Membrane damage as mechanism of photodynamic inactivation using Methylene blue and TMPyP in Escherichia coli and Staphylococcus aureus

Tim Maisch,Karl-Anton Hiller,Fabian Cieplik,Elena Brandl,Denise Muehler

doi:10.1007/s43630-021-00158-z

Tim Maisch, Karl-Anton Hiller + Show 3 more

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https://doi.org/10.1007/s43630-021-00158-z

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Abstract

The worldwide threat of antibiotic resistance requires alternative strategies to fight bacterial infections. A promising approach to support conventional antibiotic therapy is the antimicrobial photodynamic inactivation (aPDI). The aim of this work was to show further insights into the antimicrobial photodynamic principle using two photosensitizers (PS) of different chemical classes, Methylene Blue (MB) and TMPyP, and the organisms Escherichia coli and Staphylococcus aureus as Gram-negative and Gram-positive representatives. Planktonic cultures of both species were cultured under aerobic conditions for 24 h followed by treatment with MB or TMPyP at various concentrations for an incubation period of 10 min and subsequent irradiation for 10 min. Ability to replicate was evaluated by CFU assay. Accumulation of PS was measured using a spectrophotometer. The cytoplasmic membrane integrity was investigated by flow cytometry using SYBR Green and propidium iodide. In experiments on the replication ability of bacteria after photodynamic treatment with TMPyP or MB, a killing rate of 5 log10 steps of the bacteria was achieved. Concentration-dependent accumulation of both PS was shown by spectrophotometric measurements whereby a higher accumulation of TMPyP and less accumulation of MB was found for S. aureus as compared to E. coli. For the first time, a membrane-damaging effect of TMPyP and MB in both bacterial strains could be shown using flow cytometry analyses. Furthermore, we found that reduction of the replication ability occurs with lower concentrations than needed for membrane damage upon MB suggesting that membrane damage is not the only mechanism of aPDI using MB.

Highlights

Since the emergence of resistant bacteria shortly after the introduction of the first antibiotics, there has been a steady increase in antibiotic resistance worldwide [1]
The bacterial ability to replicate following Antimicrobial photodynamic inactivation (aPDI) with Methylene Blue (MB) and TMPyP was evaluated by colony-forming units (CFU) assay against planktonic cultures of E. coli and S. aureus (Fig. 1)
The exact antibacterial mechanism of the photodynamic process has not yet been completely understood. This is of great importance to estimate possible developments of tolerances and resistances and to be able to improve the effectiveness of the photosensitizers by chemical modifications

Summary

Introduction

Since the emergence of resistant bacteria shortly after the introduction of the first antibiotics, there has been a steady increase in antibiotic resistance worldwide [1]. Antimicrobial photodynamic inactivation (aPDI) represents a promising method for inactivating bacteria [8, 10,11,12]. This process generates reactive oxygen species (ROS) that cause non-specific and irreversible oxidative damage to biological molecules, which results in the killing of bacteria. ROS formed in both mechanisms lead to damage of biological molecules such as lipids, proteins, enzymes, DNA and RNA. The actual oxidative damage caused by aPDI depends on the localization of the photosensitizer to these structures and on the time-limited diffusion length of the resulting ROS [9, 13]

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