This PDF file contains the front matter associated with SPIE Proceedings Volume 12822, including the Title Page, Copyright information, Table of Contents, and Conference Committee information.

Antibiotic is one of the most important medical inventions in the 20th century ¹. However, bacterial resistance to antibiotics is becoming a global health-care problem ². One of the important measures to tackle this problem is fast detection bacterial antibiotic susceptibility ¹. In this research topic and inspired by the work report of Soares et. al. ^3,4 we were motivated to developed this study to identification of resistance to antibiotic in Staphylococcus aureus. By mean of machine learning implementation in data analyses of Fourier-Transform Infrared Spectroscopy (FTIR) spectra, we found promisor results in samples with and without antibiotic resistance develop.

In this study, we present a fluorescence imaging cytometer capable of detecting multiple biomarkers using temporally multiplexed illumination. We independently illuminated the classification and quantitative detection channels using multiwavelength LEDs. To quantify the biomarker concentrations, we employed a sandwich immunoassay with Cy5 dye, while the microbeads were internally dyed with three different intensity levels of quantum dots to distinguish among the three cytokines. We conducted separate tests with three cytokines, namely IFN-α, IL-5, and IL-6, at varying concentrations. The detection ranges for these cytokines were determined to be 10 pg/ml-2500 pg/ml, 30 pg/ml-2500 pg/ml, and 30 pg/ml-2500 pg/ml, respectively. The results of the multiplexed experiment, show that our device can independently detect three different biomarkers in a single assay. This validation demonstrates that our device has potential in diagnosing sepsis and other diseases requiring various laboratory results. With further refinement and development, our device holds great promise for enhancing point-of-care medical services.

In intensive care units (ICU), vital signs and biomarkers of critically ill patients provide a set of operational parameters for doctors to assess the severity of organ dysfunction and optimize the supporting treatment. Among those organs monitored, the gut is less accessible, and its latent risk is not manageable. There is an emerging need for sensitive and easily measured biomarkers of early intestinal injury. Here, we found plasma fluorochromics can be used to assess the severity of intestinal injury using label-free methods of quantification. In acute mesenteric ischemia-reperusion animal models, ischemia-reperfusion damage can lead to multiple times increase of NADH, flavins, and porphyrin auto-fluorescence in blood. The intensity ratio between NADH and flavin fluorescence can capture early signatures before the occurrence of shock. Using liquid chromatography and mass spectroscopy, we confirmed that riboflavin is primarily responsible for the increased flavin fluorescence. Since endogenous riboflavin in humans is absorbed from the intestine, its increase in plasma validates its association with intestinal injury. In the future, blood fluorochrome detection could serve as a time-course monitoring modality in the emergency department or ICU to assess intestinal damage in various acute illnesses and critical care conditions.

Methicillin-resistant S. aureus (MRSA) bacteria commonly found on orthopaedic implants, form treatment resistant biofilms that are difficult to manage. Creating new imaging modalities that allow us to understand biofilm development and accurately indicate the efficacy of treatments will greatly aid research in biofilm infection treatment methods. In this in vitro study, we determined the correlation between the number of MRSA CFUs and the radiance of MRSA aliquots with bioluminescent plasmids in the resolution volume of the Perkin Elmer’s IVIS Spectrum imaging system at specific imaging depths. We standardized MRSA bioluminescence curves for planktonic and biofilm-associated MRSA grown on titanium and stainless-steel orthopaedic hardware. The ability to relate measured radiance to the biofilm bioburden on a metal surface provides a critical tool for our ongoing pre-clinical studies identifying and treating biofilm-forming infections in contaminated high-energy fracture (rats) and contaminated osseointegration after amputation (rabbits).

Orthopaedic implant-associated infections cause serious complications primarily attributed to bacterial biofilm formation and often characterized by increased antibiotic resistance and diminished treatment response. There is currently a lack of imaging modalities that can directly visualize biofilms to determine the location and extent of contamination. Optical coherence tomography (OCT) is a portable, non-invasive, high-resolution imaging modality with the potential to fulfill this unmet need. In this study, we aim to evaluate the efficacy of OCT in detecting biofilms formed by life- and limb-threatening bacteria on orthopaedic implants. Bioluminescent strain SAP231 of methicillin-resistant S. aureus (MRSA) was used to grow biofilms on the surfaces of titanium and stainless-steel orthopaedic hardware situated inside custom-designed macrofluidic devices, allowing continuous nutrient broth supply and waste removal. Three-dimensional OCT images of each piece of hardware were obtained every 24 hours with subsequent bioluminescence imaging using the PerkinElmer IVIS Spectrum. OCT texture analysis based on multi-parametric fitting approach was developed and validated against IVIS quantification for accurate identification of live MRSA signatures. The monitoring of biofilm formation and measurement of film thicknesses starting at 12 micrometers and reaching 180 micrometers in 72 hours on metal hardware is demonstrated. This proof-of-concept study highlights the ability of OCT to detect and quantify the formation of MRSA bacterial biofilms in a high fidelity orthopaedic implant biofilm model in vitro, opening avenues for translation of this technique to preclinical models of contaminated orthopaedic trauma surgery and further clinical translation.

The Translational Healthcare Technologies (THT) group has evolved over the last decade to develop a translational infrastructure that is challenge-led with people focused values and embedded patient engagement. The THT champions team science and is developing and translating into patient cohorts, cutting edge tools to delineate pathophysiology with a focus on pulmonary and ocular applications. This paper describes the infrastructure within Edinburgh BioQuarter and highlights some studies where we have developed chemical compounds to characterise lung pathologies such as pneumonia, acute respiratory distress syndrome (ARDS), pulmonary fibrosis and lung cancer. We describe the bench-to-bedside assessment of these compounds for gram-negative bacterial detection, neutrophil activation, matrix metalloproteinase (in fibrosis) detection and pre-clinical targets including fibroblast activation protein (cancer detection), T cell activity and bacterial detection. We also describe the development of two next generation fibre-based imaging technologies: a LED based tri-colour imaging system and a dual channel fluorescence lifetime imaging system. Using the THT infrastructure, these systems and chemical probes are now entering clinical trials in an academic institute.

Well-organized ecosystems of bacteria colonize orthopaedic devices causing biofilm infections that are notoriously difficult to manage. Biofilms typically exhibit increased resistance to antibiotics leading to treatment failure, and tools for eradicating biofilms that do not increase antibiotic resistance are greatly needed. Antimicrobial photodynamic therapy (aPDT) is a promising form of treatment to combat clinically relevant biofilms. Exogenous provision of 5-aminolevulinic acid (5-ALA) to biofilm-forming clinical strains of E. coli, E. faecalis and S. aureus was recently shown by several research groups to result in the accumulation of sufficient quantities of endogenous photosensitizers porphyrins (protoporphyrin IX, coproporphyrin III and others), via the heme biosynthetic pathway, to produce a significant phototoxic effect when exposed to activating light. For clinical translation of this extremely promising approach, here we develop a portable light source for 5-ALA-based aPDT of orthopaedic implant biofilms, spectrally shaped for optimal porphyrin light absorption at wavelengths range approved by FDA for clinical use. After phantom calibration, we tested it on E.coli-E.faecalis biofilms grown in soft lithography-fabricated microfluidic chips and on methicillin-resistant S. aureus (MRSA) biofilms grown on titanium and stainless steel orthopaedic hardware in custom-designed macrofluidic devices. Successful in-vitro experiments allowed us to conduct a proof-of-concept validation study in a preclinical rat model of MRSA-contaminated open fracture. Following tibia fracture and two hours of wound infection development, a one hour incubation with 20% 5-ALA and treatment with either 90J/cm² or three fractions of 30J/cm² light doses demonstrated 94% and 99% overall reduction of MRSA, respectively, while the temperature of the tissue remained <39°C, below the threshold for thermal damage. The encouraging results suggest further preclinical testing of the developed light source for optimization of aPDT regimen and 5-ALA concentration to reduce the risk of long-term side effects in animal models of contaminated trauma surgery.

Bacterial antibiotic resistance poses a pressing global health crisis, challenging conventional therapies. Efflux pumps diminish antimicrobial effectiveness by expelling drugs from bacterial cells. Multidrug efflux pumps (MEPs) have been found to transport diverse compounds, including phenothiazinium dyes like methylene blue, out of bacteria. Inhibition of MEPs offers a promising strategy to bolster the efficacy of antimicrobial photodynamic inactivation (PDT). This research adopts a synergistic approach, combining the efflux pump inhibitor (EPI) , reserpine, with silver nanoparticles (Ag NPs) and methylene blue (MB) to enhance PDT efficiency. Ag NPs were synthesized via pulsed laser ablation and characterized using TEM, UV-Vis, and PL spectra. E. coli was treated with MB, Ag NPs, and reserpine, followed by LED light irradiation. MB was twice as effective, and AgNPs/MB was six times more effective with reserpine during a sixminute irradiation. Ongoing experiments on morphological changes will be presented. AgNPs/MB with reserpine could effectively combat bacterial pathogens in open wounds and prosthetic joint infections.

The rise of antimicrobial-resistant microorganisms prompts the exploration of strategies to rejuvenate traditional antibiotics. Photodynamic Inactivation (PDI) offers an alternative by combining a photosensitizer (PS) with light, generating reactive oxygen species that efficiently eliminate undesirable cells. In this study, curcumin and 450nm light are applied, resulting in reduced minimum inhibitory concentrations (MIC) for antibiotics. The application of PDI proves effective in diminishing the necessary antibiotic dosage for bacterial cell elimination, especially against resistant strains. Additionally, the interaction between the photosensitizer and antibiotics induces notable alterations in bacterial metabolism and biomolecules. These findings are crucial for advancing from in vitro studies to the clinical implementation of PDI as a promising adjunct to antibiotic therapy, offering potential solutions in the battle against antimicrobial resistance.

Due to the progression of antimicrobial resistance, Photodynamic (PDI) and Sonodynamic (SDI) Inactivation therapies arose as promising approaches for microbial control. Recently, the combination of both therapies, called Sonophotodynamic Inactivation (SPDI), have shown greater effects than the single treatments. This study evaluated the effectiveness of PDI, SDI and SPDI mediated by curcumin (Cur) against Staphylococcus aureus biofilm and the structural impact of these treatments on the biofilm community. For, this S. aureus biofilms received PDI, SDI and SPDI, mediated by Cur (80μM), LED light (450nm), and 1MHz ultrasound (20% of duty cycle, 1.5W/cm² of intensity). The effectiveness of the treatments was measured by cell viability assay (quantification of colonies - CFU/mL), metabolic activity (XTT assay) and total biomass of the biofilms was quantified by crystal violet. Additionally, the biofilm architecture after treatments was evaluated under confocal fluorescence microscopy. SPDI was more effective than PDI and SDI. The SDI, PDI and SPDI groups demonstrated reductions of 1±1, 1±1, and 3±1 log, respectively, compared to the control group. The PDI group exhibited a metabolic activity 89±1% lower than the control, while SDI and SPDI showed 82±2% and 90±1%, respectively. All treatments reduced the total biomass of the biofilms. The PDI samples exhibited a 43±9% reduction in total biomass, the SDI group showed a 25±11% reduction, and the SPDI group demonstrated a 49±11% reduction in comparison with the control group. Finally, all treatments impacted the biofilm components and structure, reducing the cells and matrix. In conclusion, SPDI was more effective in the inactivation and had greater impact on the S. aureus biofilm.

Iodine is a crucial trace element that occurs in minute amounts in nature and is necessary for the development of bones, thyroid function, and several metabolic processes. Iodine deficiency, also known as hypothyroidism, affects millions of individuals worldwide, and an overabundance of iodine in the body is known as hyperthyroidism. The early identification of iodine with high sensitivity and selectivity is crucial for physiological impact since the abnormalities caused by iodine disorder can increase the frequency of mortality and mental impairments. This work aims to detect iodine using UV-Vis and Transmission spectroscopy and utilizing selenium nanoparticles as a probe. Selenium nanoparticles (SeNPs) were synthesized by ND: YAG laser method and characterized by Dynamic light scattering (DLS), and High-resolution transmission electron microscopy (HRTEM), while the conjugation of iodine to SeNPs was confirmed by Ultravioletvisible (UV-vis) spectroscopy. For iodine detection, UV-Vis and Transmission spectroscopy were used and compared and the synthesized colloidal and spherical selenium nanoparticles were utilized as a probe to detect iodine. The absorption peaks and a red shift for SeNPs changed upon the reaction with iodine and this shift may allow for the estimation of iodine concentration. The two methods will enable the detection and monitoring of iodine at different concentrations in the body thus preventing the onset of iodine-related diseases.

In orthopedic trauma surgery, traditional socket-based prostheses are associated with functionally limiting problems affecting 1.7 million amputees in the United States. To improve post-surgical performance and minimize socket-related complications, bone-anchored (osseointegrated) prostheses have been developed. Functionally superior, their widespread implementation has been limited due to infection. In an unacceptable number of patients well-organized biofilm ecosystems of bacteria colonize the osseointegrated implant (OI) and migrate into device-tissue interface, leading to superficial and deep infections, and implant failure. Since the OI implant protrudes through the skin, the site is easily contaminated by microbes. The problem is worsened by increased resistance to antibiotics contributing significantly to surgical outcome failure. Antimicrobial photodynamic therapy (aPDT)—which uses photosensitizers excited with visible light to disrupt biofilms and kill bacteria with produced reactive oxygen species—has been proposed to address this problem. To assess biofilm formation and aPDT effectiveness, we describe a rabbit OI model and steps to investigate the ability of aPDT using 5-Aminolevulinic acid (5-ALA)-based light therapy to control methicillin-resistant S. aureus (MRSA) bacterial infection. As part of an institutionally approved survival surgery, this model involves lower limb amputation at the tibia, OI installation and MRSA inoculation. Within a week of biofilm formation, the optimal aPDT regime of light and 5-ALA dose was applied to the implant-skin interface to eradicate migrating biofilms. We have built a circumferential light source spectrally shaped for optimal photoactivation and cooled without risk of bacteria dispersal. Optical coherence tomography (skin flap healing and side-effects), micro-computed tomography (OI-bone integrity) and bioluminescence (bacterial bioburden before and after aPDT) imaging were used to monitor outcome for up to three weeks post-treatment.

Fibromyalgia, a chronic disease that causes disabling pain, is on the rise worldwide. Over the last 6 years, the University of São Paulo, in Brazil, has developed equipment capable of synergistically emitting Photobiomodulation and Ultrasound. Furthermore, a new approach to treatment methodology, using the palms of the hands as a “gateway”, has achieved better results. This new approach based on structural changes in the palms. The objective of this work is to evaluate possible differences in the intracranial compliance of patients that allow us to understand possible changes in the pain regulation center. The assessment of intracranial compliance was carried out using Brain4care technology, a mechanical extensometer that is connected to a mechanical device, in direct contact with the surface of the scalp in the temporal frontal-parietal region of the skull, being able to detect small cranial oscillations resulting from pressure. intracranial. Monitoring was carried out in periods before the intervention, during the intervention and after the intervention, totaling 20 minutes. The intervention was carried out using Laser and Ultrasound equipment. It was possible to observe the modulation of intracranial compliance, reducing values when above ideal, increasing the time considered within the ideal, Reduction of pain according to visual analogue scale and Improvement of quality of life according to the Fibromyalgia Impact Questionnaire. Based on these observations, the synergistic treatment of photobiomodulation and ultrasound promoted pain reduction and improved quality of life, allowing the patient's full reintegration into family, society and the reestablishment of their professional activities.

The objective of this study was to prepare mixed cultures of both sensitive and resistant bacteria of the same species in varying proportions (20% and 80%, 50% and 50% and 80% and 20%) We aimed to analyze the minimum inhibitory concentration (MIC) both before and after photoinactivation treatment. This treatment involved the use of the synthetic photosensitizer curcumin 5 mM (PDTPharma®) and irradiation with an LED lighting device (Biotable® - produced by MM Optics-Brasil) emitting a wavelength of 450 nm, at 40 mW/cm², and a light dose of 10 J/cm². The purpose was to examine the impact of photodynamic action on the spread of bacterial resistance. Combating the proliferation of antimicrobial resistance is a critical global health issue, necessitating treatment methods that adequately address this challenge.

Cronobacter spp., recognised as a foodborne pathogen, presents a significant risk to neonates and vulnerable groups emphasizing the necessity for effective management. This study investigates the efficacy of a dual light system, combining far-UVC (222nm) and blue light (405nm), in combating this bacterial threat. We explored the survival phenotypes of Cronobacter sakazakii and Cronobacter maloniticus under lethal dual light exposure, after 30 minutes of light treatment, strain-dependent sensitivity was observed, with 21 out of 27 strains being fully inactivated within 290mJ/cm² of far-UVC and 23800mJ/cm² of blue light. Survivors, identified as Cronobacter spp., were detected, indicating bacterial resistance. Pigment production was also examined before and after the light treatment. All tested Cronobacter sakazakii exhibited yellow colonies on tryptic soy agar, whereas creamy white colonies were seen on milk agar. Colorimetric analysis verified the difference in pigment production on the two agar types. Light treatments had minimal impact on survivor pigment production, except for one isolate that lost pigment after light treatment on tryptic soy agar, resulting in failure to grow during subsequent enrichment. This research provides new evidence to understand the impact of dual light treatment on bacterial survival, contributing to the development of safer and more effective strategies in pathogenic bacteria control.