红霉素和鼻渊舒对铜绿假单胞菌体外生物膜形成抑制作用研究

发布时间：2018-11-20 17:13

【摘要】：目的：观察大环内酯类药物红霉素及中药制剂鼻渊舒口服液在体外对铜绿假单胞菌生物膜形成的抑制作用,及对已经形成的铜绿假单胞菌生物膜的清除作用。方法：采用3株临床分离的铜绿假单胞菌,利用平板法胰酶大豆肉汤-硅胶片系统,在体外试建立细菌生物膜的模型,连续培养7天后用银染法及扫描电子显微镜鉴定,选出易形成生物膜的1株菌株进行随后的实验。分别用浓度为1/16MIC.1/4MIC.1MIC.4MIC的红霉素及以1：5、1：10、1：20、1：40倍比稀释的鼻渊舒口服液同时与此菌株在胰酶大豆肉汤-硅胶片系统中连续培养7天,使药物作用于尚未形成生物膜的铜绿假单胞菌,用银染法及震荡-活菌计数法观察上述药物对生物膜形成的抑制作用。菌落计数结果用均数±标准差表示,计数结果用SAS9.1软件进行统计,采用单因素方差分析进行两两比较的检验,以α=0.05为检验水准。及用上述不同浓度的红霉素及鼻渊舒作用于此菌株已在胰酶大豆肉汤-硅胶片系统中培养7天的成熟铜绿假单胞菌生物膜,24小时后银染法观察其对已形成的生物膜的清除作用。结果： 3株铜绿假单胞菌在胰酶大豆肉汤-硅胶片系统中培养7天后,银染结果示其中1株铜绿假单胞菌在硅胶片上形成了稳定细菌生物膜,并与扫描电镜观察结果一致。在此后的实验中,红霉素及鼻渊舒表现出在体外能抑制此铜绿假单胞菌形成成熟的细菌生物膜,银染结果光学显微镜观察示药物作用后硅胶片上的黑色絮状物较阳性对照明显减少,且抑制作用随药物浓度的增加而加强。连续稀释法菌落计数结果表明,不同浓度的红霉素及鼻渊舒均能在成熟的细菌生物膜形成前抑制细菌生长,与阳性对照组比较差异有统计学意义(P0.05)。但红霉素及鼻渊舒对已形成的铜绿假单胞菌细菌生物膜的清除作用不明显。结论： 1、用平板法胰酶大豆肉汤—硅胶片系统可以在体外建立稳定的铜绿假单胞菌生物膜。银染法可以对形成的细菌生物膜的形态进行检验。振荡一活菌计数可用于生物膜内细菌数量的定量分析。 2、鼻渊舒口服液是混合制剂,其对铜绿假单胞菌的最低抑菌浓度虽无法检测,但因其对铜绿假单胞菌生物膜的生成有抑制作用,同样可证实其对游离菌也有抑制作用。 3、红霉素及鼻渊舒对体外铜绿假单胞菌细菌生物膜的形成具有一定抑制作用,且抑制作用主要在细菌生物膜完全成熟以前。在本实验条件下,红霉素及鼻渊舒对已形成的细菌生物膜的清除作用并不显著。
[Abstract]:Aim: to observe the inhibitory effect of macrolides erythromycin and Biyuanshu oral liquid on the formation of Pseudomonas aeruginosa biofilm in vitro and the scavenging effect on the biofilm of Pseudomonas aeruginosa. Methods: three clinical isolates of Pseudomonas aeruginosa were used to establish the bacterial biofilm model in vitro by using a plate system of trypsin soybean broth and silica gel. The bacterial biofilm was identified by silver staining and scanning electron microscopy (SEM) after 7 days of continuous culture. A strain that could easily form biofilm was selected for subsequent experiments. The strain was incubated with erythromycin (1/16MIC.1/4MIC.1MIC.4MIC) at the concentration of 1: 5, 1: 10, 1: 1: 1: 20: 1: 40 times than the diluted Biyuanshu oral liquid in the trypsin soybean broth-silica gel tablet system for 7 days, respectively. The inhibitory effects of these drugs on biofilm formation of Pseudomonas aeruginosa were observed by silver staining and oscillation-living bacteria counting. The results of colony counting were expressed as mean 卤standard deviation. The counting results were counted by SAS9.1 software. The results were tested by single factor ANOVA, and 伪 = 0. 05 as the test level. The mature Pseudomonas aeruginosa biofilm was cultured in trypsin soybean broth and silica gel tablet system with different concentrations of erythromycin and Biyuanshu for 7 days. After 24 hours silver staining was used to observe the scavenging effect on the formed biofilm. Results: after three Pseudomonas aeruginosa strains were cultured in trypsin soybean broth and silica gel tablet system for 7 days, silver staining showed that one of them formed stable bacterial biofilm on silica gel sheet, and the results were consistent with those observed by scanning electron microscope. In subsequent experiments, erythromycin and Biyuanshu showed that they could inhibit the formation of mature bacterial biofilms from Pseudomonas aeruginosa in vitro. The results of silver staining optical microscope showed that the black flocculants on the silica gel tablets were significantly decreased compared with the positive control, and the inhibitory effect was enhanced with the increase of the drug concentration. Continuous dilution method showed that erythromycin and Biyuanshu at different concentrations could inhibit the growth of bacteria before the formation of mature bacterial biofilm. There was significant difference compared with the positive control group (P0.05). However, erythromycin and Biyuanshu had no obvious scavenging effect on bacterial biofilm of Pseudomonas aeruginosa. Conclusion: 1. A stable Pseudomonas aeruginosa biofilm could be established in vitro by using the plate system of trypsin soybean broth and silica gel tablets. The morphology of bacterial biofilm can be examined by silver staining. The count of oscillating live bacteria can be used for quantitative analysis of bacteria in biofilm. 2, Biyuanshu oral liquid is a mixed preparation, although its minimum inhibitory concentration against Pseudomonas aeruginosa can not be detected, it can also be proved that it has inhibitory effect on free bacteria because of its inhibitory effect on the formation of biofilm of Pseudomonas aeruginosa. 3.Erythromycin and Biyuanshu inhibited the formation of bacterial biofilm of Pseudomonas aeruginosa in vitro, and the inhibitory effect was mainly before the bacterial biofilm matured. Under the experimental conditions, erythromycin and Biyuanshu had no significant scavenging effect on the formed bacterial biofilm.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R765.4

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