發(fā)布時間：2018-11-20 17:13

【摘要】：目的： 觀察大環(huán)內(nèi)酯類藥物紅霉素及中藥制劑鼻淵舒口服液在體外對銅綠假單胞菌生物膜形成的抑制作用,及對已經(jīng)形成的銅綠假單胞菌生物膜的清除作用。 方法： 采用3株臨床分離的銅綠假單胞菌,利用平板法胰酶大豆肉湯-硅膠片系統(tǒng),在體外試建立細(xì)菌生物膜的模型,連續(xù)培養(yǎng)7天后用銀染法及掃描電子顯微鏡鑒定,選出易形成生物膜的1株菌株進(jìn)行隨后的實(shí)驗(yàn)。分別用濃度為1/16MIC.1/4MIC.1MIC.4MIC的紅霉素及以1：5、1：10、1：20、1：40倍比稀釋的鼻淵舒口服液同時與此菌株在胰酶大豆肉湯-硅膠片系統(tǒng)中連續(xù)培養(yǎng)7天,使藥物作用于尚未形成生物膜的銅綠假單胞菌,用銀染法及震蕩-活菌計數(shù)法觀察上述藥物對生物膜形成的抑制作用。菌落計數(shù)結(jié)果用均數(shù)±標(biāo)準(zhǔn)差表示,計數(shù)結(jié)果用SAS9.1軟件進(jìn)行統(tǒng)計,采用單因素方差分析進(jìn)行兩兩比較的檢驗(yàn),以α=0.05為檢驗(yàn)水準(zhǔn)。及用上述不同濃度的紅霉素及鼻淵舒作用于此菌株已在胰酶大豆肉湯-硅膠片系統(tǒng)中培養(yǎng)7天的成熟銅綠假單胞菌生物膜,24小時后銀染法觀察其對已形成的生物膜的清除作用。 結(jié)果： 3株銅綠假單胞菌在胰酶大豆肉湯-硅膠片系統(tǒng)中培養(yǎng)7天后,銀染結(jié)果示其中1株銅綠假單胞菌在硅膠片上形成了穩(wěn)定細(xì)菌生物膜,并與掃描電鏡觀察結(jié)果一致。在此后的實(shí)驗(yàn)中,紅霉素及鼻淵舒表現(xiàn)出在體外能抑制此銅綠假單胞菌形成成熟的細(xì)菌生物膜,銀染結(jié)果光學(xué)顯微鏡觀察示藥物作用后硅膠片上的黑色絮狀物較陽性對照明顯減少,且抑制作用隨藥物濃度的增加而加強(qiáng)。連續(xù)稀釋法菌落計數(shù)結(jié)果表明,不同濃度的紅霉素及鼻淵舒均能在成熟的細(xì)菌生物膜形成前抑制細(xì)菌生長,與陽性對照組比較差異有統(tǒng)計學(xué)意義(P0.05)。但紅霉素及鼻淵舒對已形成的銅綠假單胞菌細(xì)菌生物膜的清除作用不明顯。 結(jié)論： 1、用平板法胰酶大豆肉湯—硅膠片系統(tǒng)可以在體外建立穩(wěn)定的銅綠假單胞菌生物膜。銀染法可以對形成的細(xì)菌生物膜的形態(tài)進(jìn)行檢驗(yàn)。振蕩一活菌計數(shù)可用于生物膜內(nèi)細(xì)菌數(shù)量的定量分析。 2、鼻淵舒口服液是混合制劑,其對銅綠假單胞菌的最低抑菌濃度雖無法檢測,但因其對銅綠假單胞菌生物膜的生成有抑制作用,同樣可證實(shí)其對游離菌也有抑制作用。 3、紅霉素及鼻淵舒對體外銅綠假單胞菌細(xì)菌生物膜的形成具有一定抑制作用,且抑制作用主要在細(xì)菌生物膜完全成熟以前。在本實(shí)驗(yàn)條件下,紅霉素及鼻淵舒對已形成的細(xì)菌生物膜的清除作用并不顯著。
[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 鹵standard deviation. The counting results were counted by SAS9.1 software. The results were tested by single factor ANOVA, and 偽 = 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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R765.4

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