A comparison of antibacterial and antibiofilm efficacy of phenothiazinium dyes between Gram positive and Gram negative bacterial biofilm
Introduction
Microbial infections remain to be one of the main causes of mortality and the major factor of this infectious diseases caused by multiple drug resistant bacteria and their aggregation as biofilm [1]. A biofilm can be defined as a sessile community of microbes that adhere irreversibly to an inert or to living surface and are embedded in a self-producing matrix of extracellular polymeric substances (EPS) [2]. EPS is an important component of biofilm it provides structural stability as well as protection to the biofilm against antimicrobial agents leads to antimicrobial resistance [3]. Owing to the severity of these infections, there is a critical need to explore novel approaches like Antimicrobial photodynamic therapy (APDT) which appears to be the most promising alternative methodology against biofilm-related infections. APDT disrupts the biofilm and inhibits its regrowth.
In APDT, photosensitizer (PS) is excited by absorbing a light corresponding to the absorption peak of PS in presence of oxygen rich environment to produce a phototoxic response, normally via oxidative damage [4]. Two pathways of oxidative mechanisms of photoinactivation (PI) are responsible for the inactivation of the target cells. Type I pathway involves the generation of free radicals like hydroxyl radicals (HO•). Type II pathway involves the generation of excited singlet-state oxygen [5]. Type I and Type II pathways occur simultaneously however, the ratio between these two pathways depends on types of PS employed and also on the photosensitizer’s microenvironment [6].
Over the last few decades, the effectiveness of photoinactivation of Gram-positive and Gram-negative bacteria using different PS has been extensively studied but little information is available on its potential uses against bacteria organized in biofilm [7]. It has been reported that neutral, anionic and cationic photosensitizers effectively destroyed Gram-positive bacteria but only cationic photosensitizers were found to photoinactivate Gram-negative bacteria [8]. It was also found that in the case of positively charged PS, positive charge allowed it to bind to negatively charged bacterial cell membrane and in some cases penetrated to the microbial cells due to the porous nature of cell wall [9].
To the best of our knowledge, its effect on gram positive and gram negative bacterial biofilm has been less studied so far. Bridging this gap, here we examined the mechanistic insight behind photodynamic effects against both the classes of bacteria. Hence, we compared the efficacy of three phenothiazinium dyes i.e. Toluidine Blue O (TBO), Azure A (AA) and New Methylene Blue (NMB) on Enterococcus faecalis and Klebsiella pneumoniae as representative of Gram positive and Gram negative bacteria respectively. The reason behind selecting these dyes is that they show absorption in the red region of light that can penetrate tissue efficiently, limited photobleaching and less toxicity.
Section snippets
Bacterial strain
The bacterial strains used in this study were Enterococcus faecalis MTCC 2729 (Gram positive) and Klebsiella pneumoniae ATCC 700603 (Gram negative) as an experimental model. The microorganisms were subcultured in Brain Heart Infusion (BHI) broth supplemented with 1% sucrose (Himedia Labs, Mumbai, India) and was incubated at 37 °C.
Photosensitizers
Toluidine blue O (TBO), Azure A (AA), and New methylene blue (NMB) were obtained from (Sigma-Aldrich, St. Louis, MO). A stock solution of 1 mg/mL was prepared in HPLC
Eradication of planktonic bacteria
The antimicrobial activity of TBO, AA and NMB following irradiation was evaluated by plate counts method. The photoinactivation of E. faecalis by each photosensitizer (10 μM) is shown in Fig. 1. Under these conditions, 3log10 reduction was obtained after irradiation for 20 s and 8log10 reduction was found in E. faecalis after being irradiated for 80 s. The same set of experiments was done with the Gram-negative K. pneumoniae as shown in Fig. 2. There was about 3log10 reduction was observed with
Discussion
Bacterial infection has become a major problem due to increased resistance to conventional antibiotics. Bacterial biofilm or intracellular infection is a challenge for the physician. Since, it is difficult for antimicrobial to penetrate effectively through the biofilm: thus have a low activity inside the biofilm [20]. Therefore, there is an urgent need to develop alternative therapeutic strategies which may combat with drug-resistant strains and biofilm-related infections without harming the
Conclusion
We conclude that phenothiazinium group of photosensitizers induced a high photodynamic effect on planktonic cells as well as on biofilm, formed by Gram positive and Gram negative bacteria. APDT is more efficient against E. faecalis due to enhanced ROS production inside the cell, more biofilm reduction and higher PS binding efficiency.
Acknowledgements
This study was supported by the Council of scientific and Industrial Research grant sanction no. 37(1576)/13/EMR-II. The authors would like to acknowledge Mr. Md. Shamim and Mr. Abul Maaz for providing their technical assistance and university sophisticated instruments facility (USIF), AMU for providing instrumental supports. Lama Misba was supported by UGC-BSR JRF fellowship.
References (28)
- et al.
Survival strategies of infectious biofilms
Trends Microbiol.
(2005) - et al.
The phototoxicity of phenothiazinium derivatives against Escherichia coli and Staphylococcus aureus
FEMS. Immunol. Med. Microbiol.
(2003) - et al.
The antimicrobial activity of photodynamic therapy against Streptococcus mutans using different photosensitizers
J. Photochem. Photobiol. B
(2012) - et al.
Photodynamic inactivation of Staphylococcus aureus and Escherichia coli biofilms by malachite green and phenothiazine dyes: an in vitro study
Arch. Oral. Biol.
(2012) - et al.
Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material
Int. J. Antimicrob. Agents
(2014) - et al.
Phototoxicity of phenothiazinium dyes against methicillin-resistant Staphylococcus aureus and multi-drug resistant Escherichia coli
Photodiagn. Photodyn. Ther.
(2012) - et al.
Antibiotic-resistant bugs in the 21 st century—A clinical super challenge
N. Engl. J. Med.
(2009) Biofilms: microbial life on surfaces
Emerg. Infect. Dis.
(2002)- et al.
Gold nanoparticles enhance methylene blue-induced photodynamic therapy: a novel therapeutic approach to inhibit Candida albicans biofilm
Int J. Nanotechnol. Nanomed.
(2012) - et al.
Innovative stategies to overcome biofilm resistance
Int. J. Biomed. Res.
(2013)