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

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Bacterial autofluorescence; Endogenous porphyrins; NaCl concentration; Temperature; pH.

Fig. 1

Red fluorescence emitted from the selected bacteria over a 96-h period. a Fluorescence images of the selected bacteria over a 96-h period. T₁, the first appearance of red fluorescence as white rectangle shows; T₂, the maximum fluorescence intensity as red rectangle shows; T₃, the onset of fluorescence decay as blue rectangle shows. b Bacterial load, c colony diameter, and d red fluorescence intensity of the selected bacteria over time. e-g Comparison of red fluorescence intensity of the selected bacteria at T₁, T₂ and T₃. The results represent the mean ± SD of three independent experiments. ^*p < 0.05, ^**p < 0.005, ^***p < 0.001, ^****p < 0.0001

Fig. 2

Impact of culture temperature on red fluorescence intensity of A. baumannii and S. aureus. a Red fluorescence intensity of A. baumannii and S. aureus cultured at different temperatures over time. b Comparison of red fluorescence intensity of A. baumannii and S. aureus at varying culture temperatures up to T₂. c Effect of temperature on time to the maximum red fluorescence intensity in A. baumannii and S. aureus. d Linear regression of red fluorescence intensity over time for A. baumannii and S. aureus before the maximum intensity. The results represent the mean ± SD of three independent experiments. ^*p < 0.05, ^**p < 0.005, ^***p < 0.001, ^****p < 0.0001. Data of E. coli and K. pneumoniae are provided in additional Fig. 7

Fig. 3

Impact of culture salinity on red fluorescence intensity of the selected bacteria. a Red fluorescence intensity of the selected bacteria cultured at different NaCl concentrations over time. b Comparison of red fluorescence intensity of the selected bacteria cultured at different NaCl concentrations up to T₂. The results represent the mean ± SD of three independent experiments. ^*p < 0.05, ^**p < 0.005, ^***p < 0.001, ^****p < 0.0001

Fig. 4

Impact of culture pH on red fluorescence intensity of the selected bacteria. a Red fluorescence intensity of the selected bacteria cultured at different pH levels over time. b Comparison of red fluorescence intensity of the selected bacteria cultured at different pH levels up to T₂. The results represent the mean ± SD of three independent experiments. ^*p < 0.05, ^**p < 0.005, ^***p < 0.001, ^****p < 0.0001

Fig. 5

Emission spectra of porphyrins and bacteria incubated with ALA. a Normalized emission spectra of coproporphyrin I and protoporphyrin IX at 405 nm excitation. b Excitation-emission matrix spectra of coproporphyrin I and protoporphyrin IX. The emission spectra have been normalized to peak intensity. c Normalized emission spectra of the selected bacteria incubated with ALA up to T₂ at 405 nm excitation. The position of peaks measured for coproporphyrin I and protoporphyrin IX are shown in dotted lines. d Excitation-emission matrix spectra of the selected bacteria incubated with 5-ALA up to T₂. The emission spectra data have been normalized, and the relative fluorescence intensity is color-coded in the contour plot. Black contour lines indicate increments of 0.05. The scale is consistent across all plots

Fig. 6

Emission spectra of selected bacteria under 405 nm excitation over time. a Emission spectra, and b comparison of fluorescence peak values and c excitation-emission matrix spectra of the selected bacteria cultured at predefined time points under 405 nm excitation. The results represent the mean ± SD of three independent experiments. ^*p < 0.05, ^**p < 0.005, ^***p < 0.001, ^****p < 0.0001. The emission spectra data have been normalized, and the relative fluorescence intensity is color-coded in the contour plot. Black contour lines indicate increments of 0.05. The scale is consistent across all plots

Fig. 7

Fluorescence spectra of the selected bacteria cultured at different temperatures. a Emission spectra, b comparison of fluorescence peak values, and c excitation-emission matrix spectra of the selected bacteria cultured at different temperatures. The results represent the mean ± SD of three independent experiments. ^*p < 0.05, ^**p < 0.005, ^***p < 0.001, ^****p < 0.0001. The emission spectra data have been normalized, and the relative fluorescence intensity is color-coded in the contour plot. Black contour lines indicate increments of 0.05

Fig. 8

Fluorescence spectra of the selected bacteria cultured at different salinities. a Emission spectra, b comparison of fluorescence peak values, c excitation-emission matrix spectra of the selected bacteria cultured at different NaCl concentrations. The results represent the mean ± SD of three independent experiments. ^*p < 0.05, ^**p < 0.005, ^***p < 0.001, ^****p < 0.0001. The emission spectra data have been normalized, and the relative fluorescence intensity is color-coded in the contour plot. Black contour lines indicate increments of 0.05. No growth of A. baumannii was observed at 4% NaCl

Fig. 9

Fluorescence spectra of the selected bacteria cultured at different pH levels. a Emission spectra and b comparison of fluorescence peak values, and c excitation-emission matrix spectra of the selected bacteria cultured at different pH levels. ^*p < 0.05, ^**p < 0.005, ^***p < 0.001, ^****p < 0.0001. The emission spectra data have been normalized, and the relative fluorescence intensity is color-coded in the contour plot. Black contour lines indicate increments of 0.05. No growth of A. baumannii was observed at pH 5 or 9