Antimicrobial Photodynamic Inactivation Mediated by Tetracyclines in Vitro and in Vivo: Photochemical Mechanisms and Potentiation by Potassium Iodide

Weijun Xuan,Liyi Huang,Ya He,Brijesh Bhayana,Liyan Xi,Michael R Hamblin,Ying-Ying Huang,Jeffrey A Gelfand

doi:10.1038/s41598-018-35594-y

Abstract

Tetracyclines (including demeclocycline, DMCT, or doxycycline, DOTC) represent a class of dual-action antibacterial compounds, which can act as antibiotics in the dark, and also as photosensitizers under illumination with blue or UVA light. It is known that tetracyclines are taken up inside bacterial cells where they bind to ribosomes. In the present study, we investigated the photochemical mechanism: Type 1 (hydroxyl radicals); Type 2 (singlet oxygen); or Type 3 (oxygen independent). Moreover, we asked whether addition of potassium iodide (KI) could potentiate the aPDI activity of tetracyclines. High concentrations of KI (200–400 mM) strongly potentiated (up to 5 logs of extra killing) light-mediated killing of Gram-negative Escherichia coli or Gram-positive MRSA (although the latter was somewhat less susceptible). KI potentiation was still apparent after a washing step showing that the iodide could penetrate the E. coli cells where the tetracycline had bound. When cells were added to the tetracycline + KI mixture after light, killing was observed in the case of E. coli showing formation of free molecular iodine. Addition of azide quenched the formation of iodine but not hydrogen peroxide. DMCT but not DOTC iodinated tyrosine. Both E. coli and MRSA could be killed by tetracyclines plus light in the absence of oxygen and this killing was not quenched by azide. A mouse model of a superficial wound infection caused by bioluminescent E. coli could be treated by topical application of DMCT and blue light and bacterial regrowth did not occur owing to the continued anti biotic activity of the tetracycline.

Highlights

Antimicrobial photodynamic inactivation is a new approach to killing infectious pathogens, that is independent of existing antibiotic resistance status, and is not thought likely to cause any resistance to develop itself[1]
We found that in contrast to well-established PS such as MB, that was only effective when incubated with the bacteria in Phosphate-buffered saline (PBS) and had no effect in rich growth medium such as Brain Heart Infusion Broth, tetracyclines localized inside the bacterial cells and were not affected by the protein present in the medium
We continued to use blue light to excite DMCT because we had used this wavelength of light in our previous study[37], and because it was thought that UVA might be considered dangerous to use in a potential clinical application

Summary

Introduction

Antimicrobial photodynamic inactivation (aPDI) is a new approach to killing infectious pathogens, that is independent of existing antibiotic resistance status, and is not thought likely to cause any resistance to develop itself[1]. Research in this area has been driven by growing international concern about the seemingly unstoppable rise of multi-drug resistance amongst bacteria and other pathogenic microorganisms, that was highlighted in the O’Neill report. The PS forms a long-lived triplet state, that can react with oxygen to produce reactive oxygen species (ROS) including singlet oxygen and hydroxyl radicals[3] These ROS can attack critical biomolecules (lipids, proteins, nucleic acids) producing cell lysis of the microorganisms and death. We were able to show that the MICs of tetracyclines measured by a broth microplate dilution assay, were lower (more effective) when measured under 0.5 mW/cm[2] of continuous blue light than when measured in the dark

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