Susceptibility of Clostridium difficile to 405 nm light and possible mechanisms to enhance sporicidal activity

Clostridium difficile is currently one of the most significant causative agents of healthcare-associatedinfection. The ability of C. difficile to form highly resilient spores has limited the competence of current strategies for environmental decontamination, and advances to reduce environmental contamination and patient infection are urgently sought. The results of this study demonstrate the sporicidal efficacy of 405 nm violet-blue light to achieve up to 5 log₁₀ reductions in C. difficile spores. In comparison, vegetative cells demonstrated an increased susceptibility, requiring a 10-fold lower dose to achieve acomparable level of inactivation.This study progressed to demonstrate enhanced sporicidal efficacy upon combination of chlorinated disinfectants with 405 nm light at both high (up to 225 mWcm⁻²) and low (0.4 mWcm⁻²) irradiances. For decontamination of spores in suspension, a 50% increased spore susceptibility was observed upon exposure to a 33% lower light dose when incombination disinfectants. On clinically-relevant surfaces, up to 100% increased sporereductions were observed upon combination of low irradiance 405 nm light with selecteddisinfectants.Further significant findings of this study include the enhanced susceptibility of spores upon triggering germination, with inactivation achieved using up to 77% less dose. Also established was the critical requirement of oxygen for photo-inactivation of this anaerobic pathogen, thus supporting the mechanism being a result of photoexcitation of naturally-occurring porphyrins inducing ROS production, oxidative damage and ultimately cell death. This study has confirmed the fundamental sporicidal efficacy of 405 nm light and further highlights possible mechanisms to enhance sporicidal activity. 405 nm light has several advantages over current in-house cleaning procedures and novel sporicidal technologies, including its safety for human exposure permitting continuous decontamination of the patient environment, and the work of this thesis supports the potential for sporicidal efficacy to be achieved if utilised as a complementary strategy with standard cleaning regimes.Clostridium difficile is currently one of the most significant causative agents of healthcare-associatedinfection. The ability of C. difficile to form highly resilient spores has limited the competence of current strategies for environmental decontamination, and advances to reduce environmental contamination and patient infection are urgently sought. The results of this study demonstrate the sporicidal efficacy of 405 nm violet-blue light to achieve up to 5 log₁₀ reductions in C. difficile spores. In comparison, vegetative cells demonstrated an increased susceptibility, requiring a 10-fold lower dose to achieve acomparable level of inactivation.This study progressed to demonstrate enhanced sporicidal efficacy upon combination of chlorinated disinfectants with 405 nm light at both high (up to 225 mWcm⁻²) and low (0.4 mWcm⁻²) irradiances. For decontamination of spores in suspension, a 50% increased spore susceptibility was observed upon exposure to a 33% lower light dose when incombination disinfectants. On clinically-relevant surfaces, up to 100% increased sporereductions were observed upon combination of low irradiance 405 nm light with selecteddisinfectants.Further significant findings of this study include the enhanced susceptibility of spores upon triggering germination, with inactivation achieved using up to 77% less dose. Also established was the critical requirement of oxygen for photo-inactivation of this anaerobic pathogen, thus supporting the mechanism being a result of photoexcitation of naturally-occurring porphyrins inducing ROS production, oxidative damage and ultimately cell death. This study has confirmed the fundamental sporicidal efficacy of 405 nm light and further highlights possible mechanisms to enhance sporicidal activity. 405 nm light has several advantages over current in-house cleaning procedures and novel sporicidal technologies, including its safety for human exposure permitting continuous decontamination of the patient environment, and the work of this thesis supports the potential for sporicidal efficacy to be achieved if utilised as a complementary strategy with standard cleaning regimes

Synergistic efficacy of 405 nm light and chlorinated disinfectants for the enhanced decontamination of Clostridium difficile spores

The ability of Clostridium difficile to form highly resilient spores which can survive in the environment for prolonged periods causes major contamination problems. Antimicrobial 405 nm light is being developed for environmental decontamination within hospitals, however further information relating to its sporicidal efficacy is required. This study aims to establish the efficacy of 405 nm light for inactivation of C. difficile vegetative cells and spores, and to establish whether spore susceptibility can be enhanced by the combined use of 405 nm light with low concentration chlorinated disinfectants. Vegetative cells and spore suspensions were exposed to increasing doses of 405 nm light (at 70–225 mW/cm2) to establish sensitivity. A 99.9% reduction in vegetative cell population was demonstrated with a dose of 252 J/cm2, however spores demonstrated higher resilience, with a 10-fold increase in required dose. Exposures were repeated with spores suspended in the hospital disinfectants sodium hypochlorite, Actichlor and Tristel at non-lethal concentrations (0.1%, 0.001% and 0.0001%, respectively). Enhanced sporicidal activity was achieved when spores were exposed to 405 nm light in the presence of the disinfectants, with a 99.9% reduction achieved following exposure to 33% less light dose than required when exposed to 405 nm light alone. In conclusion, C. difficile vegetative cells and spores can be successfully inactivated using 405 nm light, the sporicidal efficacy can be significantly enhanced when exposed in the presence of low concentration chlorinated disinfectants. Further research may lead to the potential use of 405 nm light decontamination in combination with selected hospital disinfectants to enhance C. difficile cleaning and infection control procedures

Comparative sensitivity of Trichophyton and Aspergillus conidia to inactivation by violet-blue light exposure

The objective of this paper was to investigate the use of 405 nm light for inhibiting the growth of selected species of dermatophytic and saprophytic fungi. The increasing incidence and resilience of dermatophytic fungal infections is a major issue, and alternative treatment methods are being sought. The sensitivity of the dermatophytic fungi Trichophyton rubrum and Trichophyton mentagrophytes to 405 nm violet-blue light exposure was investigated, and the results compared with those obtained with the saprophytic fungus Aspergillus niger. Microconidia of T. rubrum and T. mentagrophytes and conidia of A. niger were seeded onto Sabauroud dextrose agar plates and irradiated with 405 nm light from an indium-gallium-nitride 99-DIE light-emitting diode (LED) array and the extent of inhibition was measured. Germination of the microconidia of the Trichophyton species was completely inhibited using an irradiance of 35 mW/cm2 for 4 h (dose of 504 J/cm2). Results: A. niger conidia showed greater resistance, and colonial growth developed after light exposure. In liquid suspension tests, 405 nm light dose levels of 360, 720, and 1440 J/cm2 resulted in complete inactivation of T. rubrum microconidia, whereas A. niger showed greater resistance, and at the highest dose level applied (1440 J/cm2 ) although A niger hyphae were completely inactivated, only a 3-log10 reduction of a 5-log10 conidial suspension was achieved. The study results demonstrate the relatively high sensitivity of Trichophyton microconidia to 405 nm violet-blue light, and this is may be of potential interest regarding the control and treatment of dermatophyte infections

Enhanced decontamination of C. difficile spores on surfaces via the synergistic action of 405nm light and disinfectants

The ability of C. difficile to form spores which can survive for prolonged periods causes significant environmental contamination problems. 405nm light has wide antimicrobial activity against vegetative bacteria, and is being developed for environmental decontamination within hospitals. As expected, spores are more resilient to inactivation. This study aims to establish whether spore susceptibility can be enhanced by combining 405nm light with low concentration chlorinated disinfectants: sodium hypochlorite, Actichlor and Tristel. Spore suspensions were seeded onto surfaces including PVC, stainless steel and vinyl flooring. Disinfectant was added to the surface, and the samples were then exposed to 405nm light at irradiances of ~0.2-225 mWcm-2. Control samples were exposed to 405nm light alone, and disinfectants alone, to establish the sporicidal activity of each agent, and to demonstrate the synergistic effect when combined. Results demonstrated increased sporicidal activity of 405nm light and low-concentration sodium hypochlorite and Actichlor against C. difficile seeded on vinyl flooring and PVC surfaces, with approximately 3-log10 reductions achieved with up to 66% lower doses than achieved with light alone. Tristel demonstrated limited synergy on vinyl and PVC, whilst all three disinfectants demonstrated minimal synergy on stainless steel. Results are also reported for lower intensity light, as used in the clinical environment. In conclusion, the sporicidal efficacy of 405nm light is enhanced when used alongside chlorinated disinfectants. Further research could potentially lead to the use of lower strength chlorinated disinfectants in combination with 405nm light to provide enhanced decontamination of C. difficile spores in the clinical environment

Antimicrobial efficacy of 405nm light against Clostridium difficile : evidence of enhanced sporicidal activity when combined with disinfectants

Clostridium difficile can cause major contamination problems due to its ability to form highly infectious and resilient spores which can survive in the environment for prolonged periods. Recent work has demonstrated the use of antimicrobial 405nm light for environmental decontamination within hospitals, however further information relating to efficacy against spores is required. The aim of this investigation was to establish the efficacy of 405nm light for inactivation of C. difficile vegetative cells and spores, and to establish whether spore susceptibility can be enhanced by the combined use of 405nm light with low concentration chlorinated and non-chlorinated disinfectants. C. difficile vegetative cells and spore suspensions were exposed to increasing doses of 405nm light (70-225mW/cm2) to establish sensitivity. Exposures were repeated with spores suspended in a range of routine hospital disinfectants at varying concentrations. A 99.9% reduction in vegetative cell population was demonstrated with a dose of 252J/cm2, however spores demonstrated higher resilience, with a 10-fold increase in dose required. Enhanced sporicidal activity was achieved when spores were exposed in the presence of low concentration disinfectant s, with 50% increase in susceptibility when exposed in the presence of 0.1% sodium hypochlorite. C. difficile vegetative cells and spores can be successfully inactivated using 405nm light, and the sporicidal efficacy can be significantly enhanc ed when exposed in the presence of low concentrations of disinfectants. Further research may lead to potential use of 405nm light decontamination in combination with hospital disinfectants to enhance C. difficile cleaning and infection control procedures

Sporicidal efficacy of the combined use of 405nm light and disinfectants for inactivation of Clostridium difficile on clinically relevant surfaces.

The ability of Clostridium difficile to form highly infectious and resilient spores which can survive in the environment for prolonged periods causes major contamination problems. Antimicrobial 405 nm light is capable of inactivating a wide range of organisms, including endospore-forming bacteria, and is being developed for environmental decontamination within hospitals. Currently, there are several drawbacks associated with the chlorinated disinfectants recommended for surface decontamination, including their corrosive nature and the release of irritating vapours affecting healthcare workers. This study aims to establish whether spore susceptibility to low concentration chlorinated disinfectants can be enhanced when used in conjunction with 405 nm light.Spores were spot inoculated and dried onto PVC, vinyl flooring and stainless steel. Samples were then simultaneously exposed to disinfectants (0.0001% Tristel, 0.001% Actichlor and 0.1% sodium hypochlorite (NaOCl)) and 405 nm light at an irradiance of 225 mWcm-2. Control samples were exposed to 405 nm light alone, and disinfectants alone, to establish the sporicidal activity of each agent, and to demonstrate the synergistic effect when combined. Results showed that spores exposed to 405 nm light alone were reduced by 1-2 log10 on all surfaces after a dose of 2.4 kJcm-2. On PVC, complete inactivation was achieved following a dose of 0.8 kJcm-2in the presence of NaOCl, and >2 log10 reduction was achieved following exposure to 0.8 kJcm-2and 1.6 kJcm-2 in combination with Actichlor and Tristel, respectively. Sporicidal activity was significantly enhanced in the presence of all three disinfectants on vinyl, with a 2.5-3 log10 reduction achieved following exposure to a dose of 1.6 kJcm-2 in the presence of both Actichlor and NaOCl. However on stainless steel, spores demonstrated higher resilience to the combined oxidative effects of 405 nm light and disinfectants, with a dose of 2.4 kJcm-2required to achieve a 1.5-2 log10 reduction for all 3 disinfectants.In conclusion, the sporicidal efficacy of commonly used chlorinated hospital disinfectants can be enhanced on a range of clinically relevant surfaces when used alongside 405 nm light. However, the extent of the enhanced sporicidal activity is dependent on the disinfectant and the surface it is applied to. This has the potential to lead to a considerable reduction in exposure time and concentrations of disinfectant required to eliminate C. difficile spores. Further research may lead to potential use of 405 nm light decontamination in combination with selected hospital disinfectants to enhance C. difficile cleaning and infection control procedures

Syngergistic efficacy of 405 nm light and hospital disinfectants for the enhanced decontamination of C. difficile on clinically relevant surfaces

The ability of Clostridium difficile to form highly infectious and resilient spores which can survive in the environment for prolonged periods causes major contamination problems. Antimicrobial 405 nm light is capable of inactivating a wide range of organisms, including endospore-forming bacteria, and is being developed for environmental decontamination within hospitals, however further information relating to its efficacy against spores is required. This study aims to establish the efficacy of 405 nm light for inactivation of C. difficile vegetative cells and spores, and to establish whether spore susceptibility can be enhanced by the combined use of 405 nm light with low concentration chlorinated and non-chlorinated disinfectants, in both liquid suspension and on surfaces. C. difficile vegetative cells and spore suspensions were exposed to increasing doses of 405 nm light (using irradiances of 70-225 mW/cm2) to establish sensitivity. Exposures were repeated with spores suspended in a range of routine hospital disinfectants at varying concentrations. Controls were exposed to 405 nm light in the absence of disinfectants, and disinfectants in the absence of 405 nm light, to establish the sporicidal activity of each agent alone, and to demonstrate the synergistic effect when combined. These experiments were repeated with spores seeded onto a range of relevant inert surfaces. A 99.9% reduction in vegetative cell population was demonstrated with a dose of 252J/cm2, however spores demonstrated higher resilience, with a 10-fold increase in dose required. Enhanced sporicidal activity was achieved when spores were exposed to 405 nm light in the presence of low concentration disinfectants, with a 50% increase in susceptibility when exposed in the presence of 0.1% sodium hypochlorite. Synergy was also noted with the other disinfectants used. In conclusion, C. difficile vegetative cells and spores can be successfully inactivated using 405nm light, and the sporicidal efficacy can be significantly enhanced when exposed in the presence of low concentrations of disinfectants. Further research may lead to potential use of 405nm light decontamination in combination with selected hospital disinfectants to enhance C. difficile cleaning and infection control procedures