本文選題：解沒(méi)食子酸鏈球菌巴氏亞種 + 分離鑒定　； 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：解沒(méi)食子酸鏈球菌巴氏亞種(Streptococcus gallolyticus subsp.pasteurianus)即牛鏈球菌Ⅱ/2(Streptococcus bovis II/2),是一種重要的醫(yī)院內(nèi)傳染病原,主要感染初生嬰兒,致嬰兒的腦膜炎、菌血癥等,甚至死亡;多種動(dòng)物也可以被解沒(méi)食子酸鏈球菌巴氏亞種感染,且死亡率較高。2010年至2013年間,湖北及廣西等地六個(gè)規(guī)�；唸�(chǎng)~10日齡雛鴨突然發(fā)病,死亡率約20%。本研究從死亡雛鴨的腦、脾組織分離了六株細(xì)菌,經(jīng)純化培養(yǎng)后進(jìn)行鑒定和動(dòng)物回歸試驗(yàn),并以AL101002菌株為代表研究其致病機(jī)制,此外還對(duì)六株菌的大環(huán)內(nèi)酯耐藥機(jī)制進(jìn)行了深入研究。研究結(jié)果如下:1.解沒(méi)食子酸鏈球菌巴氏亞種的分離鑒定分離細(xì)菌純化培養(yǎng)后,革蘭氏染色呈陽(yáng)性球菌,排列方式為單個(gè)或短鏈狀,蘭氏抗原分群鑒定為D群,生化特性鑒定為解沒(méi)食子酸鏈球菌,結(jié)合16S r RNA基因測(cè)序結(jié)果,六株分離菌鑒定為解沒(méi)食子酸鏈球菌巴氏亞種(S.gallolyticus subsp.pasteurianus),依次命名為AL101002、GX130304、GX130304、GX130630、GX130723、GX130809。采用頸部皮下途徑分別感染上述六株菌,均可以致雛鴨死亡,發(fā)病癥狀、死亡率、病理變化等與自然感染相似。以AL101002為代表進(jìn)行研究,石蠟切片革蘭氏染色發(fā)現(xiàn)在感染雛鴨肝臟的肝竇及內(nèi)皮細(xì)胞、脾臟的巨噬細(xì)胞、腎臟的腎小球內(nèi)存在大量革蘭氏陽(yáng)性細(xì)菌,用針對(duì)AL101002的兔多克隆抗體進(jìn)行免疫組織化學(xué)染色,證明存在于這些部位的細(xì)菌為解沒(méi)食子酸鏈球菌巴氏亞種;雛鴨脾臟的超微組織學(xué)病理檢測(cè)發(fā)現(xiàn),在脾臟的巨噬細(xì)胞內(nèi)可見(jiàn)復(fù)制狀的球菌。綜合上述結(jié)果,本研究分離到的六株菌為解沒(méi)食子酸鏈球菌巴氏亞種。2.解沒(méi)食子酸鏈球菌的致病機(jī)制以AL101002菌感染7日齡雛鴨,動(dòng)態(tài)研究雛鴨發(fā)病機(jī)制:感染后5d,雛鴨表現(xiàn)出典型臨床癥狀,發(fā)病率為100%,死亡率為52.38%(22/42)。AL101002菌株感染雛鴨后1-5d,各器官及血液均可以分離到接種菌,5d之后血液分離不到細(xì)菌,7d只有腎和法氏囊可以分離到細(xì)菌。剖檢病理變化可見(jiàn)脾臟腫大、心包膜增厚,外觀呈絨毛狀,肝臟表面干酪樣物質(zhì)滲出,嚴(yán)重的腦膜充血。組織病理學(xué)變化為:感染后5d,脾臟基本結(jié)構(gòu)消失,實(shí)質(zhì)性心肌炎;感染后7d,法氏囊的淋巴細(xì)胞嚴(yán)重減少;感染后3-9d,腦膜炎癥狀程度不等;其他組織出現(xiàn)異嗜性粒細(xì)胞及單核巨噬細(xì)胞浸潤(rùn)。免疫器官指數(shù)測(cè)定,發(fā)現(xiàn)該菌對(duì)脾臟的影響最大。TUNEL法檢測(cè)脾臟的細(xì)胞凋亡情況,結(jié)果表明感染組的脾臟細(xì)胞凋亡明顯,且差異顯著。感染雛鴨的超微組織學(xué)病理變化結(jié)果顯示:感染后1-5d,脾臟巨噬細(xì)胞可見(jiàn)復(fù)制狀球菌,并且在感染后5d,可見(jiàn)脾臟巨噬細(xì)胞壞死的特性;抗CD68抗體,一種巨噬細(xì)胞表面標(biāo)記物,檢測(cè)脾臟的巨噬細(xì)胞,結(jié)果表明在脾臟壞死灶周圍可見(jiàn)陽(yáng)性信號(hào)。熒光定量PCR檢測(cè)腦組織的TNF-α、IL-1β及脾組織的Caspase-3、Caspase-8、RIPK1、RIPK2-like、MLKL、TNF-α,IL-1β、IL-6的變化,結(jié)果表明:腦組織的兩個(gè)致炎因子表達(dá)上調(diào),說(shuō)明腦膜炎的發(fā)生;脾臟組織的兩個(gè)關(guān)鍵凋亡因子基本無(wú)變化,相反壞死性凋亡關(guān)鍵調(diào)控因子RIPK1和MLKL在感染后5d分別上調(diào)表達(dá)~2.5倍,炎性因子IL-1β、IL-6上調(diào)表達(dá)顯著,表明脾臟的急性炎癥。由此可見(jiàn)解沒(méi)食子酸鏈球菌巴氏亞種靶標(biāo)雛鴨的免疫器官及腦,致雛鴨脾臟巨噬細(xì)胞的壞死性凋亡,可以侵入雛鴨的腦實(shí)質(zhì),致嚴(yán)重的腦膜炎癥狀。3.解沒(méi)食子酸鏈球菌的耐藥特征及大環(huán)內(nèi)酯類耐藥機(jī)制采用瓊脂稀釋法檢測(cè)六株解沒(méi)食子酸鏈球菌巴氏亞種的MIC值,結(jié)果表明六株菌均表現(xiàn)出多重耐藥,尤其對(duì)大環(huán)內(nèi)酯類藥物呈現(xiàn)高水平耐藥,紅霉素與克拉霉素的MIC值分別高達(dá)1024 mg/L和512 mg/L。大環(huán)內(nèi)酯耐藥機(jī)制研究結(jié)果如下:A.未能檢測(cè)到任何質(zhì)粒、mef A或mef E基因,PAβN或CCCP外排泵抑制劑未能顯著影響大環(huán)內(nèi)酯MIC值,表明大環(huán)內(nèi)酯耐藥并非由外排系統(tǒng)所致;B.核糖體蛋白L4和L22無(wú)任何氨基酸的突變,23S r RNA序列上有點(diǎn)突變,而這些突變位點(diǎn)導(dǎo)致的大環(huán)內(nèi)酯耐藥MIC值￡32 mg/L,表明這些分離株中的大環(huán)內(nèi)酯高水平耐藥并非由核糖體蛋白L4和L22或23S r RNA突變導(dǎo)致;C.PCR檢測(cè)到六株菌基因組上均有核糖體甲基化酶基因erm B和erm T,以及四環(huán)素耐藥基因tet M和tet L,其中四株菌含有林可酰胺類耐藥基因Lnua,一株菌含有慶大霉素耐藥基因。對(duì)AL101002菌株進(jìn)行了全基因組測(cè)序,并在此基礎(chǔ)上擴(kuò)增erm B及erm T基因的上下游序列,發(fā)現(xiàn)AL101002基因組含有兩個(gè)耐藥基因簇,分別長(zhǎng)5.731 kb和11.244 kb,短基因簇含有erm B基因,長(zhǎng)基因簇含有erm T,二者Genbank的登錄號(hào)分別為KU511281和KU511280。D.erm B與erm T在六株菌的誘導(dǎo)表達(dá):Western blot檢測(cè)六株菌的紅霉素耐藥甲基化酶Erm B和Erm T在紅霉素存在與否時(shí)的表達(dá),結(jié)果表明在紅霉素存在時(shí),兩個(gè)紅霉素耐藥甲基化酶在六株菌中均可表達(dá),當(dāng)紅霉素不存在時(shí),六株菌均不表達(dá)這兩個(gè)甲基化酶;進(jìn)一步在E.coli里檢測(cè)含有His標(biāo)簽的這兩個(gè)甲基化酶融合蛋白的表達(dá),結(jié)果表明添加一定量的紅霉素,兩個(gè)甲基化酶均可以表達(dá),紅霉素不存在時(shí),Erm B和Erm T表現(xiàn)出有少量表達(dá),可能是和啟動(dòng)子的泄漏有關(guān)。E.erm B和erm T對(duì)大環(huán)內(nèi)酯高水平耐藥的貢獻(xiàn):測(cè)兩個(gè)耐藥基因共存和單獨(dú)存在時(shí)對(duì)大環(huán)內(nèi)酯類藥物的耐藥貢獻(xiàn),結(jié)果表明,二者共存時(shí),無(wú)論有無(wú)前導(dǎo)肽(leader peptide),所有測(cè)試的大環(huán)內(nèi)酯藥物的MIC值都高于單獨(dú)存在時(shí)的MIC值。無(wú)論二者共存還是單獨(dú)表達(dá),無(wú)leader peptide時(shí)的MIC值都是是含leader peptide時(shí)的兩倍,且二者共存并有l(wèi)eader pepetide時(shí)的MIC值與六株臨床分離株的大環(huán)內(nèi)酯MIC值相當(dāng);說(shuō)明六株菌的大環(huán)內(nèi)酯高水平耐藥是由erm B和erm T基因共存導(dǎo)致,且leader peptide對(duì)其下游的erm基因具有衰減調(diào)控的作用。綜合上述研究結(jié)果,本論文得到的結(jié)論如下:A.首次從雛鴨的腦及脾臟組織分離并鑒定了六株解沒(méi)食子酸鏈球菌巴氏亞種,發(fā)現(xiàn)解沒(méi)食子酸鏈球菌巴氏亞種可導(dǎo)致雛鴨腦膜炎及脾臟巨噬細(xì)胞壞死性凋亡,并可導(dǎo)致較高的雛鴨死亡率。B.首次發(fā)現(xiàn)解沒(méi)食子酸鏈球菌巴氏亞種基因組含有紅霉素耐藥基因核糖體甲基化酶erm B和erm T,并發(fā)現(xiàn)二者受紅霉素誘導(dǎo)表達(dá),且二者的誘導(dǎo)表達(dá)是導(dǎo)致六株解沒(méi)食子酸鏈球菌巴氏亞種分離株大環(huán)內(nèi)酯高水平耐藥的主要原因。本論文的研究結(jié)果為該類細(xì)菌的臨床診斷、耐藥監(jiān)控、臨床用藥以及抗菌藥物的研發(fā)提供了重要的理論依據(jù)。
[Abstract]:Streptococcus gallolyticus subsp.pasteurianus (Streptococcus bovis II/2) of Streptococcus gallate is an important nosocomial pathogen, which mainly infects newborn infants, causes meningitis, bacteremia and even death in infants; many animals can also be isolated from Streptococcus gallate. Subspecies infection, and the mortality rate is higher from.2010 to 2013 years to 2013, the ~10 day old ducklings in six large-scale duck farms, Hubei and Guangxi, have a sudden onset, the mortality rate is about 20%.. This study isolated six strains of bacteria from the brain of the dead ducks and the spleen tissue. After purification and culture, the test was carried out and the pathogen of AL101002 was represented to study its pathogenicity. In addition, the mechanism of drug resistance of six strains of macrolides was also studied. The results were as follows: 1. the isolation and isolation of bacteria isolated from the subspecies of Streptococcus gallate was purified and cultured. Gram-positive coccus was stained by gram-positive coccus, the arrangement was single or short chain, the LAN antigen group was identified as D group, and the biochemical characteristics were identified as the solution. Streptococcus gallate, combined with 16S R RNA gene sequencing results, six isolates were identified as S.gallolyticus subsp.pasteurianus of Streptococcus gallate (S.gallolyticus subsp.pasteurianus), which were named AL101002, GX130304, GX130304, GX130630, GX130723, GX130809. using the neck subcutaneous pathway to infect the above six strains respectively, which could cause the death of ducks. The symptoms, mortality and pathological changes were similar to those of natural infection. AL101002 was used as the representative of the study. The paraffin section gram stain found that there were a large number of Gram-positive bacteria in the liver sinus and endothelial cells infected with duck liver, the macrophages of the spleen, and the glomeruli of the kidneys, and immunized with the rabbit polyclonal antibody against AL101002. Histochemical staining showed that the bacteria existed in these parts were the subspecies of Streptococcus gallate, and the ultrahistological examination of the spleen of the ducklings found replicating cocci in the macrophages of the spleen. The results of the above results were six strains isolated from the study of Streptococcus gallate as a subspecies of.2. gallon. The pathogenic mechanism of Streptococcus acid streptococcus was infected with AL101002 bacteria 7 days old ducklings. The pathogenesis of ducklings was studied dynamically: after infection 5D, the ducklings showed typical clinical symptoms, the incidence was 100%, the mortality rate was 52.38% (22/42).AL101002 infected ducks after 1-5d, all organs and blood could be isolated from the inoculated bacteria. After 5D, the blood could not be isolated from the bacteria and 7d only. The kidney and the bursa of Fabricius could be separated into bacteria. The pathological changes showed splenomegaly, thickening of the cardiac capsule, villous appearance, exudation of the cheeselike substance in the liver, and severe meningeal congestion. The histopathological changes were: 5D after infection, the basic structure of the spleen disappeared, the parenchymal myocarditis, and 7d, the lymphocytes of the bursa of the Fabricius were severely reduced. After infection of 3-9d, the symptoms of meningitis were different, other tissues were infiltrated with eosinophils and mononuclear macrophages. The immune organ index was used to determine the effect of the bacteria on spleen by.TUNEL method. The results showed that the apoptosis of spleen cells in the infected group was obvious and the difference was significant. The pathological changes of the fabric showed: 1-5d after infection, replicating coccus in spleen macrophages and necrosis of spleen macrophages in 5D after infection; anti CD68 antibody, a surface marker of macrophage, detection of spleen macrophage, and the results showed positive signals around the spleen necrotic foci. Fluorescence quantitative PCR detection The changes of TNF- alpha, IL-1 beta and Caspase-3, Caspase-8, RIPK1, RIPK2-like, MLKL, TNF- a, IL-1 beta and IL-6 of the tissues of the spleen indicate that the expression of two inflammatory factors in the brain is up regulated, indicating the occurrence of meningitis; the two key apoptotic factors in the spleen tissue are basically unchanged, and the key regulatory factor of necrotic apoptosis is RIPK1 and MLKL. After infection, the expression of 5D was up to ~2.5 times, the inflammatory factor IL-1 beta and IL-6 were up-regulated, indicating the acute inflammation of the spleen. This shows that the immune organs and brain of the target duckling of the subspecies of Streptococcus gallate can cause necrotizing apoptosis of the spleen macrophages of ducklings, which can invade the brain of ducks and cause serious meningitis symptoms.3. The resistance characteristics of Streptococcus gallate and the mechanism of macrolide resistance were used to detect the MIC value of six strains of Streptococcus gallate subspecies by agar dilution method. The results showed that the six strains showed multiple resistance, especially the high level of macrolide drugs, and the MIC value of erythromycin and clarithromycin was up to 1024 mg/, respectively. The drug resistance mechanism of L and 512 mg/L. macrolides was studied as follows: A. failed to detect any plasmids, MEF A or MEF E genes, and the inhibitor of PA beta N or CCCP efflux did not significantly affect the MIC value of macrolide, indicating that the resistance of macrolides was not caused by the outer row system; B. ribosome protein and no amino acid mutation were found in the sequence. Point mutation, while these mutant loci led to the macrolide resistance MIC value of about 32 mg/L, indicating that the high level resistance of macrolides in these isolates is not caused by ribosomal protein L4 and L22 or 23S R RNA mutations; C.PCR has detected the ribosomal methylase gene ERM B and ERM T, and the tetracycline resistance gene on the six strains of bacteria. M and Tet L, four of which contain the lincomamide resistance gene Lnua, and one strain containing gentamycin resistance genes. The whole genome sequencing of the AL101002 strain was sequenced. On this basis, the ERM B and the upstream and downstream sequences of the ERM T gene were amplified and found that the AL101002 genome contains two resistant gene clusters, 5.731 KB and 11.244 KB, and short bases respectively. The cluster contains the ERM B gene and the long gene cluster contains ERM T. The two Genbank's login numbers are KU511281 and KU511280.D.erm B and ERM T in six strains, respectively: Western blot detected erythromycin resistant methylation enzyme and the presence or absence of erythromycin in six strains of erythromycin. The results showed that in the presence of erythromycin, two red moulds were found. The protease resistant methylation was expressed in six strains. When erythromycin did not exist, the six strains did not express the two methylation enzymes. The expression of the two methylase fusion proteins containing the His label was detected in E.coli. The results showed that the addition of a certain amount of erythromycin and two methylation enzymes could be expressed, and the erythromycin did not exist. At the time, Erm B and Erm T showed a small amount of expression, which may be related to the contribution of.E.erm B and ERM T to the high level resistance of macrolide to the.E.erm B and ERM T: measurement of the coexistence of two resistant genes and their contribution to the drug resistance of macrolides when they exist alone. The results show that, when the two coexist, no matter whether there is a preamble peptide (leader peptide), all tests The MIC value of the macrolide drug was higher than the MIC value of the single existence. The MIC value of no leader peptide was two times as high as that of leader peptide, and the MIC value of the two were equal to the MIC value of the six clinical isolates of macrolides, and the macrolide of six strains of bacteria. High level resistance is caused by coexistence of ERM B and ERM T genes, and leader peptide has the attenuation regulation of the downstream ERM gene. The results obtained in this paper are as follows: A. was first isolated from the brain and spleen of ducklings and identified six strains of Streptococcus suis pasteuric subspecies, and found the solution of gallic acid. Streptococcus pasteuri subspecies can lead to necrotic apoptosis of meningitis and spleen macrophages in ducklings, and can lead to high ducklings mortality rate.B. for the first time found that the genome of the subspecies of Streptococcus gallate contains the erythromycin resistant gene ribosomal methylase ERM B and ERM T, and two were induced by erythromycin induced expression and the induction was induced. Expression is the main cause of high level resistance of six strains of Streptococcus gallate isolate macrolides. The results of this paper provide an important theoretical basis for the clinical diagnosis, drug resistance monitoring, clinical drug use and the research and development of antimicrobial drugs.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.61

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 許彥梅;徐建國(guó);;牛鏈球菌的研究進(jìn)展[J];中國(guó)人獸共患病學(xué)報(bào);2008年09期

2 賈鑫;曹葦;張豐;;肝硬化大鼠腹腔感染時(shí)脾臟TNF-α、IL-6基因的表達(dá)[J];蘇州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2008年04期

3 商營(yíng)利,劉思當(dāng),丁寶君,肖一紅,成子強(qiáng),孫先明,褚艷麗;雞感染J亞群禽白血病病毒的免疫抑制機(jī)理[J];中國(guó)獸醫(yī)學(xué)報(bào);2005年06期

4 朱建國(guó),華修國(guó);豬鏈球菌2型致病機(jī)制研究進(jìn)展[J];中國(guó)人獸共患病雜志;2005年09期

5 白麗,錢金h?,王晶;應(yīng)用辛酸硫酸胺法提取小鼠腹水和血清中的IgG抗體[J];大理醫(yī)學(xué)院學(xué)報(bào);2000年04期

相關(guān)博士學(xué)位論文 前1條

1 李梅;脾源性炎癥反應(yīng)在重型顱腦損傷大鼠中的作用及機(jī)制研究[D];第三軍醫(yī)大學(xué);2011年

相關(guān)碩士學(xué)位論文 前2條

1 李爽;鴨源禽呼腸孤病毒感染雛鴨的免疫病理學(xué)研究[D];華中農(nóng)業(yè)大學(xué);2010年

2 張玲娟;禽白血病病毒J亞群免疫抑制機(jī)理的研究[D];山東農(nóng)業(yè)大學(xué);2006年

，

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