低水平万古霉素耐药金黄色葡萄球菌生物膜形成及其调控机制研究

发布时间：2018-05-14 01:08

本文选题：金黄色葡萄球菌 + 万古霉素　；参考：《安徽医科大学》2017年博士论文

【摘要】：金黄色葡萄球菌(金葡菌)是临床常见的条件致病菌,致病力强,可引起轻微的皮肤软组织感染,也可引起威胁生命的中毒性休克综合征、败血症、骨髓炎等多种疾病。金葡菌极易产生耐药性,其中耐甲氧西林金黄色葡萄球菌(MRSA)最为常见。万古霉素是目前临床治疗MRSA感染的首选药物,也被认为是抵御革兰阳性菌感染的最后一道防线。但随着万古霉素用量的增加,近年来出现了万古霉素耐药的金葡菌。尽管目前对万古霉素完全耐药(高水平耐药)的金葡菌仍非常罕见,但越来越多的文献报道检出低水平万古霉素耐药金葡菌,包括万古霉素中介耐药金黄色葡萄球菌(VISA)和万古霉素异质性中介耐药金黄色葡萄球菌(hVISA)。与万古霉素耐药金黄色葡萄球菌(VRSA)不同,hVISA/VISA没有明确的耐药基因介导,而是金葡菌在万古霉素选择压力下产生的适应性耐药,常伴有细胞壁增厚、生长缓慢、毒力减低等适应性改变。这些变化可导致万古霉素渗透障碍,细菌难以被清除,感染反复发作,增加了临床治疗的难度。生物膜是细菌抵御抗菌药物和宿主免疫攻击的胞外屏障,与金葡菌耐药和致病密切相关。hVISA/VISA生物膜形成是否也发生了改变尚存争议,本课题重点对hVISA/VISA生物膜形成能力和机制开展研究。目的:本研究旨在明确hVISA/VISA生物膜形成能力是否发生改变。研究生物膜形成与耐药之间的相关性,探讨生物膜形成机制和分子调控机制。研究结果将有助于进一步认识hVISA/VISA的生存方式和致病特征。方法:(1)利用含3μg/ml万古霉素的脑心浸液(BHI)琼脂平板(BHI-V3)筛选临床分离的金葡菌中万古霉素低水平耐药菌株,对两组细菌的遗传背景、生物膜形成能力进行比较。(2)随机选择一株可以在BHI-V3平板上生长的万古霉素低水平耐药菌株0534用万古霉素进行体外诱导,使其MIC值逐步升高,获得系列万古霉素梯度耐药的菌株。用微孔成膜法及激光共聚焦显微镜检测菌株的生物膜形成能力。(3)应用实时荧光定量PCR(qRT-PCR)检测0534及其系列诱导菌株的生物膜形成调控相关基因(icaA、icaR、fnbA、SarA、agr、atlA和lrgAB)及耐药调控二元信号系统VraSR转录水平。(4)使用免疫斑点印记法测定细菌的胞外多糖粘附素(PIA)产生能力。(5)利用凝胶迁移阻滞实验(EMSA)检测与金黄色葡萄球菌生物膜形成基因(icaA、icaR和fnbA)及调控基因(Agr、atlA和SarA)启动子区域的结合情况。DNase I足迹印记实验鉴定VraR蛋白与靶基因启动子区的结合位点。结果:(1)本研究共收集临床分离的金葡菌176株。其中16株金葡菌可在BHI-V3筛选平板上生长,命名为GV3。经鉴定这16株菌株均为MRSA。故我们随机挑选16株不在BHI-V3筛选平板上生长的MRSA菌株作为对照组,命名为NGV3组。两组菌株遗传背景基本一致,多为agr Ⅱ、SCCmec Ⅲ、t030型。微孔成膜法检测显示,所有GV3菌株均可形成明显的紫色斑状固着物,而NGV3组仅有部分菌株可形成一层薄薄的紫色固着物。结晶紫染色半定量分析显示,GV3组菌株平均吸光度值明显高于NGV3组(0.336±0.088 Vs 0.109±0.036)。(2)0534菌株经过万古霉素体外传代诱导后,可获得系列不同万古霉素耐药水平的菌株,最高万古霉素MIC值可达32μg/ml。与原代菌株相比,系列万古霉素梯度耐药菌株均可形成紫色斑状固着物,并且随着万古霉素MIC值的升高,颜色逐渐加深。结晶紫染色半定量分析显示,诱导菌株0534-V8(1.74±0.10)、0534-V16(2.23±0.07)和0534-V32(2.85±0.05)平均吸光度值明显高于原代菌株0534(0.41±0.03)。激光共聚焦显微镜观察菌株生物膜三维立体结构,发现较之原代菌株0534,系列万古霉素梯度耐药菌株均形成了紧凑的、厚的生物膜。其中,0534-V32菌株生物膜平均厚度为12±0.7μm,远大于0534菌株生物膜平均厚度(2±0.4μm)。(3)qRT-PCR检测结果显示,与NGV3组菌株相比,GV3菌株icaA和fnbA转录水平分别增高了1.67倍和3.44倍。而icaR和agr转录水平下降了1.88倍和2.18倍。与原代菌株相比,系列万古霉素梯度耐药诱导株icaA、fnbA、atlA和sarA转录水平均明显上升,而icaR、agr转录水平明显下降。且基因表达水平的变化与菌株万古霉素MIC升高的程度密切相关。(4)相较之于原代菌株,系列万古霉素耐药诱导株的PIA表达均明显增高。其中0534-V32菌株PIA表达量是0534菌株的5.31倍。且PIA表达水平增高与菌株万古霉素MIC升高的程度密切相关。(5)与NGV3组菌株相比,GV3菌株耐药相关双组份调控系统VraSR转录水平增高了2.20倍。与原代菌株相比,系列万古霉素梯度耐药诱导株VraSR转录水平均明显上升,与促生物膜形成基因(icaA、fnbA、atlA和sarA)表达量呈正相关,与抑制生物膜形成的icaR、agr表达量呈负相关。使用重组表达并纯化的rVraR蛋白,进行凝胶迁移阻滞实验,结果显示VraR不能与fnbA、icaADBC、sar、atlA基因的启动子区域直接结合。但可以与Agr启动子区域直接结合。DNase I足迹印记实验进一步验证表明VraR与agr启动子区的结合位点位于P2和P3启动子之间。结论:(1)hVISA/VISA生物膜形成增加,且随着万古霉素MIC值升高,生物膜形成能力逐渐增强。(2)hVISA/VISA可通过FnbA高表达和PIA依赖途径促进生物膜形成。(3)万古霉素耐药相关二元调控子VraSR可直接结合agr启动子区域,间接调控金葡菌生物膜形成。
[Abstract]:Staphylococcus aureus (Staphylococcus aureus) is a common clinical pathogen. It has strong pathogenicity, can cause mild skin soft tissue infection, and can also cause a variety of diseases such as toxic shock syndrome, septicemia and osteomyelitis, which threaten life. Staphylococcus aureus is very easy to produce drug resistance, and methicillin-resistant Staphylococcus aureus (MRSA) is the most common. Vancomycin is the first drug to treat MRSA infection and is considered to be the last line of defense against gram positive infection. However, with the increase of vancomycin, vancomycin resistant Staphylococcus aureus has emerged in recent years. Although Staphylococcus aureus is still very rare for vancomycin resistance (high level resistance), the more it is, the more The more literature reports on the detection of low level vancomycin resistant Staphylococcus aureus, including vancomycin resistant Staphylococcus aureus (VISA) and vancomycin heterogenous mediator resistant Staphylococcus aureus (hVISA). Unlike vancomycin resistant Staphylococcus aureus (VRSA), hVISA/VISA has no clear resistance gene mediated, but gold. The adaptable resistance produced by the Staphylococcus aureus under the selective pressure of vancomycin is often accompanied by adaptable changes in cell wall thickening, slow growth, and reduced virulence. These changes can lead to vancomycin osmotic disorder, the bacteria are difficult to be removed, the infection is repeated, and the difficulty of clinical treatment is increased. Biofilms are bacteria against antibacterial drugs and host immunity. The outer barrier of the attack is still controversial whether the formation of.HVISA/VISA biofilm is also related to the resistance and pathogenesis of Staphylococcus aureus. This topic focuses on the study of the ability and mechanism of hVISA/VISA biofilm formation. The purpose of this study is to clarify whether the formation of hVISA/VISA biofilm changes. The correlation between drug resistance and the mechanism of biofilm formation and molecular regulation. The results will help to further understand the way of survival and pathogenicity of hVISA/VISA. Methods: (1) the screening of low level vancomycin resistant strains of Staphylococcus aureus from the clinically isolated Staphylococcus aureus by using the BHI agar plate (BHI-V3) containing 3 Mu vancomycin (BHI-V3). The genetic background of the two groups of bacteria and the biofilm formation ability were compared. (2) a random selection of a low level vancomycin resistant strain, which could be grown on the BHI-V3 plate, was induced by vancomycin in vitro, to increase the MIC value gradually, and to obtain a series of vancomycin resistant strain resistant strains. Microporous membrane forming and laser confocal Microscopes detected the biofilm ability of the strain. (3) use real time fluorescence quantitative PCR (qRT-PCR) to detect the biofilm related genes (icaA, icaR, fnbA, SarA, agr, atlA and lrgAB) and the resistance regulation two yuan signal system VraSR transcriptional level by real time fluorescence quantitative (qRT-PCR) detection. (4) the determination of extracellular matrix of bacteria by immuno dot imprinting PIA production capacity. (5) detection of the binding sites in the promoter region of the promoter region of Staphylococcus aureus biofilm (icaA, icaR and fnbA) and the regulatory gene (Agr, atlA and SarA) using the gel migration block assay (EMSA). The binding site of the VraR protein and the target gene promoter region was determined by the.DNase I footprint test. Results: (1) the study of the binding site of the promoter region of the target gene. A total of 176 clinically isolated Staphylococcus aureus strains were collected, of which 16 strains of Staphylococcus aureus could grow on the BHI-V3 screen, and the 16 strains were identified as MRSA., so we randomly selected 16 MRSA strains that did not grow on the BHI-V3 screen as the control group and named NGV3 group. The genetic background of the two groups was basically the same, mostly agr II. SCCmec III, t030 type. Microporous membrane detection showed that all GV3 strains could form a clear purple porphyry fixation, while only part of the NGV3 group could form a thin layer of purple fixation. Semi quantitative analysis of crystal violet staining showed that the average absorbance value of the GV3 group was higher than that of the NGV3 group (0.336 + 0.088 Vs 0.109 + 0.036). (2) 0534 After the strain of vancomycin was induced, the strain of various vancomycin resistant levels could be obtained. The MIC value of the highest vancomycin can reach 32 mu g/ml.. Compared with the original strain, a series of vancomycin gradient resistant strains can form a purple porphyry solid, and with the increase of the MIC value of the vancomycin, the color gradually deepens. The semi quantitative analysis of violet staining showed that the average absorbency of the induced strain 0534-V8 (1.74 + 0.10), 0534-V16 (2.23 + 0.07) and 0534-V32 (2.85 + 0.05) was significantly higher than that of the original strain 0534 (0.41 + 0.03). The three-dimensional structure of the biofilm of the strain was observed by laser confocal microscopy, and 0534 of the original strain was found, and all vancomycin gradient resistant strains were all A compact, thick biofilm was formed. Among them, the average thickness of the biofilm of 0534-V32 was 12 + 0.7 mu m, far greater than the average thickness of the biofilm (2 + 0.4 m). (3) qRT-PCR detection showed that the transcriptional level of icaA and fnbA of GV3 strain increased by 1.67 times and 3.44 times respectively compared with the NGV3 strain, and the transcriptional level of icaR and agr decreased by 1.88. 2.18 times and 2.18 times. Compared with the original strain, the transcriptional level of icaA, fnbA, atlA and sarA in the series of vancomycin resistant gradient resistant strains increased obviously, while the icaR, agr transcriptional level decreased obviously. And the change of gene expression level was closely related to the degree of MIC increase of vancomycin. (4) compared to the original strain, a series of vancomycin resistance lure. The expression of PIA in the pilot plant increased obviously. The expression of 0534-V32 strain PIA was 5.31 times more than that of the 0534 strain. And the increased expression of PIA was closely related to the extent of the increase of vancomycin MIC. (5) the transcriptional water level of the GV3 strain related dual component regulation system was 2.20 times higher than that of the NGV3 strain. The VraSR transcriptional level of the gradient resistant strain of palaeomycin was significantly increased, which was positively correlated with the expression of icaA, fnbA, atlA and sarA, and negatively correlated with the icaR and agr expression in the biofilm formation. The gel migration block experiment was carried out with the recombinant protein and purified rVraR protein, and the results showed that VraR could not be with fnbA, icaA. The promoter region of the DBC, SAR and atlA genes is directly combined. But it can be further verified with the.DNase I footprint experiment with the Agr promoter region. The binding site of VraR and agr promoter region is between P2 and P3 promoters. Conclusion: (1) the formation of hVISA/VISA biofilm is increased and the biofilm formation energy increases with the increase of vancomycin MIC value. (2) (2) hVISA/VISA can promote biofilm formation through FnbA high expression and PIA dependent pathway. (3) vancomycin resistance related two element regulator VraSR can directly combine with agr promoter region to indirectly regulate the formation of Staphylococcus aureus biofilm.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R446.5

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