人工合成的固有防御调节肽对耐甲氧西林金黄色葡萄球菌生物被膜的影响

The research of the innate defense regulator peptide on the effects of methicillin resistant staphylococcus aureus biofilm

目的探讨不同浓度固有防御调节肽（IDR-1018）对耐甲氧西林金黄色葡萄球菌（MRSA）成熟生物被膜的破坏作用以及对MRSA生物被膜形成的抑制作用。方法将MRSA以1×10^5CFU／ml均匀接种于96孔板，48h后建成生物被膜模型。给予用胰酪胨大豆肉汤培养基（TSB）倍比稀释的不同浓度的IDR-1018溶液作为实验组，使其加入细菌悬液后的浓度达到3．75-1000mg／L。红霉素倍比稀释制成不同浓度梯度的药液后，与低浓度（15mg／L）IDR-1018联合作为混合组。等量TSB作为空白对照组。24h时结晶紫染色半定量法，测吸光度（A）值，检测生物被膜的生长情况。采用SPSS18．0统计软件对数据进行统计分析。结果与对照组（A595=1．764±0．026）相比较，IDR-1018对成熟的MRSA生物被膜发挥了较为显著的破坏作用，且IDR-1018对生物被膜的影响存在一定的剂量依赖关系，即随着药物浓度的不断降低，其对生物被膜的破坏作用出现了一定幅度的下降。当浓度低至15mg／L时，A595=0．946±0．047（t=32．955，P〈0．01）；浓度为7．5mg／L时，A595=1．211±0．054（t=12．731，P〈0．05）；浓度为3．75mg／L时，A595=1．360±0．066（t=4．843，P〈0．05），与对照组相比差异仍具有统计学意义。对于尚未成熟的生物被膜，与对照组（A595=1．689±0．068）比较，当浓度低至15mg／L（A595=0．846±0．057，t=34．127，P〈0．01）时，IDR-1018对MRSA生物被膜的形成过程仍具有较为显著的抑制作用；而浓度降至7．5mg／L（A595=1．402±0．181，t=5．240，P〈0．05）时，对生物被膜的抑制作用出现了一定幅度的下降，但与对照组比较差异仍具有统计学意义。浓度为3．75mg／L（A595=1．631±0．190，t=0．913，P〉0．05）时，这种抑制作用出现了较明显的下降。当低浓度（15mg／L）的IDR-1018和不同浓度的红霉素联合使用时，其对MRSA生物被膜的破坏与抑制作用明显高于单独使用红霉素，亦优于单独使用IDR．1018时的效果。结论在MRSA生物被膜生成过程中，IDR-1018可以起到较好地抑制作用；对于已经成熟的MRSA生物被膜，IDR-1018可以起到较好地破坏作用。

Objective To investigate the destruction of the mature biofilm and the inhibitory effect of the biofilm formation of methicillin-resistant Staphylococcus aureus （MRSA） by different concentrations of the innate defense regulatory peptide （IDR-1018）. Methods 1 × 10^5CFU /ml MRSA was inoculated uniformly into 96 well plates, the biofilm model would be completed after 48 h. Given the different concentration of IDR-1018 solution as the experimental group double diluted with tryptic soy broth （TSB）, the concentration in bacteria suspension reached 3.75 - 1 000 mg/L respectively. Erythromycin is double diluted into different concentration gradient, combined with low concentration （15 mg/L） of IDR-1018 as the mixed group. The same amount of TSB treated as the blank control group. The growth of the biofilm was measured through the measurement of the value of absorbance （A）by the semi-quantitative method of crystal violet staining at 24 h. Using SPSS 18.0 as statistical software to analyze the data. Results Compared with the control group （A595 = 1. 764 ± 0. 026）, IDR-1018 significantly damaged the mature MRSA biofilm, and function was worked in a dose-dependent method. With decreasing drug concentration, the destruction of the biofilm decreased correspondingly. When the concentration was as low as 15 mg/L, A595 = 0. 946 ± 0. 047 （t = 32. 955, P 〈 0.01）. When the concentration was 7.5 mg/L, A595 = 1. 211 ± 0. 054 （ t = 12. 731, P 〈 0.05 ）. When the concentration was 3.75 mg/L, A595 = 1. 360 ± 0. 066 （ t = 4. 843, P 〈 0. 05 ）, the difference was still statistically significant compared with the control group. For the immature biofilm, compared with the control group（A595 = 1. 689 ± 0. 068 ） ,IDR-1018 still had a significant inhibitory effect on the formation process of MRSA biofilm when the concentration was as low as 15 mg/L （A595 = 0. 846 ± 0. 057, t = 34. 127, P 〈0. 01 ）. The inhibition of biofilm had a certain decline, when the concentration was 7.5 mg/L （A595 = 1. 402 ± 0. 181, t =5. 240, P 〈0. 05）. But the difference was still statistically significant compared with the control group. However, the inhibitory effect was significantly decreased when the concentration was 3.75 mg/L （A595 = 1. 631 ±0. 190,t =0. 913, P 〉0. 05）. When the low concentration （ 15 mg/L） of IDR-1018 and different concentrations of erythromycin were used together, the destruction and inhibition of MRSA biofilm was significantly higher than using erythromycin or IDR-1018 alone. Conclusion IDR-1018 can play a good inhibitory role in the formation process of MRSA biofilm, and can play a good role in destroying MRSA biofilm.