解没食子酸链球菌巴氏亚种的分离鉴定及致病性与大环内酯耐药机制研究

发布时间：2018-06-22 02:32

本文选题：解没食子酸链球菌巴氏亚种 + 分离鉴定　；参考：《华中农业大学》2016年博士论文

【摘要】：解没食子酸链球菌巴氏亚种(Streptococcus gallolyticus subsp.pasteurianus)即牛链球菌Ⅱ/2(Streptococcus bovis II/2),是一种重要的医院内传染病原,主要感染初生婴儿,致婴儿的脑膜炎、菌血症等,甚至死亡;多种动物也可以被解没食子酸链球菌巴氏亚种感染,且死亡率较高。2010年至2013年间,湖北及广西等地六个规模化鸭场~10日龄雏鸭突然发病,死亡率约20%。本研究从死亡雏鸭的脑、脾组织分离了六株细菌,经纯化培养后进行鉴定和动物回归试验,并以AL101002菌株为代表研究其致病机制,此外还对六株菌的大环内酯耐药机制进行了深入研究。研究结果如下:1.解没食子酸链球菌巴氏亚种的分离鉴定分离细菌纯化培养后,革兰氏染色呈阳性球菌,排列方式为单个或短链状,兰氏抗原分群鉴定为D群,生化特性鉴定为解没食子酸链球菌,结合16S r RNA基因测序结果,六株分离菌鉴定为解没食子酸链球菌巴氏亚种(S.gallolyticus subsp.pasteurianus),依次命名为AL101002、GX130304、GX130304、GX130630、GX130723、GX130809。采用颈部皮下途径分别感染上述六株菌,均可以致雏鸭死亡,发病症状、死亡率、病理变化等与自然感染相似。以AL101002为代表进行研究,石蜡切片革兰氏染色发现在感染雏鸭肝脏的肝窦及内皮细胞、脾脏的巨噬细胞、肾脏的肾小球内存在大量革兰氏阳性细菌,用针对AL101002的兔多克隆抗体进行免疫组织化学染色,证明存在于这些部位的细菌为解没食子酸链球菌巴氏亚种;雏鸭脾脏的超微组织学病理检测发现,在脾脏的巨噬细胞内可见复制状的球菌。综合上述结果,本研究分离到的六株菌为解没食子酸链球菌巴氏亚种。2.解没食子酸链球菌的致病机制以AL101002菌感染7日龄雏鸭,动态研究雏鸭发病机制:感染后5d,雏鸭表现出典型临床症状,发病率为100%,死亡率为52.38%(22/42)。AL101002菌株感染雏鸭后1-5d,各器官及血液均可以分离到接种菌,5d之后血液分离不到细菌,7d只有肾和法氏囊可以分离到细菌。剖检病理变化可见脾脏肿大、心包膜增厚,外观呈绒毛状,肝脏表面干酪样物质渗出,严重的脑膜充血。组织病理学变化为:感染后5d,脾脏基本结构消失,实质性心肌炎;感染后7d,法氏囊的淋巴细胞严重减少;感染后3-9d,脑膜炎症状程度不等;其他组织出现异嗜性粒细胞及单核巨噬细胞浸润。免疫器官指数测定,发现该菌对脾脏的影响最大。TUNEL法检测脾脏的细胞凋亡情况,结果表明感染组的脾脏细胞凋亡明显,且差异显著。感染雏鸭的超微组织学病理变化结果显示:感染后1-5d,脾脏巨噬细胞可见复制状球菌,并且在感染后5d,可见脾脏巨噬细胞坏死的特性;抗CD68抗体,一种巨噬细胞表面标记物,检测脾脏的巨噬细胞,结果表明在脾脏坏死灶周围可见阳性信号。荧光定量PCR检测脑组织的TNF-α、IL-1β及脾组织的Caspase-3、Caspase-8、RIPK1、RIPK2-like、MLKL、TNF-α,IL-1β、IL-6的变化,结果表明:脑组织的两个致炎因子表达上调,说明脑膜炎的发生;脾脏组织的两个关键凋亡因子基本无变化,相反坏死性凋亡关键调控因子RIPK1和MLKL在感染后5d分别上调表达~2.5倍,炎性因子IL-1β、IL-6上调表达显著,表明脾脏的急性炎症。由此可见解没食子酸链球菌巴氏亚种靶标雏鸭的免疫器官及脑,致雏鸭脾脏巨噬细胞的坏死性凋亡,可以侵入雏鸭的脑实质,致严重的脑膜炎症状。3.解没食子酸链球菌的耐药特征及大环内酯类耐药机制采用琼脂稀释法检测六株解没食子酸链球菌巴氏亚种的MIC值,结果表明六株菌均表现出多重耐药,尤其对大环内酯类药物呈现高水平耐药,红霉素与克拉霉素的MIC值分别高达1024 mg/L和512 mg/L。大环内酯耐药机制研究结果如下:A.未能检测到任何质粒、mef A或mef E基因,PAβN或CCCP外排泵抑制剂未能显著影响大环内酯MIC值,表明大环内酯耐药并非由外排系统所致;B.核糖体蛋白L4和L22无任何氨基酸的突变,23S r RNA序列上有点突变,而这些突变位点导致的大环内酯耐药MIC值￡32 mg/L,表明这些分离株中的大环内酯高水平耐药并非由核糖体蛋白L4和L22或23S r RNA突变导致;C.PCR检测到六株菌基因组上均有核糖体甲基化酶基因erm B和erm T,以及四环素耐药基因tet M和tet L,其中四株菌含有林可酰胺类耐药基因Lnua,一株菌含有庆大霉素耐药基因。对AL101002菌株进行了全基因组测序,并在此基础上扩增erm B及erm T基因的上下游序列,发现AL101002基因组含有两个耐药基因簇,分别长5.731 kb和11.244 kb,短基因簇含有erm B基因,长基因簇含有erm T,二者Genbank的登录号分别为KU511281和KU511280。D.erm B与erm T在六株菌的诱导表达:Western blot检测六株菌的红霉素耐药甲基化酶Erm B和Erm T在红霉素存在与否时的表达,结果表明在红霉素存在时,两个红霉素耐药甲基化酶在六株菌中均可表达,当红霉素不存在时,六株菌均不表达这两个甲基化酶;进一步在E.coli里检测含有His标签的这两个甲基化酶融合蛋白的表达,结果表明添加一定量的红霉素,两个甲基化酶均可以表达,红霉素不存在时,Erm B和Erm T表现出有少量表达,可能是和启动子的泄漏有关。E.erm B和erm T对大环内酯高水平耐药的贡献:测两个耐药基因共存和单独存在时对大环内酯类药物的耐药贡献,结果表明,二者共存时,无论有无前导肽(leader peptide),所有测试的大环内酯药物的MIC值都高于单独存在时的MIC值。无论二者共存还是单独表达,无leader peptide时的MIC值都是是含leader peptide时的两倍,且二者共存并有leader pepetide时的MIC值与六株临床分离株的大环内酯MIC值相当;说明六株菌的大环内酯高水平耐药是由erm B和erm T基因共存导致,且leader peptide对其下游的erm基因具有衰减调控的作用。综合上述研究结果,本论文得到的结论如下:A.首次从雏鸭的脑及脾脏组织分离并鉴定了六株解没食子酸链球菌巴氏亚种,发现解没食子酸链球菌巴氏亚种可导致雏鸭脑膜炎及脾脏巨噬细胞坏死性凋亡,并可导致较高的雏鸭死亡率。B.首次发现解没食子酸链球菌巴氏亚种基因组含有红霉素耐药基因核糖体甲基化酶erm B和erm T,并发现二者受红霉素诱导表达,且二者的诱导表达是导致六株解没食子酸链球菌巴氏亚种分离株大环内酯高水平耐药的主要原因。本论文的研究结果为该类细菌的临床诊断、耐药监控、临床用药以及抗菌药物的研发提供了重要的理论依据。
[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.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.61

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