Evaluation of visible light and natural photosensitizers against Staphylococcus epidermidis and Staphylococcus saprophyticus planktonic cells and biofilm.

Antimicrobial photoinactivation (API) has shown some promise in potentially treating different nosocomial bacterial infections, however, its application on staphylococci, especially other than Staphylococcus aureus or methicillin-resistant S. aureus (MRSA) species is still limited. Although S. aureus is a well-known and important nosocomial pathogen, several other species of the genus, particularly coagulase-negative Staphylococcus (CNS) species such as Staphylococcus epidermidis and Staphylococcus saprophyticus, can also cause healthcare-associated infections and foodborne intoxications. CNS are often involved in resilient biofilm formation on medical devices and can cause infections in patients with compromised immune systems or those undergoing invasive procedures. In this study, the effects of chlorophyllin and riboflavin-mediated API on S. epidermidis and S. saprophyticus planktonic cells and biofilm are demonstrated for the first time. Based on the residual growth determination and metabolic reduction ability changes, higher inactivating efficiency of chlorophyllin-mediated API was determined against the planktonic cells of both tested species of bacteria and against S. saprophyticus biofilm. Some insights on whether aqueous solutions of riboflavin and chlorophyllin, when illuminated with optimal exciting wavelength (440 nm and 402 nm, respectively) generate O2-•, are also provided in this work.

Riboflavin- and chlorophyllin-based antimicrobial photoinactivation of Brevundimonas sp. ESA1 biofilms.

Some Brevundimonas spp. are globally emerging opportunistic pathogens that can be dangerous to individuals with underlying medical conditions and for those who are immunocompromised. Gram-negative Brevundimonas spp. can form resilient sessile biofilms and are found not only in different confined terrestrial settings (e.g., hospitals) but are also frequently detected in spacecraft which is inhabited by astronauts that can have altered immunity. Therefore, Brevundimonas spp. pose a serious health hazard in different environments, especially in its biofilm form. Conventional antimicrobials applied to disrupt, inactivate, or prevent biofilm formation have limited efficiency and applicability in different closed-loop systems. Therefore, new, effective, and safe biofilm control technologies are in high demand. The present work aimed to investigate antimicrobial photoinactivation (API) of Brevundimonas sp. ESA1 monocultural biofilms mediated by non-toxic, natural photosensitizers such as riboflavin (RF) and chlorophyllin (Chl) with an emphasis of this technology as an example to be safely used in closed-loop systems such as spacecraft. The present study showed that Chl-based API had a bactericidal effect on Brevundimonas sp. ESA1 biofilms at twice the lower irradiation doses than was needed when applying RF-based API. Long-term API based on RF and Chl using 450 nm low irradiance plate has also been studied in this work as a more practically applicable API method. The ability of Brevundimonas sp. ESA1 biofilms to reduce alamarBlue™ and regrowth analysis have revealed that after the applied photoinactivation, bacteria can enter a viable but non-culturable state with no ability to resuscitate in some cases.

Photoinactivation of Salmonella enterica exposed to 5-aminolevulinic acid: Impact of sensitization conditions and irradiation time.

The photodynamic inactivation (PDI) represents the potential alternative to traditional antibiotic therapy, and can be applied to treat various bacterial infections, including those caused by Gram-negative bacterial strains. One of the treatment modalities is based on the capacity of bacterial cells to synthesize the excess amounts of porphyrins after exposure to an externally applied 5-aminolevulinic acid (5-ALA), which makes them photosensitive and leads to reduced survival after irradiation with an appropriately selected light source. This study focuses on the sensitization and the photoinduced inactivation of Salmonella enterica cells in PBS containing 0.5 mM 5-ALA, incubated at 37 °C for 4 h or for 20 h and afterwards irradiated with violet LED light (11.1 mW/cm2, a peak at 400 nm). It has been found that both amounts and composition of endogenous porphyrins not only depended on the incubation duration, but also were affected by externally induced photo- and chemo-oxidation reactions. The application of different sensitization conditions has revealed that the increasing amounts of endogenously produced porphyrins do not ensure the proportional reduction of bacterial cell survival numbers. The comparative investigations also demonstrated that the presence of endogenously produced porphyrins in the medium results in secondary sensitization of bacterial cells and causes a notably stronger photoinactivation effect in comparison to their externally applied standards.

Inactivation of Opportunistic Pathogens Acinetobacter baumannii and Stenotrophomonas maltophilia by Antimicrobial Photodynamic Therapy.

Acinetobacter baumannii and Stenotrophomonas maltophilia are opportunistic pathogens causing hospital infections with limited treatment options due to bacterial multidrug resistance. Here, we report that antimicrobial photodynamic therapy (aPDT) based on the natural photosensitizers riboflavin and chlorophyllin inactivates A. baumannii and S. maltophilia. The riboflavin and chlorophyllin photostability experiments assessed the photomodifications of photosensitizers under the conditions subsequently used to inactivate A. baumannii and S. maltophilia. A. baumannii planktonic cells were more sensitive to riboflavin-aPDT, while biofilm bacteria were more efficiently inactivated by chlorophyllin-aPDT. S. maltophilia planktonic and biofilm cells were more susceptible to chlorophyllin-aPDT compared to riboflavin-aPDT. The results suggest that riboflavin- and chlorophyllin-aPDT can be considered as a potential antimicrobial treatment for A. baumannii and S. maltophilia inactivation.

Application of Antimicrobial Photodynamic Therapy for Inactivation of Acinetobacter baumannii Biofilms.

Acinetobacter baumannii is a dangerous hospital pathogen primarily due to its ability to form biofilms on different abiotic and biotic surfaces. The present study investigated the effect of riboflavin- and chlorophyllin-based antimicrobial photodynamic therapy, performed with near-ultraviolet or blue light on the viability of bacterial cells in biofilms and their structural stability, also determining the extent of photoinduced generation of intracellular reactive oxygen species as well as the ability of A. baumannii to form biofilms after the treatment. The efficacy of antimicrobial photodynamic therapy was compared with that of light alone and the role of the photosensitizer type on the photosensitization mechanism was demonstrated. We found that the antibacterial effect of riboflavin-based antimicrobial photodynamic therapy depends on the ability of photoactivated riboflavin to generate intracellular reactive oxygen species but does not depend on the concentration of riboflavin and pre-incubation time before irradiation. Moreover, our results suggest a clear interconnection between the inactivation efficiency of chlorophyllin-based antimicrobial photodynamic therapy and the sensitivity of A. baumannii biofilms to used light. In summary, all the analyzed results suggest that riboflavin-based antimicrobial photodynamic therapy and chlorophyllin-based antimicrobial photodynamic therapy have the potential to be applied as an antibacterial treatment against A. baumannii biofilms or as a preventive measure against biofilm formation.

Antimicrobial Photoinactivation Approach Based on Natural Agents for Control of Bacteria Biofilms in Spacecraft.

A spacecraft is a confined system that is inhabited by a changing microbial consortium, mostly originating from life-supporting devices, equipment collected in pre-flight conditions, and crewmembers. Continuous monitoring of the spacecraft's bioburden employing culture-based and molecular methods has shown the prevalence of various taxa, with human skin-associated microorganisms making a substantial contribution to the spacecraft microbiome. Microorganisms in spacecraft can prosper not only in planktonic growth mode but can also form more resilient biofilms that pose a higher risk to crewmembers' health and the material integrity of the spacecraft's equipment. Moreover, bacterial biofilms in space conditions are characterized by faster formation and acquisition of resistance to chemical and physical effects than under the same conditions on Earth, making most decontamination methods unsafe. There is currently no reported method available to combat biofilm formation in space effectively and safely. However, antibacterial photodynamic inactivation based on natural photosensitizers, which is reviewed in this work, seems to be a promising method.

Inactivation of Gram (-) bacteria Salmonella enterica by chlorophyllin-based photosensitization: Mechanism of action and new strategies to enhance the inactivation efficiency.

This study is focused on the enhancement of susceptibility of Gram (-) bacteria S. enterica to chlorophyllin-based (Chl) photosensitization combining it with other antimicrobial tools. In order to find best combinations, the mechanism by which Chl-based photosensitization inactivates bacteria must be identified. Data confirmed that photosensitization (Chl 1.5×10-5M, for 1-120min, 405nm, 0-46.1J/cm2) reduced S. enterica population, just by 2.05 log (CFU/ml). Fluorimetric measurements indicated that just minor part of Chl was bound to Salmonella in suspension. Addition of sodium azide (NaN3) (10mM) protected bacteria from killing, what means that 1O2 took place in photochemical reactions. Gene expression data confirmed that Chl-based photosensitization induced oxidative stress in bacteria cells, since mostly genes responsible for detoxification of ROS (OxyR, AhpC, GrxA) have been expressed in Salmonella. Moreover, the expression of genes, responsible for the inhibition of oxidative respiration (AtpC), cell division and down-regulation of metabolism (SulA) have been detected. In addition, Chl-based photosensitization induced significant release of intracellular components (absorbing at λ260 nm and λ280 nm) in bacteria that indicated increased membrane permeability. Thus, the combination of two antimicrobials (Chl-based photosensitization and chitosan (CHS)) with the same target (cellular membrane) in the presence of light drastically reduced viable Salmonella population (by 7.28 log). Combined treatment of photosensitization and high power pulsed UV light (HPPL) was also very effective, since reduced viable Salmonella by 7.5 log. Bacterial regrowth experiments clearly indicated that after both combined treatments Salmonella lost its ability to proliferate, and SEM images confirmed that after both treatments no viable bacteria have been found at all.

Effective photosensitization-based inactivation of Gram (-) food pathogens and molds using the chlorophyllin-chitosan complex: towards photoactive edible coatings to preserve strawberries.

This study is focused on the novel approaches to enhance the inactivation of the Gram (-) food pathogen Salmonella enterica and harmful molds in vitro and on the surface of strawberries using the chlorophyllin-chitosan complex. Salmonella enterica (∼1 × 10(7) CFU mL(-1)) was incubated with chlorophyllin 1.5 × 10(-5) M (Chl, food additive), chitosan 0.1% (CHS, food supplement) or the chlorophyllin-chitosan complex (1.5 × 10(-5) M Chl-0.1% CHS) and illuminated with visible light (λ = 405 nm, light dose 38 J cm(-2)) in vitro. Chlorophyllin (Chl)-based photosensitization inactivated Salmonella just by 1.8 log. Chitosan (CHS) alone incubated for 2 h with Salmonella reduced viability 2.15 log, whereas photoactivated Chl-CHS diminished bacterial viability by 7 log. SEM images indicate that the Chl-CHS complex under these experimental conditions covered the entire bacterial surface. Significant cell membrane disintegration was the main lethal injury induced in Gram (-) bacteria by this treatment. Analysis of strawberry decontamination from surface-inoculated Salmonella indicated that photoactivated Chl-CHS (1.5 × 10(-5) M Chl-0.1% CHS, 30 min incubation, light dose 38 J cm(-2)) coatings diminished the pathogen population on the surface of strawberries by 2.2 log. Decontamination of strawberries from naturally distributed yeasts/molds revealed that chitosan alone reduced the population of yeasts/molds just by 0.4 log, Chl-based photosensitization just by 0.9 log, whereas photoactivated Chl-CHS coatings reduced yeasts/molds on the surface of strawberries by 1.4 log. Electron paramagnetic resonance spectroscopy confirmed that no additional photosensitization-induced free radicals have been found in the strawberry matrix. Visual quality (color, texture) of the treated strawberries was not affected either. In conclusion, photoactive Chl-CHS exhibited strong antimicrobial action against more resistant to photosensitization Gram (-) Salmonella enterica in comparison with Gram (+) bacteria in vitro. It reduced significantly the viability of strawberry surface-attached yeasts/molds and inoculated Salmonella without any negative impact on the visual quality of berries. Experimental data support the idea that photoactivated Chl-CHS can be a useful tool for the future development of edible photoactive antimicrobial coatings which can preserve strawberries and prolong their shelf-life according to requirements of "clean green technology".

Inactivation of several strains of Listeria monocytogenes attached to the surface of packaging material by Na-Chlorophyllin-based photosensitization.

This study was focused on the possibility to inactivate thermosensitive Listeria monocytogenes ATC(L3)C 7644 and thermoresistant 56 Ly strain by Na-Chlorophyllin (Na-Chl)-based photosensitization in vitro and on the surface of packaging. Comparative analysis of antimicrobial efficiency of photosensitization with conventional surface cleaning was performed. Data indicate that both Listeria strains, after incubation with Na-Chl and following illumination (λ=400nm, 20mWcm(-2)), were inactivated by 7 log in vitro. This treatment cleaned both Listeria strains from packaging surfaces. Comparative analysis indicates that washing with water diminishes pathogens by less than 1 log, 200ppm Na-hypochlorite by 1.7 log, Na-Chl-based photosensitization by 4.5 log. Listeria biofilms were totally removed from the surface by photosensitization at higher photosensitizer concentrations and longer incubation times. In conclusion, both strains of L. monocytogenes can be effectively inactivated by photosensitization in vitro and on the surface of packaging. Listeria biofilms are susceptible to this treatment as well. Comparison of different surface decontamination treatments reveals that photosensitization is much more effective against both Listeria strains than washing with water or 200ppm Na-hypochlorite. Our data support the idea that Na-Chl-based photosensitization is an effective antimicrobial tool which may serve in the future for the development of human and environmentally friendly surface decontamination techniques.

Photosensitization-based inactivation of food pathogen Listeria monocytogenes in vitro and on the surface of packaging material.

The study was focused on the susceptibility of Listeria monocytogenes ATCL3C 7644 cells and biofilms to non-thermal antimicrobial treatment - photosensitization in vitro and after adhesion to the surface of packaging material. L. monocytogenes was incubated with 5-aminolevulinic acid (ALA) (7.5 mM) for 0-2h and illuminated with visible light. The LED-based light source used for the illumination emitted light lambda=400 nm with energy density 20 mW/cm(2). The illumination time varied 0-20 min, and a total light dose reached 0-24 J/cm(2). The obtained data indicate that L. monocytogenes produces endogenous porphyrins after incubation with 7.5mM ALA. Subsequent illumination of cells remarkably inactivates (4 log) them in vitro. Photosensitization diminished population of Listeria cells adhered onto the packaging material by 3.7 log and inactivated bacterial biofilms by 3.1 log. It was shown that antimicrobial efficiency of photosensitization depended on the illumination time, incubation with ALA time as well as on the used ALA concentration. In conclusion, cells and biofilms of L. monocytogenes ATCL3C 7644 can be effectively inactivated by ALA-based photosensitization in the solution as well as adhered onto the surface of packaging material. Obtained data support the idea, that photosensitization as non-thermal and effective antimicrobial treatment has potential to develop into environmentally safe, surface decontamination technique.