Defining Molecular Details of the Chemistry of Biofilm Formation by Raman Microspectroscopy

Abstract

Two protocols that allow for the comparison of Raman spectra of planktonic cells and biofilm formed from these cells in their growth phase have been developed. Planktonic cells are washed and flash-frozen in <1 min to reduce the time for metabolic changes during processing, prior to freeze-drying. Biofilm is formed by standing cells in 50 μL indentations in aluminum foil in an atmosphere of saturated water vapor for 24-48 h. The results for Escherichia coli type K12 cells, which do not readily form biofilm, are compared to those for Staphylococcus epidermidis cells, which prolifically synthesize biofilm. For E. coli, the Raman spectra of the planktonic and biofilm samples are similar with the exception that the spectral signature of RNA, present in planktonic cells, could not be detected in biofilm. For S. epidermidis, major changes occur upon biofilm formation. In addition to the absence of the RNA features, new bands occur near 950 cm and between 1350 and 1420 cm that are associated with an increase in carbohydrate content. Unlike the case in E. coli biofilm, the intensity of G base ring modes is reduced in but A and T base ring signatures become more prominent. For S. epidermis in the biofilm's amide III region, there is evidence of an increase in the level of β-sheet structure accompanied by a decrease in α-helical content. The presence of biofilm is confirmed by microscope-aided photography and, separately, by staining with methyl violet.

Correlation of crystal violet biofilm test results of Staphylococcus aureus clinical isolates with Raman spectroscopic read‐out

“…Mean Raman spectra with standard deviation of all investigated strains are shown in Figures S3-S5. Typical Raman bands of the biological components of bacteria can be seen in the spectra, such as vibrational bands of nucleic acid components at 787 cm À1 (purine bases cytosine, thymine, and uracil [25,30,42] ), at 805-810 cm À1 (RNA backbone stretching [20] ), at 1581 cm À1 (pyrimidine bases guanine and adenine [20,25,42] ), and at 1096 cm À1 (PO 2 À stretching [20,25,30,42] ). Interestingly, those bands had decreased relative intensity in the high biofilm producer strain S. aureus Kakl54 (Figure 1).…”

“…This spectral information can be used-in combination with statistical methods-to identify bacterial species and strains. [14] Raman spectroscopy was successfully applied not only to study clinical relevant microbial biofilms, for example, for chemical analysis of biofilm formation and composition, [15][16][17][18][19][20][21][22][23][24][25][26][27] but also to identify different bacterial species forming biofilms and to discriminate between biofilm-positive and -negative strains. [28][29][30][31] Additionally, the interaction of antibiotics with biofilms and their efficacy has been evaluated with Raman spectroscopy.…”

Mapping of a Subgingival Dual-Species Biofilm Model Using Confocal Raman Microscopy

“…The focus of these studies were based on molecular details of spectra: (1) of single species biofilms (Kusić et al, 2015;Ramirez-Mora et al, 2019;Gieroba et al, 2020;Wickramasinghe et al, 2020), (2) over time (Chao and Zhang, 2012;Carey et al, 2017;Keleştemur et al, 2020;Liu et al, 2020), and (3) under the influence of stress, mostly chemicals (Jung et al, 2014;Daood et al, 2020). All in all, these studies lacked a spatial analysis when using Raman spectra.…”

Confocal Raman microscopy to identify bacteria in oral subgingival biofilm models

“…Therefore, differentiation based on Raman spectra has significant limitations so far [16,[22][23][24][25]. To the best of our knowledge, only a few studies employed the use of CRM for environmental biofilms [26,27], but there has been very limited information in the field of oral biofilm mapping [23,28]. While surface enhanced Raman scattering (SERS) remains promising in combination with Raman microscopy [13,29,30] with the potential for higher levels of discrimination spectra, this study focuses on the analysis of chemically un-modified biofilm samples.…”