D-酪氨酸聯(lián)合萬古霉素對MRSA及其生物膜的消除作用
發(fā)布時間：2019-01-04 10:44
【摘要】：研究背景:目前人工關(guān)節(jié)置換術(shù)已成為治療終末期關(guān)節(jié)疾患、改善恢復(fù)關(guān)節(jié)功能的最有效方法之一。隨著人工關(guān)節(jié)置換手術(shù)總數(shù)的不斷增加,術(shù)后假體周圍感染(prosthetic joint infection,PJI)病人的總量也在逐漸增加,而耐甲氧西林金黃色葡萄球菌(methicillin-resistant staphylococcus aureus,MRSA)感染是PJI的治療難題。目前二期翻修手術(shù)聯(lián)合萬古霉素被認(rèn)為是假體周圍MRSA感染治療的金標(biāo)準(zhǔn),即便如此,術(shù)后仍然有感染復(fù)發(fā)的可能,而導(dǎo)致感染復(fù)發(fā)的罪魁禍?zhǔn)拙褪羌?xì)菌生物膜(biofilm,BF)。當(dāng)細(xì)菌粘附于物體表面時,細(xì)菌能通過分泌粘多糖等物質(zhì)使彼此聚集在一起,并進(jìn)一步增殖擴(kuò)散,最終形成具有屏障作用的生物膜。生物膜的屏障作用有效的阻礙了抗菌藥物對膜深層細(xì)菌的殺滅作用,在臨床中,由于生物膜對細(xì)菌的保護(hù)作用,導(dǎo)致細(xì)菌對抗生素產(chǎn)生耐藥,假體周圍一旦發(fā)生生物膜的感染,抗菌藥物很難殺滅致病菌。如果不能徹底消除生物膜,感染的復(fù)發(fā)將不可避免,因此,生物膜消除的研究越來越受到人們的重視。近年來有研究發(fā)現(xiàn),微生物能夠產(chǎn)生D-型氨基酸,這類氨基酸對枯草芽孢桿菌、銅綠假單胞菌、金黃色葡萄球菌生物膜具有分散作用,其中又以D-酪氨酸對細(xì)菌生物膜分散作用最強(qiáng),因此研究對MRSA及其生物膜有消除的方法有重要的臨床指導(dǎo)意義。目的:觀察D-酪氨酸(D-tyrosine)聯(lián)合萬古霉素對體外培養(yǎng)MRSA及其生物膜的消除作用,旨在為PJI的臨床治療提供可行性依據(jù)。方法:1.收集廣州市第一人民醫(yī)院的臨床MRSA菌株,微孔板法建立體外生物膜模型,通過結(jié)晶紫染色半定量法篩選出產(chǎn)膜能力最強(qiáng)的菌株,該菌株即為實(shí)驗(yàn)菌株。2.通過導(dǎo)片法建立體外MRSA生物膜模型,將實(shí)驗(yàn)分為四組,分別為空白組,D-酪氨酸組,萬古霉素組,聯(lián)合組(D-酪氨酸+萬古霉素),每組干預(yù)1小時、6小時、12小時、24小時后分別用結(jié)晶紫及熒光染色,分別在高倍鏡及激光共聚焦顯微鏡(CLSM)下觀察不同組MRSA及其生物膜的變化情況。結(jié)果:1.通過微孔板及結(jié)晶紫染色法篩選出編號為2913的MRSA菌株產(chǎn)膜能力最強(qiáng)。2.萬古霉素組、D-酪氨酸組、聯(lián)合組中的細(xì)菌密度會隨著時間的增加而逐漸減小,其中以D-酪氨酸組及聯(lián)合組減少明顯,且每組間具有統(tǒng)計(jì)學(xué)差異(P0.05)。3.激光共聚焦顯微鏡(CLSM)下顯示,12小時內(nèi)萬古霉素組及聯(lián)合組中死菌比例會隨著時間增加而增加,其中以聯(lián)合組增加更明顯,且聯(lián)合組與萬古霉素組比較有統(tǒng)計(jì)學(xué)意義(P0.05),12小時后萬古霉素組死菌比例無明顯變化,聯(lián)合組死菌比例進(jìn)一步增加。結(jié)論:1.D-酪氨酸對體外培養(yǎng)MRSA生物膜具有分散作用;2.D-酪氨酸聯(lián)合萬古霉素對生物膜中的MRSA具有更好的消除作用。
[Abstract]:Background: at present, artificial arthroplasty has become one of the most effective methods for the treatment of end-stage joint diseases and the improvement of joint function. With the increase of the total number of artificial joint replacement operations, the total number of patients with periprosthetic infection (prosthetic joint infection,PJI) is also increasing gradually, and methicillin-resistant Staphylococcus aureus (methicillin-resistant staphylococcus aureus,MRSA) infection is a difficult problem in the treatment of PJI. At present, the secondary revision surgery combined with vancomycin is considered to be the gold standard for the treatment of MRSA infection around the prosthesis. Even so, there is still the possibility of recurrence of infection after operation, and the main culprit causing the recurrence of infection is bacterial biofilm (biofilm,BF). When the bacteria adhere to the surface of the object, the bacteria can aggregate each other by secreting mucopolysaccharide, and further proliferate and diffuse, and finally form a barrier biofilm. The barrier effect of biofilm effectively hinders the bactericidal effect of antimicrobial agents on bacteria in deep layer of membrane. In clinic, because of the protective effect of biofilm on bacteria, bacteria become resistant to antibiotics, and once biofilm infection occurs around the prosthesis, Antimicrobial agents are difficult to kill pathogenic bacteria. If biofilm can not be eliminated completely, the recurrence of infection will be inevitable. Therefore, the study of biofilm elimination has been paid more and more attention. In recent years, it has been found that microbes can produce D- amino acids, which can disperse the biofilm of Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. Among them, Dtyrosine has the strongest effect on bacterial biofilm dispersion, so the study has important clinical significance for the elimination of MRSA and its biofilm. Aim: to observe the effect of D tyrosine (D-tyrosine) combined with vancomycin on the elimination of MRSA and its biofilm in vitro. Methods: 1. Clinical MRSA strains from the first people's Hospital of Guangzhou were collected. The biofilm model in vitro was established by microporous plate method. The strain with the strongest membrane capacity was screened by semi-quantitative method of crystal violet staining. The model of MRSA biofilm in vitro was established by the method of guide tablets. The experiment was divided into four groups: blank group, D-tyrosine group, vancomycin group and combined group (Dtyrosine vancomycin). After 24 hours, the changes of MRSA and biofilm of different groups were observed under high-power microscope and laser confocal microscope (CLSM) with crystal violet and fluorescence staining respectively. Results: 1. Through micropore plate and crystal violet staining method, the MRSA strain numbered 2913 had the strongest membrane production ability. 2. 2. The bacterial density in vancomycin group, D-tyrosine group and combination group decreased gradually with the increase of time, especially in Dtyrosine group and combined group, and there was statistical difference between each group (P0.05). Laser confocal microscope (CLSM) showed that the proportion of dead bacteria in vancomycin group and combined group increased with time within 12 hours, especially in combination group. And the combined group and vancomycin group compared with the statistical significance (P0.05), 12 hours after vancomycin group no significant change in the proportion of dead bacteria, the combined group further increased the proportion of dead bacteria. Conclusion: 1. D- tyrosine can disperse MRSA biofilm in vitro, 2. D- tyrosine combined with vancomycin can eliminate MRSA in biofilm.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R687.4
本文編號：2400178
[Abstract]:Background: at present, artificial arthroplasty has become one of the most effective methods for the treatment of end-stage joint diseases and the improvement of joint function. With the increase of the total number of artificial joint replacement operations, the total number of patients with periprosthetic infection (prosthetic joint infection,PJI) is also increasing gradually, and methicillin-resistant Staphylococcus aureus (methicillin-resistant staphylococcus aureus,MRSA) infection is a difficult problem in the treatment of PJI. At present, the secondary revision surgery combined with vancomycin is considered to be the gold standard for the treatment of MRSA infection around the prosthesis. Even so, there is still the possibility of recurrence of infection after operation, and the main culprit causing the recurrence of infection is bacterial biofilm (biofilm,BF). When the bacteria adhere to the surface of the object, the bacteria can aggregate each other by secreting mucopolysaccharide, and further proliferate and diffuse, and finally form a barrier biofilm. The barrier effect of biofilm effectively hinders the bactericidal effect of antimicrobial agents on bacteria in deep layer of membrane. In clinic, because of the protective effect of biofilm on bacteria, bacteria become resistant to antibiotics, and once biofilm infection occurs around the prosthesis, Antimicrobial agents are difficult to kill pathogenic bacteria. If biofilm can not be eliminated completely, the recurrence of infection will be inevitable. Therefore, the study of biofilm elimination has been paid more and more attention. In recent years, it has been found that microbes can produce D- amino acids, which can disperse the biofilm of Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. Among them, Dtyrosine has the strongest effect on bacterial biofilm dispersion, so the study has important clinical significance for the elimination of MRSA and its biofilm. Aim: to observe the effect of D tyrosine (D-tyrosine) combined with vancomycin on the elimination of MRSA and its biofilm in vitro. Methods: 1. Clinical MRSA strains from the first people's Hospital of Guangzhou were collected. The biofilm model in vitro was established by microporous plate method. The strain with the strongest membrane capacity was screened by semi-quantitative method of crystal violet staining. The model of MRSA biofilm in vitro was established by the method of guide tablets. The experiment was divided into four groups: blank group, D-tyrosine group, vancomycin group and combined group (Dtyrosine vancomycin). After 24 hours, the changes of MRSA and biofilm of different groups were observed under high-power microscope and laser confocal microscope (CLSM) with crystal violet and fluorescence staining respectively. Results: 1. Through micropore plate and crystal violet staining method, the MRSA strain numbered 2913 had the strongest membrane production ability. 2. 2. The bacterial density in vancomycin group, D-tyrosine group and combination group decreased gradually with the increase of time, especially in Dtyrosine group and combined group, and there was statistical difference between each group (P0.05). Laser confocal microscope (CLSM) showed that the proportion of dead bacteria in vancomycin group and combined group increased with time within 12 hours, especially in combination group. And the combined group and vancomycin group compared with the statistical significance (P0.05), 12 hours after vancomycin group no significant change in the proportion of dead bacteria, the combined group further increased the proportion of dead bacteria. Conclusion: 1. D- tyrosine can disperse MRSA biofilm in vitro, 2. D- tyrosine combined with vancomycin can eliminate MRSA in biofilm.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R687.4
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