Label-Free SERS Discrimination and In Situ Analysis of Life Cycle in Escherichia coli and Staphylococcus epidermidis

Abstract

Surface enhanced Raman spectroscopy (SERS) has been proven suitable for identifying and characterizing different bacterial species, and to fully understand the chemically driven metabolic variations that occur during their evolution. In this study, SERS was exploited to identify the cellular composition of Gram-positive and Gram-negative bacteria by using mesoporous silicon-based substrates decorated with silver nanoparticles. The main differences between the investigated bacterial strains reside in the structure of the cell walls and plasmatic membranes, as well as their biofilm matrix, as clearly noticed in the corresponding SERS spectrum. A complete characterization of the spectra was provided in order to understand the contribution of each vibrational signal collected from the bacterial culture at different times, allowing the analysis of the bacterial populations after 12, 24, and 48 h. The results show clear features in terms of vibrational bands in line with the bacterial growth curve, including an increasing intensity of the signals during the first 24 h and their subsequent decrease in the late stationary phase after 48 h of culture. The evolution of the bacterial culture was also confirmed by fluorescence microscope images.

Keywords: E. coli; S. epidermidis; SERS; biofilm; metal-dielectric nanostructures.

Figure 1

Scheme of the surface enhanced Raman spectroscopy (SERS) analysis of E. coli and S. epidermidis grown on brain heart infusion (BHI) agar and collected after different times of culture.

Figure 2

SERS spectra of E. coli (a) and S. epidermidis (b) in the 600 to 1800 cm⁻¹ range, collected after 24 h of culturing. The spectrum was divided into three distinct zones (labeled zones I to III), while the band frequencies were labeled according to Table 1.

Figure 3

SERS spectra of E. coli (a) and S. epidermidis (b) in the 600 to 1800 cm⁻¹ range, collected after 12, 24, and 48 h of culturing. The spectrum was divided into three distinct zones (labeled zones I to III), while the band frequencies were labeled according to Table 1.

Figure 4

Fluorescence micrograph after propidium iodide (PI), 5(6)-carboxyfluorescein diacetate (CFDA), and 4′,6-diamidino-2-phenyilindole (DAPI) staining of E. coli cultured for 12, 24, and 48 h. Dead and living cells are labelled in red and green, respectively, while the nucleic acids are displayed in blue.

Figure 5

Fluorescence micrograph after PI, CFDA, and DAPI staining of S. epidermidis cultured for 12, 24, and 48 h. Dead and living cells are labelled in red and green, respectively, while the nucleic acids are displayed in blue.