This PDF file contains the front matter associated with SPIE Proceedings Volume 10863, including the Title Page, Copyright information, Table of Contents, and Author and Conference Committee lists.

Infections caused by drug resistant bacteria poses a significant threat to global human health, with predicted annual mortality of 10 million by 2050. While much attention is focused on developing better therapies, improving diagnosis would allow for rapid initiation of optimal treatment, reducing unnecessary antibiotic use and enhancing therapeutic outcomes. There are currently no whole body imaging techniques in clinical use that are capable of specifically identifying bacterial infections. We have developed antibiotic-derived fluorescent probes that bind and illuminate either Gram-positive or Gram-negative bacteria with high specificity and selectivity over mammalian cells. Antibiotics are functionalised with an azide substituent in a position that minimises effects on antibiotic activity. These are reacted by facile 1,3-dipolar cycloaddition with alkyne-substituted imaging components such as visible or near-infrared fluorophores. The resulting adducts can be used as tools to image bacteria in vitro and in vivo. We have successfully functionalised representatives of seven major antibiotic classes. These derivatives retain antibacterial activity, and have been coupled with a range of fluorophores. Fluorescent versions of vancomycin and polymyxin B are particularly useful for specific labelling of G+ve and G-ve bacteria, respectively. Preliminary studies have now extended the visualisation component to include moieties compatible with PET imaging.

Infection of burns and traumatic wounds leads to delayed healing, chronic wounds, worse scarring, and increased health care costs. Infection continues to be a burden on patients and healthcare systems around the world, there is a great need for timely and appropriate intervention in these wounds. However, infection remains extremely challenging to identify due to subjective standard examinations for clinical symptoms and signs of infection and suboptimal, delayed microbiologic samples. Historically, wound care has suffered from a lack of imaging technology to assist with proper identification of infection. This manuscript reviews the MolecuLight i:X fluorescence imaging device, an innovative tool which can be used at the bedside to visualize endogenous fluorescence of tissue and bacteria, facilitating real-time detection of clinically significant bacterial loads. The MolecuLight i:X’s capability to visualise bacteria, not seen in a standard examination, leads to improved sampling and enables treatment and debridement specifically targeted to regions of bacterial load. In our experience, having this technology has reduced the burden on the patient and has guided clinicians to the best course of action. This can prevent antibiotic over-usage through the appropriate use of targeted therapies and can lead to health economic benefits by preventing unwarranted, costly therapies. The use of this simple device, with tremendous implications for the future of burn and trauma wound assessment and treatment, will be discussed. Begin the abstract two lines below author names and addresses. The abstract summarizes key findings in the paper.

Optical systems have shown their potential in non-invasive medical diagnostics over the last years. While most imaging systems use information on wavelength or phase, e.g. OCT, in our approach we focus on the polarization properties of biotissue. We designed a Mueller matrix (MM) measurement setup for in vivo investigations on skin tissue. The MM describes the polarization-changing properties of a sample.. Thus, it is possible to calculate the MM from images taken with different polarization states of the illuminating and the observed light. For medical application, an important requirement is that the process is fast to enable in vivo measurement, avoid motion artifacts, and reduce stress for patients. In our setup, we use a combination of two polarizers and four liquid crystal retarders to quickly change between polarization states. The system is able to measure the location dependent MM of a target for different wavelengths. It is designed for measurement in reflection mode, however, upon simple modifications, it can be used in transmission mode as well. One interesting field of application is diagnostics for inflammatory skin diseases. Here, for example, changes in the structure and concentration of collagen could provide diagnostically valuable information. We evaluated our system on different skin phantoms to investigate the diagnostic advantages compared to standard approaches. In the future, our system could be part of a non-contact dermatoscopic device and provide extra information for the physician.

In this paper, we show that plasmonic nanostructures, aka, metasurfaces, have robust performance in killing bacteria via the photothermal mechanism. Due to the highly enhanced local electric field, they can simultaneously provide impressive electric field enhancement for molecular sensing using various spectroscopic techniques.

Microbial community behavior is coupled to a set of genetically-regulated chemical signals that correlate with cell density – the quorum sensing (QS) system – and there is growing appreciation that the QS-regulated behavior of bacteria is chemically, spatially, and temporally complex. In addition, while it has been known for some time that different species use different QS networks, we are beginning to appreciate that different strains of the same bacterial species also differ in their QS networks. Here, we combine mass spectrometric imaging (MSI) and confocal Raman microscopy (CRM) approaches to investigate co-cultures involving different strains (FRD1 and PAO1C) of the same species (Pseudomonas aeruginosa) as well as those involving different species (P. aeruginosa and E. coli). Combining MSI and CRM makes it possible to supersede the limits imposed by individual imaging approaches and enables the spatial mapping of individual bacterial species and their microbial products within a mixed bacterial community growing in situ on surfaces. MSI is used to delineate the secretion of a specific rhamnolipid surfactant as well as alkyl quinolone (AQ) messengers between FRD1 and PAO1C strains of P. aeruginosa, showing that the spatial distribution and production of AQ messengers in PAO1C differs substantially from that of FRD1. In the case of multiple species, CRM is used to show that the prolific secretion of AQs by the PAO1C strain of P. aeruginosa is used to mediate its interaction with cocultured E. coli.

Ag+ ions are a well-known antibacterial agent, and Ag nanoparticles act as a reservoir of these Ag+ ions for targeted therapy of bacterial infections. However, there are no tools to effectively trigger and monitor the release of Ag+ ions from Ag nanoparticles. Photoacoustic (PA) imaging is an emerging noninvasive imaging tool, and gold nanorods (AuNRs) are an excellent contrast agent for PA imaging. In this work, we developed Au/Ag hybrid nanoparticles by coating AuNRs with silver (Ag), which decreased their photoacoustic signal. The as-prepared, Ag-coated Au nanorods (Au/AgNRs) are stable under ambient conditions, but the addition of ferricyanide solution (1 mM) results in oxidative etching of the silver shell. The PA contrast is simultaneously recovered as the silver is released, and this PA signal offers noninvasive monitoring of localized release of Ag+ ions. The released Ag+ ions exhibit a strong bactericidal efficacy similar to equivalent free Ag+ ions (AgNO3), and the nanoparticles killed >99.99% of both (Gram-positive) methicillin- resistant Staphylococcus aureus (MRSA, 32 μM Ag+ equivalent) and (Gram-negative) Escherichia coli (8 μM Ag+ equivalent). The theranostic potential of these nanoparticles was demonstrated in a pilot in vivo study. Mice were inoculated with MRSA and Au/AgNRs were subcutaneously implanted followed by silver etching. There was a 730% increase in the PA signal (p < 0.01) pre- and post-etching, and the bacterial counts in infected tissues of the treated group were reduced by 1000-fold (log CFU/g = 4.15 vs 7.75) versus the untreated control; this treatment efficacy was confirmed with histology. We further showed that these hybrid nanoparticles could release Ag+ after stimulation by reactive oxygen species including hydrogen peroxide and peroxynitrite. These hybrid Au/Ag nanoparticles are a useful theranostic agent for the photoacoustic imaging and treatment of bacterial infections.

Scleroderma (SD) is a rare autoimmune disease, which is divided into two categories: the localized SD and systemic SD. The localized SD mainly causes skin thickening of the fingers, whereas the systemic SD can further affect the blood vessels and internal organs. In this pilot study, the multispectral photoacoustic elastic tomography (PAET) imaging technique was used to recover quantitative physiological and elastic parameters of biological tissues for the diagnosis of SD. Three healthy subjects and three scleroderma patients were recruited and clinically examined by a rheumatologist, and then their hand /fingers were scanned by the both the commercial MRI and our home-made photoacoustic imaging system. Physiological parameters including oxygen saturation (S_TO₂), deoxy-hemoglobin (Hb) and oxy-hemoglobin (HbO₂) concentrations and mechanical properties such as bulk modulus images were reconstructed by using the developed PAET reconstruction method. Our imaging results demonstrated that the physiological and elastic parameters exhibit striking differences between the SD and healthy fingers, indicating that these indicators can serve as molecular signatures for the early detection of SD. These quantitative physiological properties and bulk modulus may also pave a new path for improved understanding the pathological mechanism of SD.

Diabetes is a chronic disease that affects millions of people every year worldwide. Patients with diabetes have high levels of glucose in their blood, since their bodies cannot produce or adequately use the insulin produced. Identification of diabetes is usually performed by tests of glucose in the blood by means of colorimetric reactions, which are time consuming and use a considerable amount of reagents. Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy has been used in clinical research as a potential tool to obtain spectrochemical information of biological materials. The infrared spectra can be used as source of information for classiciation models and biomarker extraction by using specific computational tools. In this paper, a semi-portable Bruker Alpha ATR-FTIR was employed to analyse urine samples of 7 patients (3 normal, 2 diabetics and 2 pre-diabetics) in order to distinguish these three groups based on their spectrochemical information. Cross-validated principal component analysis, coupled with linear discriminant analysis was applied to the spectral dataset, resulting in 94% total accuracy. Sensitivities were observed to be 95%, 96% and 100% for normal, pre-diabetics and diabetics patients, respectively, with specificities of 93%, 91% and 100%. These findings show the potential of ATR-FTIR as a new possible tool for identification of diabetics in clinical environments, whereby the diagnosis can be performed in a rapid, non-invasive and automated way.

Diabetic retinopathy is a degenerative disease that can lead to irreversible blindness in patients with long-term diabetes. The mechanism of tissue damage is through inflammatory response to high blood sugar levels degrade blood vessels throughout the body, and in the retina, this can lead to microbleeds and damage to photoreceptors. It is hypothesized that a change in vascular permeability could be an early indicator of an eventual progression to retinopathy, yet no clinical methods exist to date that are capable of measuring vascular permeability accurately. We have developed mathematical models that aim to quantify blood flow and vascular permeability in the retina using clinically collectable fluorescein videoangiography data. Recently, the method was demonstrated to be effective identifying early levels of retina damage in a rat model of diabetic retinopathy. Here we present a sensitivity analysis in a simulation study and the first results from a clinical study involving 4 diabetic patients and 3 healthy controls. While there were no significant differences in measured blood flow between the groups, the “extraction fraction” (a surrogate parameter of vascular permeability) was found to be significantly higher in diabetic patients than controls (0.082 ± 0.041 vs. 0.001 ± 0.001, p < 0.001). These results highlight the potential for kinetic modeling applied to fluorescein videoangiography to identify early signs of retinopathy in diabetic patients, such that therapy can be enacted at an earlier stage of the disease when the damage is not irreversible.

The initial biological response to spinal cord injury is initiated by intra- and extracellular chemical signals. We compare Raman spectra of injured spinal cord obtained minutes after injury to those of uninjured spinal cord to obtain chemical information that precedes the biological response. We studied 29 rats including both Injured and Control using Raman spectra of spinal cords in vivo. Principal Component Analysis (PCA) indicates that <99% of the variation of these spectra across both Injured and Control groups is accounted for with 3 components. The first component does not vary significantly representing structural materials. The second and third components reflect the variation in the chemistry of the cerebrospinal fluid. We demonstrate the first noninvasive in vivo measurement of pH in the CSF using only Raman spectra. We hypothesize that the earliest inflammatory response to mild contusive injury reflects the chemistry of inorganic phosphate present at abnormally high concentrations, likely due to physical disruption of the blood-brain barrier in the choroid plexus and/or mitochondrial release of phosphate, reacting with CSF water.

As I'm currently held by a confidentiality agreement regarding our latest data (towards patent and manuscript submission), I cannot submit a paper at this point. Please kindly refer to our most recently published work on the topic: Liesbeth Vanherp, Jennifer Poelmans, Amy Hillen, Kristof Govaerts, Sarah Belderbos, Tinne Buelens, Katrien Lagrou, Uwe Himmelreich, Greetje Vande Velde. Bronchoscopic fibered confocal fluorescence microscopy for longitudinal in vivo assessment of pulmonary fungal infections in free-breathing mice. Scientific Reports, 2018, 14; 8(1): 3009. Jennifer Poelmans, Uwe Himmelreich, Liesbeth Vanherp, Luca Zhai, Amy Hillen, Bryan Holvoet, Sarah Belderbos, Matthias Brock, Johan Maertens, Greetje Vande Velde*, Katrien Lagrou*. A multimodal imaging approach enables in vivo assessment of antifungal treatment in a mouse model of invasive pulmonary aspergillosis. Antimicrobial Agents and Chemotherapy [accepted, 2018] Vande Velde G, Kucharíková S, Van Dijck P, Himmelreich U. Bioluminescence imaging increases in vivo screening efficiency for antifungal activity against device-associated Candida albicans biofilms. Int J Antimicrob Agents. 2018 [Epub ahead of print]

There is a need for the development of diagnostic and analytical models in experimental infection models. We performed in vivo cell-tracking of S. aureus functionalized in vitro with a hybrid antimicrobial peptide tracer 99mTc-UBI29−41-Cy5, containing both a fluorescent and radioactive moiety. To create an invasive infection in mice, viable 99mTc-UBI29−41-Cy5 functionalized bacteria were inoculated in a thigh muscle. Thereafter, the mice were imaged using SPECT and fluorescence imaging modalities at various intervals for a 28h time period. In addition, biodistribution studies were performed at all intervals for quantitative analysis of the colonization and dissemination of the bacteria. SPECT and fluorescence imaging in mice revealed clear uptake of the tracer in the thigh muscle localization, decreasing over time from 52%ID/g at 4h to 44%ID/g (15% decrease) at 28h p.i. There was little uptake of the tracer in the urinary bladder only at 2-4h p.i.;. Since viable bacteria S. aureus were cultured in the urine samples obtained from the infected mice at all time-points it seems that this reduction is the result of bacterial dissemination. For the other tissues, no substantial accumulation of radioactivity or fluorescence was noticed. This non-GMO approach of imaging bacteria allowed us to accurately map the distribution of the labeled bacteria in a non-invasive manner. Given the versatility of the approach we are confident that this will pave the way for the development of diagnostic and analytical options in fundamental and translational research on experimental infection models.

The clinical use of photodynamic therapy (PDT) with rose bengal (RB) is emerging as an effective t reatment for a range of applications given its non-invasive and localised mode of delivery. In particular, rose bengal PDT has shown promising antifungal action in vitro. While focus has largely been on the physical and chemical impacts of PDT on the cell, an understanding of the role of genetics underpinning the cellular response is still limited. We have, therefore, reported a screen of the entire non-essential gene library of the model organism, Saccharomyces cerevisiae, using rose bengal PDT to ascertain the key genetic pathways affecting fungal tolerance to PDT. We also investigated the dosage of PDT required to eradicate Trichophyton rubrum spores, the main causative organism of onychomycosis infection. Following this, we conducted a pilot patient study of six patients (seven toenails) for the treatment of onychomycosis using rose bengal PDT (140 μM RB and ~763 J/cm² green light), where the clinical treatment protocol was developed on the basis of the in vitro outcomes. The key biochemical pathways identified by the genetic screen as having altered tolerance to PDT included ergosterol biosynthesis, vacuolar acidification, and purine/S-adenosyl-L-methionine biosynthesis. The subsequent pilot patient study saw the complete cure of onychomycosis for all patients within three to five treatment sessions in the absence of pain or other local side effects. The outcome of the genetic screen for tolerance may thus inform the development of efficient clinical treatments using rose bengal PDT.

Introduction: The prevalence and diversity of methicillin-resistant Staphylococcus aureus (MRSA) virulent factors as well as the host inflammatory response makes managing MRSA keratitis challenging. Alternative treatments are being investigated that can both neutralize the MRSA toxins while also reducing the host immune response. One such alternative is Photodynamic Antimicrobial Therapy (PDAT); however, limited research has been performed to understand the impact of PDAT on the host immune response during MRSA-related infections. Purpose: To measure the immune response (Interleukin(IL)-1, 6 and 8) of human corneal epithelial cells in an ex-vivo tissue model following MRSA infection and rose bengal PDAT. Methods: EpiCorneal tissue models (Maktek) were prepared according to manufacturer’s protocol. All groups were tested in duplicate: Control, Infection, Infection-PDAT. Infections were created in the tissue with three separate MRSA inocula (10E5 CFU/mL). For the Infection-PDAT group, 0.1% rose bengal solution was applied and wells were irradiated with a custom-made green LED light source. After 30 minutes, fluid from all of the tissue model wells was collected and IL-1, 6 and 8 were quantified with ELISA kits (Thermo Fisher Scientific). Results: The EpiCorneal Tissue model simulated the immune response of human corneal epithelial cells during MRSA infection and treatment with PDAT. Compared to the control, the immune response increased in all three MRSA infection groups. Of the cytokines tested, IL-8 showed the greatest response in the tissue models, followed by IL-6 and IL-1. In the Infection-PDAT groups, immune response was mixed depending on the MRSA strain. The largest downregulation of immune factors was observed in the community-associated MRSA strain.

Catheter–associated urinary tract infections (CAUTIs) cause millions of infections in the US every year, with direct medical costs exceeding billions of dollars and resulting in > 1 million ER visits and hospitalizations, resulting in thousands of deaths. CAUTI is thought to be a major reservoir both containing and creating highly drug-resistant infections, due to the chronicity of infection, biofilm formation, and the setting of institutionalization with chronic exposure to antibiotics, thus enabling resistance. To reduce antibiotic resistance developing in this setting, we are attempting to apply both antimicrobial photodynamic therapy (aPDT), antimicrobial blue light (aBL), and combinations of both with minimal use of antibiotics. To this end, we established a rat model of UTI. In this model, we catheterized female rats, infected them with a standard uropathogenic E.Coli (UPEC; UTI89), infused the bladder with methylene blue (MB) and potassium Iodide (KI), and illuminated the bladder once with a diffusing fiber connected to a 1W 660nm laser. Multilog killing was observed, but given the transient nature of ROS generation, regrowth of UPEC was seen 24 hours later. To this end, we are experimenting with the combination of illumination with antibiotics. When tetracyclines are illuminated by aBL, we have found > 6 log(10) steps of microbial killing in vitro, and significant drops in the antibiotic MIC effected by the combination of light and drug. Multiple treatments with aBL and aPDT both with and without limited intravesical application of antimicrobials may light the way to solving this problem.

The survival of pathogens on surfaces is a major contributor to infection transmission, and drives the development of selfsterilizing surfaces. Here, we have investigated if manganese-doped zinc sulfide quantum dots (Mn:ZnS QDs) can be used as photosensitizers for their potential application in surface disinfection via antimicrobial photodynamic inactivation (aPDI). A small library of Mn:ZnS QDs capped with 3-mercaptopropionic acid was synthesized using a hydrothermal approach in which both the amount of manganese (0-30 at.%) and heating period (9 – 20 h) were varied. The resultant Mn:ZnS QDs were shown by transmission electron microscopy to vary in size from 2.6-3.9 Å as a function of heating time, and exhibited a strong emission band at ~598 nm (λ_ex = 325 nm). Upon excitation of 5%-Mn:ZnS QDs at 514 nm, a near-IR emission band attributable to singlet oxygen phosphorescence was observed at 1278 nm, confirming that these QDs may function as photosensitizers via a Type II mechanism. The aPDI efficacy of the Mn:ZnS QDs was evaluated against both Gram-positive [methicillin-resistant S. aureus (MRSA; ATCC-44), vancomycin-resistant E. faecium (VRE; ATCC-2320)], and Gram-negative [multidrug-resistant A. baumannii (MDRAB; ATCC-1605), NDM-1 positive K. pneumoniae (KP; ATCC-2146)] bacteria. Our best results demonstrated detection limit photodynamic inactivation (6 log units reduction) of KP, MDRAB, and MRSA upon illumination (30 min; 65±5 mW/cm²; 400-700 nm), but only a ~1 log unit reduction against VRE. Together with antiviral studies of Zika virus that showed ~3 log units of inactivation, these findings demonstrate the utility of Mn:ZnS QDs as photosensitizers for aPDI.

The increased incidence of antibiotic-resistant gram-positive bacteria, like methicillin-resistant S. aureus (MRSA), necessitates treatments that eliminate the potential of developing further resistance. Antimicrobial photodynamic therapy (aPDT) has shown promise as gram-positive infections can be specifically photosensitized by inducing the accumulation of coproporphyrin III (CPIII) through the administration of VU0038882 (‘882), a small-molecule activator of coproporphyrinogen oxidase, and delta-aminolevulinic acid hydrochloride (ALA). While the phototoxic effects of CPIII are most pronounced when stimulated with 395nm light, corresponding to its Soret absorption-band, the high absorption of the skin at that wavelength reduces the efficacy in vivo by three orders of magnitude as compared to in vitro. Although the issue of light penetrance can be mitigated by using red-shifted wavelengths targeting the Q-bands of CPIII (λpeak=498/530/565/619nm), the efficiency of cytotoxic reactive oxygen species (ROS) production and bacterial killing drastically reduces. Though this inefficiency can be partially overcome through an increased light dose, photoinactivation of CPIII and oxygen depletion limits this process to a maximum effective light dose. To overcome these limitations and improve the overall efficacy of CPIII-targeted aPDT, we designed and built a novel multi-LED light source and explored the effect of simultaneously targeting the Soret-band and Q-bands. We present that lower radiant exposures of blue light in conjunction with a higher exposure of green or red light increases the amount of bacterial killing by 1 to 3 logs in vitro as compared to either treatment alone. This enhancement is expected to increase when utilized in vivo due to differences in penetrance.

We previously showed that antimicrobial photodynamic inactivation (aPDI) of Gram-positive and Gram-negative bacteria mediated by the phenothiazinium dye, methylene blue (MB) was potentiated by addition of potassium thiocyanate (10mM). The mechanism was suggested to involve a singlet oxygen mediated reaction with SCN- to form sulfite and cyanide and then to produce sulfur trioxide radical anion. We now report that potassium selenocyanate (KSeCN) (concentrations up to 100 mM) can also potentiate (up to 6 logs of killing) aPDI mediated by a number of different photosensitizers: MB, Rose Bengal, and TPPS4 (as low as 200 nM). When a mixture of selenocyanate with these PS in solution was illuminated and then bacteria were added after the light, there was up to 6 logs killing (Gram-negative > Gram-positive) but the antibacterial species decayed rapidly (by 20 min). Our hypothesis to explain this antibacterial activity is the formation of selenocyanogen (SeCN)2 by reaction with singlet oxygen (1O2) as shown by quenching of 1O2 by SeCN- and increased photoconsumption of oxygen. The fact that lead tetra-acetate reacted with SeCN- (literature preparation of (SeCN)2) also produced a short-lived antibacterial species supports this hypothesis.

Antimicrobial resistance in Neisseria gonorrhoeae is a major issue of public health, and there is a critical need for the development of new anti-gonococcal strategies. In this study, we investigated the effectiveness of antimicrobial blue light (aBL; 405 nm wavelength), an innovative non-pharmacological approach, for the inactivation of N. gonorrhoeae. Our findings indicated that aBL preferentially inactivated N. gonorrhoeae, including antibiotic-resistant strains, over human vaginal epithelial cells in vitro. Furthermore, no genotoxicity of aBL to the vaginal epithelial cells was observed at the radiant exposure for inactivating N. gonorrhoeae. aBL also effectively inactivated N. gonorrhoeae that had attached to and invaded into the vaginal epithelial cells in their co-cultures. No gonococcal resistance to aBL developed after 15 successive cycles of sub-therapeutic aBL inactivation. Taken together, aBL represents a potent potential treatment for antibiotic-resistant gonococcal infection.

In recent studies, we showed that pulsed blue light is more potent in suppressing bacterial growth than continuous wave blue light. The potency of pulsed blue light makes it a viable antimicrobial for suppressing bacteria growth in biofilms, where the protective cover of the biofilm makes it is tougher to suppress bacteria. Consequently, we studied the efficacy of pulsed 450 nm light in suppressing the growth of MRSA and P. acnes biofilms. The results showed 100% bacterial suppression in planktonic cultures of MRSA irradiated with 7.6 J/cm² three times a day, using 3 mW/cm² irradiance, and in P. acnes planktonic cultures irradiated with 5 J/cm² thrice daily for 3 days, using 2 mW/cm² irradiance. However, a similar 100% suppression was not attained in MRSA or P. acnes biofilms irradiated thrice daily for 3 days at various fluences; but LIVE/DEAD assay showed a degree of bacterial suppression, with more live cells in controls than irradiated biofilms, and more dead cells in irradiated than control biofilms. In addition, while control biofilms had intact biofilm networks, irradiated biofilms had disrupted biofilm. The higher the dose, the more bacterial suppression and biofilm disruption. These findings confirm our previous reports that 100% bacterial suppression is attainable with pulsed blue light, and suggests further modification of the treatment protocol in order to achieve 100% bacterial suppression in biofilms.

The prevalence of antibiotic resistance and the presence of bacterial persisters increasingly challenge the successful treatment of Staphylococcus aureus infections, and thus poses a great threat to the global health. Here, we present a photonic approach to revive a broad spectrum of antibiotics for eradication of MRSA persisters via photo-disassembly of functional membrane microdomains. Membrane microdomains on MRSA cells are enriched in staphyloxanthin-derived lipids as constituent lipids with co-localized and oligomerized multimeric protein complexes including PBP2a to execute various cellular processes and cell virulence. We demonstrated that the membrane-bound staphyloxinthin is prone to photobleaching by blue light due to triplet-triplet annihilation and thus compromises the membrane integrity. Using high-intensity 460 nm pulsed laser (wide-field illumination, dosage far below human safety limit), we achieved strikingly high staphyloxanthin bleaching efficiency and depth when compared to low-level light sources (quantified by resonance Raman spectroscopy). More importantly, such efficient and selective photolysis of constituent lipids leads to catastrophic disassembly of membrane microdomains, yielding highly compromised cell membrane with nanometer-scale pores created and PBP2a unanchored from cell membrane or dispersed (proved and quantified by immunofluorescence, fluorescence assay, confocal, super-resolution imaging, and Western blotting). The disruption renders MRSA persisters highly traumatized, thus no longer in dormant state (verified by stimulated Raman scattering microscopy). Consequently, cells with compromised membrane are found highly susceptible to a broad spectrum of antibiotics: beta-lactam antibiotics, such as penicillin and cephalosporins, due to PBP2a disassembly; antibiotics that inhibit intracellular activities enabled by effective diffusion via nanometer-scale pores, such as quinolones, aminoglycosides and sulfonamides. These synergistic therapies are validated both in vitro and in clinically relative models including biofilm and mice skin infection model. Collective, our findings unveil the underlying mechanism of photo-disassembly of MRSA membrane microdomains and highlight this photonic approach as a novel platform to revive a broad spectrum of conventional antibiotics and guide the development of new antibiotics for treatment of MRSA infections.

Research on photobiomodulation (PBM) has led to the development of various light-generating devices that can benefit a wide range of clinical indications. A novel approach of inducing PBM is through application of a Fluorescence Biomodulation (FB) System consisting of a blue light (peak wavelength between 440 and 460 nm) which activates topical photoconverter substrates containing specialized chromophores that generate fluorescence light energy (FLE). In clinical trials, FLE has been shown to modulate both healthy and disease-affected skin/soft tissue, providing a unique method for managing inflammatory skin conditions and accelerating healing. To better understand the biological impact of FB-induced FLE, we studied this system in vitro on dermal human fibroblasts (DHFs) and in vivo in canine deep pyoderma. In vitro data from stimulated DHFs exposed to an FB System showed a significant decrease in IL-6 production by 130.14% after 24 hr (p<0.001), compared to control groups. In canines with chronic deep pyoderma, the use of FB plus standard of care (SOC) treatment significantly reduced time to clinical resolution compared to controls that received SOC alone (p<0.001). Biopsies from lesional areas showed enhanced mitochondrial biogenesis in the FB lesions versus the SOC lesions, as supported by a significant increase in the number and size of mitochondria (89.31% and 90.15% respectively; p<0.0001). Significant modulation of inflammatory pathways, epithelialization, and angiogenesis were also demonstrated. These results support the use of FB Systems for skin conditions impacted by inflammation and offer a promising therapeutic solution to support its use in other medical conditions.

Dental biofilms are produced by bacterial communities. During the first 24 h of colonization, oral streptococci compose 60% to 90% of the supragingival plaque biomass. Mutans streptococci are biofilm-forming bacteria and are considered to be the primary etiologic agents of human dental caries. They possess a variety of abilities to colonize tooth surfaces and under certain conditions are present in large quantities in cariogenic biofilms and also form biofilms with other organisms, including other streptococci and bacteria. To reduce microbial, like biofilm, can be performed by using photodynamic therapy. Successful of this kind of therapy is induced by penetration of light and photosensitizer into target cells. The sonodynamic therapy offers greater penetrating capability into tissues. Then, the purpose of this study was to evaluate the antibacterial effect of photodynamic inactivation (PDI) associated with ultrasound (U) using curcumin solution irradiated by LED light source over Streptococcus mutans biofilm. Initially, minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were performed, The concentrations of 40 and 80 μM were selected for the next experiments. Streptococcus mutans biofilm were induced using a 96-well plates for 7 days according to Groups G1 (negative control, L0D0), G2 (L + U +), G3 (L + D0), G4 (L0D40-80), G5 (chlorhexidine, positive control), G6 (L + D40-80 U +), G7 (L + D40-80) and G8 (D40-80 U +). For the dark cytotoxicity, curcumin was incubated for 5 minutes. For PDI, the groups were incubated in the dark for 5minutes (pre-irradiation time) and irradiated by blue LED at 15J/cm2 (36mW/cm2) for 7minutes and 55seconds. Ultrasound was used for 5min under 0.16KW of power output and 1.5A (pre-irradiation). After treatment, the strains were seeded on BHI agar and incubated at 37°C for 48 hours to determine the number of CFU/mL. The results were transformed into log10 and submitted to analysis of variance (ANOVA) and Tukey test at the 5% level. Significant reductions in the number of viable cells of S. mutans were observed in groups G6 (6log10) providing 4log10 of bacterial reduction when compared to group G1 (2log10) (p <0.05). The association of PDI and ultrasound can be an effective method to control microorganisms in the oral cavity, especially S. mutans, which causes dental caries.

With the effectiveness of antimicrobials waning because of antimicrobial resistance, it is imperative that novel strategies are investigated for the treatment of infections. Antimicrobial blue light (aBL) is an innovative strategy that has proven efficacy against an array of pathogens, albeit, with different species having variable susceptibilities to the therapy. Quinine was discovered during the mid-17^th century as a plant-derived potent antimalarial. More recently, its bactericidal properties were revealed, illustrating its potential as an antimicrobial adjuvant. Here we report a novel combination therapy, aBL+quinine hydrochloride (Q-HCL) for the treatment of multi-drug resistant infections. QHCL successfully potentiated the antimicrobial effects of aBL in numerous microbial pathogens of different etiologies, in vitro and in vivo. In addition, it synergistically improved the antimicrobial effects of aBL against bacterial biofilms. Raman spectroscopy revealed that concurrent exposure of aBL and Q-HCL improved uptake of Q-HCL into bacterial cells, when compared to the non aBL exposed sample. In addition, ultra-pure liquid chromatography (UPLC) revealed that Q-HCL increased the relative abundance of porphyrins in bacteria, suggesting the mechanism of this synergistic interaction is through increased production of intermediate photosensitizing porphyrins arising through perturbation of the heme biosynthesis pathway by Q-HCL. Genotoxic potential of the combination therapy against mouse skin tissue, was evaluated using the TUNEL assay, where it was revealed that a high dose exposure of aBL+Q-HCL (<3x the therapeutic dose) was not genotoxic to mouse skin tissue. In conclusion, the findings strongly suggest the potential of aBL+Q-HCL combination therapy as an alternative to traditional antibiotics for the treatment of localized infections.

Background and Objectives: Antimicrobial resistance is a rapidly evolving and emerging threat to modern medicine. Non-antibiotic approaches can be alternatives to minimize the development of antimicrobial resistance. The present study aims at investigation of synergistic effects of antimicrobial blue light (aBL) at 405 nm with oregano oil on bacterial inactivation, both of which are non-antibiotic approaches and possess different and multiple targets.

Bacteria preferentially grow as colonies surrounded by a complex matrix, together called a biofilm. Biofilms are highly resistant to antibiotics and the immune system, making them difficult to treat at best, and nearly impossible to eradicate at worst. This presentation will describe a method which combines low energy, non-focused ultrasound with blue/violet light. Staphylococcus epidermidis, S. aureus, E. coli, and P. acnes biofilms were grown on hanging inserts (i.e. transwells) at 37°C for up to 72 hours before exposure. Following exposure to simultaneous ultrasound (at less than 100mW/cm2) and light energy (405nm), bacteria killing was quantified by serial dilution and plating on media agar. Killing was dose dependent with exposure time, and could be optimized with tuning of either the light or ultrasound energy. There was >1 log10 reduction after 5 min and >3 log10 reduction after 30 min treatment (p<0.05). Importantly, the two energies had to be delivered coincidentally for optimal effect. These initial results validated the basic mechanism: low intensity ultrasound “activates” the bacteria within the biofilm such that they become susceptible to the antimicrobial effects of blue light. A clinical system was then developed, safety tested, and submitted for IRB approval. Several clinical trials have been conducted which demonstrated not only a significant (~90%) reduction in facial P. acnes bacteria, but also a significant improvement in the appearance of acne vulgaris in test subjects. Bacteria reduction was dose dependent and statistically significant, and was not influenced by subject skin type. No adverse events were recorded.

Biofilms are collections of microorganisms that attach to a surface and are covered with extracellular matrices produced by these microorganisms from the environment. A biofilm is an ideal place for plasmid exchange, where plasmids can carry genes that regulate resistance to antibiotics so that biofilms play a role in the spread of bacterial resistance to antibiotics. This allegedly due to changes and rearrangement of cell walls so that antibiotics do not easily penetrate it. An alternative method for reducing biofilm S.aureus is photodynamic inactivation. PDI is a method of inactivation of microorganisms that utilize light, photosensitizers, and Reactive Oxygen Species. This present work aims to determine the potential of blue diode laser as an activator of nano doxycycline to reduce Staphylococcus aureus biofilm. The treatment was divided into six groups, the control group without any treatment, the control group with doxycycline photosensitizer, the control group with nano doxycycline photosensitizer, laser treatment group, laser, and doxycycline treatment group, laser, and nano doxycycline treatment groups. The laser treatment group has a variation of exposure time 30s (4.37 J / cm²), 60s (8.73 J / cm²), 90s (13.09 J / cm²), 120s (17.47 J / cm²), and 150s ( 21.83 J / cm²). Biofilm reduction was measured using an ELISA reader and analyzed using factorial ANOVA. The results showed that 403 nm blue diode laser exposure for 150s with energy density 21.83 J / cm² could reduce biofilms up to (31.74 ± 1.67)% for the laser treatment group, (65.01 ± 1.67)% for laser and doxycycline treatment groups, (80.25 ± 1.67)% for the laser treatment group and nano doxycycline. So, the exposure of blue diode laser has the potential to activate nano doxycycline to increase the percentage of Staphylococcus aureus biofilm death.

The World Health Organization (WHO) published a catalogue of 12 families of antibiotic-resistant bacteria which pose an alarming threat to human health in 2017. These bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (P. aeruginosa), could cause a wide range of infections from minor subcutaneous infection to toxic shock syndrome, and bacteremia. As the body’s second line of host defense, phagocytosis could eliminate the majority of the invasive bacteria. However, the survival of microbial pathogens within the macrophage cells which act as ‘Trojan horses’ largely provides a reservoir relatively related protected from antibiotics, thus causing recurrent infections from the dissemination of intracellular bacteria. Moreover, the pace of antibiotic development can’t keep with the resistance acquisition from bacteria. Therefore, there is an unmet need for alternative approaches to eradicate multi-drug resistant intracellular bacteria. Here, we develop an effective photonic approach to assist macrophage cell (RAW 264.7) to efficiently eradicate intracellular MRSA, P. aeruginosa along with Salmonella enterica. This approach selectively targets intracellular bacteria without damaging macrophage cells through photoinactivation of a microbial detoxifying enzyme existing in most of the bacteria. Moreover, we utilize advanced nonlinear optical imaging methods to record the in situ photoinactivation process and to visualize the real-time phagocytosis difference with or without photoinactivation of this enzyme. Our findings and approach reported here could provide an effective method to eliminate multi-drug resistant intracellular bacteria, and also treat the clinical bacterial infection in the future.

Clostridium difficile spore transmission from contaminated surfaces is a continuing problem for health care facilities. In this study high-intensity UV LED was used to rapidly inactivate Clostridium difficile spores. Wavelengths were specifically chosen to target protein stability through disulfide functional groups rather than nucleic acids. Clostridium difficile spores, at 3.09 × 10⁶ CFU/sq.in. carrier, were illuminated by hi-intensity UV LED. Irradiance is specified for the target distance, 25 mm. Spores were exposed to either 275nm alone or to a 275nm+365nm combination for 30, 88, or 147 seconds. For 275nm alone, spores were exposed to 491.5 mW/cm², 245.7 mW/cm², or 122.9 mW/cm². Irradiance varied for 365nm (720.3 mW/cm², 360.2 mW/cm², or 180.1 mW/cm²) while 275 irradiance was constant (245.7mW/cm²) for the 275nm+365nm combination. High-intensity 275 nm UV LED was consistently effective at doses above 36.1 J/cm², the highest irradiance (491.5 mW/cm²) achieved maximum log reduction at 14.7 J/cm². Addition of high-intensity 365 nm UV LED resulted in maximum log reduction for all 365 nm intensities and doses tested. Maximum reduction of 5.79 log was achieved. Simultaneous use of two specific UV wavelengths reduced time required for inactivation. This is consistent with our previous findings on protein inactivation using RNase A¹, suggesting that targeting protein is viable for inactivation of bacterial spores, even in the absence of wavelengths specifically targeting nucleic acids.

Recently, efforts have been made to create a transparent ceramic cranial implant comprised of nanocrystalline yttriastabilized zirconia (nc-YSZ) that will provide optical access to the brain. This has been referred to as Window to the Brain (WttB) in the literature. WttB will allow the use of laser and photonic treatments and diagnostics in areas with difficult optical access in the brain. For example, the WttB platform would allow for non-invasive antibacterial treatments based on laser light. This is important as conventional antibiotics are prevented to reach the brain tissue due to the blood brain barrier. Moreover, infection is still one of the frequent cranial implant complications. In most cases a second surgery is required to replace the infectious implant. To address potential infections in the WttB platform, we have studied the antibacterial effect of commercial Zinc Oxide (ZnO) nanoparticles and femtosecond laser light on bacterial solutions of Staphylococcus aureus (S. aureus). Infrared (1030nm) laser light was used to enhanced the antibacterial effect of ZnO nanoparticles by irradiating bacterial solutions of S. aureus with low power ultrashort laser pulses (890mW, 230fs).

With the increasing number of pathogenic microbes that are becoming resistant to routinely used antimicrobials, it is important to look to non-traditional approaches for the treatment of infections. Antimicrobial blue light (aBL;405 nm) is a novel strategy for the treatment of infections. Here we report an investigation into the potential for resistance development to aBL in three clinically important Gram-negative bacteria, through sequential exposure in vitro and in vivo. We found that 20 cycles of aBL exposure, in vitro, did not incur resistance development, in any of the species tested (Acinetobacter baumanii, Pseudomonas aeruginosaor Escherichia coli). In addition, sub-curative sequential aBL treatment of a wound infected with a bioluminescent variant of the P. aeruginosa PAO1 strain, did not influence sensitivity to aBL. In conclusion, it is unlikely that sequential treatment of aBL will result in resistance generation, suggesting that multiple treatments of aBL may be administered without resistance development becoming a concern.

It has been found that the oral cavity and tongue are the second abundant habitats of bacteria in the body. The oral microbiome is essential to maintain oral health, while it also has the potential to cause disease due to having pathogens simultaneously. In this study, seven oral bacterial strains which are known to be associated with an oral disease were selected to evaluate the antibiotic susceptibility to four different photosensitizers (PSs). Stock solution (1 mg/mℓ) of four PSs was prepared by dissolving the powder in dimethyl sulfoxide for curcumin and protoporphyrin IX, and distilled water for resazurin and riboflavin. The inoculation of 20 μℓ of a 24 h cultured bacterial suspension mixed with 1,980 μℓ each test solution and then a light source was placed in front of the mixed solution. The LED excitation wavelength was 405 nm and its energy was 84.5 mW/cm² for 300 s. Bacterial viability was determined by measuring the absorbance at 655 nm with a microplate reader. It was found that all PSs affect the viability of some oral bacteria by irradiated with light. The antibacterial susceptibility on the different PSs and visible blue light irradiation were different depending on each bacterial strain. Moreover, the same bacterial strain showed different effects depending on the type of the PSs. From the above results, antibacterial photodynamic therapy composed of light source and photosensitizers can be used to control the oral bacteria strains which are related to oral disease.