A comparison of antibacterial and antibiofilm efficacy of phenothiazinium dyes between Gram positive and Gram negative bacterial biofilm

https://doi.org/10.1016/j.pdpdt.2017.01.177Get rights and content

Highlights

  • Phenothiazinium dyes are highly effective against Gram positive and Gram negative bacteria.

  • Gram-positive bacteria are more susceptible due to enhanced ROS generation, higher photosensitizers binding efficiency and DNA degradation.

  • Preformed biofilm of both the bacteria were reduced after APDT by 3log10. E. faecalis formed dense and deep biofilm than K. pneumoniae.

  • CLSM and SEM revealed the efficient antibiofilm effect of phenothiazinium dyes.

Abstract

Background

Antimicrobial photodynamic therapy (APDT) is a process that generates reactive oxygen species (ROS) in presence of photosensitizer, visible light and oxygen which destroys the bacterial cells. We investigated the photoinactivation efficiency of phenothiazinium dyes and the effect of ROS generation on Gram positive and Gram negative bacterial cell as well as on biofilm.

Material and methods

Enterococcus faecalis and Klebsiella pneumonia were incubated with all the three phenothiazinium dyes and exposed to 630 nm of light. After PDT, colony forming unit (CFU) were performed to estimate the cell survival fraction. Intracellular reactive oxygen species (ROS) was detected by DCFH-DA. Crystal violet (CV) assay and extracellular polysaccharides (EPS) reduction assay were performed to analyze antibiofilm effect. Confocal laser electron microscope (CLSM) scanning electron microscope (SEM) was performed to assess the disruption of biofilm.

Results

8log10 reduction in bacterial count was observed in Enterococcus faecalis while 3log10 in Klebsiella pneumoniae. CV and EPS reduction assay revealed that photodynamic inhibition was more pronounced in Enterococcus faecalis. In addition to this CLSM and SEM study showed an increase in cell permeability of propidium iodide and leakage of cellular constituents in treated preformed biofilm which reflects the antibiofilm action of photodynamic therapy.

Conclusion

We conclude that Gram-positive bacteria (Enterococcus faecalis) are more susceptible to APDT due to increased level of ROS generation inside the cell, higher photosensitizer binding efficiency and DNA degradation. Phenothiazinium dyes are proved to be highly efficient against both planktonic and biofilm state of cells.

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)

  • F. Vatansever et al.

    Antimicrobial strategies centered around reactive oxygen species—bactericidal antibiotics photodynamic therapy, and beyond

    FEMS Microbiol. Rev.

    (2013)
  • L. Huang et al.

    Type I and type II mechanisms of antimicrobial photodynamic therapy: an in vitro study on gram-negative and gram-positive bacteria

    Lasers Surg. Med.

    (2012)
  • L. Huang et al.

    Stable synthetic cationic bacteriochlorins as selective antimicrobial photosensitizers

    Antimicrob. Agents Chemother.

    (2010)
  • K.R. Kasimova et al.

    Potentiation of photoinactivation of Gram-positive and Gram-negative bacteria mediated by six phenothiazinium dyes by addition of azide ion

    Photochem. Photobiol. Sci.

    (2014)
  • Cited by (0)

    View full text