本文關鍵詞： 單核細胞增生李斯特菌 數(shù)值鑒定系統(tǒng) 污染調查 遺傳多樣性 蛋白質組　出處：《華南理工大學》2016年博士論文　論文類型：學位論文

【摘要】：李斯特菌屬(Listeria spp.)為革蘭氏陽性菌,兼性厭氧。目前,該屬包含了16個種和2個亞種,其中6種李斯特菌與食品相關。單增細胞增生李斯特菌(Listeria monocytogenes)是李斯特菌屬最具危害的一種食源性致病菌,它對人有致病性,能引起人和動物患腦膜炎、敗血癥、流產(chǎn)和局部的膿性損傷,造成孕婦流產(chǎn)、死胎等疾病,發(fā)病者死亡率可達20%~30%,被世界衛(wèi)生組織定義為全球四大食源性病原菌之一。本論文基于細菌數(shù)值鑒定法,結合計算機輔助技術,自主研發(fā)了食源性李斯特菌數(shù)值鑒定系統(tǒng)(Numide-Listeria v1.0)。在此基礎上,系統(tǒng)調查全國不同地區(qū)不同類型的食品中單增李斯特菌的分布情況,在驗證本鑒定系統(tǒng)的同時,進一步研究了單增李斯特菌分離株的遺傳多樣性、血清型、毒力現(xiàn)狀和耐藥性等遺傳特性。此外,本論文還通過iTRAQ蛋白組學技術建立了李斯特菌全菌蛋白數(shù)據(jù)庫,為尋找種間差異物質,進一步完善鑒定系統(tǒng)提供了依據(jù)。本論文的主要內容和研究結果如下:1.利用李斯特菌種間生化反應的差異,篩選了11種不同于目前市面上同等產(chǎn)品的生化試驗組合。對本實驗室前期分離到近2000株李斯特菌進行相關實驗,獲得各生化反應陽性率,建立了這11種生化反應陽性率數(shù)據(jù)庫。基于細菌數(shù)值鑒定法的原理,運用C++語言等計算機技術編寫相關軟件,初步構建了食源性李斯特菌數(shù)值鑒定系統(tǒng)(Numide-Listeria v1.0),可有效區(qū)分六種食源性李斯特菌。2.對1400份零售食品進行了全國單增李斯特菌污染調查研究,共檢出232份陽性樣品,平均污染率16.6%,平均污染水平15.72 MPN/g。七大類食品的檢出率從高到低依次為:速凍食品(44.9%)食用菌(23.2%)肉與肉制品(20.9%)即食食品(8.2%)水產(chǎn)品(6.4%)蔬菜(5.7%)乳制品(2.4%)。從采樣城市來看,北京市的污染率最高,為34%,污染水平也達到了42.7 MPN/g。以長江為界,北方幾大城市的整體污染率和污染水平要顯著高于南方地區(qū)。同時,對分離的880株疑似李斯特菌分別進行了MID-67、Numide-Listeria v1.0和分子鑒定,分子鑒定顯示99.8%的菌株含有種特異性基因,而這其中99.3%的單增李斯特菌、96.3%的英諾克李斯特菌、100%的格式李斯特菌、100%的西式李斯特菌、100%的綿羊李斯特菌和80%威爾士李斯特菌顯示兩種鑒定系統(tǒng)結果一致,而本論文研制的Numide-Listeria v1.0在鑒定單增李斯特菌和英諾克李斯特菌方面表現(xiàn)出優(yōu)于MID-67的能力。3.對全國食品污染調查中分離的單增李斯特菌分離株進行了遺傳多樣性和毒力特性等研究。耐藥菌株普遍存在,僅32.5%的即食食品分離株和23.8%的生鮮分離株對所選的14種抗生素全部敏感,30.8%的菌株為多重耐藥菌株,其中13株分離株對10種以上抗生素具有耐藥性。菌株在血清型、ERIC-PCR、REP-PCR甚至是耐藥性上均有著一定的關聯(lián)性,表現(xiàn)出相似的遺傳關系。本研究中選取的毒力基因,除llsX是Lineage I菌株所特有的毒力基因,大部分的菌株均攜帶有hly(100%)、inlA(99.7%)、inlB(99.1%)、inlC(98.5%)、inlJ(99.4%)。Epidemic Clones(ECs)作為食源性李斯特菌病流行暴發(fā)的可能標志物,也在26.3%的即食食品分離株和6.7%的生鮮分離株中檢測到。對即食食品分離株進行分子遺傳特性研究,也進一步詮釋了相關菌株的毒力現(xiàn)狀。MLST分型結果顯示除新發(fā)現(xiàn)的ST型外(ST804,ST805,ST806,ST807),其余ST型均在臨床菌株和李斯特菌病的暴發(fā)中出現(xiàn)過;毒力基因inlA的全長測序,顯示91.4%的菌株具有編碼InlA全長蛋白并侵染細胞的能力,發(fā)現(xiàn)的3個提前終止密碼子(PMSC),其中1個型為首次出現(xiàn),位于326號氨基酸位置(GAA→TAA)。4.運用iTRAQ蛋白組學技術,獲得了L.monocytogenes ATCC19115和L.innocua ATCC33090全菌蛋白圖譜。通過LC-MS/MS技術,分別在單增李斯特菌和英諾克李斯特菌中鑒定出1650和1732個可信蛋白。經(jīng)蛋白豐度比分析后,鑒定出121個獨有蛋白(包括49個單增李斯特菌特有蛋白和72個英諾克李斯特菌特有蛋白)。對這些差異蛋白進行Gene Ontology(GO)分析,發(fā)現(xiàn)它們主要參與了兩種李斯特菌的細胞內碳氮化合物的生物合成、分子轉運、糖類運輸和代謝等生物學途徑。進一步的生物學功能分析,發(fā)現(xiàn)大部分(75%)蛋白為功能未知的假設蛋白,其余則涉及酶、DNA結合蛋白和離子結合蛋白等相關蛋白。綜上所述,本論文始終圍繞食源性單增李斯特菌進行研究,構建了食源性李斯特菌數(shù)值鑒定系統(tǒng)(Numide-Listeria v1.0),首次全面掌握了我國不同地區(qū)不同類型食品中單增李斯特菌的污染情況和分布規(guī)律;建立在此基礎上的單增李斯特菌遺傳多樣性和毒力特征研究,為我們進一步了解該菌在我國的流行趨勢和風險識別提供了堅實的基礎。通過比較蛋白組學獲得的種間特異性酶,對于進一步研發(fā)特異性的酶底物有效區(qū)分有害和無害李斯特菌提供了依據(jù)。此外,其他獨有蛋白的發(fā)現(xiàn),也為研究單增李斯特菌的生物學功能提供了基礎和依據(jù)。
[Abstract]:Lester (Listeria spp.) is a genus of gram positive bacteria, facultative anaerobic. At present, the genus contains 16 species and 2 subspecies, including 6 species of bacteria associated with food. Lester List Rand Listeria cell proliferation (Listeria monocytogenes) is a genus of Lester most foodborne pathogen, it has pathogenicity of human and animal, can cause meningitis, septicemia, abortion and purulent partial injury, cause miscarriage, stillbirth and other diseases, the incidence rate of up to 20%~30%, the WHO is defined as the world's four largest food source pathogenic bacteria of venereal disease. The bacteria identification method based on the combination of numerical and computer aided technology independent research and development of food borne bacteria Lester numerical identification system (Numide-Listeria v1.0). On this basis, the distribution of Listeria bacteria Lester of different types of food in different regions of the country in the survey system, verify the The identification system at the same time, further research on Listeria isolates from Lester genetic diversity, serotype, virulence and drug resistance status of genetic characteristics. In addition, this paper also through iTRAQ proteomics technology established Lester bacteria whole cell protein database, the difference of material for species, provide a basis to further improve the identification system. The main content of this thesis and the research results are as follows: 1. by Lester strains biochemical differences, screened 11 different from the currently available on the same products. The combination of biochemical tests on laboratory isolated 2000 strains of bacteria in Lester related experiments to obtain the positive rate of each of these 11 biochemical reactions. Biochemical reactions and the positive rate of bacteria identification database. Numerical method based on the principle, the use of C++ language programming and computer technology, constructs a foodborne bacteria Lester numerical discrimination The system (Numide-Listeria v1.0), which can effectively distinguish between six kinds of food borne bacteria Lester.2. for Listeria contamination of Lester national survey of 1400 retail food, were detected in 232 positive samples, the average pollution rate was 16.6%, the average pollution level of 15.72 MPN/g. seven kinds of food detection rate from high to low are: frozen food (44.9%) edible fungus (23.2%) meat and meat products (20.9%) instant food (8.2%) aquatic products (6.4%) and vegetables (5.7%) dairy products (2.4%). From the sampling of pollution in the city of Beijing City, the highest rate is 34%, the pollution level has reached 42.7 MPN/g. is bounded by the Yangtze River, the whole the contamination rate and the pollution level in several northern city was significantly higher than in the south. At the same time, the separation of the 880 strains of suspected Lester bacteria were analyzed by MID-67, v1.0 and Numide-Listeria molecular identification, molecular identification showed that 99.8% strains contain a specific gene, and this 鍏朵腑99.3%鐨勫崟澧炴潕鏂壒鑿，

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