Autofluorescence properties of wound-associated bacteria cultured under various temperature, salinity, and pH conditions

Bacterial autofluorescence plays a vital role in photodiagnosis (PD) and antimicrobial photodynamic therapy (aPDT), yet the autofluorescence properties of wound-associated bacteria and their responses to the physicochemical microenvironment, remain underexplored. Here, we investigated the bacterial autofluorescence of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus under various culture conditions, including different temperatures, NaCl concentrations, and pH levels found within wounds. Fluorescence imaging was employed to quantify red fluorescence intensity, while fluorescence spectrometry was used to correlate the observed fluorescence with spectral profiles, benchmarking them against coproporphyrin and protoporphyrin IX. Our results revealed that the selected bacteria emitted red fluorescence in vitro, consistent with their known porphyrin biosynthesis capabilities. The intensity of red fluorescence was primarily dependent on the bacterial species, growth phase, and culture conditions. Elevated culture temperature accelerated the fluorescence metabolism, whereas increasing NaCl concentrations and alkaline pH levels inhibited red fluorescence in a dose-dependent manner. Linear regression revealed a strong positive correlation between red fluorescence intensity and peak fluorescence emission when excited at 405 nm. The biosynthesis of endogenous porphyrins varied both across and within bacterial species, with distinct porphyrins produced under specific conditions. The emission spectra of the gram-positive S. aureus consistently aligned with coproporphyrin, while the gram-negative A. baumannii, E. coli, and K. pneumoniae typically displayed fluorescence peaks characteristic of protoporphyrin IX. Notably, K. pneumoniae shifted to coproporphyrin with extended culture duration or more favourable conditions, and E. coli exhibited a similar transition as the pH increased to 9. We concluded that the local physicochemical conditions found within wounds could affect the autofluorescence and endogenous porphyrins of the bacteria, which may, in turn, have connotations for PD and aPDT.