Jung Gyeong Bok, Nam Seong Won, Choi Samjin, Lee Gi-Ja, Park Hun-Kuk
Department of Biomedical Engineering & Healthcare Industry Research Institute, College of Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, South Korea ; These authors contributed equally to this work.
Department of Biomedical Engineering & Healthcare Industry Research Institute, College of Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, South Korea ; Program of Medical Engineering, Kyung Hee University, Seoul 130-701, South Korea.
Biomed Opt Express. 2014 Aug 28;5(9):3238-51. doi: 10.1364/BOE.5.003238. eCollection 2014 Sep 1.
We investigate the mode of action and classification of antibiotic agents (ceftazidime, patulin, and epigallocatechin gallate; EGCG) on Pseudomonas aeruginosa (P. aeruginosa) biofilm using Raman spectroscopy with multivariate analysis, including support vector machine (SVM) and principal component analysis (PCA). This method allows for quantitative, label-free, non-invasive and rapid monitoring of biochemical changes in complex biofilm matrices with high sensitivity and specificity. In this study, the biofilms were grown and treated with various agents in the microfluidic device, and then transferred onto gold-coated substrates for Raman measurement. Here, we show changes in biochemical properties, and this technology can be used to distinguish between changes induced in P. aeruginosa biofilms using three antibiotic agents. The Raman band intensities associated with DNA and proteins were decreased, compared to control biofilms, when the biofilms were treated with antibiotics. Unlike with exposure to ceftazidime and patulin, the Raman spectrum of biofilms exposed to EGCG showed a shift in the spectral position of the CH deformation stretch band from 1313 cm(-1) to 1333 cm(-1), and there was no difference in the band intensity at 1530 cm(-1) (C = C stretching, carotenoids). The PCA-SVM analysis results show that antibiotic-treated biofilms can be detected with high sensitivity of 93.33%, a specificity of 100% and an accuracy of 98.33%. This method also discriminated the three antibiotic agents based on the cellular biochemical and structural changes induced by antibiotics with high sensitivity and specificity of 100%. This study suggests that Raman spectroscopy with PCA-SVM is potentially useful for the rapid identification and classification of clinically-relevant antibiotics of bacteria biofilm. Furthermore, this method could be a powerful approach for the development and screening of new antibiotics.
我们使用包括支持向量机（SVM）和主成分分析（PCA）在内的多变量分析拉曼光谱，研究了抗生素（头孢他啶、棒曲霉素和表没食子儿茶素没食子酸酯；EGCG）对铜绿假单胞菌生物膜的作用方式和分类。该方法能够以高灵敏度和特异性对复杂生物膜基质中的生化变化进行定量、无标记、非侵入性且快速的监测。在本研究中，生物膜在微流控装置中生长并用各种试剂处理，然后转移到涂金的基底上进行拉曼测量。在此，我们展示了生化特性的变化，并且该技术可用于区分使用三种抗生素在铜绿假单胞菌生物膜中诱导的变化。与对照生物膜相比，当生物膜用抗生素处理时，与DNA和蛋白质相关的拉曼带强度降低。与暴露于头孢他啶和棒曲霉素不同，暴露于EGCG的生物膜的拉曼光谱显示CH变形伸缩带的光谱位置从1313 cm(-1) 移至1333 cm(-1)，并且在1530 cm(-1)（C = C伸缩，类胡萝卜素）处的带强度没有差异。PCA - SVM分析结果表明，抗生素处理的生物膜能够以93.33%的高灵敏度、100%的特异性和98.33%的准确度被检测到。该方法还基于抗生素诱导的细胞生化和结构变化，以100%的高灵敏度和特异性区分了三种抗生素。本研究表明，结合PCA - SVM的拉曼光谱对于细菌生物膜临床相关抗生素的快速鉴定和分类具有潜在用途。此外，该方法可能是开发和筛选新抗生素的有力途径。
