Influence of Photosensitizer Solvent on the Mechanisms of Photoactivated Killing of Enterococcus faecalis Saji George and Anil Kishen* Restorative Dentistry, Faculty of Dentistry, National University of Singapore, Singapore Received 19 August 2007, accepted 8 October 2007, DOI: 10.1111 ⁄ j.1751-1097.2007.00244.x ABSTRACT This study evaluated the mechanisms involved and the influence of photosensitizer solvent in the killing of Enterococcus faecalis using photodynamic therapy (PDT). Enterococcus faecalis cells incubated with 100 lM methylene blue dissolved in water and in MIX (a mixture of glycerol:ethanol:water) were irradiated with 664 nm diode laser (63.69 J cm )2 ). The effect of PDT on the viability of bacteria, and the functional integrity of cell wall, chromosomal DNA and membrane proteins were analyzed. The bactericidal action of PDT was significantly higher when a MIX-based photosensitizer solvent was used (P < 0.001). Fluorimetric and fluorescence microscopy-based analysis showed the functional impairment of E. faecalis cell wall which was significantly higher when a MIX-based photosensitizer solvent was used (P < 0.001). PDT with MIX-based photosensitizer solvent showed extensive damage to chromosomal DNA. However, both PDT conditions showed similar trend in the degradation of membrane proteins, although cross-linked pro- teins were evident only in PDT conducted with MIX-based photosensitizer solvent. The findings from our study showed that PDT destroyed the functional integrity of cell wall, DNA and membrane proteins of E. faecalis. The degrees of damage on these targets were influenced by the photosensitizer solvent used during PDT. INTRODUCTION The emergence of antibiotic-resistant clinical strains of infec- tious bacteria necessitates an effective alternate treatment strategy (1). Lethal photosensitization ⁄ photodynamic therapy (PDT) of bacteria has been recognized as a promising alternative to conventional antibacterial strategies based on antibiotics (2–4). Unlike antibiotics, PDT acts on multiple targets in bacterial cells such as membrane lipids, genomic DNA, proteins and enzymes that reduce the chance of bacteria acquiring resistance to the treatment (2,3,5). In addition, a cumulative assault on bacterial cell insures instantaneous killing of bacteria. In principle, the photoactivated killing of bacteria involves the activation of a photosensitizer by irradiation with a visible light of appropriate wavelength. The activated photosensitizer can either interact with oxygen to produce singlet oxygen (Type 2 reaction) or can directly react with molecules present in the immediate vicinity (Type 1 reaction) (6). Although the bacterial killing can be mediated by either of the abovementioned reactions, singlet oxygen is the predominant chemical entity causing cell death. The short half- life and limited diffusion length of singlet oxygen necessitate the close association of photosensitizers with the target site (7). Further, the effectiveness of photoactivation and subsequent free radical generation are greatly influenced by factors such as (1) the interaction of photosensitizer molecules, (2) the physicochemical environment at the site of application, (3) half-life of the free radicals generated, and (4) oxygen availability at the site of application (6,7). All these factors may be influenced by the solvent in which the photosensitizer is dispersed. Earlier studies have shown that the photophysical proper- ties of photosensitizers can be influenced by the polarity and viscosity of the solvent (8). The half-life of singlet oxygen which influences the antibacterial effect is extremely dependent on the nature of the solvent used (9). Although a considerable amount of work has been done in achieving bacterial kill using the photodynamic approach, only a few studies describe the mechanism of action on bacterial cells. Many researchers have looked at the feasibility of targeting photosensitizers to specific bacteria to minimize adverse side effects. However, only very few attempts have been made to enhance the efficacy of PDT by modifying the photosensitizer solvent. Enterococcus faecalis has been associated with a wide range of human infections comprising endocarditis, urinary tract infections, persistent endodontic infection and biomaterial- centered infections in humans (10,11). This bacterium pro- duces biofilm on anatomic sites which are highly resistant to conventional treatment strategies (12–14). PDT has been suggested as a possible alternative to conventional antibiotic treatment in combating biofilm-mediated infections (1,15). Previous research has shown that the susceptibility of E. faecalis to PDT varied in different photosensitizer solvents of methylene blue (MB) (16). MB dissolved in MIX (etha- nol:glycerol:water 20:30:50) photosensitizer solvents enhanced disinfection potential of PDT on biofilm-infected root canal of tooth (16). MIX-based photosensitization solvents prevented MB aggregation, and enhanced the photo-oxidation potential of MB during irradiation, contributing toward the effective inactivation of biofilm bacteria (16). The use of such a solvent system to improve the photophysical property of indocyanine green was also reported earlier (17). However, the effect of photosensitizer solvents on the mechanisms of bacterial killing has not yet been established. This study aimed *Corresponding author email: rsdak@nus.edu.sg (Anil Kishen) Ó 2007 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/08 Photochemistry and Photobiology, 2008, 84: 734–740 734