An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Summary

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

Abstract

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

Introduction

S. aureus is an important Gram-positive pathogen that colonizes the skin and mucosa of human hosts. Its ability to form biofilms is considered an important aspect of its pathogenesis¹. Bacterial biofilms are a community of bacteria embedded in a self-produced matrix, which is composed of extracellular polymeric substances, including polysaccharide, DNA, and protein. This matrix plays a significant role in the persistence of bacterial infections, contributing to a high degree of resistance to the human immune system and current anti-microbial therapies². Antibiotics are still the major treatment for biofilm infec....

Protocol

1. Biofilm Formation

1. Biofilm formation in 96-well microplates
   1. Retrieve the S. aureus strain USA300 and 3 biofilm-forming clinical strains (C1 - C3) stored at -80 °C.
      NOTE: The ability of the clinical strains to form biofilms was determined by the microtiter plate assay described previously¹⁵.
   2. Inoculate the bacterium in 5 mL tryptone soya broth (TSB) medium, and cultivate in an incubator with shaking at 37 °C overnight to the stationary phase.
   3. Centrifuge the overnight bacterial culture at 4,000 x g for 10 min at 25 °C and then discard the supernatant. ....

Results

The viability of the bacteria in the biofilms was decreased after ALA-PDT treatment when compared to the controls (ALA-LED-, ALA+LED-, and ALA-LED+) in both USA300 and the three clinical strains (Figure 1).

To confirm the results from the CFU assay and observe the antibacterial effect of ALA-PDT on the S. aureus biofilm in situ, the USA300 biofilms were visualized by CLSM with viab.......

Discussion

PDT has been a well-studied therapy for the treatment of cancer since it was invented more than 100 years ago¹⁸. Over the last decade, PDT has been applied as an antimicrobial strategy and has shown effectiveness against some antibiotic-resistant pathogenic bacteria¹². Compared to the planktonic state, bacterial biofilms appear to be more resistant to antibiotic treatment³, while the effect of ALA-PDT on biofilms has not been fully investigated yet.<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tryptone Soya Broth (TSB)	OXOID	CM0129B
Tryptone Soya Agar (TSA)	OXOID	CM0131
SYTO9	Thermo Fisher Scientific	L7012	The LIVE/DEAD BacLight Bacterial Viability Kits
Propidium iodide (PI)	Thermo Fisher Scientific	L7012	The LIVE/DEAD BacLight Bacterial Viability Kits
Pancreatin	Sigma-Aldrich	P3292
5-aminolevulinic acid (ALA)	Fudan Zhangjiang Bio-Pharm	3.1
Staphylococcus aureus strain USA300	/	/	The source of USA 300 references “Tenover FC, Goering RV. J Antimicrob Chemother. 2009 Sep; 64(3):441-6”.
Staphylococcus aureus clinical strains (C1-C3)	/	/	All clinical strains were isolated from patients with chronic rhinosinusitis in the Department of Otorhinolaryngology-Head and Neck Surgery, Eye and ENT Hospital of Fudan University [Zhang QZ, Zhao KQ, Wu Y, et al. PLoS One. 2017 Mar; 12(3): e0174627].
96-well microplate	Corning Inc	3599	Clear Flat Bottom Polystyrene TC-Treated Microplates, Individually Wrapped, with Lid, Sterile
Fluorodish	NEST Biotechnology	801001	Glass bottom, Non-pyrogenic
Eppendorf Safe-Lock Tubes, 1.5 mL	Eppendorf	0030120086
Eppendorf microcentrifuge 5417	Eppendorf	Z365998 | SIGMA
Incubator	Thermo Fisher Scientific	SHKE4000	MaxQ 4000 Benchtop Orbital Shakers
Light emitting diode (LED)	Wuhan Yage Optic and Electronic Technique CO	LED-IB
Leica TCS SP8 confocal laser-scanning microscope	Leica Microsystems
Leica LAS AF software	Leica Microsystems
IMARIS software	Bitplane