Analysis of Pathogenic Bacterial and Yeast Biofilms Using the Combination of Synchrotron ATR-FTIR Microspectroscopy and Chemometric Approaches

Abstract

Biofilms are assemblages of microbial cells, extracellular polymeric substances (EPS), and other components extracted from the environment in which they develop. Within biofilms, the spatial distribution of these components can vary. Here we present a fundamental characterization study to show differences between biofilms formed by Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative Pseudomonas aeruginosa, and the yeast-type Candida albicans using synchrotron macro attenuated total reflectance-Fourier transform infrared (ATR-FTIR) microspectroscopy. We were able to characterise the pathogenic biofilms' heterogeneous distribution, which is challenging to do using traditional techniques. Multivariate analyses revealed that the polysaccharides area (1200-950 cm^-1) accounted for the most significant variance between biofilm samples, and other spectral regions corresponding to amides, lipids, and polysaccharides all contributed to sample variation. In general, this study will advance our understanding of microbial biofilms and serve as a model for future research on how to use synchrotron source ATR-FTIR microspectroscopy to analyse their variations and spatial arrangements.

Keywords: ATR; biofilms; chemometrics; infrared; spatial heterogeneity; synchrotron.

Figure 1

SEM and CLSM images showing the morphology of pathogenic biofilms formed by MRSA, P. aeruginosa and C. albicans. An example of an early pseudohyphal cell (yellow) and yeast cell (red) are highlighted in the C. albicans biofilm. Scale bars in the insets of the electron micrographs are 1 µm for the two bacterial species and 5 µm for C. albicans.

Figure 2

Average macro ATR-FTIR spectra for the three biofilms. MRSA, PA (P. aeruginosa) and CA (C. albicans). Regions characteristic of chemical species of interest are highlighted.

Figure 3

Synchrotron macro ATR-FTIR spectral maps of the biofilms formed by MRSA, P. aeruginosa and C. albicans. All scale bars are 2 µm.

Figure 4

HCA maps of biofilm samples to identify and group regions high in amide I. The FTIR chemical image of the Amide I band (left), the HCA map of the 5 clusters (middle) and the average spectra of the clusters (right). The black arrows refer to the average spectrum, representative of the same colored cluster, which was chosen for further PCA analysis.

Figure 5

PCA analysis of the 2nd derivative spectra for each biofilm sample. (A) Second derivative spectra for each biofilm sample. (B) PCA score plots showing projections against the first 3 PCs that explain the majority of the spectral variation. (C) PC loading plots for the first 3 PCs. The wavenumbers at which significant change occurs are designated.