流式细胞术应用于铜绿假单胞菌体外药敏实验的研究

发布时间：2018-01-21 23:41

  本文关键词： 流式细胞术(FCM) 碘化丙啶阳性百分率(PI%) 平均荧光强度(MFI) 流式细胞荧光术抗生素敏感试验(FCST) 铜绿假单胞菌(PA)　出处：《苏州大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：传统的抗生素敏感试验包括：稀释法(肉汤稀释法和琼脂稀释法)、纸片扩散法和E-试验，均需要将细菌在药物作用下培养过夜才能观察结果，相当费时费力。目前多采用比浊法目测结果，提供的结果代表细菌生长的均数，对亚菌群的敏感性无法判断。流式细胞仪可同时、快速、多参数地分析单个细胞的生物学特性，已经被应用于生物医学的许多领域。铜绿假单胞菌(Pseudomonas Aeruginosa PA)是医院感染的三大病原菌之一，探索一种快速检测此类细菌及其药物敏感性的方法，对于重症感染性疾病的快速诊断和治疗，预防和治疗医院感染，减少多重耐药菌株的产生与传播，减少患者和医院的负担有重要的经济效益与社会效益。 本试验选择能够通过破损的细胞膜进入细胞并与核酸相结合的荧光染料碘化丙啶(PI)，使其着染于经过抗生素处理后的细菌，用流式细胞仪检测细菌荧光信息的变化，推断细菌对抗生素的敏感性和药物的最低抑菌浓度(Minimal inhibitory concentration，MIC)。并与传统的试管稀释法检测结果进行比较，客观判断流式细胞荧光术抗生素敏感试验(Flow cytofluorometric antibiotic susceptibility test，FCST)的优缺点，为该方法在临床上的应用积累实验依据。 方法：我们收集了2003-2004年苏州大学附属儿童医院外科及重症监护病房医院感染患儿的临床标本中分离出的铜绿假单胞菌共22株，并购买了质控标准菌株ATCC27853。将细菌转种于血琼脂平板，35℃孵育过夜，次日从平板上挑取1～2个菌落，接种于M-H肉汤培养基中，于35℃恒温水浴箱中培养2小时，用VITEK比浊仪调整菌液浓度至0.5麦氏单位(相当于1.5×10~8CFU／ml)。在测定管中加入亚胺培南1.0ml使其终浓度为32.0、16.0、8.0……0.25、0.125ug／ml，然后在测定管中加入细菌悬液1.0ml，调整使其终浓度为7.5×10~6CFU／ml，37.0℃孵育3小时后加入染料PI 10ul(最终浓度为10ug／m)室温避光5分钟后，以流式细胞仪检测10~4以上个细菌。根据荧光信息的变化判断细菌对药物的敏感性。随着药物浓度的增加，如果碘化丙啶荧光阳性百分率(PI％)或平均荧光强度(MFI)均在狭窄范围内，无明显上升，判断为菌株耐药：若PI％逐渐增至75％或以上，则此时的最低药物浓度为MIC，，
[Abstract]:Objective: traditional antibiotic sensitivity tests include: broth dilution and Agar dilution test, paper diffusion method and E- test, bacteria need to be cultured overnight in order to observe the results. It is rather time-consuming and laborious. At present, most of the results are measured by turbidimetry. The results provided represent the mean number of bacteria growth, and the sensitivity to subflora can not be judged. Flow cytometry can be used simultaneously and quickly. The biological characteristics of single cell were analyzed with multiple parameters. Pseudomonas Aeruginosa PAA is one of the three major pathogens of nosocomial infection. To explore a method for rapid detection of these bacteria and their drug sensitivity, for the rapid diagnosis and treatment of severe infectious diseases, to prevent and treat nosocomial infections, and to reduce the production and transmission of multidrug resistant strains. Reducing the burden of patients and hospitals has important economic and social benefits. In this experiment, we selected a fluorescent dye, pyridine iodide, which can enter the cell through damaged cell membrane and bind to nucleic acid, so that it can be infected with bacteria treated with antibiotics. The changes of bacterial fluorescence information were detected by flow cytometry. The sensitivity of bacteria to antibiotics and the minimum inhibitory concentration (MEC) of bacteria to antibiotics were inferred. The results were compared with the traditional test tube dilution method. Objective judgement of antibiotic sensitivity test by flow cytometry (FCM). Flow cytofluorometric antibiotic susceptibility test. The advantages and disadvantages of FCSTs provide experimental basis for the clinical application of FCSTs. Methods: from 2003 to 2004, 22 strains of Pseudomonas aeruginosa were isolated from surgical and intensive care unit patients with nosocomial infection in the Children's Hospital affiliated to Suzhou University. The standard strain ATCC27853 was purchased. The bacteria were transferred to the blood Agar plate at 35 鈩

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