本文選題：金黃色葡萄球菌 + 生物膜　； 參考：《廣西醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 金黃色葡萄球菌(Staphylococcus aureus, S. aureus)是常見(jiàn)的革蘭染色陽(yáng)性條件致病菌之一,是醫(yī)學(xué)相關(guān)材料、呼吸機(jī)相關(guān)肺炎、支氣管擴(kuò)張、肺膿腫等疾病的常見(jiàn)病原體。在急性感染過(guò)程中,S. aureus對(duì)常用抗生素耐藥與其自身特征有關(guān)；而在慢性感染過(guò)程中,其耐藥則與細(xì)菌生物膜(bacterial biofilm, BF)形成關(guān)系密切。 參閱近年眾多國(guó)內(nèi)外文獻(xiàn)資料,目前尚無(wú)MRSA與MSSA,以及同源性S. aureus的BF形成能力差異報(bào)道；同時(shí),目前尚無(wú)能形成BF的S. aureus國(guó)際公認(rèn)菌株,為減少實(shí)驗(yàn)偏倚,使研究結(jié)果穩(wěn)定、可靠、可重復(fù)性強(qiáng),有必要篩選出具有代表性可形成BF的S. aureus,作為后繼科學(xué)研究用菌株。 基于此,我課題組已對(duì)廣西醫(yī)科大學(xué)第一附屬醫(yī)院臨床微生物檢驗(yàn)中心2007年12月分離出的21株S. aureus進(jìn)行了耐藥譜分型及蛋白A基因(spa)分型。在此基礎(chǔ)上,本課題首先建立S. aureus的BF體外靜置模型；然后,比較其BF的形成能力；最后,探討鹽酸氨溴索協(xié)同萬(wàn)古霉素對(duì)S. aureus生物膜的干預(yù)作用。 第一部分金黃色葡萄球菌生物膜形成能力比較 目的建立S. aureus的BF體外靜置模型,比較臨床分離21株S.aureus的BF形成能力差異。 方法(1)以醫(yī)學(xué)材料(聚氯乙烯)為載體,采用胰蛋白胨大豆肉湯(tryptic soy broth, TSB)培養(yǎng)基,建立S. aureus體外靜置BF模型,分別于建模第3天和第7天,用銀染、掃描電子顯微鏡·(scanning electron microscope,SEM)觀察載體表面形成的BF。(2)探討TSB中不同濃度葡萄糖(2.5 g／L5.0 g／L、7.5 g／L、10 g／L)對(duì)S. aureus粘附及其BF形成能力的影響。(3)采用剛果紅平板法定性能形成BF的菌株,對(duì)能形成BF的S. aureus采用結(jié)晶紫染色BF半定量法,比較MRSA與MSSA、同源性S. aureus的粘附及其BF形成能力。 結(jié)果(1)培養(yǎng)3天,載體銀染后光鏡下觀察,可見(jiàn)團(tuán)塊狀、棉絮樣的BF,SEM觀察載體表面可見(jiàn)早期BF；培養(yǎng)7天,載體銀染后光鏡下觀察,可見(jiàn)體積更大的團(tuán)塊狀、棉絮樣的BF,SEM觀察載體表面可見(jiàn)具有立體結(jié)構(gòu)的成熟BF。(2)含5.0g／L葡萄糖的TSB,能明顯促進(jìn)S. aureus粘附于載體表面,并促進(jìn)其BF形成；但是加入更高濃度(7.5g／L、10g／L)的葡萄糖其促進(jìn)S. aureus粘附和BF形成能力并不呈遞增關(guān)系。(3)臨床分離21株S. aureus株中,有20株為剛果紅平板法定性BF形成陽(yáng)性菌株,陽(yáng)性率為95.2%(20／21)。(4)MRSA的粘附及早期BF形成能力明顯強(qiáng)于MSSA；但隨著培養(yǎng)時(shí)間的延長(zhǎng),至形成成熟BF結(jié)構(gòu)時(shí),MSSA的BF形成能力明顯強(qiáng)于MRSA。(5)同源性S. aureus之間粘附及早期BF形成能力有差異：與其它S. aureus比較,編號(hào)為17546的S. aureus粘附及BF形成能力最強(qiáng),編號(hào)為17422的S. aureus粘附及BF形成能力最弱,差異有統(tǒng)計(jì)學(xué)意義,P0.001；在成熟BF階段,與其他S. aureus相比,編號(hào)為17546、17642的S. aureus生物膜形成能力最強(qiáng),差異有統(tǒng)計(jì)學(xué)意義,P0.001；編號(hào)為17422的S. aureus生物膜形成能力弱,與編號(hào)為17431、18541-2、18558、18565、18719的S. aureus相比,差異無(wú)統(tǒng)計(jì)學(xué)意義,P0.05。結(jié)論1.以聚氯乙烯為載體,含5%葡萄糖的TSB為培養(yǎng)基,能成功建立S. aureus的BF體外靜置模型；2.我院形成BF的S. aureus檢出率高,且MRSA與MSSA之間,同源性S. aureus之間BF的形成能力有差異。 第二部分鹽酸氨溴索協(xié)同萬(wàn)古霉素對(duì)金黃色葡萄球菌生物膜破壞及協(xié)同殺菌作用 目的探討鹽酸氨溴索協(xié)同萬(wàn)古霉素對(duì)S. aureus的BF破壞作用,以及協(xié)同萬(wàn)古霉素對(duì)BF內(nèi)S. aureus的殺菌作用。 方法(1)首先檢測(cè)萬(wàn)古霉素對(duì)S. aureus的MIC、MBC。(2)分為空白對(duì)照組、鹽酸氨溴索組、萬(wàn)古霉素組、鹽酸氨溴索+萬(wàn)古霉素組。于建模第3天、第7天分別加入上述藥物及其組合,藥物作用24小時(shí)后,SEM、銀染法觀察載體表面BF；藥物作用24小時(shí)后,采用MTT法行載體表面活菌計(jì)數(shù)。 結(jié)果(1)萬(wàn)古霉素對(duì)S. aureus的MIC、MBC分別是1μg／mL、2μg／mL。(2)無(wú)論是培養(yǎng)3天,還是培養(yǎng)7天,藥物作用24小時(shí)后,SEM觀察發(fā)現(xiàn),萬(wàn)古霉素組、鹽酸氨溴索+萬(wàn)古霉素組載體表面BF較空白對(duì)照組,BF結(jié)構(gòu)明顯破壞,且鹽酸氨溴索+萬(wàn)古霉素組明顯于萬(wàn)古霉素組。(3)無(wú)論是培養(yǎng)3天,還是培養(yǎng)7天,MTT活菌計(jì)數(shù)法結(jié)果顯示,藥物作用24小時(shí)后,鹽酸氨溴索+萬(wàn)古霉素組、萬(wàn)古霉素組活菌數(shù)少于空白對(duì)照組、鹽酸氨溴索組,且鹽酸氨溴索+萬(wàn)古霉素組少于萬(wàn)古霉素組。 結(jié)論鹽酸氨溴索具有協(xié)同萬(wàn)古霉素破壞S. aureus的BF作用,并能協(xié)同萬(wàn)古霉素減少BF內(nèi)的S. aureus。
[Abstract]:Staphylococcus aureus (Staphylococcus aureus, S. aureus) is one of the common pathogens of gram-positive positive conditions. It is a common pathogen of medical related materials, ventilator related pneumonia, bronchiectasis, and lung abscess. In the course of acute infection, the resistance of S. aureus to common antibiotics is related to its own characteristics; but in the slow process, it is slow. In the course of sexual infection, the drug resistance is closely related to the bacterial biofilm (BF).
Referring to many domestic and foreign literature in recent years, there is no MRSA and MSSA, and the difference of BF formation ability of homologous S. aureus. At the same time, there is no internationally recognized S. aureus strain to form BF, in order to reduce experimental bias and make the research result stable, reliable and repeatable, and it is necessary to screen out S. a that can represent BF. UREUS, as a successor scientific research strain.
Based on this, we have classified 21 strains of S. aureus isolated from the clinical microbiology test center of the First Affiliated Hospital of Guangxi Medical University in December 2007. On the basis of this, we first set up BF in vitro S. model of S. aureus; then, compare the formation ability of BF; finally, explore the formation ability of BF. Objective to explore the intervention effect of ambroxol hydrochloride combined with vancomycin on S. aureus biofilm.
Part 1 Comparison of biofilm forming ability of Staphylococcus aureus
Objective to establish a BF static model of S. aureus in vitro and compare the difference in BF formation ability between 21 S.aureus isolates.
Methods (1) the S. aureus in vitro BF model was established by using tryptic soy broth (TSB) culture medium as the carrier of medical material (polyvinyl chloride) and the S. aureus in vitro BF model was established. The BF. (2) was studied by silver staining, scanning electron microscope (scanning electron microscope, SEM), respectively. The effect of concentration of glucose (2.5 g / L5.0 g / L, 7.5 g / L, 10 g / L) on the adhesion of S. aureus and its BF formation ability. (3) using the legal performance of Congo red plate to form a BF strain.
Results (1) for 3 days, it was observed under the light microscope of the carrier silver dye that the early BF was visible on the surface of the BF, and the surface of the carrier was observed on the surface of the carrier. For 7 days, the bulk of the bulk, the cotton like BF, the SEM observation carrier showed the mature BF. (2) containing 5.0g / L glucose in the surface of the carrier, and the TSB of 5.0g / L was visible on the surface of the carrier. Obviously, S. aureus adhered to the surface of the carrier and promoted the formation of BF, but the glucose added to the higher concentration (7.5G / L, 10g / L) did not increase the S. aureus adhesion and BF formation ability. (3) in 21 S. aureus strains, 20 strains formed a positive strain for Congo red plate, with a positive rate of 95.2% (20 / 2). 1) (4) the adhesion and early BF formation ability of MRSA is stronger than that of MSSA, but with the prolongation of culture time and the formation of mature BF structure, the BF formation ability of MSSA is significantly stronger than that of MRSA. (5) homologous S. aureus and the ability of early BF formation. The strongest, 17422 S. aureus adhesion and BF formation ability was the weakest, the difference was statistically significant, P0.001; in the mature BF stage, the S. aureus biofilm with the number of 1754617642 was the strongest, and the difference was statistically significant, P0.001; the S. aureus biofilm numbered 17422 was weak, and the number was 17. Compared with 43118541-2185581856518719 S. aureus, the difference was not statistically significant. P0.05. conclusion 1. using polyvinyl chloride as the carrier and the TSB containing 5% glucose as the medium, the BF in vitro static model of S. aureus can be successfully established. 2. the S. aureus detection rate of BF in our hospital is high, and between MRSA and MSSA. There is a difference.
The second part is the destruction and synergistic bactericidal effect of ambroxol hydrochloride combined with vancomycin on Staphylococcus aureus biofilm.
Objective to explore the destruction effect of ambroxol hydrochloride combined with vancomycin on BF of S. aureus and the bactericidal effect of vancomycin combined with vancomycin on BF S. aureus.
Method (1) first test vancomycin S. aureus MIC, MBC. (2) divided into blank control group, ambroxol hydrochloride group, vancomycin group, ambroxol HCl + vancomycin group. After modeling for third days, seventh days respectively added to the drugs and their combination, after 24 hours of drug effect, SEM, silver staining method to observe the carrier surface BF; after 24 hours of drug effect, the use of drugs, the use of the drug The MTT method was used to count the living bacteria on the surface of the carrier.
Results (1) vancomycin for S. aureus MIC, MBC was 1 g / mL, 2 mu g / mL. (2) no matter for 3 days, or 7 days of culture, and after 24 hours of drug effect, SEM observation found that vancomycin group, ambroxol + vancomycin group carrier surface BF compared with the blank control group, BF structure obviously destroyed, and ambroxol + vancomycin group was obvious ten thousand The group of palaeomycin. (3) no matter for 3 days or 7 days of culture, the results of MTT living bacteria count showed that after 24 hours of drug action, the number of ambroxol + vancomycin hydrochloride and vancomycin group was less than that of the blank control group, ambroxol hydrochloride group and ambroxol and vancomycin hydrochloride group were less than the vancomycin group.
Conclusion ambroxol hydrochloride has synergistic effect on vancomycin destroying S. aureus, and can synergy with vancomycin to reduce S. aureus. in BF BF.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378

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