本文選題：金黃色葡萄球菌 + 萬古霉素��； 參考：《安徽醫(yī)科大學(xué)》2017年博士論文

【摘要】：金黃色葡萄球菌(金葡菌)是臨床常見的條件致病菌,致病力強(qiáng),可引起輕微的皮膚軟組織感染,也可引起威脅生命的中毒性休克綜合征、敗血癥、骨髓炎等多種疾病。金葡菌極易產(chǎn)生耐藥性,其中耐甲氧西林金黃色葡萄球菌(MRSA)最為常見。萬古霉素是目前臨床治療MRSA感染的首選藥物,也被認(rèn)為是抵御革蘭陽性菌感染的最后一道防線。但隨著萬古霉素用量的增加,近年來出現(xiàn)了萬古霉素耐藥的金葡菌。盡管目前對(duì)萬古霉素完全耐藥(高水平耐藥)的金葡菌仍非常罕見,但越來越多的文獻(xiàn)報(bào)道檢出低水平萬古霉素耐藥金葡菌,包括萬古霉素中介耐藥金黃色葡萄球菌(VISA)和萬古霉素異質(zhì)性中介耐藥金黃色葡萄球菌(hVISA)。與萬古霉素耐藥金黃色葡萄球菌(VRSA)不同,hVISA/VISA沒有明確的耐藥基因介導(dǎo),而是金葡菌在萬古霉素選擇壓力下產(chǎn)生的適應(yīng)性耐藥,常伴有細(xì)胞壁增厚、生長緩慢、毒力減低等適應(yīng)性改變。這些變化可導(dǎo)致萬古霉素滲透障礙,細(xì)菌難以被清除,感染反復(fù)發(fā)作,增加了臨床治療的難度。生物膜是細(xì)菌抵御抗菌藥物和宿主免疫攻擊的胞外屏障,與金葡菌耐藥和致病密切相關(guān)。hVISA/VISA生物膜形成是否也發(fā)生了改變尚存爭(zhēng)議,本課題重點(diǎn)對(duì)hVISA/VISA生物膜形成能力和機(jī)制開展研究。目的:本研究旨在明確hVISA/VISA生物膜形成能力是否發(fā)生改變。研究生物膜形成與耐藥之間的相關(guān)性,探討生物膜形成機(jī)制和分子調(diào)控機(jī)制。研究結(jié)果將有助于進(jìn)一步認(rèn)識(shí)hVISA/VISA的生存方式和致病特征。方法:(1)利用含3μg/ml萬古霉素的腦心浸液(BHI)瓊脂平板(BHI-V3)篩選臨床分離的金葡菌中萬古霉素低水平耐藥菌株,對(duì)兩組細(xì)菌的遺傳背景、生物膜形成能力進(jìn)行比較。(2)隨機(jī)選擇一株可以在BHI-V3平板上生長的萬古霉素低水平耐藥菌株0534用萬古霉素進(jìn)行體外誘導(dǎo),使其MIC值逐步升高,獲得系列萬古霉素梯度耐藥的菌株。用微孔成膜法及激光共聚焦顯微鏡檢測(cè)菌株的生物膜形成能力。(3)應(yīng)用實(shí)時(shí)熒光定量PCR(qRT-PCR)檢測(cè)0534及其系列誘導(dǎo)菌株的生物膜形成調(diào)控相關(guān)基因(icaA、icaR、fnbA、SarA、agr、atlA和lrgAB)及耐藥調(diào)控二元信號(hào)系統(tǒng)VraSR轉(zhuǎn)錄水平。(4)使用免疫斑點(diǎn)印記法測(cè)定細(xì)菌的胞外多糖粘附素(PIA)產(chǎn)生能力。(5)利用凝膠遷移阻滯實(shí)驗(yàn)(EMSA)檢測(cè)與金黃色葡萄球菌生物膜形成基因(icaA、icaR和fnbA)及調(diào)控基因(Agr、atlA和SarA)啟動(dòng)子區(qū)域的結(jié)合情況。DNase I足跡印記實(shí)驗(yàn)鑒定VraR蛋白與靶基因啟動(dòng)子區(qū)的結(jié)合位點(diǎn)。結(jié)果:(1)本研究共收集臨床分離的金葡菌176株。其中16株金葡菌可在BHI-V3篩選平板上生長,命名為GV3。經(jīng)鑒定這16株菌株均為MRSA。故我們隨機(jī)挑選16株不在BHI-V3篩選平板上生長的MRSA菌株作為對(duì)照組,命名為NGV3組。兩組菌株遺傳背景基本一致,多為agr Ⅱ、SCCmec Ⅲ、t030型。微孔成膜法檢測(cè)顯示,所有GV3菌株均可形成明顯的紫色斑狀固著物,而NGV3組僅有部分菌株可形成一層薄薄的紫色固著物。結(jié)晶紫染色半定量分析顯示,GV3組菌株平均吸光度值明顯高于NGV3組(0.336±0.088 Vs 0.109±0.036)。(2)0534菌株經(jīng)過萬古霉素體外傳代誘導(dǎo)后,可獲得系列不同萬古霉素耐藥水平的菌株,最高萬古霉素MIC值可達(dá)32μg/ml。與原代菌株相比,系列萬古霉素梯度耐藥菌株均可形成紫色斑狀固著物,并且隨著萬古霉素MIC值的升高,顏色逐漸加深。結(jié)晶紫染色半定量分析顯示,誘導(dǎo)菌株0534-V8(1.74±0.10)、0534-V16(2.23±0.07)和0534-V32(2.85±0.05)平均吸光度值明顯高于原代菌株0534(0.41±0.03)。激光共聚焦顯微鏡觀察菌株生物膜三維立體結(jié)構(gòu),發(fā)現(xiàn)較之原代菌株0534,系列萬古霉素梯度耐藥菌株均形成了緊湊的、厚的生物膜。其中,0534-V32菌株生物膜平均厚度為12±0.7μm,遠(yuǎn)大于0534菌株生物膜平均厚度(2±0.4μm)。(3)qRT-PCR檢測(cè)結(jié)果顯示,與NGV3組菌株相比,GV3菌株icaA和fnbA轉(zhuǎn)錄水平分別增高了1.67倍和3.44倍。而icaR和agr轉(zhuǎn)錄水平下降了1.88倍和2.18倍。與原代菌株相比,系列萬古霉素梯度耐藥誘導(dǎo)株icaA、fnbA、atlA和sarA轉(zhuǎn)錄水平均明顯上升,而icaR、agr轉(zhuǎn)錄水平明顯下降。且基因表達(dá)水平的變化與菌株萬古霉素MIC升高的程度密切相關(guān)。(4)相較之于原代菌株,系列萬古霉素耐藥誘導(dǎo)株的PIA表達(dá)均明顯增高。其中0534-V32菌株P(guān)IA表達(dá)量是0534菌株的5.31倍。且PIA表達(dá)水平增高與菌株萬古霉素MIC升高的程度密切相關(guān)。(5)與NGV3組菌株相比,GV3菌株耐藥相關(guān)雙組份調(diào)控系統(tǒng)VraSR轉(zhuǎn)錄水平增高了2.20倍。與原代菌株相比,系列萬古霉素梯度耐藥誘導(dǎo)株VraSR轉(zhuǎn)錄水平均明顯上升,與促生物膜形成基因(icaA、fnbA、atlA和sarA)表達(dá)量呈正相關(guān),與抑制生物膜形成的icaR、agr表達(dá)量呈負(fù)相關(guān)。使用重組表達(dá)并純化的rVraR蛋白,進(jìn)行凝膠遷移阻滯實(shí)驗(yàn),結(jié)果顯示VraR不能與fnbA、icaADBC、sar、atlA基因的啟動(dòng)子區(qū)域直接結(jié)合。但可以與Agr啟動(dòng)子區(qū)域直接結(jié)合。DNase I足跡印記實(shí)驗(yàn)進(jìn)一步驗(yàn)證表明VraR與agr啟動(dòng)子區(qū)的結(jié)合位點(diǎn)位于P2和P3啟動(dòng)子之間。結(jié)論:(1)hVISA/VISA生物膜形成增加,且隨著萬古霉素MIC值升高,生物膜形成能力逐漸增強(qiáng)。(2)hVISA/VISA可通過FnbA高表達(dá)和PIA依賴途徑促進(jìn)生物膜形成。(3)萬古霉素耐藥相關(guān)二元調(diào)控子VraSR可直接結(jié)合agr啟動(dòng)子區(qū)域,間接調(diào)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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R446.5

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