Intrinsic fluorescence spectroscopy coupled with chemometrics for characterizing Listeria innocua and Escherichia coli binary mixtures: a proof-of-concept study.

Abstract

Intrinsic fluorescence spectroscopy provides a rapid and label-free approach for bacterial characterization, but its application to mixed bacterial populations remains less explored. In this study, the ability of intrinsic fluorescence to detect changes in the relative composition of binary mixtures of Escherichia coli and Listeria innocua was investigated. Bacterial suspensions were adjusted to approximately 1 × 106 CFU/mL and combined at controlled volumetric ratios. Excitation-emission matrices (EEMs) were acquired for 11 compositions ranging from pure L. innocua to pure E. coli, while the two pure cultures and the 10:90 and 90:10 E. coli:L. innocua mixtures were investigated using 15 independent preparations per condition. Fluorescence emission spectra were recorded from 300 to 500 nm following excitation at 266 nm. The overall mean emission spectrum reached its maximum intensity at 338 nm, where E. coli-dominated samples exhibited significantly higher fluorescence intensities than L. innocua-dominated samples (Welch's ANOVA, p < 0.001). EEMs provided a descriptive visualization of composition-dependent changes in fluorescence patterns across the bacterial mixture series. Principal component analysis accounted for 95% of the total spectral variance within the first two components, and the score plot showed a distribution of samples broadly associated with the dominant bacterial species. Cross-validated PCA-LDA achieved an overall classification accuracy of 81.67%.These findings provide a proof of concept that intrinsic fluorescence fingerprints are sensitive to changes in bacterial mixture composition under controlled conditions.

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