- 學位論文
畜產相關抗藥性金黃色葡萄球菌分子特性與親緣動態
Molecular Characterization and Phylodynamics of Livestock-associated Methicillin-Resistant Staphylococcus aureus
摘要
畜產相關型抗藥性金黃色葡萄球菌 (livestock-associated methicillin-resistant Staphlococcus aureus, LA-MRSA)自2003年首度於歐洲被發現後已成為一新興的人畜共通傳染病病原。本研究之目的在於探討由臺灣肥育豬隻及畜產環境所分離之LA-MRSA的分子特性及其親緣動態。本研究分四部份，研究目的分別為: (1) 比較MRSA相關抗藥性基因在都市廢水及豬隻屠宰廢水的動態分布，並比較由都市廢水與豬隻屠宰廢水所分離出MRSA的分子與表現型特性；(2) 比較臺灣LA-MRSA ST9與人類臨床MRSA ST9的毒性因子與毒素基因圖譜及親緣關係；(3) 比較由豬隻與豬隻屠宰廢水所分離出臺灣LA-MRSA的微生物黏附分子分布及生物膜之生成能力及(4) 分析臺灣LA-MRSA在臺灣的族群動態與分布。 針對研究目的(1)，本研究取樣臺灣第二大肉品市場附設廢水處理廠各處理單元的廢水，另取樣臺灣最大都市二級污水處理廠的廢水作為比較樣本。總計96個廢水樣本經離心濃縮後以即時定量聚合酵素連鎖反應定量抗藥性基因(mecA, qacEΔ1與intI1)，並分析113 株MRSA分離株(都市廢水27株與豬隻屠宰廢水86株)的抗藥性、最小抑菌濃度及Staphylococcal cassette chromosome mec (SCCmec)分型。研究目的(2)偵測152株MRSA ST9 (豬隻來源147株與人類臨床菌株5株)的葡萄球菌附屬基因調控系統(accessory gene regulator, agr)、20種葡萄球菌腸毒素基因(staphylococcal enterotoxin, se)、脫皮毒素基因(exfoliatoxin gene)與毒性休克症候群毒素(toxic shock syndrome toxin-1, TSST-1)的分布，並以分子親緣法分析臺灣MRSA ST9葡萄球菌凝固酵素(Staphylocoagulase, SC)之親緣關係。研究目的(3) 比較233株不同來源LA-MRSA分離株 (豬隻鼻腔拭子147株與豬隻屠宰廢水來源86株)的生物膜生成基因，10種與移生及感染相關的MRSA黏附分子的分布及比較不同來源菌株的生物膜生成能力。最後，本研究以140株MRSA 的SCCmec-associated direct repeat unit (dru)建構來自不同地理區，包括西臺灣(96株,包括人類臨床5株)、東臺灣中部(22株)與澎湖(22株)LA-MRSA族群的遺傳結構，以最短距離網狀圖推估可能的祖先族群，同時利用貝氏天際線法推估臺灣LA-MRSA過往的演化動態。 研究結果顯示，與MRSA有關的抗藥性基因廣泛且穩定存在於豬隻屠宰廢水與都市廢水(6.1–10.8 log10 copies/mL)中，進一步分析發現超過60%由豬隻屠宰廢水所分離之LA-MRSA帶有四級胺消毒劑抗性基因(qacEΔ1)且為多重抗藥性菌株，並對多種抗菌劑呈現高濃度的最小抑菌濃度。此外，都市廢水分離株的菌株分型為SCCmec II, III, IV 與 VII，豬隻屠宰廢水的分離株則屬於SCCmec V與IX。毒性基因分析發現臺灣LA-MRSA同時帶有第二型腸毒素基因簇與毒性休克症候群基因，以及屬於新型的重組凝固酵素SC XIc，並且與人類臨床菌株在毒性因子圖譜上具有高度的相似性。從豬隻鼻腔或是從豬隻屠宰廢水所分離的臺灣LA-MRSA皆攜帶多種MRSA黏附分子，並且豬隻鼻腔分離株的生物膜生成能力顯著高於豬隻屠宰廢水的分離株。最後，族群遺傳分析顯示臺灣LA-MRSA ST9的亞族群中總計有28種dru 型 (其中24型為本研究發現)，不同的族群間有個別分化的現象。依據最短距離網狀圖及結合分離株地理資料發現，臺灣LA-MRSA ST9族群存在兩個祖先型。貝氏天際線顯示臺灣LA-MRSA ST9具有穩定的豬隻宿主族群。 本研究證實臺灣LA-MRSA及其抗藥性基因廣泛分布於豬隻屠宰廢水，並攜帶多種毒性因子及具有生物膜的生成能力，顯示此新型態的MRSA具有潛在的公共衛生威脅。親緣關係與族群遺傳分析顯示，臺灣LA-MRSA具有穩定的豬隻宿主族群且可能已演化出地域性的亞族群，顯示此LA-MRSA在地化發展的趨勢，因此有必要持續進行監測以瞭解此菌的演化與其對人類社群的可能衝擊。
並列摘要
Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has emerged as a potential zoonotic pathogen since it was first identified in Europe in 2003. The overall objective of this dissertation was to investigate the molecular characteristics and phylodynamics of Taiwan LA-MRSA isolated from finishing pig and livestock environments. The specific aims were: (1) to compare the dynamics of MRSA-related resistance genes and the genotypic and phenotypic characteristics of MRSA isolates in municipal and swine wastewater; (2) to investigate and compare the virulence factors, toxic gene profiles, and phylogenetic relationships between Taiwan LA-MRSA ST9 and human clinical ST9; (3) to compare the presence of adhesion factors in LA-MRSA populations isolated directly from pigs and from swine slaughterhouse wastewater, and to compare their abilities to produce biofilms and (4) to investigate the effects of the LA-MRSA Taiwan clone on the population dynamics. For aim (1), the concentrations of resistance genes (mecA, intI1, and qacEΔ1) were quantified using real-time quantitative polymerase chain reaction (real-time qPCR) in 72 samples taken from a swine slaughterhouse (the second largest swine auction market for pigs ready to slaughter in Taiwan) and 24 samples from the largest secondary municipal wastewater treatment plant in Taiwan. One hundred and thirteen MRSA isolates (86 from municipal wastewater and 27 from swine slaughterhouse wastewater) were recovered and were characterized by antimicrobial susceptibility testing, minimum inhibitory concentrations (MICs), and staphylococcal cassette chromosome mec (SCCmec) typing. For aim (2), a total of 152 MRSA ST9 isolates, including 147 LA-MRSA isolates and 5 human clinical isolates, were screened for the presence of the accessory gene regulator (agr) and 20 enterotoxin genes (se), the exfoliatoxin gene, and toxic shock syndrome toxin-1 (TSST-1) gene. The evolutionary history of staphylocoagulase (SC) in Taiwan MRSA ST9 was reconstructed using phylogenetic methods. For aim (3), this study investigated the dynamics of biofilm formation gene, 10 microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) - MRSA infection and colonization, and biofilm expression in MRSA from 147 asymptomatic nasal swine isolates and 86 swine slaughterhouse wastewater isolates. Finally, 140 MRSA (135 piggery and 5 human clinical) isolates which collected from 3 populations of western Taiwan, central eastern Taiwan, and Penghu Island were used to reconstruct the population structure by analysis of the mec-associated direct repeat unit (dru). The ancestral population dynamics of the dru type were estimated by constructing a minimum spanning network (MSN). Past population dynamics of LA-MRSA isolates were estimated using a Bayesian skyline plot (BSP) model. The results revealed that resistance genes are highly abundant throughout the year in swine slaughterhouse wastewater (6.1–10.8 log10 copies/mL). More than 60% of the MRSA isolates from swine slaughterhouse wastewater carried the antiseptic resistance gene (qacE△1) with multidrug resistance (MDR) and high MICs. The municipal MRSA isolates harbored SCCmec II, III, IV, and VII, whereas those isolates from the swine slaughterhouse harbored only SCCmec V and IX. Most Taiwan LA-MRSA ST9 harbored agr-II with a combination of the enterotoxin gene cluster (egc)-2 and the tst gene. Further, Taiwan LA-MRSA ST9 and human clinical MRSA ST9 belonged to a novel SC XIc subtype with similar genetic toxin profiles. Taiwan LA-MRSA isolates from swine and swine slaughterhouse wastewater carried many MSCRAMMs. Biofilm production was higher in piggery nasal swab samples than in swine-related wastewater samples. Finally, population genetic analyses identified 28 dru types (24 novel types) and 15 dru-Clonal Complexes (CCs) in Taiwan LA-MRSA ST9 subpopulation. MSN and analysis of the genetic structure of sub-populations revealed that dru types in Taiwan LA-MRSA ST9 had at least of two origins. BSP analysis indicated that MRSA ST9 is a stable population with a long evolutionary history in Taiwan. In conclusions, Taiwan LA-MRSA and its resistance genes are widely distributed in swine populations and swine-related environments. These Taiwan LA-MRSA isolates carry several virulence factors and are able to form biofilm- pose potential threat to public health. Phylogenetic and population genetic analyses indicate a large and stable population of LA-MRSA clone and may have evolved unique regional subpopulations in Taiwan. Further epidemiological and evolutionary studies of these novel LA-MRSA are needed in order to assess future public health implications.