The effects of 405 nm light on bacterial membrane integrity determined by salt and bile tolerance assays, leakage of UV-absorbing material and SYTOX green labelling

Abstract

Bacterial inactivation by 405 nm light is accredited to the photoexcitation of intracellular porphyrin molecules resulting in energy transfer and the generation of reactive oxygen species that impart cellular oxidative damage. The specific mechanism of cellular damage, however, is not fully understood. Previous work has suggested that destruction of nucleic acids may be responsible for inactivation; however, microscopic imaging has suggested membrane damage as a major constituent of cellular inactivation. This study investigates the membrane integrity of Escherichia coli and Staphylococcus aureus exposed to 405 nm light. Results indicated membrane damage to both species, with loss of salt and bile tolerance by S. aureus and E. coli, respectively, consistent with reduced membrane integrity. Increased nucleic acid release was also demonstrated in 405 nm light-exposed cells, with up to 50 % increase in DNA concentration into the extracellular media in the case of both organisms. SYTOX green fluorometric analysis, however, demonstrated contradictory results between the two test species. With E. coli, increasing permeation of SYTOX green was observed following increased exposure, with >500 % increase in fluorescence, whereas no increase was observed with S. aureus. Overall, this study has provided good evidence that 405 nm light exposure causes loss of bacterial membrane integrity in E. coli, but the results with S. aureus are more difficult to explain. Further work is required to gain greater understanding of the inactivation mechanism in different bacterial species, as there are likely to be other targets within the cell that are also impaired by the oxidative damage from photo-generated reactive oxygen species.

Figures

Fig. 1.

Inactivation kinetics of E. coli and S. aureus with exposure to 405 nm light, with an irradiance of 65 mW cm−2. (a and b) E. coli at 107 and 109 c.f.u. ml−1, respectively; (c and d) S. aureus at 107 and 109 c.f.u. ml−1, respectively. Non-exposed control samples showed no significant change. An asterisk (*) represents significant bacterial inactivation, when compared to associated non-exposed control (P≤0.05). Each data point is a mean value±sem (n≥6).

Fig. 2.

Demonstration of lethal and sub-lethal damage of (a) E. coli and (b) S. aureus exposed to 405 nm light by comparison of post-exposure survivors plated on non-selective and selective media. Bacterial populations were exposed to an irradiance of 65 mW cm−2. VRBA and MSA were selected for use due to their high bile and salt concentrations, respectively, to provide selective pressures which discourage growth of bacteria with reduced membrane integrity. NA was used as the non-selective growth medium. An asterisk (*) represents significant bacterial inactivation on selective agar, when compared to associated non-selective counts (P≤0.05). Each data point is a mean value±sem (n≥6).

Fig. 3.

Absorbance measurements of (a) E. coli and (b) S. aureus cell supernatants at 260 nm following 405 nm light exposure (65 mW cm−2). An asterisk (*) represents a significant increase in absorbance reading when compared to the equivalent non-exposed control samples (P≤0.05). Each data point is a mean value±sem (n≥3).

Fig. 4.

SYTOX green fluorescence at 523 nm of (a) E. coli and (b) S. aureus cells following increasing doses of 405 nm light exposure (65 mW cm−2), using an excitation wavelength of 490 nm. Results are measured as the percentage increase compared to non-exposed control samples. An asterisk (*) represents a significant increase in fluorescence when compared to non-exposed control samples (P≤0.05). Each data point is a mean value±sem (n≥3).

Fig. 5.

Fluorescence microscopy of SYTOX green stain labelled bacteria. Cells were exposed to a dose of 234 and 117 J cm−2, for E. coli and S. aureus, respectively (first data point in inactivation curve Fig. 1). Cells were stained with 5 mM SYTOX green and brightness in fluorescence between exposed and non-exposed samples was compared visually using an epifluorescent microscope. Images (a) and (b) represent MosaiX images (8×8) of non-exposed and 405 nm exposed E. coli. Images (c) and (d) represent non-exposed and 405 nm exposed S. aureus. Excitation and emission were 490 nm and >520 nm, respectively, for all samples.