本文選題：奇異變形桿菌 + 毒力因子�。� 參考：《南方醫(yī)科大學(xué)》2016年博士論文

【摘要】：奇異變形桿菌(Proteus mirabilis)是一種能引起泌尿系統(tǒng)感染的常見(jiàn)致病菌,但該菌已知的毒力基因與腹瀉關(guān)系尚不明確,如何引起腹瀉尚無(wú)定論。目前國(guó)外針對(duì)奇異變形桿菌的研究主要集中于引起泌尿系統(tǒng)感染的菌株中,對(duì)耐藥研究較為多,但對(duì)奇異變形桿菌致腹瀉的相關(guān)機(jī)制研究,Pubmed收錄的論文很少。我們?cè)诓糠质澄镏卸臼录?從病人腹瀉標(biāo)本和剩余食品中都分離到奇異變形桿菌,未分離到其他致病菌。同一批次分離株皆具有相同的脈沖場(chǎng)凝膠電泳(PFGE)型別,感染小鼠后能造成腹瀉或死亡。而從健康人群與外環(huán)境分離的菌株不引起小鼠出現(xiàn)以上癥狀,其PFGE圖譜間差異較大。由此提出假設(shè),除了已知的毒力基因外,致腹瀉的奇異變形桿菌的基因組中可能還存在著其它特異的毒力因子,是造成食物中毒的主要原因。圍繞以上線索,本文主要開(kāi)展以下幾方面的研究：(1)致腹瀉奇異變形桿菌病原學(xué)和分子生物學(xué)特征分析；(2)全基因組測(cè)序分析比較獲得僅存在于致腹瀉菌株中的特異基因；(3)在致腹瀉菌株C02011中新發(fā)現(xiàn)的Ⅳ型分泌系統(tǒng)(T4SS)毒力島編碼基因virBl-11基本特性分析；(4)構(gòu)建奇異變形桿菌C02011的virB9基因缺失株及回補(bǔ)株,鑒定其基本屬性；(5)黏附和侵襲實(shí)驗(yàn)敏感細(xì)胞株的篩選,體外細(xì)胞模型的建立；(6)體外比較野生株C02011、virB9基因缺失株及回補(bǔ)株對(duì)人結(jié)腸癌Lovo細(xì)胞的黏附和侵襲水平；(7)腹瀉動(dòng)物模型的建立和初步實(shí)驗(yàn)；(8)明確virB9基因是否影響奇異變形桿菌對(duì)小鼠的致病能力。(9)特異基因的熒光PCR診斷方法的建立與菌株篩查。方法與結(jié)果：一、致腹瀉奇異變形桿菌病原學(xué)、分子生物學(xué)特征1、選擇6株奇異變形桿菌作為實(shí)驗(yàn)研究菌株,包括2株標(biāo)準(zhǔn)菌株：ATCC29906和HI4320;2株食物中毒分離株和2株健康人群和外環(huán)境拭子分離株。2、分析比較了不同來(lái)源的奇異變形桿菌的病原學(xué)特征和分子特征,不同來(lái)源的奇異變形桿菌生化特征基本相同,且均具有常見(jiàn)的毒力基因ureC、rsmA、 hpmA和zapA。說(shuō)明這些基因沒(méi)有特異性,不能區(qū)分致病菌株和非致病菌株。3、利用PFGE方法對(duì)奇異變形桿菌分子分型,同一起食物中毒分離株(病人標(biāo)本和剩余食品分離株)的PFGE圖譜一致,說(shuō)明可能是由奇異變形桿菌引起的食物中毒。4、發(fā)現(xiàn)奇異變形桿菌中分別存在著1-3個(gè)不同大小的質(zhì)粒。最大的質(zhì)粒為10100bp左右,在所有菌株中普遍存在。而C05028和C02034中都存在著一個(gè)2683bp的小質(zhì)粒,有四個(gè)開(kāi)放讀碼框,其中645bp的qnrD基因?yàn)猷Z酮耐藥基因,與耐藥表型一致,可見(jiàn)該質(zhì)粒與菌株的耐藥性相關(guān)。5、環(huán)境分離菌株C02034攜帶的質(zhì)粒最多(3個(gè)),其耐藥性也最強(qiáng),對(duì)喹諾酮類、磺胺類、氨基糖苷類和p-內(nèi)酰胺類青霉素等均為耐藥,由此推論其攜帶的另2個(gè)質(zhì)粒也可能編碼耐藥基因,有待進(jìn)一步驗(yàn)證。6、研究表明,憑借傳統(tǒng)的生化分析及常見(jiàn)的毒力基因鑒定不能區(qū)分菌株的來(lái)源,無(wú)法確定是食物中毒菌株還是健康人群自身攜帶(或外環(huán)境中存在)的菌株；但PFGE和質(zhì)粒分析可區(qū)分菌株來(lái)源。本研究還發(fā)現(xiàn)了奇異變形桿菌菌株中普遍存在的質(zhì)粒,為奇異變形桿菌耐藥及致病性研究提供了理論依據(jù)。二、奇異變形桿菌全基因組測(cè)序和序列比對(duì)1、為了進(jìn)一步明確致腹瀉菌株中的特異基因,收集了2株典型的致腹瀉菌株(C05028和C02011),與1株健康人分離株(B02005)及1株外環(huán)境對(duì)照株(C02034)一起進(jìn)行全基因組測(cè)序。2、通過(guò)序列比對(duì)分析,發(fā)現(xiàn)了僅存在于食物中毒株而非致病菌株中不存在的特異基因有7個(gè),其功能預(yù)測(cè)結(jié)果顯示這些基因有些編碼溶血素,有些編碼DNA核苷酸轉(zhuǎn)移酶或糖基轉(zhuǎn)移酶,有些是與真核細(xì)胞結(jié)合相關(guān)的轉(zhuǎn)錄調(diào)控原件,具體的功能有待進(jìn)一步研究。這些基因?yàn)楸狙芯渴状伟l(fā)現(xiàn),未見(jiàn)相關(guān)文獻(xiàn)報(bào)道。3、在食物中毒分離株C02011中還發(fā)現(xiàn)了編碼Ⅳ型分泌系統(tǒng)(T4SS)的特異基因,在奇異變形桿菌中目前尚無(wú)相關(guān)文獻(xiàn)報(bào)道,是本課題組在食物中毒株中的首次發(fā)現(xiàn)。三、C02011中的Ⅳ型分泌系統(tǒng)(T4SS)基本特性分析針對(duì)C02011中的Ⅳ型分泌系統(tǒng)(T4SS)的11個(gè)基因virB1-virBll,通過(guò)同源比對(duì)、蛋白質(zhì)功能域、三維結(jié)構(gòu)預(yù)測(cè)、進(jìn)化樹(shù)、點(diǎn)陣圖、基因結(jié)構(gòu)預(yù)測(cè)、基因島結(jié)構(gòu)分析等,研究了整體的水平基因轉(zhuǎn)移事件的進(jìn)化關(guān)系。奇異變形桿菌C02011的T4SS主要的成分是：裂解糖基轉(zhuǎn)移酶(VirB1),能量轉(zhuǎn)移(VirB3-4, VirB11),融合通道(VirB2, VirB5-VirB10,脂蛋白),以及細(xì)胞黏附(VirB3-4)。結(jié)論：T4SS所有的11個(gè)基因在蛋白質(zhì)和核苷酸水平上與肺炎克雷伯菌PMK1和大腸埃希菌ECOR31的T4SS基因高度相似,但與8株已測(cè)序的奇異變形桿菌菌株的核苷酸不存在同源。因此可以推斷：該基因島是近期從與其密切相關(guān)的腸桿菌科微生物大腸埃希菌和肺炎克雷伯菌中水平轉(zhuǎn)移而來(lái)的。而這個(gè)事件至少發(fā)生在奇異變形桿菌C02011從其他奇異變形桿菌菌株分化之后。四、奇異變形桿菌C02011株virB9基因敲除及特性分析為了研究virB基因在奇異變形桿菌致病中的相關(guān)功能,選擇編碼分泌系統(tǒng)融合通道的virB9作為研究對(duì)象,構(gòu)建virB9基因敲除株P(guān)mi/AvirB9。利用基因同源重組的原理,通過(guò)自殺質(zhì)粒PCVD442及中間宿主SM10λpir,成功構(gòu)建了缺失virB9基因的突變株。并構(gòu)建攜帶有virB9基因的Pmi/△virB9回補(bǔ)株,為下一步研究打下基礎(chǔ)。在營(yíng)養(yǎng)豐富條件下,C02011及Pmil△virB菌體的菌落形成和生長(zhǎng)能力、生化反應(yīng)并無(wú)顯著差異。因而突變株適用于研究該基因?qū)Ω腥鞠嚓P(guān)毒力的影響。五、黏附和侵襲實(shí)驗(yàn)敏感細(xì)胞株的篩選,體外細(xì)胞模型的建立選用人結(jié)腸癌細(xì)胞(Lovo)與人結(jié)腸腺癌細(xì)胞(Caco-2)進(jìn)行體外研究,分別比對(duì)奇異變形桿菌C02011(食物中毒病人嘔吐物分離株)和B02005(健康人糞便分離株)對(duì)細(xì)胞的侵襲性與黏附性,選擇確定合適的細(xì)胞實(shí)驗(yàn)?zāi)Ｐ汀＝Y(jié)果顯示,食物中毒病人嘔吐物分離株C02011對(duì)Lovo、Caco-2細(xì)胞具有很強(qiáng)的黏附性,健康人糞便分離株B02005對(duì)Lovo、 Caco-2細(xì)胞黏附性弱,兩菌株間的黏附率差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。侵襲性實(shí)驗(yàn)也顯示了類似的結(jié)果,兩菌株對(duì)Lovo和Caco-2細(xì)胞的侵襲率差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。本研究證實(shí)分離自食物中毒伴腹瀉癥狀病人的嘔吐物中的奇異變形桿菌C02011具有很強(qiáng)的黏附力和侵襲力,在引起人和動(dòng)物腸道疾病中發(fā)揮重要的作用。人結(jié)腸癌細(xì)胞(Lovo)具有更高的敏感性,適宜于研究奇異變形桿菌對(duì)其的黏附和侵襲能力。六、virB9缺失降低了奇異變形桿菌C02011對(duì)Lovo細(xì)胞的黏附和侵襲能力體外模型采用Lovo單層細(xì)胞,與野生株C02011的黏附率相比,Pmil△virB9黏附Lovo單層細(xì)胞能力顯著降低,二者黏附率差異具有統(tǒng)計(jì)學(xué)意義(P0.001)。慶大霉素保護(hù)實(shí)驗(yàn)結(jié)果表明,突變株在侵襲水平上也表現(xiàn)出明顯的降低,二者侵襲率差異具有統(tǒng)計(jì)學(xué)意義(P0.001)。Pmil△virB9轉(zhuǎn)化入回補(bǔ)質(zhì)粒后,突變株黏附能力和侵襲能力基本得到恢復(fù)。七、腹瀉動(dòng)物模型的建立和初步實(shí)驗(yàn)為了證明部分奇異變形桿菌的確能夠引起腹瀉并具有不同的毒力,并建立合適的動(dòng)物模型,分別采用6株不同來(lái)源的奇異變形桿菌感染小鼠,觀察糞便性狀及小鼠死亡情況。在經(jīng)口灌胃實(shí)驗(yàn)中,所有菌株均未引起小鼠死亡,但食物中毒分離株引起小鼠糞便形狀改變。腹腔注射實(shí)驗(yàn)中,7小時(shí)后,從腹瀉病人嘔吐物中分離的奇異變形桿菌作用的小鼠全部死亡,且小鼠的糞便稀軟,小鼠的大腸切片染色結(jié)果也證實(shí)了病理改變。而從健康人群糞便及外環(huán)境分離的奇異變形桿菌作用的小鼠未死亡,且小鼠形態(tài)活力都正常。本研究成功建立了奇異變形桿菌的小鼠腹瀉模型,為后續(xù)毒力基因的功能研究提供了較成熟的實(shí)驗(yàn)動(dòng)物模型。本研究證明了奇異變形桿菌食物中毒分離株的毒力比健康人群及外環(huán)境分離株的毒力強(qiáng),能引起小鼠腹瀉甚至死亡,造成大腸的病理學(xué)改變。八、virB9缺失降低了奇異變形桿菌C02011對(duì)小鼠的致病能力為研究確認(rèn)virB9基因的功能,在已建立好的小鼠動(dòng)物模型上進(jìn)行體內(nèi)試驗(yàn),分別采用C02011野生株、virB9基因敲除株及回補(bǔ)株感染小鼠,觀察感染動(dòng)物后的腹瀉癥狀及病理改變,鑒別比較三者的致病能力差異。實(shí)驗(yàn)結(jié)果證明：C02011野生株和回補(bǔ)株的毒力比virB9基因敲除株的毒力強(qiáng),能引起小鼠腹瀉,導(dǎo)致腸組織含水量增多,與生理鹽水對(duì)照株相比有統(tǒng)計(jì)學(xué)差異(t=2.7764,P=0.0193),并能造成大腸的病理學(xué)改變。virB9基因敲除株未能引起小鼠腹瀉,腸組織含水量與生理鹽水對(duì)照株無(wú)統(tǒng)計(jì)學(xué)差異(t=0.2979,P=0.7783)。說(shuō)明virB9缺失降低了奇異變形桿菌C02011對(duì)小鼠的致病能力。但大腸的病理切片顯示仍有一定的炎性反應(yīng),說(shuō)明virB9基因不是該菌株的唯一的毒力基因,缺失virB9的敲除株仍具備一定的侵襲小鼠腸組織的毒力。有待進(jìn)一步實(shí)驗(yàn)證實(shí)。九、致腹瀉奇異變形桿菌熒光PCR檢測(cè)方法的建立本研究在新發(fā)現(xiàn)的特異的毒力因子基礎(chǔ)上,通過(guò)PCR篩查選取了4個(gè)在食物中毒菌株中普遍存在的毒力基因作為靶基因,采用相同標(biāo)簽輔助-無(wú)引物二聚體(Hand, Homo-Tag Assisted Non-Dimer)系統(tǒng)與改良分子信標(biāo)熒光探針,建立1管同時(shí)檢測(cè)奇異變形桿菌4種毒力基因的多重實(shí)時(shí)熒光PCR方法。所建立的針對(duì)致腹瀉奇異變形桿菌的多重?zé)晒釶CR方法可廣泛用于食物中毒或食品污染物的快速檢測(cè),區(qū)分正常人群攜帶株(外環(huán)境污染菌株)和致腹瀉菌株,簡(jiǎn)化和完善現(xiàn)有的國(guó)標(biāo)診斷方法,節(jié)省人力物力,滿足食物中毒快速準(zhǔn)確的診斷和傳染來(lái)源追蹤,減少誤判和漏檢,保障食品安全和人體健康。統(tǒng)計(jì)學(xué)分析：數(shù)據(jù)均表示為均數(shù)±標(biāo)準(zhǔn)差.兩組間的比較采用t檢驗(yàn),多重比較采用單因素方差分析。動(dòng)物實(shí)驗(yàn)陽(yáng)性率的比較采用Pearson卡方檢驗(yàn)。P0.05認(rèn)為差異有統(tǒng)計(jì)學(xué)意義,檢驗(yàn)水準(zhǔn)為a=0.05。數(shù)據(jù)分析采用SPSS 13.0軟件。結(jié)論：引起腹瀉的奇異變形桿菌與健康人群及外環(huán)境分離的奇異變形桿菌經(jīng)過(guò)全基因組測(cè)序和比對(duì)分析,發(fā)現(xiàn)了僅存在于致腹瀉菌株中的一些特異的毒力基因,尤其是發(fā)現(xiàn)了存在于腹瀉伴嘔吐的食物中毒病人分離株C02011中存在著Ⅳ型分泌系統(tǒng)(T4SS)。該T4SS所有的11個(gè)基因(virB1-11)在蛋白質(zhì)和核苷酸水平上與肺炎克雷伯菌PMK1和大腸埃希菌ECOR31的T4SS高度相似,但與8株已測(cè)序的奇異變形桿菌菌株的核苷酸不存在同源。因此推斷：該基因島是近期從與其密切相關(guān)的腸桿菌科微生物大腸埃希菌和肺炎克雷伯菌中水平轉(zhuǎn)移而來(lái)的。而這個(gè)事件至少發(fā)生在奇異變形桿菌C02011從其他奇異變形桿菌菌株分化之后。食物中毒病人嘔吐物分離株C02011對(duì)Lovo、Caco-2細(xì)胞均具有很強(qiáng)的黏附性和侵襲性,健康人糞便分離株B02005對(duì)Lovo、 Caco-2細(xì)胞黏附性和侵襲性弱,兩菌株間的黏附率和侵襲率差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。人結(jié)腸癌細(xì)胞(Lovo)具有更高的敏感性,適宜于研究奇異變形桿菌對(duì)其的黏附和侵襲能力。通過(guò)基因敲除構(gòu)建的virB9基因缺失株對(duì)Lovo細(xì)胞的黏附和侵襲力下降,對(duì)小鼠的致病性降低,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。因此推論T4SS系統(tǒng)是奇異變形桿菌C02011的特異毒力因子,該毒力島在與真核生物的黏附和侵襲作用中發(fā)揮重要作用,可能是導(dǎo)致人體產(chǎn)生腹瀉和嘔吐的決定性因子。構(gòu)成T4SS系統(tǒng)的其他幾個(gè)組成基因的功能將是我們后續(xù)研究中需要探索的問(wèn)題。
[Abstract]:Proteus mirabilis is a common pathogenic bacterium that can cause urinary system infection. However, the relationship between the known virulence gene and diarrhea is not clear. The research on how to cause diarrhea is not conclusive. At present, the research on Proteus mirabilis mainly focuses on the strains causing urinary system infection, and the study of drug resistance is more important. Many, however, the research on the related mechanism of diarrhoea caused by Proteus mirabilis, few papers included in Pubmed. In some food poisoning events, we separated from the patient's diarrhea specimen and the remaining food to the Proteus mirabilis and the other pathogenic bacteria. The same batch isolates have the same pulse field gel electrophoresis (PFGE) type. It is suggested that there may be other specific virulence factors in the genome of Proteus mirabilis that caused diarrhoea in the genome of a healthy population and the external environment, which does not cause the above symptoms in mice, and thus suggests that there are other specific virulence factors in the genome of Proteus mirabilis, which causes diarrhea, which is the cause of food. The main reasons for the toxicosis. Around the above clues, this article mainly carried out the following research: (1) pathogenic and molecular biological characteristics analysis of Bacillus proteus diarrhoea; (2) complete genome sequencing analysis and comparison of the specific genes only existed in diarrhoea strains; (3) new type of genotypes found in diarrhoea strain C02011 Analysis of the basic characteristics of T4SS gene virBl-11 coding gene; (4) construction of virB9 gene deletion strain and remedial strain of Proteus mirabilis C02011, identification of its basic properties; (5) screening of sensitive cell lines of adhesion and invasion experiments, establishment of cell model in vitro; (6) comparison of wild strain C02011, virB9 gene deletion and recharge in vitro The adhesion and invasion level of the Lovo cells to human colon cancer cells; (7) the establishment and preliminary experiment of the animal model of diarrhea; (8) to determine whether the virB9 gene affects the pathogenic ability of the Proteus mirabilis to mice. (9) the establishment of the specific gene fluorescence PCR diagnosis method and the strain screening. Method and results: 1. Cause the pathogen of Bacillus proteus diarrhoea Study and molecular biological characteristics 1, 6 strains of Proteus mirabilis were selected as experimental strains, including 2 strains of standard strains, ATCC29906 and HI4320, 2 strains of food poisoning and 2 healthy populations and external environmental swab isolates,.2. The pathogenic and molecular characteristics of different sources were analyzed and compared. The biochemical characteristics of Bacillus proteus are basically the same, and they all have common virulence genes ureC, rsmA, hpmA and zapA. show that these genes are not specific and can not distinguish between the pathogenic bacteria and the non pathogenic strain.3. The PFGE method is used to classify the molecules of Proteus mirabilis and the PFG in the same food (the patient specimen and the remaining food isolate). The E map was consistent with the food poisoning.4 caused by Proteus mirabilis. It was found that there were 1-3 different sizes of plasmids in Proteus mirabilis. The largest plasmid was around 10100bp and was common in all strains. There was a small 2683bp plasmid in C05028 and C02034, with four open reading frames, of which 645 The qnrD gene of BP is the quinolone resistant gene, which is the same as that of the resistant phenotype. It can be seen that the plasmid is related to the resistance of the strain.5, and the environment isolate C02034 carries the most plasmids (3) and its resistance is the strongest. It is resistant to quinolones, sulfonamides, aminoglycosides and p- lactam penicillin, and thus deduces the other 2 substances carried by the strain. It is also possible to encode drug resistant genes and further verify.6. Studies have shown that traditional biochemical analysis and common virulence gene identification can not distinguish the source of the strain. It is impossible to determine the strain of food poisoning or the strain of the healthy population itself (or in the external environment); but PFGE and plasmid analysis can distinguish the source of the strain. The research also found the ubiquitous plasmids in Proteus mirabilis strain, which provided a theoretical basis for the study of resistance and pathogenicity of Proteus mirabilis. Two, the whole genome sequencing and sequence alignment of Proteus mirabilis were 1. In order to further clarify the specific genes of diarrhoea strains, 2 typical strains of diarrhoea (C05028 and C02011) were collected. The whole genome sequencing.2 was carried out with 1 healthy human isolates (B02005) and 1 external environmental control strains (C02034). Through sequence alignment analysis, 7 specific genes were found that existed only in food poisoning and non pathogenic strains. Their functional prediction results showed that some of these genes encode hemolysin and some encoded DNA nucleosides. Acid transferase or glycosyltransferase, some of which are transcriptional regulatory originals associated with eukaryotic cells, and specific functions need to be further studied. These genes were first discovered in this study and have not been reported in the relevant literature.3. The specific genes encoding the type IV secretory system (T4SS) in the food poisoning isolates C02011 are also found in the strange deformable rod. For the first time, there is no related literature in the bacteria. Three, three, the basic characteristics of type IV secretory system (T4SS) in C02011 are analyzed for the 11 gene virB1-virBll of type IV secretory system (T4SS) in C02011, through homologous comparison, egg white matter domain, three-dimensional structure prediction, evolutionary tree, lattice map, gene Structural prediction, gene island structure analysis, etc., studied the evolutionary relationships of the overall horizontal gene transfer events. The main components of T4SS of Proteus C02011 are: VirB1, VirB3-4, VirB11, VirB2, VirB5-VirB10, lipoprotein, and cell adhesion (VirB3-4). Conclusion: T4SS all The 11 genes are highly similar to the T4SS gene of Klebsiella pneumoniae PMK1 and Escherichia coli ECOR31 at protein and nucleotide levels, but have no homologous with the nucleotide of 8 strains of Proteus mirabilis. Therefore, it is inferred that this gene island is a recent Enterobacteriaceae microorganism Escherichia coli which is closely related to it. And the horizontal transfer in Klebsiella pneumoniae. This event occurred at least after the differentiation of Bacillus Proteus C02011 from other strains of Proteus mirabilis. Four, virB9 gene knockout and characterization of Proteus mirabilis C02011 strain in order to study the related functions of the virB gene in the pathogenicity of Proteus mirabilis, and select the coded secretory line VirB9 as the research object, the virB9 gene knockout strain Pmi/AvirB9. was constructed by using the principle of homologous recombination of gene and by suicide plasmid PCVD442 and the intermediate host SM10 lambda PIR, the mutant virB9 gene deletion mutant was constructed successfully, and the Pmi/ Delta virB9 recharge strain with virB9 gene was constructed to lay the foundation for the next step of the study. Under nutrient enrichment conditions, the colony formation and growth ability of C02011 and Pmil Delta virB have no significant difference in biochemical reaction. Therefore, the mutant strain is suitable for studying the effect of the gene on infection related virulence. Five, screening of sensitive cell lines in adhesion and invasion experiments, the establishment of human colon cancer cells (Lovo) and human colon gland in vitro cell model establishment Cancer cells (Caco-2) were studied in vitro to determine the cell invasiveness and adhesion of Proteus mirabilis C02011 (food intoxication patient) and B02005 (healthy human feces isolate). The results showed that the vomited isolate C02011 of the patients with food poisoning was used to Lovo, Caco-2 cells. The adhesion of healthy human fecal isolates B02005 to Lovo and Caco-2 cells was weak, and the adhesion rate difference between two strains was statistically significant (P0.01). The invasive experiment also showed similar results, and the difference of invasion rate between two strains and Lovo and Caco-2 cells was statistically significant (P0.01). This study confirmed that it was isolated from the food. C02011 has strong adhesion and invasiveness in the vomit of patients with diarrhea and diarrhea. It plays an important role in human and animal intestinal diseases. Human colon cancer cell (Lovo) has higher sensitivity. It is suitable for the study of the adhesion and invasion ability of Proteus mirabilis. Six, the loss of virB9 is reduced. The adhesion and invasion ability of Bacillus Proteus C02011 to Lovo cells in vitro model used Lovo monolayer cells. Compared with the adhesion rate of wild strain C02011, the ability of Pmil Delta virB9 to adhere to Lovo monolayer was significantly decreased, and the difference of adhesion rate between the two groups was statistically significant (P0.001). It also showed a significant decrease. The difference of invasion rate between the two was statistically significant (P0.001).Pmil Delta virB9 was converted into the supplementation plasmid, and the adhesion and invasiveness of the mutant strain were basically recovered. Seven. The establishment and preliminary experiment of the animal model of diarrhoea in order to prove that some proteus mirabilis can cause diarrhea and have different effects. The virulence and the establishment of a suitable animal model were established to infect mice with 6 different sources of Proteus mirabilis, respectively, to observe the fecal traits and the death of mice. In the oral gavage experiment, all the strains did not cause the death of the mice, but the food poisoning isolated strain caused the shape change of the feces in the mice. In the intraperitoneal injection experiment, after 7 hours, diarrhea from diarrhea. All the mice that were isolated from the vomit of the patient's vomit were all dead, and the mice's feces were thin and soft. The coloring results of the mice were also confirmed by the pathological changes. The mice that were isolated from the feces and external environment of the healthy population did not die, and the morphologic activity of the mice was normal. The study was successfully established. The mice diarrhea model of Proteus mirabilis has provided a mature experimental animal model for the follow-up of the function of the subsequent virulence genes. This study proved that the virulence of the isolated strains of Proteus mirabilis food poisoning is stronger than that of the healthy and external environmental isolates, which can cause abdominal diarrhea and even death in mice, and cause the pathological changes of the large intestine. Eight VirB9 deletion reduced the pathogenicity of Proteus mirabilis C02011 to mice and confirmed the function of virB9 gene. In the established mice animal model, the body test was carried out in vivo, with C02011 wild strain, virB9 gene knockout strain and remedial strain infected mice respectively. The diarrhea symptoms and pathological changes of the infected animals were observed and the identification ratio was compared. The experimental results showed that the virulence of the C02011 wild strain and the remedial plant was stronger than that of the virB9 gene knockout strain, causing diarrhea in mice and increasing the water content of the intestinal tissue, compared with the saline control strain (t=2.7764, P=0.0193), and could cause the pathological changes of the large intestine to change the.VirB9 gene knockout. The strain did not cause diarrhea in mice, and there was no statistical difference between the water content of intestinal tissue and normal saline (t=0.2979, P=0.7783). It indicated that the deletion of virB9 reduced the pathogenicity of Proteus mirabilis C02011 to mice. But the pathological section of the large intestine showed that there was still a certain inflammatory response, and that the virB9 gene was not the only virulence gene of the strain. The virB9 knockout strains still have a certain toxicity to the intestinal tissue of mice. Nine, the establishment of a fluorescence PCR detection method for Bacillus proteus diarrhoea, based on the newly discovered specific virulence factors, 4 virulence genes commonly found in the food poisoning strains were selected by PCR screening. For the target gene, the multiplex real-time fluorescent PCR method for simultaneous detection of 4 virulence genes of Proteus mirabilis with the same label assisted Hand (Hand, Homo-Tag Assisted Non-Dimer) system and the modified molecular beacon fluorescent probe was established. The multiplex fluorescence PCR method for the Bacillus proteus caused by diarrhoea was widely used. The rapid detection of food poisoning or food contaminants can be used to distinguish normal population and diarrhea strains, to simplify and improve the existing national standard diagnosis methods, to save manpower and material resources, to meet the rapid and accurate diagnosis of food poisoning and to trace the source of infection, to reduce misjudgement and leakage, and to ensure food safety and human health. Statistical analysis: the data were all expressed as mean + standard deviation. T test was used for comparison between the two groups, and single factor variance was used for multiple comparisons.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R378

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