (10株)及ST2198 (2株)四型。葡萄球菌蛋白A基因(spa)則以ST2250的t5078 (55株)佔最大宗,亦發現多達11種新spa型別,其中8株ST2793帶有新型t19483。輔助基因調節基因(agr)分別為第I型(ST2250)、第III型(ST1223與ST2793)及第IV型(ST2198)。進行PFGE、dnaJ及groEL基因序列分析,演化圖顯示相同ST菌株之基因型也較接近。凝固酶基因(coa)分屬4種基因型。 在分子異質性部分,CRISPR/Cas基因僅發現於72株ST2250,其重複序列(direct repeat, DR)及間隔序列(spacer)具有多樣性,較特別的是所有12株ST2793均帶有膠原蛋白結合基因(cna),其他ST則全無此基因,僅有的2株ST2198則帶有骨橋蛋白基因(bone sialoprotein-binding protein, bbp)基因。8株ST2793帶有腸毒素C基因(sec),未有文獻發表。所有10株ST1223帶有腸毒素基因群(enterotoxin gene cluster, egc),亦發現4株(3株ST1223與1株ST2198)帶有腸毒素B基因(seb)。MSCRAMMs (Microbial surface components recognizing adhesive matrix molecules)各種吸附因子基因的分析結果顯示,S. argenteus具生物膜生成及吸附相關的大多數基因(如eno、ebpS、fnbA、fnbB、fib、clfA及clfB)。 表現型分析顯示所有96株S. argenteus均為methicillin-susceptible (MS),且僅對非常少數之抗生素具抗藥性,大多數菌株具青黴素(benzylpenicillin)及部分菌株具四環素(tetracycline)抗藥性。生化分析結果發現N-acetyl-D-glucosamine (NAG)、urea及D-ribose等試驗或部分脂肪酸之比例似可輔助傳統生化鑑定。另進行線蟲試驗、H2O2氧化壓力測試、全血殺菌能力測試及生物膜(biofilm)生成能力測試等,結果顯示S. argenteus具生物膜生成能力且部分菌株生長速度稍慢,恐與最近多篇文獻報導之慢行感染相關。 選取20菌株(ST2250 4株、ST2793全部12株、ST1223 2株及ST2198全部2株)進行全基因定序(whole genome sequencing, WGS),以比較不同ST之差異或特殊基因標誌,另一方面可確認前述各種PCR分子檢驗結果之正確性,發現可能有少部分之基因特異性引子須重新設計。亦確認ST2793帶有之sec位於vSa基因島上,ST1223帶有之egc位於vSa基因島,seb位於致病島(SaPI)。在其他基因的分析比對上,發現所有20株帶有完整或部份第VII型分泌系統,各種ST型別在vSa基因島上帶有數量不一之脂蛋白基因(lpl)及超抗原蛋白基因(ssl),亦發現各種毒力因子散佈於各型,例如:selw僅發現於ST1223、selx發現於所有型別、sely則僅在ST2250;vSa基因於ST2250帶有sel26(seq)、sel27(sec-bovine)、bsaG(bacitracin相關基因)及lukDE,而在ST2793帶有hyaluronate lyase、bsaG及lukDE。ST1223之跳耀基因(transposon, Tn)帶有sagB(streptolysin S相關基因)亦未有文獻報告於S. argenteus,18株(90%)有嗜菌體Sa3嵌入並帶有scn、chp或sak基因。17株(85%)帶有質體,大致分成6種樣態,分別帶有不同rep、blaZ、tet(L)、aph、czcD或cadD等基因。 本研究發現北台灣96株S. argenteus 之四種ST各有其獨特的基因型,部分型別帶有之特殊毒力基因亦尚未有文獻報告。但基因型與表現型關聯仍不清楚,有待未來進一步深入研究及探討。 Staphylococcus argenteus, a newly species renamed from a branching and divergent lineage of S. aureus displaying white colonies due to lacking crtOPQMN gene for the production of staphyloxanthin. The increasing worldwide occurrence of S. argenteus is a considerable concern. However, knowledge about its genomic characteristics or virulence is limited. In the present study, we investigate genotyping and phenotyping for 96 S. argenteus isolated from northern Taiwan. Whole genome sequencing from 20 selected isolates was also performed. Multilocus sequence typing (MLST) identified four sequence types (ST2250, ST2793, ST1223, and ST2198 with 72, 12, 10, and 2 isolates, respectively). The majority of spa (staphylococcal protein A gene) type was t5078 in ST2250. A total of 11 novel spa types were found. All 12 ST2793 isolates harboured newly spa types, in which eight belonged to t19483. The ST2250, ST2793, ST1223 and ST2198 belonged to agr type I, III, III, and IV, respectively, and displayed four staphylocoagulase (coa) types. The CRISPR/Cas gene was solely found in ST2250. Collagen binding protein gene (cna) was exclusively detected in all ST2793, and eight of them carried enterotoxin type C gene (sec) which was located in vSa genomic island. Two ST2198 carried bbp gene. All of ten ST1223 uniquely harboured enterotoxin gene cluster (egc) located in vSa genomic island. The enterotoxin type B gene (seb) in SaPI was found in three ST1223 isolates and one ST2198 isolate. Detection of MSCRAMMs (Microbial surface components recognizing adhesive matrix molecules) by PCR displayed common carriage of adhesion and biofilm-related genes, which may be linked to chronic infections. Phenotypic characteristics indicated all S. argenteus are methicillin-susceptible (MS). Most isolates were positive for N-acetyl-D-glucosamine (NAG), urea, or D-ribose tests. A slight difference in cellular fatty acid composition among four STs was observed. In addition, we performed C. elegans pathogenicity assay, H2O2 oxidative stress study, whole blood bactericidal activities assay, and biofilm formation test for selected isolates. WGS from 20 selected S. argenteus isolates including ST2250 (n = 4), ST2793 (n = 12), ST1223 (n = 2), and ST2198 (n = 2) was performed. All isolates harboured a complete or partial type VII secretion system and staphylococcal enterotoxin-like X gene (selx). The lipoprotein genes (lpl) and super-antigen-like protein genes (ssl) were located in vSa genomic islands. Each ST or isolate may display its native specialty or acquired genes by phage integration. For example, selw or sely was exclusively detected in ST1223 or ST2250. The vSa genomic island of ST2250 harboured sel26 (seq), sel27 (sec-bovine), bsaG (bacitracin related genes), and lukDE while that of ST2793 possessed hyaluronate lyase, bsaG and lukDE. The sagB (streptolysin S related genes) located in transposon (Tn) in the ST1223 lineage was first found in S. argenteus. The 18 isolates (90%) harboured Sa3 which was mainly consisted of scn, chp, or sak genes. Most isolates (85%, 17 out of 20) born one plasmid, which can be classified into six patterns. In conclusion, each ST of S. argenteus isolates in northern Taiwan carried specific genetic markers, in which some have not been previously reported. Our study unveils the molecular heterogeneity of S. argenteus. However, the correlation of genotypes and phenotypes is not clear, which needs further studies. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74894 |
DOI: | 10.6342/NTU202100303 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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