Ultraviolet germicidal irradiation is effective against SARS-CoV-2 in contaminated makeup powder and lipstick.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is mainly transmitted by airborne droplets generated by infected individuals. Since this and many other pathogens are able to remain viable on inert surfaces for extended periods of time, contaminated surfaces play an important role in SARS-CoV-2 fomite transmission. Cosmetic products are destined to be applied on infection-sensitive sites, such as the lips and eyelids. Therefore, special biosafety precautions should be incorporated into the routine procedures of beauty parlors and shops. Indeed, innovative cosmetics companies are currently searching for disinfection protocols that ensure the customers' safety in makeup testing. Here, we propose an ultraviolet germicidal irradiation (UVGI) strategy that can be used to reduce the odds of COVID-19 fomite transmission by makeup testers. It is well-known that UVGI effectively inactivates pathogens on flat surfaces and clear fluids. However, ultraviolet-C (UVC) radiation at 254 nm penetrates poorly in turbid and porous materials, such as makeup and lipstick formulations. Thus, we investigated the virucidal effect of UVGI against SARS-CoV-2 deposited on such substrates and compared their performance to that of flat polystyrene surfaces, used as controls. Concentrated infectious SARS-CoV-2 inoculum (106 PFU/mL) deposited on lipstick and makeup powder was completely inactivated (>5log10 reduction) following UVC exposures at 1,260 mJ/cm2, while flat plastic surfaces required 10 times less exposure (126 mJ/cm2) to reach the same microbicidal performance. We conclude that UVGI comprises an effective disinfection strategy to promote biosafety for cosmetics testers. However, appropriate UVC dosimetry must be implemented to overcome inefficiencies caused by the optical properties of turbid materials in lipsticks and makeup powders.

Antimicrobial photodynamic inactivation mediated by methylene blue: Analysis of inactivation kinetics and biochemical mechanisms / Inativação fotodinâmica antimicrobiana mediada por azul de metileno: Análises de cinética de inativação e mecanismos bioquímicos

The widespread use of antimicrobial chemotherapy in medicine and livestock production imposed an evolutive selection of drug-resistant strains worldwide. As a result, the effectiveness of our current antimicrobial armamentarium is constantly being reduced to alarming levels. Therefore, novel antimicrobial therapeutic strategies are urgently needed. Antimicrobial photodynamic therapy (APDT) comes to this scenario as a powerful tool to counteract the emergence of microbial drug-resistance. Its mechanisms of action are based on simultaneous oxidative damage of multiple targets and, therefore, it is much less likely to allow any type of microbial resistance. Therefore, the objectives of this study were focused into establishing 1) a mathematical tool to allow precise analysis of microbial photoinactivation; 2) a broad analysis of APDT effectiveness against global priority drug-resistant pathogens; 3) inhibition of ßlactamase enzymes; and 4) how the biochemical mechanisms of APDT avoid emergence of resistance. The main results obtained through the investigation led by this thesis were divided into 4 scientific articles regarding each of the above-mentioned objectives. In summary, we discovered that 1) a power-law function can precisely fit all microbial inactivation kinetics data and provide insightful information of tolerance factors and lethal doses; 2) there is no correlation between drug-resistance and APDT sensitivity, i.e., extensively drug resistant microorganisms are killed in the same kinetics as drug-sensitive controls; 3) ß-lactamases are very sensitive to photodynamic inhibition; 4) biochemical mechanisms of APDT promote oxidative damages to external cell membranes, DNA and proteins whereas the main cause of microbial death seems to be directly associated with protein degradation. Thus, we conclude that APDT is effective against a broad-spectrum of pathogens and has minimum chances of promoting resistance mechanisms

O amplo uso da quimioterapia antimicrobiana impôs uma seleção evolutiva de cepas resistentes a medicamentos. Como resultado, a eficácia dos fármacos antimicrobianos tem sido reduzida a níveis alarmantes. Portanto, novas estratégias terapêuticas antimicrobianas são urgentemente necessárias. A terapia fotodinâmica antimicrobiana (TFDA) entra neste cenário como uma ferramenta poderosa para combater a resistência microbiana. Seus mecanismos de ação são baseados no dano oxidativo sobre múltiplos alvos e, portanto, é muito menos provável que permita o surgimento de qualquer tipo de resistência. Os objetivos deste estudo foram focados ao estabelecimento de 1) modelo matemático para análise precisa da fotoinativação microbiana; 2) ampla análise da eficácia da TFDA contra patógenos resistentes a fármacos antimicrobianos de prioridade global; 3) inibição de ß-lactamases por TFDA; e 4) como os mecanismos bioquímicos da TFDA evitam o surgimento de resistência. Os principais resultados obtidos através da investigação conduzida por esta tese foram divididos em 4 artigos científicos. Em resumo, descobrimos que 1) uma função de lei de potência pode ajustar com precisão todos os dados de cinética de inativação microbiana e fornecer informações detalhadas sobre fatores de tolerância e doses letais; 2) não há correlação entre resistência à quimioterapia antimicrobiana e sensibilidade à TFDA, isto é, cepas extensivamente resistentes aos antimicrobianos são inativadas sob a mesma cinética que controles sensíveis aos antimicrobianos; 3) ß-lactamases são altamente sensíveis à inibição fotodinâmica; 4) os mecanismos bioquímicos da TFDA promovem danos oxidativos às membranas celulares e DNA, porém, a principal causa de morte microbiana é diretamente associada à degradação das proteínas. Assim, concluímos que a TFDA é eficaz contra um amplo espectro de patógenos e tem chances mínimas de promover mecanismos de resistência

Global priority multidrug-resistant pathogens do not resist photodynamic therapy.

Microbial drug-resistance demands immediate implementation of novel therapeutic strategies. Antimicrobial photodynamic therapy (aPDT) combines the administration of a photosensitizer (PS) compound with low-irradiance light to induce photochemical reactions that yield reactive oxygen species (ROS). Since ROS react with nearly all biomolecules, aPDT offers a powerful multitarget method to avoid selection of drug-resistant strains. In this study, we assayed photodynamic inactivation under a standardized method, combining methylene blue (MB) as PS and red light, against global priority pathogens. The species tested include Acinetobacter baumannii, Klebsiella aerogenes, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, Candida albicans and Cryptococcus neoformans. Our strain collection presents resistance to all tested antimicrobials (>50). All drug-resistant strains were compared to their drug-sensitive counterparts. Regardless of resistance phenotype, MB-aPDT presented species-specific dose-response kinetics. More than 5log10 reduction was observed within less than 75 s of illumination for A. baumannii, E. coli, E. faecium, E. faecalis and S. aureus and within less than 7 min for K. aerogenes, K. pneumoniae, P. aeruginosa, C. albicans and C. neoformans. No signs of correlations in between drug-resistance profiles and aPDT sensitivity were observed. Therefore, MB-aPDT can provide effective therapeutic protocols for a very broad spectrum of pathogens. Hence, we believe that this study represents a very important step to bring aPDT closer to implementation into mainstream medical practices.

Natural anthraquinones as novel photosentizers for antiparasitic photodynamic inactivation.

BACKGROUND: Cutaneous leishmaniasis (CL) is a vector-borne disease caused by obligate protist parasites from the genus Leishmania. The potential toxicity as well as the increased resistance of standard treatments has encouraged the development of new therapeutical strategies. Photodynamic inactivation (PDI) combines the use of a photosensitizer and light to generate reactive oxygen species and kill cells, including microorganisms. Vegetal kingdom constitutes an important source of bioactive compounds that deserve to be investigated in the search of naturally occurring drugs with leishmanicidal activity. PURPOSE: The purpose of this study was to test the antiparasitic activity of PDI (ApPDI) of five natural anthraquinones (AQs) obtained from Heterophyllaea lycioides (Rusby) Sandwith (Rubiacae). To support our results, effect of AQ mediated-PDI on parasite´s morphology and AQ uptake were studied. Cytotoxicity on fibroblasts was also evaluated. STUDY DESIGN/METHODS: Two monomers, soranjidiol (Sor) and 5-chlorosoranjidiol (5-ClSor) plus three bi-anthraquinones (bi-AQs), bisoranjidiol (Bisor), 7-chlorobisoranjidiol (7-ClBisor) and Lycionine (Lyc) were selected for this study. Recombinant L. amazonensis promastigote strain expressing luciferase was subjected to AQs and LED treatment. Following irradiation with variable light parameters, cell viability was quantified by bioluminescence. Alteration on parasite's morphology was analyzed by scanning electron microscopy (SEM). In addition, we verified the AQ uptake in Leishmania cells by fluorescence and their toxicity on fibroblasts by using MTT assay. RESULTS: Bisor, Sor and 5-ClSor exhibited photodynamic effect on L. amazonensis. SEM showed that promastigotes treated with Bisor-mediated PDI exhibited a significant alteration in shape and size. Sor and 5-ClSor presented higher uptake levels than bi-AQs (Bisor, Lyc and 7-ClBisor). Finally, Sor and Bisor presented the lowest toxic activity against fibroblasts. CONCLUSION: Taking together, our results indicate that Sor presents the highest specificity towards Leishmania cells with no toxicity on fibroblasts.

Algicidal effect of blue light on pathogenic Prototheca species.

Prototheca spp. are pathogenic algae with important zoonotic potential. Most importantly, these algae often infect dairy cattle. Since there is no effective therapy against the algae, the standard recommendation is the disposal or culling of infected cows to avoid outbreaks. This study investigated the ability of blue light to inactivate pathogenic Prototheca species. Blue LED light (λ = 410 nm) was used to inactivate in vitro suspensions of P. zopfii genotypes 1 and 2, and P. blaschkeae. Our results showed that blue light irradiation induced a strain-specific dose-dependent algicidal effect against all tested strains. P. zopfii genotype 1, was more sensitive than genotype 2 and P. blaschkeae was the most tolerant. Even though we observed different inactivation kinetics, all strains presented significant photoinactivation levels within feasible procedure periods. Therefore, we conclude that blue light irradiation offers promising potential for the development of novel technologies that control contaminations and infections caused by Prototheca spp.

Glucose modulates antimicrobial photodynamic inactivation of Candida albicans in biofilms.

Candida albicans biofilm is a main cause of infections associated with medical devices such as catheters, contact lens and artificial joint prosthesis. The current treatment comprises antifungal chemotherapy that presents low success rates. Photodynamic inactivation (PDI) involves the combination of a photosensitizing compound (PS) and light to generate oxidative stress that has demonstrated effective antimicrobial activity against a broad-spectrum of pathogens, including C. albicans. This fungus senses glucose inducing an upregulation of membrane transporters that can facilitate PS uptake into the cell. The aim of this study was to evaluate the effects of glucose on methylene blue (MB) uptake and its influence on PDI efficiency when combined to a red LED with central wavelength at λ=660nm. C. albicans biofilms were grown on hydrogel disks. Prior to PDI assays, MB uptake tests were performed with and without glucose-sensitization. In this system, the optimum PS administration was determined as 500µM of MB in contact with the biofilm during 30min before irradiation. Irradiation was performed during 3, 6, 9, 12, 15 and 18min with irradiance of 127.3mW/cm2. Our results showed that glucose was able to increase MB uptake in C. albicans cells. In addition, PDI without glucose showed a higher viability reduction until 6min; after 9min, glucose group demonstrated a significant decrease in cell viability when compared to glucose-free group. Taken together, our data suggest that glucose is capable to enhance MB uptake and modulate photodynamic inactivation of C. albicans biofilm.

Photobiomodulation reduces abdominal adipose tissue inflammatory infiltrate of diet-induced obese and hyperglycemic mice.

Systemic inflammation is closely related to the development of insulin resistance and type-2 diabetes, since the activation of pro-inflammatory pathways leads to inhibition of insulin signaling. Although photobiomodulation (PBM) has proven beneficial effects on the treatment of inflammatory disorders, the phototherapeutic approach to manage the chronic inflammatory component of obesity and hyperglycemia had never been explored. In this work, obese and hyperglycemic mice are treated with PBM, and their body mass, glycemia and inflammatory infiltrate of abdominal adipose tissue are evaluated. During four weeks, irradiated animals are exposed to six irradiation sessions using an 843 nm LED (5.7 J cm-2 at 19 mW cm-2 per session). Non-irradiated control animals display inflammatory areas almost five times greater than the treated group (p < 0.001). This result on inflammatory infiltrate may have caused impacts on the significant lower blood glucose level from irradiated animals (p = 0.04), twenty-four hours after the last irradiation session. PBM on obese and hyperglycemic mice reduced five times the areas of inflammatory infiltrate within abdominal adipose tissue (a, b), whereas dense inflammatory regions were a common finding amidst non-irradiated animals (c). The asterisks on (c) correspond to the inflammatory infiltrate permeating adipocytes.

In vitro photoinactivation of bovine mastitis related pathogens.

BACKGROUND: Bovine mastitis is considered the most important disease of worldwide dairy industry. Treatment of this disease is based on the application intramammary antibiotic, which favors an increase in the number of resistant bacteria in the last decade. Photodynamic inactivation (PDI) has been investigated in different areas of Health Sciences, and has shown great potential for inactivating different pathogens, without any selection of resistant microorganisms. The objective of this study was to investigate the efficacy of PDI in the inactivation of pathogens associated with bovine mastitis. METHODS: We tested the effectiveness of PDI against antibiotic resistant strains, isolated from bovine mastitis, from the following species: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium bovis, and the alga Prototheca zopfii. Nine experimental groups were evaluated: control, no treatment; light only, irradiation of a red light-emitting diode (λ=662 (20) nm) for 180 s; exposure to 50 µM methylene blue alone for 5 min; and PDI for 5, 10, 30, 60, 120 and 180 s. RESULTS: S. dysgalactiae, S. aureus, and C. bovis were inactivated after 30s of irradiation, whereas S. agalactiae was inactivated after 120 s and P. zopfii at 180 s of irradiation. CONCLUSION: These results show that PDI can be an interesting tool for inactivating pathogens for bovine mastitis.

Photodynamic therapy for pododermatitis in penguins.

Pododermatitis is currently one of most frequent and important clinical complications in seabirds kept in captivity or in rehabilitation centers. In this study, five Magellanic penguins with previous pododermatitis lesions on their footpad were treated with photodynamic therapy (PDT). All PDT treated lesions successfully regressed and no recurrence was observed during the 6-month follow-up period. PDT seems to be an inexpensive and effective alternative treatment for pododermatitis in Magellanic penguins encouraging further research on this topic.

Antimicrobial photodynamic inactivation inhibits Candida albicans virulence factors and reduces in vivo pathogenicity.

The objective of this study was to evaluate whether Candida albicans exhibits altered pathogenicity characteristics following sublethal antimicrobial photodynamic inactivation (APDI) and if such alterations are maintained in the daughter cells. C. albicans was exposed to sublethal APDI by using methylene blue (MB) as a photosensitizer (0.05 mM) combined with a GaAlAs diode laser (λ 660 nm, 75 mW/cm(2), 9 to 27 J/cm(2)). In vitro, we evaluated APDI effects on C. albicans growth, germ tube formation, sensitivity to oxidative and osmotic stress, cell wall integrity, and fluconazole susceptibility. In vivo, we evaluated C. albicans pathogenicity with a mouse model of systemic infection. Animal survival was evaluated daily. Sublethal MB-mediated APDI reduced the growth rate and the ability of C. albicans to form germ tubes compared to untreated cells (P < 0.05). Survival of mice systemically infected with C. albicans pretreated with APDI was significantly increased compared to mice infected with untreated yeast (P < 0.05). APDI increased C. albicans sensitivity to sodium dodecyl sulfate, caffeine, and hydrogen peroxide. The MIC for fluconazole for C. albicans was also reduced following sublethal MB-mediated APDI. However, none of those pathogenic parameters was altered in daughter cells of C. albicans submitted to APDI. These data suggest that APDI may inhibit virulence factors and reduce in vivo pathogenicity of C. albicans. The absence of alterations in daughter cells indicates that APDI effects are transitory. The MIC reduction for fluconazole following APDI suggests that this antifungal could be combined with APDI to treat C. albicans infections.