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Abstract In the age of antimicrobial resistance, the urgency by which novel therapeutic approaches need to be introduced into the clinical pipeline has reached critical levels. Antimicrobial blue light (aBL), as an alternative approach, has demonstrated promise as a stand‐alone therapeutic method, albeit with a limited window of antimicrobial activity. Work by others indicates that treatment with antibiotics increases the production of reactive oxygen species (ROS) which may, in part, contribute to the bactericidal effects of antibiotics. These findings suggest that there may be potential for synergistic interactions with aBL, that similarly generates ROS. Therefore, in this study, the mechanism of aBL is investigated, and the potential for aBL to synergistically promote antibiotic activity is similarly evaluated. Furthermore, the translatability of using aBL and chloramphenicol in combination within a mouse model of Acinetobacter baumanii burn infection is assessed. It is concluded that porphyrins and hydroxyl radicals driven by “free iron” are paramount to the effectiveness of aBL; and aBL is effective at promoting multiple antibiotics in different multidrug‐resistant bacteria. Moreover, rROS up‐regulation, and promoted antibiotic uptake are observed during aBL+antibiotic exposure. Lastly, aBL combined with chloramphenicol appears to be both effective and safe for the treatment of A. baumannii burn infection. In conclusion, aBL may be a useful adjunct therapy to antibiotics to potentiate their action.

Despite decades of efforts, state-of-the-art synthetic burn dressings to treat partial-thickness burns are still far from ideal. Current dressings adhere to the wound and necessitate debridement. This work describes the first “supramolecular hybrid hydrogel (SHH)” burn dressing that is biocompatible, self-healable, and on-demand dissoluble for easy and trauma-free removal, prepared by a simple, fast, and scalable method. These SHHs leverage the interactions of a custom-designed cationic copolymer via host-guest chemistry with cucurbit[7]uril and electrostatic interactions with clay nanosheets coated with an anionic polymer to achieve enhanced mechanical properties and fast on-demand dissolution. The SHHs show high mechanical strength (>50 kPa), self-heal rapidly in ∼1 min, and dissolve quickly (4–6 min) using an amantadine hydrochloride (AH) solution that breaks the supramolecular interactions in the SHHs. Neither the SHHs nor the AH solution has any adverse effects on human dermal fibroblasts or epidermal keratinocytes in vitro. The SHHs also do not elicit any significant cytokine response in vitro. Furthermore, in vivo murine experiments show no immune or inflammatory cell infiltration in the subcutaneous tissue and no change in circulatory cytokines compared to sham controls. Thus, these SHHs present excellent burn dressing candidates to reduce the time of pain and time associated with dressing changes.

A metagenome contains all DNA sequences from an environmental sample, including viruses, bacteria, archaea, and eukaryotes. Since viruses are of huge abundance and have caused vast mortality and morbidity to human society in history as a type of major pathogens, detecting viruses from metagenomes plays a crucial role in analyzing the viral component of samples and is the very first step for clinical diagnosis. However, detecting viral fragments directly from the metagenomes is still a tough issue because of the existence of a huge number of short sequences. In this study a hybrid Deep lEarning model for idenTifying vIral sequences fRom mEtagenomes (DETIRE) is proposed to solve the problem. First, the graph-based nucleotide sequence embedding strategy is utilized to enrich the expression of DNA sequences by training an embedding matrix. Then, the spatial and sequential features are extracted by trained CNN and BiLSTM networks, respectively, to enrich the features of short sequences. Finally, the two sets of features are weighted combined for the final decision. Trained by 220,000 sequences of 500 bp subsampled from the Virus and Host RefSeq genomes, DETIRE identifies more short viral sequences (

Wong et al. describe a machine learning approach for visual auditing of lateral flow tests for SARS-CoV-2 antibodies. Their automated analysis shows strong agreement with experts and consistently better performance than non-expert study participants at classifying positive results.

In recent years, with the increases in microorganisms that express a multitude of antimicrobial resistance (AMR) mechanisms, the threat of antimicrobial resistance in the global population has reached critical levels. The introduction of the COVID-19 pandemic has further contributed to the influx of infections caused by multidrug-resistant organisms (MDROs), which has placed significant pressure on healthcare systems. For over a century, the potential for light-based approaches targeted at combatting both cancer and infectious diseases has been proposed. They offer effective killing of microbial pathogens, regardless of AMR status, and have not typically been associated with high propensities of resistance development. To that end, the goal of this review is to describe the different mechanisms that drive AMR, including intrinsic, phenotypic, and acquired resistance mechanisms. Additionally, the different light-based approaches, including antimicrobial photodynamic therapy (aPDT), antimicrobial blue light (aBL), and ultraviolet (UV) light, will be discussed as potential alternatives or adjunct therapies with conventional antimicrobials. Lastly, we will evaluate the feasibility and requirements associated with integration of light-based approaches into the clinical pipeline.

Vibrio vulnificus is an invasive marine bacterium that causes a variety of serious infectious diseases. With the increasing multidrug-resistant variants, treatment of V. vulnificus infections is becoming more difficult. In this study, we explored antimicrobial blue light (aBL; 405 nm wavelength) for the treatment of V. vulnificus infections. We first assessed the efficacy of aBL against five strains of V. vulnificus in vitro. Next, we identified and quantified intracellular porphyrins in V. vulnificus to provide mechanistic insights. Additionally, we measured intracellular reactive oxygen species (ROS) production and bacterial membrane permeabilization following aBL exposures. Lastly, we conducted a preclinical study to investigate the efficacy and safety of aBL for the prevention and treatment of burn infections caused by V. vulnificus in mice. We found that aBL effectively killed V. vulnificus in vitro in both planktonic and biofilm states, with up to a 5.17- and 4.57-log10 CFU reduction being achieved, respectively, following an aBL exposure of 216 J/cm2. Protoporphyrin IX and coproporphyrins were predominant in all the strains. Additionally, intracellular ROS was significantly increased following aBL exposures (P < 0.01), and there was evidence of aBL-induced permeabilization of the bacterial membrane (P < 0.0001). In the preclinical studies, we found that female mice treated with aBL 30 min after bacterial inoculation showed a survival rate of 81% following 7 days of observation, while only 28% survival was observed in untreated female mice (P < 0.001). At 6 h post-inoculation, an 86% survival was achieved in aBL-treated female mice (P = 0.0002). For male mice, 86 and 63% survival rates were achieved when aBL treatment was given 30 min and 6 h after bacterial inoculation, respectively, compared to 32% survival in the untreated mice (P = 0.0004 and P = 0.04). aBL did not reduce cellular proliferation or induce apoptosis. We found five cytokines were significantly upregulated in the males after aBL treatment, including MCSF (P < 0.001), MCP-5 (P < 0.01), TNF RII (P < 0.01), CXCL1 (P < 0.01), and TIMP-1 (P < 0.05), and one in the females (TIMP-1; P < 0.05), suggesting that aBL may induce certain inflammatory processes. In conclusion, aBL may potentially be applied to prevent and treat V. vulnificus infections.

Abstract Confronted with the severe situation that the pace of resistance acquisition is faster than the clinical introduction of new antibiotics, health organizations are calling for effective approaches to combat methicillin‐resistant Staphylococcus aureus (MRSA) infections. Here, an approach to treat MRSA through photolysis of staphyloxanthin, an antioxidant residing in the microdomain of S. aureus membrane, is reported. This photochemistry process is uncovered through transient absorption imaging and quantitated by absorption spectroscopy, Raman spectroscopy, and mass spectrometry. Photolysis of staphyloxanthin transiently elevates the membrane permeability and renders MRSA highly susceptible to hydrogen peroxide attack. Consequently, staphyloxanthin photolysis by low‐level 460 nm light eradicates MRSA synergistically with hydrogen peroxide and other reactive oxygen species. The effectiveness of this synergistic therapy is well validated in MRSA planktonic culture, MRSA‐infected macrophage cells, stationary‐phase MRSA, persisters, S. aureus biofilms, and two mice wound infection models. Collectively, the work demonstrates that staphyloxanthin photolysis is a new therapeutic platform to treat MRSA infections.

Polymicrobial biofilms, in which mixed microbial species are present, play a significant role in persistent infections. Furthermore, polymicrobial biofilms promote antibiotic resistance by allowing interspecies transfer of antibiotic resistance genes. In the present study, we investigated the effectiveness of antimicrobial blue light (aBL; 405 nm), an innovative non-antibiotic approach, for the inactivation of polymicrobial biofilms. Dual-species biofilms with Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) as well as with P. aeruginosa and Candida albicans were reproducibly grown in 96-well microtiter plates or in the CDC biofilm reactor for 24 or 48 h. The effectiveness of aBL inactivation of polymicrobial biofilms was determined through colony forming assay and compared with that of monomicrobial biofilms of each species. aBL-induced morphological changes of biofilms were analyzed with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). For 24-h old monomicrobial biofilms formed in 96-well microtiter plates, 6.30-log10 CFU inactivation of P. aeruginosa, 2.33-log10 CFU inactivation of C. albicans and 3.48-log10 CFU inactivation of MRSA were observed after an aBL exposure of 500 J/cm2. Under the same aBL exposure, 6.34-log10 CFU inactivation of P. aeruginosa and 3.11-log10 CFU inactivation of C. albicans were observed, respectively, in dual-species biofilms. In addition, 2.37- and 3.40-log10 CFU inactivation were obtained in MRSA and P. aeruginosa, dual-species biofilms. The same aBL treatment of the biofilms developed in the CDC-biofilm reactor for 48 h significantly decreased the viability of P. aeruginosa monomicrobial and polymicrobial biofilm when cocultured with MRSA (3.70- and 3.56-log10 CFU inactivation, respectively). 2.58-log10 CFU inactivation and 0.86-log10 CFU inactivation was detected in MRSA monomicrobial and polymicrobial biofilm when cocultured with P. aeruginosa. These findings were further supported by the CLSM and SEM experiments. Phototoxicity studies revealed a no statistically significant loss of viability in human keratinocytes after an exposure to 216 J/cm2 and a statistically significant loss of viability after 500 J/cm2. aBL is potentially an alternative treatment against polymicrobial biofilm-related infections. Future studies will aim to improve the efficacy of aBL and to investigate aBL treatment of polymicrobial biofilm-related infections in vivo.

Antimicrobial blue light (aBL) has attracted increasing interest for its antimicrobial properties. However, the underlying bactericidal mechanism has not yet been verified. One hypothesis is that aBL causes the excitation of intracellular chromophores; leading to the generation of reactive oxygen species (ROS) and the resultant oxidization of various biomolecules. Thus, monitoring the levels of redox-sensitive intracellular biomolecules such as coproporphyrins, as well as singlet oxygen and various ROS may help to uncover the physiological changes induced by aBL and aid in establishing the underlying mechanism of action. Furthermore, the identification of novel targets of ROS, such as fatty acids, is of potential significance from a therapeutic perspective. In this study, we sought to investigate the molecular impact of aBL treatment on methicillin-resistant Staphylococcus aureus (MRSA). The results showed that aBL (5–80 J/cm2) exhibited a bactericidal effect on MRSA, and almost no bacteria survived when 80 J/cm2 had been delivered. Further studies revealed that the concentrations of certain intracellular molecules varied in response to aBL irradiation. Coproporphyrin levels were found to decrease gradually, while ROS levels increased rapidly. Moreover, imaging revealed the emergence and increase of singlet oxygen molecules. Concomitantly, the lipid peroxidation product malondialdehyde (MDA) increased in abundance and intracellular K+ leakage was observed, indicating permeability of the cell membrane. Atomic force microscopy showed that the cell surface exhibited a coarse appearance. Finally, fatty acid profiles at different illumination levels were monitored by GC-MS. The relative amounts of three unsaturated fatty acids (C16:1, C20:1, and C20:4) were decreased in response to aBL irradiation, which likely played a key role in the aforementioned membrane injuries. Collectively, these data suggest that the cell membrane is a major target of ROS during aBL irradiation, causing alterations to membrane lipid profiles, and in particular to the unsaturated fatty acid component.

Development of non-antibiotic alternatives to treat infections caused by multidrug-resistant (MDR) microbes represents one of the top priorities in healthcare and community settings, especially in the care of combat trauma-associated wound infections. Here, we investigate efficacy of oregano oil against pathogenic bacteria including MDR isolates from the combat casualties in vitro and in a mouse burn model. Oregano oil showed a significant anti-bacterial activity against 11 MDR clinical isolates including four Acinetobacter baumannii, three Pseudomonas aeruginosa, and four methicillin-resistant Staphylococcus aureus (MRSA) obtained from combat casualties and two luminescent strains of PA01 and MRSA USA300, with a MIC ranging from 0.08 mg/ml to 0.64 mg/ml. Oregano oil also effectively eradicated biofilms formed by each of the 13 pathogens above at similar MICs. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed that oregano oil damaged bacterial cells and altered the morphology of their biofilms. While efficiently inactivating bacteria, there was no evidence of resistance development after up to 20 consecutive passages of representative bacterial strains in the presence of sublethal doses of oregano oil. In vivo study using the third-degree burn wounds infected with PA01 or USA300 demonstrated that oregano oil, topically applied 24 h after bacterial inoculation, sufficiently reduced the bacterial load in the wounds by 3 log10 in 1 h, as measured by drastic reduction of bacterial bioluminescence. This bactericidal activity of oregano oil concurred with no significant side effect on the skin histologically or genotoxicity after three topical applications of oregano oil at 10 mg/ml for three consecutive days. The investigation suggests potentials of oregano oil as an alternative to antibiotics for the treatment of wound-associated infections regardless of antibiotic susceptibility.

Antimicrobial resistance is a threat to public health that requires our immediate attention. With increasing numbers of microbes that are becoming resistant to routinely used antimicrobials, it is vital that we look to other, non-traditional therapies for the treatment of infections. Antimicrobial blue light (aBL) is an innovative approach that has demonstrated efficacy for the inactivation of an array of microbial pathogens. In the present study, we investigated the potential for resistance development to aBL in Gram-negative pathogenic bacteria by carrying out multiple aBL exposures on bacteria. In the first aBL exposure, clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, and uropathogenic Escherichia coli [107 colony forming units/mL (CFU/mL)] were irradiated in phosphate-buffered saline with aBL at 405 nm until a >99.99% reduction in bacterial viability was achieved. Irradiation was then repeated for each bacterial species over 20 cycles of aBL exposure. The potential for resistance development to aBL was also investigated in vivo, in superficial mouse wounds infected with a bioluminescent strain of P. aeruginosa (PAO1; 108 CFU) and irradiated with a sub-curative radiant exposures of 108 or 216 J/cm2 aBL over 5 cycles of treatment (over 5 days) prior to bacterial isolation from the animal tissue. PAO1 isolated from infected tissue were treated with aBL at 216 J/cm2, in vitro, in parallel with unexposed PAO1 or PAO1 isolates from mouse wound infections not treated with aBL. No statistically significant correlation was found between the aBL-susceptibility of bacteria in vitro and the number of cycles of aBL exposure any bacterial species (P ≥ 0.26). In addition, serial exposure of infected mouse wounds to aBL did not result in any change in the susceptibility to aBL of PAO1 (P = 0.97). In conclusion, it is unlikely that sequential exposure to aBL will result in aBL-resistance in Gram-negative bacteria. Also, multiple aBL treatments may potentially be administered to an infected wound without resistance development becoming a concern.

Low-level laser (light) therapy (LLLT) has been clinically applied around the world for a spectrum of disorders requiring healing, regeneration and prevention of tissue death. One area that is attracting growing interest in this scope is the use of transcranial LLLT to treat stroke and traumatic brain injury (TBI). We developed a mouse model of severe TBI induced by controlled cortical impact and explored the effect of different treatment schedules. Adult male BALB/c mice were divided into 3 broad groups (a) sham-TBI sham-treatment, (b) real-TBI sham-treatment, and (c) real-TBI active-treatment. Mice received active-treatment (transcranial LLLT by continuous wave 810 nm laser, 25 mW/cm(2), 18 J/cm(2), spot diameter 1 cm) while sham-treatment was immobilization only, delivered either as a single treatment at 4 hours post TBI, as 3 daily treatments commencing at 4 hours post TBI or as 14 daily treatments. Mice were sacrificed at 0, 4, 7, 14 and 28 days post-TBI for histology or histomorphometry, and injected with bromodeoxyuridine (BrdU) at days 21-27 to allow identification of proliferating cells. Mice with severe TBI treated with 1-laser Tx (and to a greater extent 3-laser Tx) had significant improvements in neurological severity score (NSS), and wire-grip and motion test (WGMT). However 14-laser Tx provided no benefit over TBI-sham control. Mice receiving 1- and 3-laser Tx had smaller lesion size at 28-days (although the size increased over 4 weeks in all TBI-groups) and less Fluoro-Jade staining for degenerating neurons (at 14 days) than in TBI control and 14-laser Tx groups. There were more BrdU-positive cells in the lesion in 1- and 3-laser groups suggesting LLLT may increase neurogenesis. Transcranial NIR laser may provide benefit in cases of acute TBI provided the optimum treatment regimen is employed.

Local microbial infections induced by multiple-drug-resistant bacteria in the orthopedic field can be intractable, therefore development of new therapeutic modalities is needed. Photodynamic therapy (PDT) is a promising alternative modality to antibiotics for intractable microbial infections, and we recently reported that PDT has the potential to accumulate neutrophils into the infected site which leads to resolution of the infection. PDT for cancer has long been known to be able to stimulate the innate and adaptive arms of the immune system.In the present study, a murine methicillin-resistant Staphylococcus aureus (MRSA) arthritis model using bioluminescent MRSA and polystyrene microparticles was established, and both the therapeutic (Th-PDT) and preventive (Pre-PDT) effects of PDT using methylene blue as photosensitizer were examined. Although Th-PDT could not demonstrate direct bacterial killing, neutrophils were accumulated into the infectious joint space after PDT and MRSA arthritis was reduced. With the preconditioning Pre-PDT regimen, neutrophils were quickly accumulated into the joint immediately after bacterial inoculation and bacterial growth was suppressed and the establishment of infection was inhibited.This is the first demonstration of a protective innate immune response against a bacterial pathogen produced by PDT.

Transcranial low-level laser therapy (LLLT) using near-infrared light can efficiently penetrate through the scalp and skull and could allow non-invasive treatment for traumatic brain injury (TBI). In the present study, we compared the therapeutic effect using 810-nm wavelength laser light in continuous and pulsed wave modes in a mouse model of TBI.TBI was induced by a controlled cortical-impact device and 4-hours post-TBI 1-group received a sham treatment and 3-groups received a single exposure to transcranial LLLT, either continuous wave or pulsed at 10-Hz or 100-Hz with a 50% duty cycle. An 810-nm Ga-Al-As diode laser delivered a spot with diameter of 1-cm onto the injured head with a power density of 50-mW/cm(2) for 12-minutes giving a fluence of 36-J/cm(2). Neurological severity score (NSS) and body weight were measured up to 4 weeks. Mice were sacrificed at 2, 15 and 28 days post-TBI and the lesion size was histologically analyzed. The quantity of ATP production in the brain tissue was determined immediately after laser irradiation. We examined the role of LLLT on the psychological state of the mice at 1 day and 4 weeks after TBI using tail suspension test and forced swim test.The 810-nm laser pulsed at 10-Hz was the most effective judged by improvement in NSS and body weight although the other laser regimens were also effective. The brain lesion volume of mice treated with 10-Hz pulsed-laser irradiation was significantly lower than control group at 15-days and 4-weeks post-TBI. Moreover, we found an antidepressant effect of LLLT at 4-weeks as shown by forced swim and tail suspension tests.The therapeutic effect of LLLT for TBI with an 810-nm laser was more effective at 10-Hz pulse frequency than at CW and 100-Hz. This finding may provide a new insight into biological mechanisms of LLLT.