LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

Summary

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

Abstract

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

Introduction

Photodynamic therapy (PDT) is a minimally invasive therapeutic approach that has shown promising results in recent years, particularly in dermatological clinical treatment¹. One particularly interesting area of application of this therapeutic modality is the treatment of microbial infections, a process known as antimicrobial photodynamic inactivation (aPDI)². Although initially overlooked, mostly because of the remarkable efficiency of antibiotics, light-triggered eradication of bacterial growth underwent a renewed interest over the recent years, driven by the emergence of antimicrobial multidrug resistance (AMR) and the....

Protocol

An overall description of the protocol is illustrated in Figure 1. The details of the reagents and equipment used are listed in the Table of Materials.

1. Bacterial culture preparation

1. Grow S. aureus (ATCC 29213) bacteria on Mueller-Hinton agar (MH) from stock cultures. Incubate the MH plates at 37 °C for 24 h prior to the assay to obtain fresh cultures.
2. Inoculate bacteria: Collect 2-3 fresh colonies from the ag.......

Discussion

Although this protocol serves as an initial stage testing platform, it is important to consider more meaningful testing conditions for the treatment of microbial infections in a more advanced study phase. When assessing the effectiveness of aPDI, experiments on biofilm-type bacterial organizations rather than planktonic bacteria should be conducted. Many clinical cases involve microorganisms organized in this more resistant conformation, such as chronic wounds, cystic fibrosis, and on implanted medical devices

Materials

Name	Company	Catalog Number	Comments
Cary 60 UV-Vis Spectrophotometer	Agilent	G6860A
Dimethyl Sulfoxide	Sigma-Aldrich	D8418
Disposable cuvettes PMMA	BRAND GMBH + CO KG	759030
Eppendorfs (500 mL)	Fisher Scientific	15625367
Falcon tubes (15 mL)	Corning	430791
Falcon tubes (50 mL)	Corning	430291
LED panel IP65 50W
Micropippete (100 uL)	Transferpette S	705874
Micropippete (1000 uL)	Transferpette S	705880
Mueller Hinton agar	Oxoid	CM0405
Multichannel pippete 12-channel	Transferpette S
Nunc MicroWell 96-Well Microplates	Thermo Scientific	260844
Phospate Buffered Saline tablets pH 7.4	Panreac Applichem	A9177
Serological Pipets (10 mL)	Thermo Scientific	170356N
Serological Pipets (5 mL)	Thermo Scientific	170366N
Tissue Culture Dish	TPP Techno Plastic Products AG	93150