The 2023 Orthopedic Research Society's international consensus meeting on musculoskeletal infection: Summary from the in vitro section.

Antimicrobial strategies for musculoskeletal infections are typically first developed with in vitro models. The In Vitro Section of the 2023 Orthopedic Research Society Musculoskeletal Infection international consensus meeting (ICM) probed our state of knowledge of in vitro systems with respect to bacteria and biofilm phenotype, standards, in vitro activity, and the ability to predict in vivo efficacy. A subset of ICM delegates performed systematic reviews on 15 questions and made recommendations and assessment of the level of evidence that were then voted on by 72 ICM delegates. Here, we report recommendations and rationale from the reviews and the results of the internet vote. Only two questions received a ≥90% consensus vote, emphasizing the disparate approaches and lack of established consensus for in vitro modeling and interpretation of results. Comments on knowledge gaps and the need for further research on these critical MSKI questions are included.

Blue Light Potentiates Antibiotics in Bacteria via Parallel Pathways of Hydroxyl Radical Production and Enhanced Antibiotic Uptake.

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.

Antimicrobial Resistance: Is There a 'Light' at the End of the Tunnel?

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.

DETIRE: a hybrid deep learning model for identifying viral sequences from metagenomes.

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 (<1,000 bp) than the three latest methods, such as DeepVirFinder, PPR-Meta, and CHEER. DETIRE is freely available at Github (https://github.com/crazyinter/DETIRE).

Supramolecular hybrid hydrogels as rapidly on-demand dissoluble, self-healing, and biocompatible burn dressings.

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.

New Insights into the Bacterial Targets of Antimicrobial Blue Light.

Antimicrobial blue light (aBL) offers efficacy and safety in treating infections. However, the bacterial targets for aBL are still poorly understood and may be dependent on bacterial species. Here, we investigated the biological targets of bacterial killing by aBL (λ = 410 nm) on three pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Initially, we evaluated the killing kinetics of bacteria exposed to aBL and used this information to calculate the lethal doses (LD) responsible for killing 90 and 99.9% of bacteria. We also quantified endogenous porphyrins and assessed their spatial distribution. We then quantified and suppressed reactive oxygen species (ROS) production in bacteria to investigate their role in bacterial killing by aBL. We also assessed aBL-induced DNA damage, protein carbonylation, lipid peroxidation, and membrane permeability in bacteria. Our data showed that P. aeruginosa was more susceptible to aBL (LD99.9 = 54.7 J/cm2) relative to S. aureus (LD99.9 = 158.9 J/cm2) and E. coli (LD99.9 = 195 J/cm2). P. aeruginosa exhibited the highest concentration of endogenous porphyrins and level of ROS production relative to the other species. However, unlike other species, DNA degradation was not observed in P. aeruginosa. Sublethal doses of blue light (LD99.9). We conclude that the primary targets of aBL depend on the species, which are probably driven by variable antioxidant and DNA-repair mechanisms. IMPORTANCE Antimicrobial-drug development is facing increased scrutiny following the worldwide antibiotic crisis. Scientists across the world have recognized the urgent need for new antimicrobial therapies. In this sense, antimicrobial blue light (aBL) is a promising option due to its antimicrobial properties. Although aBL can damage different cell structures, the targets responsible for bacterial inactivation have still not been completely established and require further exploration. In our study, we conducted a thorough investigation to identify the possible aBL targets and gain insights into the bactericidal effects of aBL on three relevant pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. This research not only adds new content to blue light studies but opens new perspectives to antimicrobial applications.

Antimicrobial Blue Light for Prevention and Treatment of Highly Invasive Vibrio vulnificus Burn Infection in Mice.

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.

Machine learning to support visual auditing of home-based lateral flow immunoassay self-test results for SARS-CoV-2 antibodies.

Background: Lateral flow immunoassays (LFIAs) are being used worldwide for COVID-19 mass testing and antibody prevalence studies. Relatively simple to use and low cost, these tests can be self-administered at home, but rely on subjective interpretation of a test line by eye, risking false positives and false negatives. Here, we report on the development of ALFA (Automated Lateral Flow Analysis) to improve reported sensitivity and specificity. Methods: Our computational pipeline uses machine learning, computer vision techniques and signal processing algorithms to analyse images of the Fortress LFIA SARS-CoV-2 antibody self-test, and subsequently classify results as invalid, IgG negative and IgG positive. A large image library of 595,339 participant-submitted test photographs was created as part of the REACT-2 community SARS-CoV-2 antibody prevalence study in England, UK. Alongside ALFA, we developed an analysis toolkit which could also detect device blood leakage issues. Results: Automated analysis showed substantial agreement with human experts (Cohen's kappa 0.90-0.97) and performed consistently better than study participants, particularly for weak positive IgG results. Specificity (98.7-99.4%) and sensitivity (90.1-97.1%) were high compared with visual interpretation by human experts (ranges due to the varying prevalence of weak positive IgG tests in datasets). Conclusions: Given the potential for LFIAs to be used at scale in the COVID-19 response (for both antibody and antigen testing), even a small improvement in the accuracy of the algorithms could impact the lives of millions of people by reducing the risk of false-positive and false-negative result read-outs by members of the public. Our findings support the use of machine learning-enabled automated reading of at-home antibody lateral flow tests as a tool for improved accuracy for population-level community surveillance.

Understanding a defensive response of methicillin-resistant Staphylococcus aureus after exposure to multiple cycles of sub-lethal blue light.

Blue light (BL) has shown bactericidal effectiveness against methicillin-resistant Staphylococcus aureus (MRSA), one of the major clinical pathogens with antibiotic resistance. Bacteria likely respond to the oxidative stress induced by BL; however, the defensive response is still unclear. This study aimed to reveal the phenotypic change in MRSA after being exposed to 15 cycles of sub-lethal BL illumination. The comparative transcriptomic results showed that the expression of peptidoglycan (PG) synthesis gene glmS was significantly upregulated in the cells after the multiple cycle light treatment, and the biochemical analysis determined that the content of PG synthesized was increased by 25.86% when compared with that in control cells. Furthermore, significant thickening of the cell wall was observed under a transmission electron microscope (P < .05). The light sensitivity of the tested MRSA strain was reduced after the multiple cycle light treatment, indicating the possibility of MRSA being more adaptive to the BL stress. The present study suggested that multiple cycles of sub-lethal BL could change the light susceptibility of MRSA through thickening the cell wall.

Potentiation of Antimicrobial Photodynamic Therapy by Curcumin-loaded Graphene Quantum Dots.

Increasing resistance to existing antibiotics by microbes is currently the biggest dilemma. Antimicrobial photodynamic therapy is a promising alternative for the treatment of multidrug-resistant infections. The aim of the current study was to fabricate graphene quantum dots loaded with curcumin as photosensitizer for improved antimicrobial photodynamic therapy. The study involved fabrication of blank and curcumin-loaded graphene quantum dots, their characterizations (TEM, UV-visible and fluorescence emission spectra), cytotoxicity assay, ROS assay and investigation of enhanced antimicrobial photodynamic effect against resistant microbes. The fabrication of blank and loaded graphene quantum dots was confirmed by the observation of peak shift and changes in peak intensity of blank graphene quantum dots, curcumin alone compared with curcumin-loaded graphene quantum dots in UV-visible and fluoresce emission spectra. Cytotoxicity assay showed that 100 µm concentration was not toxic to NIH/3t3 fibroblasts. In ROS assay, the curcumin-loaded formulation showed three-fold increase in ROS production. Blue-light (405 nm) irradiance of 30 J cm-2 and photosensitizer concentration of 100 µm showed ~3.5 log10 enhanced CUF reduction against Pseudomonas aeruginosa, MRSA, Escherichia coli and Candida albicans. In conclusion, curcumin-loaded graphene quantum dots shoed enhanced antimicrobial photodynamic effects and can be used as an alternative effective treatment for resistant infections.

Antimicrobial blue light: A 'Magic Bullet' for the 21st century and beyond?

Over the past decade, antimicrobial blue light (aBL) at 400 - 470 nm wavelength has demonstrated immense promise as an alternative approach for the treatment of multidrug-resistant infections. Since our last review was published in 2017, there have been numerous studies that have investigated aBL in terms of its, efficacy, safety, mechanism, and propensity for resistance development. In addition, researchers have looked at combinatorial approaches that exploit aBL and other traditional and non-traditional therapeutics. To that end, this review aims to update the findings from numerous studies that capitalize on the antimicrobial effects of aBL, with a focus on: efficacy of aBL against different microbes, identifying endogenous chromophores and targets of aBL, Resistance development to aBL, Safety of aBL against host cells, and Synergism of aBL with other agents. We will also discuss our perspective on the future of aBL.

How can biophotonics help dentistry to avoid or minimize cross infection by SARS-CoV-2?

Biophotonics is defined as the combination of biology and photonics (the physical science of the light). It is a general term for all techniques that deal with the interaction between biological tissues/cells and photons (light). Biophotonics offers a great variety of techniques that can facilitate the early detection of diseases and promote innovative theragnostic approaches. As the COVID-19 infection can be transmitted due to the face-to-face communication, droplets and aerosol inhalation and the exposure to saliva, blood, and other body fluids, as well as the handling of sharp instruments, dental practices are at increased risk of infection. In this paper, a literature review was performed to explore the application of Biophotonics approaches in Dentistry focusing on the COVID-19 pandemic and how they can contribute to avoid or minimize the risks of infection in a dental setting. For this, search-related papers were retrieved from PubMED, Scielo, Google Schoolar, and American Dental Association and Centers for Disease Control and Prevention databases. The body of evidence currently available showed that Biophotonics approaches can reduce microorganism load, decontaminate surfaces, air, tissues, and minimize the generation of aerosol and virus spreading by minimally invasive, time-saving, and alternative techniques in general. However, each clinical situation must be individually evaluated regarding the benefits and drawbacks of these approaches, but always pursuing less-invasive and less aerosol-generating procedures, especially during the COVID-19 pandemic.

Efficacy of combinational therapy using blue light and benzoyl peroxide in reducing Cutibacterium acnes bioburden at the deltopectoral interval: a randomized controlled trial.

BACKGROUND: The purpose of this study was to compare the efficacy of blue light therapy (BLT) and 5% topical benzoyl peroxide (BPO) gel in combination with standard chlorhexidine (CHX) preparation in eradicating Cutibacterium acnes at the deltopectoral interval measured by positive, quantitative culture findings. METHODS: Adult male volunteers were randomized to 1 of 3 treatment groups: BPO, BLT, and BPO followed by BLT. Contralateral shoulders served as matched controls. Volunteers randomized to BPO applied the gel for a total of 5 treatments. In the BLT group, a single 23-minute treatment was administered at an estimated irradiance of 40 mW/cm2 (radiant exposure, 55.2 J/cm2). In the BPO-BLT group, volunteers received both treatments as described earlier. After treatment with either BPO, BLT, or both, a single swab culture was taken from the treatment shoulder. Next, control and treatment shoulders were prepared with CHX, and cultures were taken from each shoulder. Cultures were sent for anaerobic quantitative growth analysis with both polymerase chain reaction and Sanger sequencing confirmation of presumptive C acnes colonies. RESULTS: This study enrolled 60 male volunteers, 20 per group, with no loss to follow-up. After treatment but prior to CHX administration, all culture samples in the BPO group and BLT group grew C acnes. Prior to CHX, 16 samples (80%) in the BPO-BLT group grew C acnes. On quantitative analysis, the BPO group and BPO-BLT group had significantly less growth of C acnes compared with the BLT group after treatment but prior to CHX (P < .05 for each). Following CHX administration, the BPO and BPO-BLT groups had significantly fewer positive culture findings (odds ratios of 0.03 and 0.29, respectively) and less quantity of growth compared with their control arms (P < .05). This was not seen in the BLT group. For quantitative between-group analysis, no significant synergistic effects were seen with BPO-BLT compared with BPO alone (P = .688). There was no difference in side effects between groups. CONCLUSION: The combination of topical BPO and CHX was effective at eliminating C acnes in most cases. BLT alone did not demonstrate effective antimicrobial properties against C acnes at the radiant exposure administered in this study. Combining BPO and BLT did not lead to significant synergistic antimicrobial effects. Both BPO and BLT are safe with few, transient side effects reported. More work is needed to determine whether BLT at higher radiant exposures or serial treatment results in bactericidal effects against C acnes in vivo.

Corrigendum: Bactericidal Property of Oregano Oil Against Multidrug-Resistant Clinical Isolates.

[This corrects the article DOI: 10.3389/fmicb.2018.02329.].

Can microorganisms develop resistance against light based anti-infective agents?

Recently, there have been increasing numbers of publications illustrating the potential of light-based antimicrobial therapies to combat antimicrobial resistance. Several modalities, in particular, which have proven antimicrobial efficacy against a wide range of pathogenic microbes include: photodynamic therapy (PDT), ultraviolet light (UVA, UVB and UVC), and antimicrobial blue light (aBL). Using these techniques, microbial cells can be inactivated rapidly, either by inducing reactive oxygen species that are deleterious to the microbial cells (PDT, aBL and UVA) or by causing irreversible DNA damage via direct absorption (UVB and UVC). Given the multi-targeted nature of light-based antimicrobial modalities, it has been hypothesised that resistance development to these approaches is highly unlikely. Furthermore, with the exception of a small number of studies, it has been found that resistance to light based anti-infective agents appears unlikely, irrespective of the modality in question. The concurrent literature however stipulates, that further studies should incorporate standardised microbial tolerance assessments for light-based therapies to better assess the reproducibility of these observations.

Effective Treatment of Cutaneous Mold Infections by Antimicrobial Blue Light That Is Potentiated by Quinine.

BACKGROUND: Cutaneous mold infections commonly result from an array of traumatic injuries that involve direct inoculation of contaminated soil into wounds. Here, we explored the use of antimicrobial blue light (aBL; 405 nm wavelength) and the combination of aBL with quinine hydrochloride (aBL + Q-HCL) for the treatment of cutaneous mold infections. METHODS: Efficacy of aBL and aBL + Q-HCL in killing clinically important pathogenic molds (Aspergillus fumigatus, Aspergillus flavus, and Fusarium oxyprorum) was investigated. Ultraperformance liquid chromatography identified and quantified endogenous porphyrins in the mold conidia. Finally, a mouse model of dermabrasion wound infected with a bioluminescent variant of A. fumigatus was developed to investigate the efficacy of aBL in treating cutaneous mold infections. RESULTS: We demonstrated that mold conidia are tolerant to aBL, but Q-HCL enhances efficacy. Transmission electron microscopy revealed intracellular damage by aBL. aBL + Q-HCL resulted in intracellular and cell wall damage. Porphyrins were observed in all mold strains, with A. fumigatus having the highest concentration. aBL and aBL + Q-HCL effectively reduced the burden of A. fumigatus within an established dermabrasion infection and limited recurrence posttreatment. CONCLUSIONS: aBL and aBL + Q-HCL may offer a novel approach for the treatment of mold infections.

Potentiated antimicrobial blue light killing of methicillin resistant Staphylococcus aureus by pyocyanin.

As antimicrobial resistance continues to threaten the efficacy of conventional antibiotic therapy, it is paramount that we investigate innovative approaches to treat infectious diseases. In this study, we investigated the antimicrobial capabilities of the innovative combination of antimicrobial blue light (aBL; 405 nm wavelength) with the Pseudomonas aeruginosa pigment pyocyanin against methicillin resistant Staphylococcus aureus (MSRA. We explored the effects of different radiant exposures of aBL and increasing concentrations of pyocyanin against planktonic cells and those within biofilms. In addition, we investigated the effect of the aBL/pyocyanin on the endogenous staphyloxanthin pigment, as well as the role of hydrogen peroxide and singlet oxygen scavenging in the efficacy of this combination. Lastly, we investigated the potential for the aBL/pyocyanin to reduce the MRSA burden within a proof-of-principle mouse abrasion infection model. We found pyocyanin to be a powerful potentiator of aBL activity under all in vitro conditions tested. In addition, we serendipitously discovered the capability of the aBL/pyocyanin combination to bleach staphyloxanthin within colonies of MRSA. Furthermore, we established that singlet oxygen is an important mediator during combined aBL/pyocyanin exposure. Moreover, we found that the combination of aBL and pyocyanin could significantly reduce the viability of MRSA within a proof-of-principle early onset MRSA skin abrasion infection. Exposure to the treatment did not have deleterious effects on skin tissue. In conclusion, the combination of aBL and pyocyanin represents a potentially powerful therapeutic modality for the treatment of infections caused by MRSA.