There is a need for alternative “treatments” for killing microbes in wound and other body surfaces. Antimicrobial Photodynamic Therapy (APDT) and Electrochemically Activated Solutions (ECAS) are two recent developments in the field of biocide research that in the future may have widespread applications, replacing conventional antibiotics or disinfectants. The aims of the work described in this thesis are 5-fold. (1) Develop an in vitro model and determine the kill rates of bioluminescent species using bioluminescence light output (2) Measure the comparative effects of heavy water and reactive oxygen species (ROS) inhibitory agents against appropriate controls for both APDT and ECAS treatment and gain insight into general killing mechanisms (3) Develop methods to compare kill rates of target microbe (prokaryotic cells) against mammalian eukaryotic cells, by comparison of kill rates under the same treatment conditions and thus, assess the likelihood of cytotoxic damage to the host (4) Measure the potential of treatments to induce genotoxic damage to mammalian cells using a COMET assay (5) Measure the effects of treatments when the target is growing in biofilm mode (using a continuous matrix perfusion model) and compare standard treatments for fast and slow growing cells.
A standard assay was developed containing target cell suspensions with killing agent ECAS or APDT (methylene blue (MB) combined with polychromatic light) and bioluminescence was measured using luminometer and viable count methods. For mechanistic study, the assay was repeated in the presence of ROS scavenger molecules. Fluorescence responses by probes singlet oxygen sensor green (SOSG), 3’-(p-aminophenyl) fluorescein (APF), 3’-(p-hydroxyphenyl) fluorescein (HPF) were measured using a fluorimeter to detect ROS in the presence or absence of specific ROS inhibitory agents. The potential cytotoxicity of APDT and ECAS against keratinocytes (H103) and lymphocytes (Jurkat cell) was measured using the neutral red test (for keratinocytes) and MTS assay (for lymphocytes). Trevigen’s comet assay kit was used to measure genotoxicity produced by ECAS and APDT towards lymphocytes, DNA damage was determined using epifluorescence microscopy. An in vitro flat-bed perfusion biofilm model was used to compare the effects of ECAS and APDT against biofilms, using a low light camera to measure bioluminescence within the biofilm. All data were compared using appropriate statistical analyses.
The light output from the bioluminescent target species was highly proportional to the viable counts with high correlation (R2> 0.9). Order of killing susceptibility was S. aureus > E. coli > P. aeruginosa > MRSA. Kill rates measured using luminescent light output from lux-modified target species are more accurate than conventional viable count. The rapid assay method, coupled with the use of D2O, ROS-scavengers and fluorescent probes provided key insights into mechanisms of APDT and ECAS. It was confirmed that singlet oxygen is the main cytotoxic species for ADPT whilst a mixed system (hydroxyl radical plus singlet oxygen, and possibly other species) was involved for ECAS. APDT and ECAS are not particularly cytotoxic to mammalian cells (keratinocytes or lymphocytes); therefore a large safety margin of dose may exist to reduce the microbial cells without harm to mammalian cells. APDT is more genotoxic to lymphocytes than is ECAS but both are less toxic compared to H2O2 positive control. The in vitro flat-bed perfusion biofilm model was suitable to study both APDT and ECAS against biofilm cells. The results showed that biofilm was resistant compared to planktonic cells, and was able to recover easily post treatment. Slower growing cells take longer to recover following APDT.
Kamil, Z. In vitro studies comparing activities of antimicrobial photodynamic therapy and electrochemically activated solutions. (Thesis). University of the West of England