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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

McKenzie, Karen and MacLean, Michelle and Grant, M. Helen and Ramakrishnan, Praveen and MacGregor, Scott J. and Anderson, John G. (2016) 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. Microbiology, 162 (9). pp. 1680-1688. ISSN 1350-0872

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Abstract

Bacterial inactivation by 405nm light is accredited to the photo-excitation of intracellular porphyrin molecules which results in energy transfer and the generation of reactive oxygen species (ROS) which 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 405nm 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 405nm 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 405nm 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 which are also impaired by the oxidative damage from photo-generated ROS.