Characterization and analysis of mycobacteria and Gram-negative bacteria and co-culture mixtures by Raman microspectroscopy, FTIR, and atomic force microscopy

Abstract

The molecular composition of mycobacteria and Gram-negative bacteria cell walls is structurally different. In this work, Raman microspectroscopy was applied to discriminate mycobacteria and Gram-negative bacteria by assessing specific characteristic spectral features. Analysis of Raman spectra indicated that mycobacteria and Gram-negative bacteria exhibit different spectral patterns under our experimental conditions due to their different biochemical components. Fourier transform infrared (FTIR) spectroscopy, as a supplementary vibrational spectroscopy, was also applied to analyze the biochemical composition of the representative bacterial strains. As for co-cultured bacterial mixtures, the distribution of individual cell types was obtained by quantitative analysis of Raman and FTIR spectral images and the spectral contribution from each cell type was distinguished by direct classical least squares analysis. Coupled atomic force microscopy (AFM) and Raman microspectroscopy realized simultaneous measurements of topography and spectral images for the same sampled surface. This work demonstrated the feasibility of utilizing a combined Raman microspectroscopy, FTIR, and AFM techniques to effectively characterize spectroscopic fingerprints from bacterial Gram types and mixtures.

An Overview of the Evolution of Infrared Spectroscopy Applied to Bacterial Typing

“…Sulphate and thiosulphate reducing bacteria Genus FTIRS, FTIRS-ATR HCA, PCA [16,17] Alicylobacillus Genus FTIRS PCA, SIMCA [20] Strain FTIRS-HATR LDA, PCA [75] Photorhabdus Genus FTIRS-ATR HCA, PCA [18] Xenorhabdus Genus FTIRS-ATR HCA, PCA [18] Arcanobacterium Genus FTIRS ANN [19] Actinomyces Genus FTIRS ANN [19] Trueperella Genus FTIRS ANN [19] Species FTIRS ANN [19] Bacillus Genus FTIRS PCA, SIMCA [20] Species FTIRS, FTIRS-ATR, Diffuse Reflectance-FTIRS PCA, HCA, DFA, CVA, SIMCA [26][27][28][29][30]49,50,54,55] Vegetative/Sporulated FTIRS PCA, CART [83] Escherichia Genus FTIRS-ATR HCA, PCA [18] Species FTIRS, FTIRS-ATR HCA, ANN, PCA, SIMCA, CVA [49][50][51][52][53][54]86,92] Escherichia coli Sequence type FTIRS, FTIRS-ATR PC-DFA, PLSDA, HCA, SIMCA [67,68] Strain FTIRS PCA, SIMCA, CVA [49,50,52,73] MLVA profile FTIRS HCA, CVA [72] Listeria spp. Species FTIRS, FTIRS-ATR, FTIR microspectroscopy CVA, ANN, SCDA, PLSRDA, PCA [5,[21][22][23][24][25]…”

“…Species FTIRS -- [41] Microbacterium spp. Species FTIRS -- [41,86] Brachybacterium spp. Species FTIRS -- [41] (Continued) Rathayibacter tritici Species FTIRS -- [41] Lactococcus cremoris Species FTIRS PLS, KPLS [42] Rhodococcus erythropolis Species FTIRS -- [38] Acinetobacter spp.…”

“…Species FTIRS, FTIRS-ATR HCA, PCA, DFA, ANN [43][44][45] Acinetobacter baumannii Sequence type FTIRS-ATR PLSDA [69,70] Burkholderia cepacia complex Species FTIRS PLS, PCA, SIMCA, ANN [45,46] Ribopatterns FTIRS PCA, PLS-DA [46] Pseudomonas spp. Species FTIRS HCA, ANN, PCA, SIMCA [45,49,53,86,92] Stenotrophomonas maltophilia Species FTIRS PCA, ANN [45,55] Ralstonia pickettii Species FTIRS ANN [45] Achromobacter spp Species FTIRS ANN [45] Yersinia spp. Species FTIRS, FTIRS-ATR ANN, SIMCA, PCA, CVA [47,50,52] Yersinia enterocolitica Serotype FTIRS ANN [47] Biotype FTIRS ANN [47] Yersinia ruckeri Biotype FTIRS ANN [80] Oscillatoria limosa Species FTIRS-ATR PCA, ANN [48] Arthrospira platensis Species FTIRS-ATR PCA, ANN [48] Phormidium spp.…”

“…IR is highly limited when it concerns the processing of aqueous samples (e.g., bacterial suspensions). [86] The lack of IR spectroscopy based libraries does not allow the utilization of this method for routine analysis. In general, the reported methods based on IR spectroscopy do not provide enough information for establishing the method for routine analysis essentially because they do not account for the intrinsic variability of clones.…”

“…The results obtained with IR spectroscopy were slightly better (98.8% versus 92.2% of correct assignments). Tang and coworkers [86] compared FTIRS, Raman microspectroscopy, and atomic force microscopy to characterize a collection of mycobacteria and Gram-negative bacteria. [86] The authors concluded that a combined approach, including the three techniques, will lead to a more complete characterization of the isolates (making advantage on the distinct outputs of the techniques).…”