This PDF file contains the front matter associated with SPIE Proceedings Volume 10479, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Host-pathogen interactions orchestrate the human innate immune system triggering responses from the immune cells which are usually beneficial. However, in some cases the innate immune system overreacts and turns against the host leading to a systematic inflammatory response, especially in immune compromised patients. Major players of the innate immune system are leukocytes, in particular neutrophils, monocytes and lymphocytes. During leukocytes’ confrontation with the pathogens and the associated molecular motifs, various chemical combat strategies are presented by the cell to clear the invading infection. Understanding this pathogen specific chemical signalling cascade will allow early recognition of invading pathogens and help physicians in their therapeutic decision. To probe the cells in their native environment a label-free and non-invasive method is required. One such upcoming method in the diagnostic field is Raman spectroscopy. This technique enables to capture changes within the leukocytes which are triggered upon their encounter with various bacterial and fungal stimuli. For fast and high throughput screening of cells an in-house built high content screening Raman spectrometer has been employed which allows fast recording of Raman spectra from 100,000 cells within a time span of a few hours yielding spectra with high S/N ratio and retaining cell specific information. This platform has been utilized for in-vitro investigation of the leukocytes subtypes’ interaction with fungi, gram-positive and gram-negative bacteria. Raman spectral analysis with aid of the chemometric methods provides information on the leukocyte subtypes’ selective response towards particular pathogens.

Publisher’s Note: This conference presentation, originally published on 14 March 2018, was withdrawn per author request on 10 April 2018.

We have developed a rapid phenotypic antimicrobial susceptibility testing (AST) in which photonic 2D silicon microarrays are employed as both the optical transducer element and as a preferable solid−liquid interface for bacterial colonization. We harness the intrinsic ability of the micro-architectures to relay optical phase-shift reflectometric interference spectroscopic measurements (termed PRISM) and incorporate it into a platform for culture-free, label-free tracking of bacterial accumulation, proliferation, and death. This assay employs microfluidic channels interfaced with PRISM chips and is carried out in a two-stage process, namely bacteria seeding and antibiotic incubation. Bacteria proliferation within the microtopologies results in an increase in refractive index of the medium, yielding an increase in optical path difference, while cell death or bacteriostatic activity results in decreasing or unchanged values. The optical responses of bacteria to various concentrations of relevant antibiotics have been tracked in real time, allowing for accurate determination of the minimum inhibitory concentration (MIC) values within 2-3 hours. We further extended this work to analyze antibiotic susceptibilities of clinical isolates and direct urine samples derived from patients at neighboring hospitals in newly designed, disposable microfluidic devices. This has opened the door to the observation of unique bacterial behaviors, as we can evaluate bacterial adhesion, growth, and antibiotic resistance on different microarchitectures, different surface chemistries, and even different strains. Motility, charge, and biofilm abilities have been explored for their effect of bacterial adhesion to the microstructures as we further develop our method of rapid, label-free AST for full clinical application.

The standard laboratory procedure for determining the antibiotic susceptibility of a pathogen (an antimicrobial susceptibility test, AST) measures the inhibition of growth, and requires several days. This can delay effective therapy and lead to antibiotic overuse and misuse. Recent work (Wei Hou et al, Lab on a Chip 2015) has shown that resistant and susceptible pathogens will have very different gene expression profiles shortly following antibiotic exposure, and that these expression biomarkers may be used to accurately identify the pathogen species, strain and antibiotic susceptibility without growth. We therefore developed an ultrasensitive ‘digital microarray’ for performing rapid & quantitative gene expression analysis as part of a rapid AST. The digital microarray uses plasmonic gold nanorods (GNRs) functionalized with DNA to specifically label each target RNA that binds to the microarray. Each GNR on the array is then individually detected based on its light scattering, with an interferometric microscopy technique called SP- IRIS. Our optimized high-throughput version of SP-IRIS is able to scan a typical array of 500 spots in less than 10 minutes. Due to its single molecule readout, the assay has a limit of detection of less than 1 femtomolar following just 2 hours of incubation. Altogether, digital microarrays are about 10,000-fold more sensitive than fluorescence microarrays, yet maintain all of the strengths of the platform including low cost and high multiplexing. The reproducibility and robustness of the multiplexed assay will next be evaluated with clinically relevant pathogenic strains of E. coli as part of a functional rapid AST.

Elastic Light Scattering (ELS) is an innovative technique to identify bacterial pathogens directly on culture plates. Compelling results have already been reported for agri-food applications. Here, we have developed ELS for clinical diagnosis, starting with Staphylococcus aureus early screening. Our goal is to bring a result (positive/negative) after only 6 h of growth to fight surgical-site infections. The method starts with the acquisition of the scattering pattern arising from the interaction between a laser beam and a single bacterial colony growing on a culture medium. Then, the resulting image, considered as the bacterial species signature, is analyzed using statistical learning techniques. We present a custom optical setup able to target bacterial colonies with various sizes (30-500 microns). This system was used to collect a reference dataset of 38 strains of S. aureus and other Staphyloccocus species (5459 images) on ChromIDSAID/ MRSA bi-plates. A validation set from 20 patients has then been acquired and clinically-validated according to chromogenic enzymatic tests. The best correct-identification rate between S. aureus and S. non-aureus (94.7%) has been obtained using a support vector machine classifier trained on a combination of Fourier-Bessel moments and Local- Binary-Patterns extracted features. This statistical model applied to the validation set provided a sensitivity and a specificity of 90.0% and 56.9%, or alternatively, a positive predictive value of 47% and a negative predictive value of 93%. From a clinical point of view, the results head in the right direction and pave the way toward the WHO’s requirements for rapid, low-cost, and automated diagnosis tools.

In this presentation we demonstrate how light can be useful both for sensing as well as for treating different types of inflammations and infections. In respect to sensing we show how a laser and a camera based system can be used in order to “hear” from a distance the “sounds” of e.g. the lungs while realizing a non-contact stethoscope which can be useful for detection of inflammable lungs diseases such as asthma. In respect to treating inflammations and infections with light, we will show how special light sources can be used in order to destroy candida nail infection or how combination of laser light together with gentamicin antibiotic can significantly enhancement the susceptibility of a bacteria such as P. aeruginosa.

Tissue inflammation is often accompanied by fever and edema, which are common and troublesome problems that probably trigger disability, lymphangitis, cosmetic deformity and cellulitis. Here we developed a device, which can measure concentration and temperature variations of water in local human body by extended near infrared spectroscopy in 900~1000 nm wavelength range. An experiment of four steps incremental cycling exercise was designed to change tissue water concentration and temperature of subjects. Body temperature was also estimated by tympanic thermometer and surface thermometer as comparisons during the experiment. In the stage of recovery after exercise, the signal detected by custom device is similar to tympanic thermometer at the beginning, but it is closer to the temperature of surface later. In particular, this signal shows a better linearity, and a significant change when the exercise was suspended. This study demonstrated the potential of optical touch-sensing for inflammation severity monitoring by measuring water concentration and temperature variations in local lesions.

Influenza viruses cause seasonal epidemics and frequent pandemics such as avian influenza viruses H5N1 and H7N9 that resulted in significant morbidity and fatality in humans. We engineered influenza A viruses to carry a gene encoding Gaussia luciferase (IAV-luci) while remaining replication competent. Influenza A virus has 17 serological hemagglutinin (HA) subtypes, we thus generated a series of IAV-luci with different HAs including pandemic H1/California/7/2009, avian influenza H5/Hongkong/482/1997, and H7/Anhui/1/2013. Through the detection of bioluminescent signals, we showed that: 1) Infection of animals with IAV-luci enabled real time visualization of infection status or assessment of drug treatment in living animals; 2) Cultured cells infected with as little as one IAV-luci could be detected for its bioluminescent signals. 3) Neutralizing antibodies to influenza virus from the serum of H7N9 infected patients could be detected within 6 hours for diagnosis and prediction of disease outcome; 4) Neutralizing antibodies secreted by one antibody producing B cell became detectable using IAV-luci, therefore enabled the cloning of monoclonal antibodies at single cell level. 5) An ultrasensitive microneutralization assay was developed that allows the epidemiological study for “hidden” avian influenza virus infections in human population. We believe that engineered bioluminescent influenza viruses will greatly facilitate the serological monitoring of infection, epidemicological study, antiviral drug discovery, especially the search for anti-influenza neutralizing antibodies.

The modern medicine requires the new type of drug delivery carriers that will combine functions of in vivo navigation and visualization ability to deploy drug in controllable manner, including external triggering. This combination can be realized by multifunctional carriers produced by layer-by-layer assembly method. The carrier biodistribution can be controlled by a chosen mode of in vivo administration. Realization for chemical targeted delivery based on surface modification are not working well in vivo due to the corona effect [Kreyling W. et al., Nature Nanotech., 2015, 619]. Thus, physical targeting of drug delivery is more promising approach. It can be realized by gradient of magnetic field [Voronin D. et al., ACS App. Mater. & Interfaces, 2017, 6885], optical tweezers [Stetciura I. et al., Analyst, 2015,4981]. It was demonstrated that the sensitivity of nanostructured carriers to external influences as laser irradiation, ultrasound treatment can be changed by variation of volume fraction and chemical composition of inorganic nanoparticles in the carrier shell [Korolovych V. et al., PCCP, 2016,2389]. Same approach is applied for nanostructured carriers (NCs) imaging by MRI [German S. et al., PCCP, 2016, 32238], OCT [Genina E. et al., Biomed.Opt.Express, 2016, 2082] and photoacoustic method [Yashchenok A. et al., J. Biophotonics, 2016, 792] using magnetite and gold nanoparticles as contrast agents, respectively. Obtained NCs can be used as drug delivery systems including drug depot, combined much functionalities as navigation and visualization, in vivo monitoring of biochemical process, remote activated release of bioactive substances.

Due to the growing global threat of antibiotic resistance, there is a critical need for the development of alternative therapeutics for infectious diseases. Antimicrobial blue light (aBL), as an innovative non-antibiotic approach, has attracted increasing attention. This paper discussed the basic concepts of aBL and recent findings in the studies of aBL. It is commonly hypothesized that the antimicrobial property of aBL is attributed to the presence of endogenous photosensitizing chromophores in microbial cells, which produce cytotoxic reactive oxygen species upon light irradiation. A wide range of important microbes are found to be susceptible to aBL inactivation. Studies have also shown there exist therapeutic windows where microbes are selectively inactivated by aBL while host cells are preserved. The combination of aBL with some other agents result in synergistically improved antimicrobial efficacy. Future efforts should be exerted on the standardization of study design for evaluating aBL efficacy, further elucidation of the mechanism of action, optimization of the technical parameters, and translation of this technique to clinic.

Pseudomonas aeruginosa may be isolated from skin wounds of burn patients, bedsore and diabetic ulcers. The healing of wounds is often impaired by the intrinsic antibiotic resistance, the tolerance to many antimicrobials and the ability to form biofilm of this opportunistic pathogen. Finding new topical treatments to combine with antibiotics is thus essential. Among natural products, the antimicrobial properties of honeys have been known for millennia. In this study honey and visible light have been combined to control the growth of P. aeruginosa PAO1. The irradiation by a broad spectrum light source of bacteria inoculated onto 2 % w/v fir and forest honeydew (HD) honeys caused a killing effect that the honeys alone or the light alone did not show. This antimicrobial activity was light energy-dose and honey-concentration dependent. Among the tested honeys, the fir and forest HD honeys were the most efficient ones. In particular, the irradiation by blue LED (λ_max = 466 nm) yielded good rates of killing, that were significantly higher in comparison to irradiation alone and honey alone. Interestingly, a similar effect was obtained by plating bacteria on blue LED pre-irradiated HD honeys. The combined use of honey and blue light was also successful in inhibiting the biofilm formation of P. aeruginosa. The blue LED irradiation of PAO1 administered with 10 % w/v forest HD honey significantly enhanced the inhibition of biofilm formation in comparison to dark incubated honey.

The photoinactivation properties of 405 (violet) and 470 nm (blue) light have been studied by many research groups within the last few years. Both wavelengths are capable of disinfecting bacteria and fungi, with 405 nm radiation being more efficient. The basic photoinactivation mechanism is understood for 405 nm. Violet light is absorbed by endogenous porphyrins that act as photosensitizers and generate reactive oxygen species, subsequently destroying the microorganisms from within. The underlying photobiological mechanism for 470nm radiation is still unclear though porphyrins and flavins are widely believed to be involved endogenous photosensitizer.

We performed own measurements of disinfection efficacy and additionally did a meta-analysis of published photoinactivation data. The disinfection experiments were performed with LEDs at peak wavelengths between 440 and 490 nm in an interval of about 10 nm. Staphylococcus auricularis was irradiated with doses of 70, 140 and 210 J/cm² and peak efficacy was observed at 470 nm while the impact of irradiation decreases steeply to lower and higher wavelengths. These observations are supported by the meta-analysis results and rather contradictory to the porphyrin and flavin hypothesis so that our conclusion is that there may be another unknown photosensitizer involved.

Surgical site infections (SSIs) are a leading cause of morbidity and mortality and a significant expense to the healthcare system and hospitals. The majority of these infections are preventable; however, increasing bacterial resistance, biofilm persistence, and human error contribute to the occurrence of these healthcare-associated infections. We present a flexible antimicrobial blue-light emitting bandage designed for use on postoperative incisions and wounds. The photonic device is designed to inactivate bacteria present on the skin and prevent bacterial colonization of the site, thus reducing the occurrence of SSIs. This antimicrobial light emitting bandage uses blue light’s proven abilities to inactivate a wide range of clinical pathogens regardless of their resistance to antibiotics, inactivate bacteria without harming mammalian cells, improve wound healing, and inactivate bacteria in biofilms. The antimicrobial bandage consists of a thin 2”x2” silicone sheet with an array of 77 LEDs embedded in multiple layers of the material for thermal management. The 405 nm center wavelength LED array is designed to be a wearable device that integrates with standard hospital infection prevention protocols. The device was characterized for irradiance of 44.5 mW/cm². Methicillin-resistant Staphylococcus aureus seeded in a petri dish was used to evaluate bacterial inactivation in vitro. Starting with a concentration of 2.16 x 10⁷ colony forming units (CFU)/mL, 45% of the bacteria was inactivated within 15 minutes, 65% had been inactivated by 30 minutes, 99% was inactivated by 60 minutes, and a 7 log reduction and complete sterilization was achieved within 120 minutes.

Antibiotic resistance is one of the most serious threats to public health. It is estimated that at least 23,000 people die each year in the USA as a direct result of antibiotic-resistant infections. In addition, many antibiotic-resistant microorganisms develop biofilms, surface-associated microbial communities that are extremely resistant to antibiotics and the immune system. A light-based approach, antimicrobial blue light (aBL), has attracted increasing attention due to its intrinsic antimicrobial effect without the involvement of exogenous photosensitizers. In this study, we investigated the effectiveness of this non-antibiotic approach against biofilms formed by multidrug-resistant (MDR) microorganisms. MDR Acinetobacter baumannii, Escherichia coli, Candida albicans, and Pseudomonas aeruginosa biofilms were grown either in 96-well microtiter plates for 24 h or in a CDC biofilm reactor for 48 h, and then exposed to aBL at 405 nm emitted from a light-emitting diode (LED). We demonstrated that, for the biofilms grown in the CDC biofilm reactor, approximately 1.88 log₁₀ CFU reduction was achieved in A. baumannii, 2.78 log10 CFU in E. coli and 3.18 log₁₀ CFU in P. aeruginosa after 162 J/cm² , 576 J/cm² and 500 J/cm² aBL were delivered, respectively. For the biofilms formed in the 96-well microtiter plates, 5.67 and 2.46 log₁₀ CFU reduction was observed in P. aeruginosa and C. albicans polymicrobial biofilm after an exposure of 216 J/cm² . In conclusion, aBL is potentially an alternative non-antibiotic approach against MDR biofilm-related infections. Future studies are warranted to investigate other important MDR microorganisms, the mechanism of action of aBL, and aBL efficacy in vivo.

Neisseria gonorrhoeae is a human-adapted, gram-negative diplococcus that infects human reproductive tracts and causes gonorrhea, a sexually transmitted disease, resulting in discharge and inflammation at the urethra, cervix, pharynx, or rectum. Over the years, N. gonorrhoeae has developed resistance to nearly every drug ever used to treat it, including sulfonamides, penicillin, tetracycline, and fluoroquinolones. Drug-resistant N. gonorrhoeae is now considered by the Centers for Disease Control and Prevention (CDC) as an urgent threat. The present study aimed to evaluate the efficacy of antimicrobial blue light (aBL) at 405 and 470 nm for inactivating N. gonorrhoeae and reveal the mechanism of action. Our results showed that an exposure of 45 J/cm² aBL at 405 nm reduced the bacterial CFU by 7.16-log₁₀. When the aBL exposure was increased to 54 J/cm², eradication of bacterial CFU was achieved. When the bacteria were exposed to aBL at 470 nm, 3-log₁₀ reduction of CFU was observed at an aBL exposure of higher than 126 J/cm². Absorption and fluorescence spectroscopic analyses revealed the presence of endogenous porphyrins and flavins in N. gonorrhoeae cells. The present study indicated that aBL is a potential strategy to control N. gonorrhoeae infections. Endogenous porphyrins play a vital role in the killing effects of aBL. In vivo experiments are ongoing in our laboratory to treat genital tract infections in mice using aBL and explore the potential clinical applications.

Given that the dearth of new antibiotic development loads an existential burden on successful infectious disease therapy, health organizations are calling for alternative approaches to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report a drug-free photonic approach to eliminate MRSA through photobleaching of staphyloxanthin, an indispensable membrane-bound antioxidant of S. aureus. The photobleaching process, uncovered through a transient absorption imaging study and quantitated by absorption spectroscopy and mass spectrometry, decomposes staphyloxanthin, and sensitizes MRSA to reactive oxygen species attack. Consequently, staphyloxanthin bleaching by low-level blue light eradicates MRSA synergistically with external or internal reactive oxygen species. The effectiveness of this synergistic therapy is validated in MRSA culture, MRSAinfected macrophage cells. Collectively, these findings highlight broad applications of staphyloxanthin photobleaching for treatment of MRSA infections.

Natural anthraquinones (AQs) isolated from Heterophyllaea lycioides (Rusby) Sandwith (Rubiaceae) demonstrated to have photodynamic properties: soranjididol (Sor), 5-Chlorosoranjidiol (5-ClSor), bisoranjidiol (Bisor), 7-Chlorobisoranjidiol (7-ClBisor) and lycionine (Lyc). Sor, 5-ClSor and Bisor exhibited photodynamic inactivation on bacteria and parasites. As they could be used in topical application, the aim of this work was to study their photodynamic activity on fibroblasts.

AQs were tested at 2.5 μM in darkness and under irradiation conditions. They were photoactivated with violet-blue LED (λ = 410 ± 10 nm; fluence rate =50 mW/cm2) and exposure time corresponded to a fluence of 27 J/cm². Negative and positive control (−C and +C, respectively) were included. Mitochondrial activity was determined by using MTT assay that is a measure of the cell viability and it was expressed as a percentage respect to −C (% CV).

Results showed that AQs in darkness conditions showed similar metabolic activity as −C, except for 5-ClSor (about 75% CV). Under irradiation, AQs exhibited dissimilar results. Sor and 7-ClBisor maintained cell viability at approximately 100%, Bisor and Lyc around 70%, whereas 5-ClSor reduced cell viability by 90%. Taken together, our results suggest that Sor could mediate photodynamic therapy (PDT) in cutaneous infections since no toxicity was observed in fibroblasts. On the other hand, 5-ClSor could be used for topical PDT of keloids and hypertrophic scars.

We have discovered that antimicrobial photodynamic inactivation (aPDI) can be strongly potentiated by addition of the non-toxic salt potassium iodide. This approach works with a wide variety of different photosensitizers including those possessing cationic charges that bind to microbial cells, and those neutral or anionic compounds that are completely ineffective in photoinactivating Gram-negative cells, but can kill > 6 logs in presence of 100 mM KI. The approach is broad spectrum in nature and works with MRSA, a range of Gram-negative bacteria and Candida. The major mechanism is likely to involve the addition of singlet oxygen to iodide to form peroxyiodide, which then decomposes via two possible routes: (a) formation of the stable species, free iodine and hydrogen peroxide; (b) formation of short-lived radicals I2•- + HOO•. When the PS binds to the microbial cells, killing by the short-lived radicals becomes significant, while for Gram-negative cells with Photofrin or Rose Bengal, killing by I3- and H2O2 are dominant. This can be studied by comparing “in” (all ingredients together, “after” cells added after light, and “spin” KI and light added after cells were incubated with PS and centrifuged. We have recently studied two porphyrins TMPyP4 (tetracationic) and TPPS4 (tetraanionic). Surprisingly TPPS4 was an excellent PS for MRSA and Candia, and could eradicate Gram-negative species when KI and light were added after a spin, showing it was bound to the surface. Another tetraanionic phthalocyanine (ClAlPCS4) did not show this behavior. We conclude that TPPS4 behaves as if it has some cationic character in the presence of bacteria. KI could potentiate RB-PDT in a mouse model of skin abrasions infected with bioluminescent P. aeruginosa demonstrating possible in vivo applications.

Drug resistance of pathogenic microbes is a quickly growing and extremely dangerous health threat. Bacterial infections are one of the most important causes of skin ulcers. The significant increases in the rates of infection with antibiotic- and multi-drug-resistant bacteria in recent years have hindered clinical treatment, but the effect of antibiotics is very disappointing. Therefore, we have applied ALA-PDT in the treatment of refractory infectious ulcers in clinical practice, and find that ALA-PDT is a potential modality to control Pseudomonas aeruginosa infection and can effectively kill Pseudomonas aeruginosaon chronic skin ulcers, and promote healing of chronic skin ulcers. Through in vitro experiments, we have further confirmed the elimination effect of Pseudomonas aeruginosa biofilm by ALA-PDT and its related mechanisms. And our results shows that ALA-PDT kills planktonic P. aeruginosa and viable cells in the biofilm, destroys biofilm structure, reduces virulence factor secretion, and affects QS system gene expression. These results provide further experimental evidence for the use of PDT for the clinical treatment of P. aeruginosa infections.

Dental caries is an infectious disease caused by acidogenic bacteria. Effective removal and/or inactivation of the cariogenic biofilm is crucial for the prevention and treatment of dental caries. Thus, the purpose of this study was to evaluate the susceptibility of S. mutans biofilm to photodynamic inactivation using two photosensitizers based on curcumin. Suspensions of S. mutans were exposed to LED at 440nm (BioTable®) under 36.1mW, 15J/cm2 and 5 min of pre-irradiation time with synthetic and commercial curcumin at different concentrations (160, 80, 40, 20, 10, 5, 2.5, 1.25, 0625 and 0.3μM) to determine minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC). After that, biofilm was induced for 7 days over hydroxyapatite discs (5mmx1.8mm). Serial dilutions were seeded onto BHI agar to determine viability by CFU/mL. Additionally, confocal laser scanning microscopy (CLSM) was performed using LIVE/DEAD® BacLight™ system to the distribution of non-viable viable/cells. Different Groups were analyzed: L-D- (negative control), L-D+ (drug Group), L+D- (light Group), L+D+ (PDI Group) and chlorhexidine at 0.2% (positive control). In addition, the mechanisms involved were determined before and after irradiation by absorption and fluorescence spectra. The results were analyzed by two-way ANOVA and Tukey’s test (p<0.05). Statistically significant difference in all PDI and chlorhexidine Groups compared to negative control and light Group (p<0.05) was observed. For the dark cytotoxicity, no significant difference was observed compared to the negative control Group (p>0.05). Photodynamic inactivation using curcumin can be an adjunct and effective method to control Streptococcus mutans biofilm responsible to dental caries.

Introduction: In the past 25 years, methicillin-resistant Staphylococcus aureus (MRSA) strains have grown in both magnitude and diversity, making it more difficult for healthcare providers to treat these types of infections. Virulence factors of MRSA allow it to adapt to its environment and develop antibiotic resistance rapidly. One alternative treatment to combat these resistant pathogens is PDAT. Previous studies demonstrated the inhibitory effect of PDAT against MRSA isolates; however, there is limited knowledge regarding the effect of PDAT on virulence factors (toxins, defense mechanisms, stress response). Purpose: To evaluate the impact of rose bengal (RB) and riboflavin (RI) PDAT on the virulence factors of six different ocular species of MRSA. Methods: Suspensions were made with six separate MRSA species inocula (108CFU/mL) with either water (control), 0.1% RB, or 0.1% RI solutions. Each suspension was aliquoted onto an agar plate and exposed to either dark or a 5.4J/cm2 irradiation dose with custom-made LED irradiation sources [λ= 525nm (RB) or 375nm (RI)]. Plates were incubated for 48 hours and then photographed for percent growth measurement. Microbial samples were collected from each plate from which DNA was extracted and sent for full genome sequencing at CosmosID. Results: Rose bengal PDAT completely inhibited the growth of all six MRSA species within the irradiation zone, while riboflavin had minimal inhibition. The dark conditions for both photosensitizers showed minimal inhibition. Full genome sequencing revealed that the virulence factors had varying responses to PDAT, depending on the MRSA species, photosensitizer used, and light exposure.

We report a novel class of highly water-soluble decacationic methano[60]fullerene decaiodides C60[>M(C3N6+C3)2]-(I−)10 [1-(I−)10] capable of co-producing singlet oxygen (Type-II) and highly reactive hydroxyl radicals, formed from superoxide radicals in Type-I photosensitizing reactions, upon illumination at both UVA and white light wavelengths. The O2‒·-production efficiency of 1-(I−)10 was confirmed by using a O2‒·-reactive bis(2,4-dinitrobenzenesulfonyl)tetrafluorofluorescein probe and correlated to the photoinduced electron-transfer event going from iodide anions to 3C60*[>M(C3N6+C3)2] leading to C60‒·[>M(C3N6+C3)2]. Incorporation of a defined number (ten) of quaternary ammonium cationic charges per C60 in 1 was aimed to enhance its ability to target pathogenic Gram-positive and Gram-negative bacterial cells. We used the well-characterized malonato[60]fullerene diester monoadduct C60[>M(t-Bu)2] as the starting fullerene derivative to provide a better synthetic route to C60[>M(C3N6+C3)2] via transesterification reaction under trifluoroacetic acid catalyzed conditions. These compounds may be used as effective photosensitizers and nano-PDT drugs for photoinactivation of pathogens.

A new cationic modified coumarin derivative, 7-diethylamino-3-(3-(4-(trimethylbenzenaminium iodide) phenyl) acryloyl)-2H-chromen-2-one (1), was synthesized and characterized by ¹H NMR and mass spectra. It had a strong intramolecular charge transfer absorption band around 460 nm with large molar extinction coefficients of 3.94 × 10⁴ M^-1 cm^-1 in DMF and 3.86 × 10⁴ M^-1 cm^-1 in PBS, respectively. Moreover, a moderate singlet oxygen quantum yield of 0.16 was obtained for 1 in DMF. Using methylene blue (MB) under a 630 nm laser as reference, the in vitro antimicrobial photodynamic therapy (aPDT) activity of 1 against three strains, gram positive bacteria methicillin-resistant staphylococcus aureus (MRSA), negative bacteria acinetobacter baumannii (A. baumannii) and fungus Candida albicans (C. albicans), was carried out by irradiation with a 457 nm laser. It was shown that 1 had no dark toxicity to these bacteria when its concentration was up to 100 μM, while under the 457 nm laser it could kill them effectively with an over 3 log CFU/ml decrease of the bacterial viability with its concentration up to 5 μM. The aPDT capability of 1 against MRSA and A. baumannii was equivalent to that of MB. For C. albicans, 1 exhibited much better aPDT effect than MB.

Rose Bengal (RB) is a halogenated xanthene dye that has been used to mediate antimicrobial photodynamic inactivation. While highly active against Gram-positive bacteria, RB is largely inactive in killing Gram-negative bacteria. We have discovered that addition of the non-toxic salt potassium iodide (100mM) potentiates green light (540nm)-mediated killing by up to six extra logs with Gramnegative bacteria Escherichia coli and Pseudomonas aeruginosa,Gram-positive methicillin resistant Staphylococcus aureus, and fungal yeast Candida albicans. The mechanism is proposed to be singlet oxygen addition to iodide anion to form peroxyiodide, which decomposes into radicals, finally forms hydrogen peroxide and molecular iodine. The effects of these different bactericidal species can be teased apart by comparing killing in three different scenarios: (1) cells+RB+KI are mixed together then illuminated with green light; (2) cells+RB are centrifuged then KI added then green light; (3) RB+KI+green light then cells added after light. We showed that KI could potentiate RBPDT in a mouse model of skin abrasions infected with bioluminescent P.aeruginosa.

Photodynamic therapy (PDT) uses photosensitizers (PS) that are excited with light to generate ROS in the presence of oxygen for treating various diseases. PS also has the potential use as photodynamic insecticides (PDI) and for light-inactivation of Leishmania for photodynamic vaccination (PDV). PDT-inactivated Leishmania are non-viable, but remain immunologically competent as whole-cell vaccines against leishmaniasis, and as a universal carrier for delivery of add-on vaccines against other infectious and malignant diseases. We have screened novel PS, including Zn- and Si-phthalocyanines (PC) for differential PDT activities against Leishmania, insect and mammalian cells in vitro to assess their PDI and PDV potential. Here, Zn-PC were conjugated with various functional groups. The conjugates were examined for uptake by cells as a prerequisite for their susceptibility to light-inactivation. PDT sensitivity was found to vary with cell types and PS used. PDI potential of several PS was demonstrated by their mosquito larvicidal PDT activities in vitro. PDT-inactivated Leishmania were stored frozen for PDV in several ongoing studies: [1] Open label trial with 20 sick dogs for immunotherapy of canine leishmaniasis after chemotherapy in Naples, Italy. Clinical follow-up for >3 years indicate that the PDV prolongs their survival; [2] PDV of murine models with a human lung cancer vaccine showed dramatic tumor suppression; [3] Open label trial of multiple PDV via compassionate access to 4 advanced cancer patients showed no clinically adverse effects. Two subjects remain alive. Genetic modifications of Leishmania are underway to further enhance their safety and efficacy for PDV by installation of activable mechanisms for self-destruction and spontaneous light-emission.

Photodynamic therapy is a non-invasive therapeutic modality that has gained much attention in the last few decades. It involves administration of a photoactivatable substance, called photosensitizer (PS) into the body, and this substance, upon receiving light can generate reactive oxygen species (ROS), which could subsequently destroy nearby malignancies. The technique has been widely used for cancer treatment, but is also of significance to fight against infectious diseases. For the purpose of this study, Enterococcus faecalis, a bacterial strain common in oral cavities, gastrointestinal system, and wound infections, was selected and its antimicrobial photodynamic inactivation (aPDI) effect with the phenothiazinium dye Toluidine Blue Ortho (TBO) using a 635 nm diode laser was observed. To investigate the possibility of aPDI enhancement, potassium iodide (KI), an inorganic salt which has been proven to have antimicrobial use in dermatology, was used as a potentiating mediator, in order to give a comparative account of the aPDI effect. Laser power was set at 300 mW with irradiation times of 30, 60, and 180 s. Photosensitizer and inorganic potentiator concentration ranges were selected to be 100 μM(TBO) and 100 mM(KI) respectively. Noticeable potentiation of aPDI effect was observed, especially for 60 and 180 s irradiation groups.

Antimicrobial photodynamic inactivation (aPDI) is a new approach to killing microbial cells involving the excitation of photosensitizers (PS) by the correct wavelength of light to produce microbicidal reactive oxygen species. aPDI is independent of the antibiotic resistance status of the target cells, and is thought unlikely to produce resistance itself. Among a wide range of antimicrobial PS that have so far been investigated, tetracyclines occupy a unique niche. They are potentially dual-action compounds that can both kill bacteria under illumination, and prevent bacterial regrowth by inhibiting ribosomes. Demeclocycline (DMCT) can be efficiently activated by blue light (405 nm), while doxycycline (DOTC) is excited best by UVA light (360 nm). Both compounds were able to eradicate Gram-positive and Gram-negative bacteria at concentrations up to (100µM) and fluences up to 10J/cm2. The addition of potassium iodide (400mM) potentiates bacteria killing by up to six extra logs with little amount of DMCT and DOTC (5µM). In contrast to methylene blue (MB), tetracyclines can photoinactivate bacteria in rich growth medium. Bacteria regrowth inhibition and even further bacterial killing were observed when bacteria were partially killed with photosensitized DOTC or DMCT, while MB allowed complete regrowth. MIC studies were carried out either in the dark or exposed to continuous blue light (0.5mW/cm2). Up to 3 extra steps (8-fold) of antibiotic activity was found in the light compared to dark, while 5 extra steps (32-fold) of that was observed with 200mM KI. The mechanism is proposed to be singlet oxygen addition to iodine anion to form peroxyiodine which can decomposes into hydrogen peroxide and molecular iodine.

We previously explored the use of antibody-conjugated, antibiotic-loaded gold nanocages for the treatment of bacterial infections. Using Staphylococcus aureus as a proof-of-principle pathogen, we confirmed that nanocages coated with polydopamine and loaded with daptomycin could be effectively targeted to bacterial cells using an antibody targeting S. aureus surface-associated protein A. We also confirmed that laser irradiation could then be used to achieve a lethal photothermal effect and localized release of the antibiotic, the synergistic effect of which was capable of eradicating viable bacteria even from a therapeutically recalcitrant biofilm. To assess the possibility that this comes at the cost of adverse side effects, we used multispectral optoacoustic tomography (MSOT) to track the biodistribution of our nanocages following intravenous administered and determined whether their administration was associated with toxic side effects. The results of our MSOT analysis confirmed that our nanocages accumulate primarily in the liver, spleen and kidney irrespective of infection status. However, in an infected animal, they also confirmed that nanocages ultimately do reach the site of infection. MSOT results were consistent with studies involving the direct analysis of these tissues, which confirmed the correlation between MSOT signals and the presence of gold nanocages. More importantly, they also demonstrated that the presence of nanocages was not associated with appreciable histopathology in the spleen, liver, kidney, lung or heart. This suggests that our use of antibody-conjugated, antibiotic-loaded gold nanocages for the treatment of infection offers significant promise that would not be compromised by systemic toxicity.

An endotracheal tube (ETT) is required for the management of critically ill, mechanically ventilated patients. Ventilatorassociated pneumonia (VAP) affects patients hospitalized in intensive care units; its risk of occurrence is 1% to up 3% for each day of mechanical ventilation. The polymicrobial nature of VAP is established with mixed bacterial-fungal biofilms colonizing the ETT. The microbial interaction enhances the microbial pathogenesis contributing to high indexes of morbidity/mortality. Antimicrobial Photodynamic Therapy (aPDT) could be a suitable therapy for decontamination of oral cavity and ETT at the same time, but the use of a fiber optics inside the ETT seems to not be appropriated since a cannula for secretion aspiration has to be introduced into the ETT to keep it´s lumen. The aim of this study is to proof the concept that an external light source from a LED is capable of reach all areas of the ETT. We use a commercial ETT, 60μM methylene blue (MB), and a 660nm diode laser and calculated the transmission coefficient of light in different situations as only tube, tube with biofilm and biofilm+MB. The results prove that is possible to transmit light through the tube even in the presence of MB and biofilm although a high attenuation of about 60% was measured depending on the tested condition.

Potassium iodide can potentiate antimicrobial photodynamic inactivation (aPDI) of a broad-spectrum of microorganisms, producing many extra logs of killing. We compared two charged porphyrins, TPPS4 (thought to be anionic and not able to bind to Gram-negative bacteria) and TMPyP4 (considered cationic and well able to bind to bacteria). As expected TPPS4 + light did not kill Gram-negative Escherichia coli, but surprisingly when 100 mM KI was added, it was highly effective at mediating aPDI (eradication at 200 nM + 10 J/cm² of 415 nm light). TPPS4 was more effective than TMPyP4 in eradicating the Gram-positive bacteria, methicillin-resistant Staphylococcus aureus and the fungal yeast Candida albicans (regardless of KI). TPPS4 was also highly active against E. coli after a centrifugation step when KI was added, suggesting that the supposedly anionic porphyrin bound to bacteria and Candida. We conclude that TPPS4 behaves as if it has some cationic character in the presence of bacteria, which may be related to its supply from vendors in the form of a dihydrochloride salt.

The World Health Organization has been alerting that the “post-antibiotic era” is approaching, making the search for alternative antimicrobial therapies mandatory. Antimicrobial photodynamic therapy (aPDT) has been gaining prominence due to its non-specific mechanism of action, which relies on the generation of reactive oxygen species upon the activation of a photosensitizer (PS) by light of a specific wavelength in the presence of oxygen. However, the discussion of whether or not aPDT can induce bacterial resistance is controversial. In that sense, the aim of this study was to determine if multiple cycles of suboptimal doses of aPDT could induce resistance in Enterococcus faecalis. First, we determined optimal and suboptimal conditions of aPDT employing chlorin-e6 (Ce6) and methylene (MB) for planktonic E. faecalis. The combinations of light dose and PS concentration that induced bacterial reductions between 1 log10 and 3 log10 of CFU/mL were selected to start the cycles (21 µM of Ce6 + 45 J/cm²; and 78 µM of MB + 80 J/cm²). The cycling consisted of treatment, plating on PS-free blood agar and recovering grown colonies to start again. By the end of four cycles, cells treated with both Ce6-aPDT or MB-aPDT were completely eliminated, after sustaining a stable survival rate through the first three cycles. We employed two different PS and observed the same outcomes for both of them, indicating the results were not PS-dependent. Our findings are of paramount importance since they are in the way to prove that bacterial resistance cannot be induced by this approach.

Healthcare Acquired Infections (HAIs) pose a significant health risk to our nation, especially to those most in need of healthcare. One in every 25 people admitted to a hospital will be infected by one or more HAIs. Significant reductions in HAI risks can be advanced through innovative technologies, such as UV antimicrobial disinfecting devices. Development of such technologies, along with the associated Behavioral, Chemical and Technological protocols to combat infectious HAIs is a worthwhile pursuit for the public good. A significant good will be accomplished by engaging optical scientists and engineers as well as healthcare professionals in opportunities to advance light-driven antimicrobial devices to halt infections. Fundamental change can be effected through a path of advancing standards and methods including optical measurements, and testing the efficacy of UV light antimicrobial devices and related technologies.

Healthcare associated infections (HAI) affect approximately 1 of every 25 hospitalized patients, lead to substantial morbidity and mortality, degrade patient experience and are costly. Risks for HAI are multifactorial and it is known that microbial contamination of the healthcare environment increases risk for HAI. Portable ultraviolet-C (UVC) surface disinfection as an adjunct to standard hospital disinfection has been shown to decrease both surface microbial contamination and HAI. However, there remain significant gaps in the understanding of the efficient and effective application of UVC in healthcare. Specific barriers identified are: 1) the variability in size, shape, and surface materials of hospital rooms as well as the presence of medical devices and furniture, which impacts the amount of UVC energy delivered to surfaces and its disinfection efficiency; 2) the significant resources needed to acquire and efficiently use UVC equipment and achieve the desired patient benefits- a particular challenge for complex healthcare facilities with limited operating margins; and 3) the lack of implementation guidance and industry standard methods for measuring the UVC output and antimicrobial effects from the multiple commercial UVC options available. An improved understanding of the efficient and effective use of UVC surface disinfection in healthcare and the implementation of standard device industry metrics may lead to increased use and decrease the burden of HAI.

Infections with Chlamydia trachomatis are the major cause for infectious blindness and still represent the most common bacterial sexually transmitted disease worldwide. Considering the possible side effects of antibiotic therapy and the increasing threat of antibiotic resistance, alternative therapeutic strategies are needed.

Previous studies showed a reduction of C. trachomatis infectivity after irradiation with water filtered infrared A alone (wIRA) or in combination with visible light (wIRA/VIS).

In this study, we aimed to gain further insight into the working mechanism of wIRA/VIS by analyzing cytokine and chemokine levels of infected and non-infected HeLa cells following triple dose irradiation at 24, 36 and 40 hours post infection. Subsequently, we examined the influence of cytokines on irradiation and chlamydial infection using a cytokine/chemokine inhibitor (Azelastine) and by IL-6 and IL-8 gene silencing.

A triple dose irradiation significantly reduced chlamydial infectivity in HeLa cells without inducing the chlamydial stress response. The reducing effect was present regardless of the addition of cycloheximide (CHX), a host protein synthesis inhibitor. Chlamydial infection, wIRA/VIS treatment and the combination of both revealed a similar release pattern of a subset of pro-inflammatory cytokines (IL-6, IL-8, RANTES, Serpin E1). The addition of Azelastine induced the chlamydial stress response in non-irradiated samples. This effect was even more pronounced in wIRA/VIS-treated conditions. Silencing of IL-6 and IL-8 resulted in a lower chlamydial infectivity. However, wIRA/VIS treatment of infected and silenced cells reduced the chlamydial infectivity similar to wIRA/VIS treated control cells. Further studies are needed to elucidate the working mechanism of wIRA/VIS.

Methicillin-resistant Staphylococcus aureus (MRSA) and influenza A virus are two of the major targets for new antimicrobial technologies. In contrast to conventional germicidal lamps emitting primarily at 254 nm, which are both carcinogenic and cataractogenic, recent work has shown the potential of far-UVC technology, mainly between 207 and 222 nm, to be an effective means of sterilization of pathogens without apparent harm to mammalian cells. This is because, due to its strong absorbance in biological materials, far-UVC light cannot penetrate even the outer (non living) layers of human skin or eye; however, because bacteria and viruses are of micrometer or smaller dimensions, far-UVC can penetrate and inactivate them. With this report, we present progress on in vitro tests to inactivate MRSA on a surface using far-UVC light from a laser delivered using an optical diffuser. Qualitative and quantitative results show that this means of far-UVC exposure is adequate to inactivate MRSA with a dose comparable to that which would be required using a conventional germicidal lamp. Also included is a report on progress on inactivation of aerosolized influenza A virus. A custom benchtop aerosol exposure chamber was constructed and used to determine the effectiveness of far- UVC. Results indicate that far-UVC efficiently inactivates airborne aerosolized viruses, with a very low dose of 2 mJ/cm² of 222-nm light inactivating >95% of aerosolized H1N1 influenza virus. Together these studies help to further establish far-UVC technology as a promising, safe and inexpensive tool for sterilization in many environments.

Airborne transmission of infectious organisms is a considerable concern within the healthcare environment. A number of novel methods for ‘whole room’ decontamination, including antimicrobial 405 nm blue light, are being developed. To date, research has focused on its effects against surface-deposited contamination; however, it is important to also establish its efficacy against airborne bacteria. This study demonstrates evidence of the dose-response kinetics of airborne bacterial contamination when exposed to 405 nm light and compares bacterial susceptibility when exposed in three different media: air, liquid and surfaces. Bacterial aerosols of Staphylococcus epidermidis, generated using a 6-Jet Collison nebulizer, were introduced into an aerosol suspension chamber. Aerosolized bacteria were exposed to increasing doses of 405 nm light, and air samples were extracted from the chamber using a BioSampler liquid impinger, with viability analysed using pour-plate culture. Results have demonstrated successful aerosol inactivation, with a 99.1% reduction achieved with a 30 minute exposure to high irradiance (22 mWcm^-2) 405 nm light (P=0.001). Comparison to liquid and surface exposures proved bacteria to be 3-4 times more susceptible to 405 nm light inactivation when in aerosol form. Overall, results have provided fundamental evidence of the susceptibility of bacterial aerosols to antimicrobial 405 nm light treatment, which offers benefits in terms of increased safety for human exposure, and eradication of microbes regardless of antibiotic resistance. Such benefits provide advantages for a number of applications including ‘whole room’ environmental decontamination, in which reducing levels of airborne bacteria should reduce the number of infections arising from airborne contamination.

Osteoarthritis (OA) is the most common disease of the knee joints in adults throughout the world. Photobiomodulation (PBM) and physical exercise have been studied for clinical treatment of OA, even though the effects and action mechanisms have not yet been clarified. The aim of this study was to evaluate the effects of PBM and aerobic exercise (associated or not) on degenerative modifications and inflammatory mediators in articular cartilage using an experimental model of knee OA. Forty male Wistar rats were randomly divided into 4 groups: OA animals without treatment (OAC); OA plus aerobic exercise training (OAT); OA animals plus PBM treatment (OAP); OA plus aerobic exercise training and PBM treatment (OATP). The exercise training (treadmill; 16m/min; 50 min/day) and the PBM treatment started 4 weeks after the surgery, 3 days/week for 8 weeks. The results showed that all treated groups showed a lower degenerative process measured by OARSI system and higher thickness values. Moreover, aerobic exercise and PBM (associated or not) decreased iNOS expression and increased IL-10 expression in OAT and OATL compared to OAC. Furthermore, a lower TGF-β expression was observed in associated therapies. These results suggest that PBM and aerobic exercise training were effective in modulating inflammatory process and preventing cartilage degeneration in knees in OA rats.

Diabetes Mellitus is a chronic disease that can lead to lower-limb ulceration. The photodynamic therapy (PDT) is based on light interaction with a photosensitizer capable to promote bacterial death and tissue repair acceleration. This study analyzed the effects of PDT in the repair of human diabetic ulcers, by means of microbiological assessment. The clinical study was composed of 12 patients of both sexes with diabetic ulcers in lower limbs that were divided into two groups, control group (n=6) and PDT group (n=6). All patients were treated with collagenase/chloramphenicol during the experimental period, in which 6 of them have received PDT with methylene blue dye (0.01%) associated with laser therapy (660 nm), dose of 6 J/cm^2¨ and 30 mW laser power. PDT group received ten treatment sessions. Wounds were evaluated for micro-organisms analysis. It was found a reduction in the occurrence of Staphylococcus aureus in both groups, being that reduction more pronounced in the PDT group. Microbial count was performed on PDT group, showing a statistical difference reduction (p<0.05) when compared before and after the treatment. It is concluded that PDT seems to be effective in microbial reduction of human diabetic wounds, promoting acceleration and improvement of tissue repair quality.ty.