Evaluation of antibiotic effects on Pseudomonas aeruginosa biofilm using Raman spectroscopy and multivariate analysis

Abstract

Abstract: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.

Raman spectroscopy: techniques and applications in the life sciences

“…Some of these include the evaluation of antibiotic treatments [488], identification of circulating tumor cells [489], and classification of inflammatory bowel disease [490]. Stöckel et al used a hierarchy of SVM and LDA classifiers to identify bacterial species [491].…”

Raman and Surface-Enhanced Raman Scattering for Biofilm Characterization

“…The effects of antibiotics on Pseudomonas aeruginosa biofilms have also been shown [46]. Biofilms grown in a microfluidic device were monitored with CLSM and their composition was characterized by Raman spectroscopy in the presence of three antibiotics (ceftazidime, patulin, and epigallocatechin gallate; EGCG).…”

Label-free optical detection of age-related and diabetic oxidative damage in human aqueous humors

“…Such rapid, nondestructive, label‐free analysis has been used effectively in real‐time monitoring of subtle cellular changes. We previously investigated microstructural and biochemical changes in tissues from in vivo and in vitro human and animal studies detected by Raman spectroscopy (Ahn et al, ; Choi et al, ; Jung et al, ; Kim et al, ; Lee et al, ; Shin et al, ). Our results showed that distinctive Raman peaks were observed in different treatment methods and tissues and could be used as biomarkers to detect abnormalities.…”