A Functional Photosensitizer Designed For Photodynamic Therapy Of Methicillin-resistant Staphylococcus Aureus Infection

Background and objectiveIn recent years, methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most common multi-drug resistant bacteria in both hospital and community. MRSA accounts for larger than 60% of clinical Staphylococcus aureus strains isolated from intensive care units and burn units in many countries. Most patients acquire MRSA infection in a hospital setting. However, community-acquired MRSA strains have emerged, and rapidly become the dominant pathogens in the community. Infection due to MRSA imposes a high and increasing burden on health care resources in most countries. As the multi-drug resistance grows, patients with MRSA infection are usually difficult to treat due to limited therapeutic options. For a long time, clinicians have relied on vancomycin in the management of serious MRSA infection patients. However, it is becoming clear that vancomycin is losing its potency against MRSA now. Therefore it is necessary to continuously search for alternative methods against MRSA towards which no resistance can develop so quickly. One of the most promising and innovative approaches in this respect is antimicrobial photodynamic therapy (PACT), which generates cytotoxic reactive molecules and kills microorganisms by combining photosensitizer, light of appropriate wavelength, and oxygen. The aim of this study is to investigate the selective inhibition of MRS A by a modified photosensitizer (LAEtNBS) in vitro and the efficacy of MRSA infection treatment by photodynamic therapy with LAEtNBS in vivo.Methods1. LAEtNBS was synthesized by adding a cationic photosensitizer molecule (EtNBS-COOH) and a quencher molecule (BHQ3) to two side chains of cephalosporin (ACLE). The absorption and emission spectra of LAEtNBS and EtNBS-COOH were measured in methanol. And also the fluorescence quantum yield and singlet oxygen quantum yield were determinated and compared to EtNBS-COOH.2. The selective inactivation and less phototoxicity of LAEtNBS in vitro, compared to that of EtNBS-COOH, were assessed and confirmed by conducting PACT to two Staphylococcus aureus strains (MRSA and MSSA) and human skin cells at a fluence of 15 J/cm2 with 640±10 nm LED light.3. Using mouse skin wound model infected with 108 CFU of MRS A, LAEtNBS, EtNBS-COOH, and placebo were applied in the wounds and illuminated with a 640± 10 nm LED light,90 J/cm2. The MRS A within the wounds and blood were detected by culturing and CFU counting. Also bioluminescent MRSA infection was assessed through measuring luminescence with a CCD luminescent camera. Wound healing was determinated by calculating healing ratio, and histological examination of wounds 14 days after infection. The temperature of wound was also detected on each mouse during the procedure of PACT.Results1. LAEtNBS was shown to have similar absorption and emission wavelengths with EtNBS-COOH, but tremendously suppressed fluorescence quantum yield and less potency in singlet oxygen production. The reduction of singlet oxygen yield of LAEtNBS was not as significant as the reduction of fluorescence quantum yield.2. In vitro selectively inactivation of MRSA and less inhibition to MSSA by PACT with LAEtNBS were demonstrated using susceptibility tests, while PACT with EtNBS-COOH produced both growth inhibition to MRSA and MSSA. In addition, LAEtNBS exerted less phototoxicity to human skin fibroblasts and keratinocytes than EtNBS-COOH.3. Both LAEtNBS and EtNBS-COOH were shown to be able to reduce MRSA infection in mouse skin wound and enhance tissue repair. However, there was no significant difference in the two photo sensitizers that might be due to the environment in vivo.ConclusionModification of the photosensitizer will be very beneficial for developing new strategies to treat drug resistant bacterial infection with less harm to host tissue. We anticipate that the modified photosensitizer LAEtNBS based on-lactamase targeting with high selectivity will be able to be applied in combination with standard antibiotic treatment to destroy specific bacterial infections especially infections caused by multi-drug resistant bacteria.