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Endolysins (lysins), a novel class of antibacterial agents derived from bacteriophages, efficiently lyse bacteria by degrading the peptidoglycan layer within the bacterial wall. Colistin, a classic peptide antibiotic with the ability to permeabilize the outer membrane, has recently shown great promise in synergizing with lysins against gram-negative bacteria. However, the exact mechanisms responsible for their synergy remain unclear. Here, we first demonstrated the synergistic bacterial killing of various lysin and colistin combinations. With a model lysin, LysAB2, we then confirmed that there is a threshold concentration of colistin causing sufficient permeabilization of the outer membrane for lysin to access the peptidoglycan layer and subsequently exert its lytic ability. The threshold colistin concentrations were found to range 0.2-0.8 μM for the tested bacteria, with the exact value largely depending on the density of lipopolysaccharides on the outer membrane. Beyond the threshold colistin level, LysAB2 could synergize with colistin at a concentration as low as 0.31 μM. Next, we proved for the first time that lysin-induced degradation of the peptidoglycan layer facilitated the disruption of cytoplasmic membrane by colistin, elevated the level of reactive oxygen species in bacterial cells, and boosted the killing effect of colistin. Additionally, the colistin-lysin combination could effectively eliminate established biofilms due to the biofilm dispersal ability of lysin. The in-vivo efficacy was preliminary confirmed in a Galleria mellonella infection model for combination with colistin doses (≥ 1.8 μg/larvae), which could reach beyond the threshold concentration, and a fixed LysAB2 dose (10 μg/larvae). In summary, our study provided the first experimental evidence unravelling the mechanisms behind the synergy of colistin and lysins. All these findings provided important insights in guiding the dosing strategy for applying this combination in future development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Asthma is a common chronic disease amongst children. Epidemiological studies showed that the mortality rate of asthma in children is still high worldwide. Asthma control is therefore essential to minimize asthma exacerbations, which can be fatal if the condition is poorly controlled. Frequent monitoring could help to detect asthma progression and ensure treatment effectiveness. Although subjective asthma monitoring tools are available, the results vary as they rely on patients' self-perception. Emerging evidence suggests several objective tools could have the potential for monitoring purposes. However, there is no consensus to standardise the use of objective monitoring tools. In this review, we start with the prevalence and severity of childhood asthma worldwide. Then, we detail the latest available objective monitoring tools, focusing on their effectiveness in paediatric asthma management. Publications of spirometry, fractional exhaled nitric oxide (FeNO), hyperresponsiveness tests and electronic monitoring devices (EMDs) between 2016 and 2023 were included. The potential advantages and limitations of each tool were also discussed. Overall, this review provides a summary for researchers dedicated to further improving objective paediatric asthma monitoring and provides insights for clinicians to incorporate different objective monitoring tools in clinical practices., (© 2024. The Author(s).)

Chronic skin wounds are often associated with multidrug-resistant bacteria, impeding the healing process. Bacteriophage (phage) therapy has been revitalized as a promising strategy to counter the growing concerns of antibiotic resistance. However, phage monotherapy also faces several application drawbacks, such as a narrow host spectrum, the advent of resistant phenotypes and poor stability of phage preparations. Phage-antibiotic synergistic (PAS) combination therapy has recently been suggested as a possible approach to overcome these shortcomings. In the present study, we employed a model PAS combination containing a vB_AbaM-IME-AB2 phage and colistin to develop stable wound dressings of PAS to mitigate infections associated with Acinetobacter baumannii . A set of thermosensitive hydrogels were synthesized with varying amounts of Pluronic® F-127 (PF-127 at 15, 17.5 and 20 w/w%) modified with/without 3 w/w% hydroxypropyl methylcellulose (HPMC). Most hydrogel formulations had a gelation temperature around skin temperature, suitable for topical application. The solidified gels were capable of releasing the encapsulated phage and colistin in a sustained manner to kill bacteria. The highest bactericidal effect was achieved with the formulation containing 17.5% PF-127 and 3% HPMC (F5), which effectively killed bacteria in both planktonic (by 5.66 log) and biofilm (by 3 log) states and inhibited bacterial regrowth. Good storage stability of F5 was also noted with negligible activity loss after 9 months of storage at 4 °C. The ex vivo antibacterial efficacy of the F5 hydrogel formulation was also investigated in a pork skin wound infection model, where it significantly reduced the bacterial burden by 4.65 log. These positive outcomes warrant its further development as a topical PAS-wound dressing.

Bacteriophage (phage) therapy, exploiting phages which are the natural enemies of bacteria, has been re-introduced to treat multidrug-resistant (MDR) bacterial infections. However, some intrinsic drawbacks of phages are overshadowing their clinical use, particularly the narrow host spectrum and rapid emergence of resistance upon treatment. The use of phage-antibiotic combinations exhibiting synergistic bacterial killing [termed 'phage-antibiotic synergy' (PAS)] has therefore been proposed. It is well reported that the types and doses of phages and antibiotics are critical in achieving PAS. However, the impact of treatment order has received less research attention. As such, this study used an Acinetobacter baumannii phage vB_AbaM-IME-AB2 and colistin as a model PAS combination to elucidate the order effects in-vitro. While application of the phage 8 h before colistin treatment demonstrated the greatest antibacterial synergy, it failed to prevent the development of phage resistance. On the other hand, simultaneous application and antibiotic followed by phage application were able to suppress/delay the development of resistance effectively, and simultaneous application demonstrated superior antibacterial and antibiofilm activities. Further in-vivo investigation is required to confirm the impact of treatment order on PAS., (Copyright © 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Antimicrobial resistance is a normal evolutionary process for microorganisms. Antibiotics exerted accelerated selective pressure that hasten bacterial resistance through mutation, and acquisition external genes. These genes often carry multiple antibiotic resistant determinants allowing the recipient microbe an instant "super-bug" status. The extent of Antimicrobial Resistance (AMR) has reached a level of global crisis, existing antimicrobials are no long effective in treating infections caused by AMR pathogens. The great majority of clinically available antimicrobial agents are administered through oral and intra-venous routes. Overcoming antibacterial resistance by novel drug delivery approach offered new hopes, particularly in the treatment of AMR pathogens in sites less assessible through systemic circulation such as the lung and skin. In the current review, we will revisit the mechanism and incidence of important AMR pathogens. Finally, we will discuss novel drug delivery approaches including novel local antibiotic delivery systems, hybrid antibiotics, and nanoparticle-based antibiotic delivery systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bacteriophages (Phages) are antibacterial viruses that are unaffected by antibiotic drug resistance. Many Phase I and Phase II phage therapy clinical trials have shown acceptable safety profiles. However, none of the completed trials could yield data supporting the promising observations noted in the experimental phage therapy. These trials have mainly focused on phage suspensions without enough attention paid to the stability of phage during processing, storage, and administration. This is important because in vivo studies have shown that the effectiveness of phage therapy greatly depends on the ratio of phage to bacterial concentrations (multiplicity of infection) at the infection site. Additionally, bacteria can evade phages through the development of phage-resistance and intracellular residence. This review focuses on the use of phage therapy against bacteria that survive within the intracellular niches. Recent research on phage behavior reveals that some phage can directly interact with, get internalized into, and get transcytosed across mammalian cells, prompting further research on the governing mechanisms of these interactions and the feasibility of harnessing therapeutic phage to target intracellular bacteria. Advances to improve the capability of phage attacking intracellular bacteria using formulation approaches such as encapsulating/conjugating phages into/with vector carriers via liposomes, polymeric particles, inorganic nanoparticles, and cell penetrating peptides, are summarized. While promising progress has been achieved, research in this area is still in its infancy and warrants further attention., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

With the emergence of multidrug resistance (MDR) bacteria, wound infection continues to be a challenging problem and represents a considerable healthcare burden. This study aims to evaluate the applicability of a phage loaded thermosensitive hydrogel in managing wound infections caused by MDR Acinetobacter baumannii, using IME-AB2 phage and MDR-AB2 as the model phage and bacteria, respectively. Excellent storage stability of the IME-AB2 phage in a ~18 wt% Poloxamer 407 (P407) hydrogel solution was first demonstrated with negligible titer loss (~0.5 log) in 24 months at 4 °C. The incorporated phage was released in a sustained manner with a cumulative release of 60% in the first 24 h. The in vitro bacterial killing efficiency of phage gel and phage suspension at 37 °C demonstrated >5 log 10 CFU/ml reduction against A. baumannii. A comparable biofilm elimination capacity was also noted between the phage gel and phage suspension (59% and 45% respectively). These results suggested that the incorporation of phage into the hydrogel not only had insignificant impacts on the bacterial killing efficiency of phage, but also act as a phage depot to maintain higher phage titer at the infectious site for a prolong period for more effective treatment. We also found that the hydrogel formulation significantly suppressed microbial survival in an ex vivo wound infection model using pig skin (90% reduction in bacterial counts was achieved after 4 h treatment). In summary, our results demonstrated that the P407-based phage-loaded thermosensitive hydrogel is a simple and promising phage formulation for the management of wound infections., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Challenges of ophthalmic drug delivery arise not only from the limited solubility of hydrophobic therapeutics, but also the restricted permeability and fast clearance of drugs due to the complex anatomy and physiology of eyes. Excellent biocompatibility of a thermosensitive polymer, PLGA-PEG-PLGA (1800-1500-1800, LA:GA ratio = 3:1), as an ophthalmic delivery system was demonstrated in our previous work. In this study, delivery of dexamethasone using this thermogel via a single subconjunctival injection for prolonged treatment was evaluated with corneal neovascularization using an alkali-burn diseased model in rat. Solubility of dexamethasone in the polymeric solution was increased by 5.2-fold and the resulting drug-loaded solution formed in situ rigid gel at body temperature. Prolonged in vitro release of dexamethasone from the gel structure was noted. Dexamethasone gel formulation was demonstrated to be more effective in reducing the burn stimulus and neovascularization in the rat diseased model. The findings suggest the PLGA-PEG-PLGA in situ gelling system can be applied for ophthalmic drug delivery to achieve sustained drug release and improved efficacy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aims: Cisplatin is the mainstay of first-line treatment for advanced non-small cell lung cancer (NSCLC). Accumulating evidence suggests that flavonoids inhibit histone deacetylase (HDAC) to mediate their anticancer effect in various cancer types. The study was conducted to investigate the inhibition of HDAC and the modulation of apoptotic and cell cycle regulatory genes by selected flavonoids to potentiate the anticancer effect of cisplatin., Main Methods: Combinations of cisplatin and selected flavonoids were investigated in three NSCLC cell lines (A549, H460, and H1299). Sulforhodamine B assay was used to evaluate cytotoxicity of drug combinations. Western blot analysis was conducted to evaluate histone acetylation. Flow cytometric assays were used to investigate the apoptotic and cell cycle effect. Chromatin immunoprecipitation assay was performed to elucidate the binding of transcription factors to promoters of selected apoptotic and cell cycle regulatory genes., Key Findings: Apigenin was found to exhibit the strongest HDAC inhibitory effect among all flavonoids tested. Cisplatin-apigenin combination was shown to produce significantly more S phase prolongation and G2/M cell cycle arrest, and apoptosis compared with cisplatin or apigenin alone, by inducing p21 and PUMA, respectively. More pronounced effect was observed in p53-proficient than p53-null NSCLC cells. Mechanistically, apigenin was found to reduce the binding of HDAC1 but increase the association of RNA polymerase II and Sp1 to p21 and PUMA promoters., Significance: Our findings provide a better insight about the mechanism contributing to the HDAC inhibitory effect of apigenin to potentiate anticancer effect of cisplatin by inducing apoptosis and cell cycle arrest., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: Osteosynthesis-associated infection is a challenging complication post fracture fixation, burdening the patients and the orthopaedic surgeons alike. A clinically relevant animal model is critical in devising new therapeutic strategies. Our aim was to perform a systematic review to evaluate existing preclinical models and identify their applications in aspects of animal selection, bacterial induction, fracture fixation and complications., Methods: A systematic literature research was conducted in PubMed and Embase up to February 2020. A total of 31 studies were included. Information on the animal, bacterial induction, fracture fixation, healing result and complications were extracted., Results: Animals selected included murine (23), rabbit (6), ewe (1) and goat (1). Larger animals had enabled the use of human-sized implant, however small animals were more economical and easier in handling. Staphylococcus aureus (S. aureus) was the most frequently chosen bacteria for induction. Bacterial inoculation dose ranged from 10 2-8  ​CFU. Consistent and replicable infections were observed from 10 4  ​CFU in general. Methods of inoculation included injections of bacterial suspension (20), placement of foreign objects (8) and pretreatment of implants with established biofilm (3). Intramedullary implants (13), plates and screws (18) were used in most models. Radiological (29) and histological evaluations (24) in osseous healing were performed. Complications such as instability of fracture fixation (7), unexpected surgical death (5), sepsis (1) and persistent lameness (1) were encountered., Conclusion: The most common animal model is the S. aureus infected open fracture internally fixated. Replicable infections were mainly from 10 4  ​CFU of bacteria. However, with the increase in antibiotic resistance, future directions should explore polymicrobial and antibiotic resistant strains, as these will no doubt play a major role in bone infection. Currently, there is also a lack of osteoporotic bone infection models and the pathophysiology is unexplored, which would be important with our aging population., The Translational Potential of This Article: This systematic review provides an updated overview and compares the currently available animal models of osteosynthesis-associated infections. A discussion on future research directions and suggestion of animal model settings were made, which is expected to advance the research in this field., Competing Interests: The authors have no conflicts of interest to disclose in relation to this article., (© 2020 The Authors.)

Due to the emergence of antibiotic resistance, antimicrobial photodynamic therapy (aPDT) has recently been demonstrated as a promising alternative to antibiotics to treat wound infections caused by multidrug-resistant (MDR) pathogens. This study aimed to evaluate the bacterial killing efficiency of aPDT mediated by methylene blue (MB) loaded thermosensitive hydrogels against methicillin-resistant Staphylococcus aureus (MRSA). Box-Behnken Design method was employed to investigate the impacts of the polymer compositions, Poloxamer 407, Poloxamer 188 and Carbopol 934P, on the gelation temperature (T sol-gel ) and release rate of MB. The viscosity and in vitro bacterial killing efficiency of three selected formulations with T sol-gel ranged 25-34 °C and MB release in 2 h (the incubation time used for aPDT experiment) ≥ 70%, were assessed. The viscosity was found to increase with increasing P407 content and increasing total gel concentration. In the in vitro aPDT experiment, all tested MB-hydrogels demonstrated >2.5 log 10 colony forming unit (CFU) reduction against three clinical relevant MRSA strains. Interestingly, the bacterial reduction increased with decreasing amount of gel added (reduced MB concentration). This was possibly attributed to the increased viscosity at higher gel concentration reducing the diffusion rate of released MB towards bacterial cells leading to reduced aPDT efficiency. In summary, aPDT with the thermosensitive MB hydrogel formulations is a promising treatment strategy for wound infections., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows reproducible mixing in miliseconds on the nanoliter scale. Here we investigated the feasibility of a microfluidic flow focusing setup built from commercially available fittings to encapsulate phages into liposomes. Two types of Pseudomonas phages, PEV2 (Podovirus, ∼65 nm) and PEV40 (Myovirus, ∼220 nm), were used as model phages. A mixture of soy phosphatidylcholine and cholesterol at a ratio of 4:1 dissolved in absolute ethanol with a total solid content of 17.5 mg/mL was injected through the center inlet channel of a cross mixer. Phage suspensions were injected into the cross mixer from the two side channels intersecting with the center channel. The total flow rate (TFR) varied 160-320 µL/min and the organic/aqueous flow rate ratio (FRR) varied 1:3-2:3. The size of liposomes and the encapsulation efficiency both increased with increasing FRR and slightly decreased with increasing TFR. Due to the different size of the two studied phages, the size of liposomes encapsulating PEV2 were smaller (135-218 nm) than those encapsulating the Myovirus PEV40 (261-448 nm). Highest encapsulation efficiency of PEV2 (59%) and PEV40 (50%) was achieved at a TFR of 160 µL/ml and a FRR of 2:3. Generally, the encapsulation efficiency was slightly higher than that obtained from the conventional thin film hydration followed by extrusion method. In summary, the proposed microfluidic technique was capable of encapsulating phages of different size into liposomes with reasonable encapsulation efficiency and minimal titer reduction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study aimed to assess the robustness of using a spray drying approach and formulation design in producing inhalable phage powders. Two types of Pseudomonas phages, PEV2 (Podovirus) and PEV40 (Myovirus) in two formulations containing different amounts of trehalose (70% and 60%) and leucine (30% and 40%) were studied. Most of the surface of the produced powders was found to be covered in crystalline leucine. The powders were stored at 4 °C and 20 °C under vacuum. The phage stability and in vitro aerosol performance of the phage powders were examined on the day of production and after 1, 3 and 12 months of storage. A minor titer loss during production was observed for both phages (0.2-0.8 log 10 pfu/ml). The storage stability of the produced phage powders was found to be phage and formulation dependent. No further reduction in titer occurred for PEV2 powders stored at 4 °C across the study. The formulation containing 30% leucine maintained the viability of PEV2 at 20 °C, while the formulation containing 40% leucine gradually lost titer over time with a storage reduction of ∼0.9 log 10 pfu/ml measured after 12 months. In comparison, the PEV40 phage powders generally had a ∼ 0.5 log 10 pfu/ml loss upon storage regardless of temperature. When aerosolized, the total in vitro lung doses of PEV2 were of the order of 10 7  pfu, except the formulation containing 40% leucine stored at 20 °C which had a lower lung dose. The PEV40 powders also had lung doses of 10 6 -10 7  pfu. The results demonstrate that spray dried Myoviridae and Podoviridae phage in a simple formulation of leucine and trehalose can be successfully stored for one year at 4 °C and 20 °C with vacuum packaging., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study aimed to develop inhalable powders containing phages active against antibiotic-resistant Pseudomonas aeruginosa for pulmonary delivery. A Pseudomonas phage, PEV2, was spray dried into powder matrices comprising of trehalose (0-80%), mannitol (0-80%) and l-leucine (20%). The resulting powders were stored at various relative humidity (RH) conditions (0, 22 and 60% RH) at 4°C. The phage stability and in vitro aerosol performance of the phage powders were examined at the time of production and after 1, 3 and 12 months storage. After spray drying, a total of 1.3 log titer reduction in phage was observed in the formulations containing 40%, 60% and 80% trehalose, whereas 2.4 and 5.1 log reductions were noted in the formulations containing 20% and no trehalose, respectively. No further reduction in titer occurred for powders stored at 0 and 22% RH even after 12 months, except the formulation containing no trehalose. The 60% RH storage condition had a destructive effect such that no viable phages were detected after 3 and 12 months. When aerosolised, the total lung doses for formulations containing 40%, 60% and 80% trehalose were similar (in the order of 10 5 pfu). The results demonstrated that spray drying is a suitable method to produce stable phage powders for pulmonary delivery. A powder matrix containing ≥40% trehalose provided good phage preservation and aerosol performances after storage at 0 and 22% RH at 4°C for 12 months., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Inhaled delivery of drugs incorporated into poly (lactic-co-glycolic acid) (PLGA) microparticles allows a sustained lung concentration and encourages phagocytosis by alveolar macrophages that harboring Mycobacterium tuberculosis. However, limited data are available on the effects of physicochemical properties of PLGA, including the monomer ratio (lactide:glycide) and molecular weight (MW) on the aerosol performance, macrophage uptake, and toxicity profile. The present study aims to address this knowledge gap, using PLGAs with monomer ratios of 50:50, 75:25 and 85:15, MW ranged 24 - 240kDa and an anti-tuberculosis (TB) drug, rifapentine. The PLGA-rifapentine powders were produced through a solution spray drying technique. The particles were spherical with a smooth surface and a volume median diameter around 2μm (span ~2). When the powders were dispersed using an Osmohaler(®) at 100L/min for 2.4s, the fine particle fraction (FPFtotal, wt.% particles in aerosol <5μm relative to the total recovered drug mass) was ranged between 52 and 57%, with no significant difference between the formulations. This result suggests that the monomer ratio and MW are not crucial parameters for the aerosol performance of PLGA. The phagocytosis analysis was performed using Thp-1 monocyte-derived macrophages. The highest rate of uptake was observed in PLGA 85:15 followed by 75:25 and 50:50 with about 90%, 80% and 70%, respectively phagocytosis over 4h of exposure. Furthermore, the cytotoxicity analysis on Thp-1 and human lung adenocarcinoma epithelial cells demonstrated that PLGA concentration up to 1.5mg/mL, regardless of the monomer composition and MW, were non-toxic. In conclusion, the monomer ratio and MW are not crucial in determining the aerosol performance and cytotoxicity profile of PLGA however, the particles with high lactide composition have a superior tendency for macrophage uptake., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Currently bronchial provocation testing (BPT) using mannitol powder cannot be performed in children under 6 years. A primary reason is it is challenging for children at this age to generate a consistent inspiratory effort to inhale mannitol efficiently from a dry powder inhaler. A prototype system, which does not require any inhalation training from the pediatric subject, is reported here. It uses an external source of compressed air to disperse mannitol powder into a commercial holding chamber. Then the subject uses tidal breathing to inhale the aerosol., Method: The setup consists of a commercially available powder disperser and Volumatic™ holding chamber. Taguchi experimental design was used to identify the effect of dispersion parameters (flow rate of compressed air, time compressed air is applied, mass of powder, and the time between dispersion and inhalation) on the fine particle dose (FPD). The prototype was tested in vitro using a USP throat connected to a next generation impactor. The aerosols from the holding chamber were drawn at 10 L/min. A scaling factor for estimating the provoking dose to induce a 15% reduction in forced expiratory volume in 1 second (FEV1) (PD15) was calculated using anatomical dimensions of the human respiratory tract at various ages combined with known dosing values from the adult BPT., Results: Consistent and doubling FPDs were successfully generated based on the Taguchi experimental design. The FPD was reliable over a range of 0.8 (±0.09) mg to 14 (±0.94) mg. The calculated PD15 for children aged 1-6 years ranged from 7.1-30 mg. The FPDs generated from the proposed set up are lower than the calculated PD15 and therefore are not expected to cause sudden bronchoconstriction., Conclusion: A prototype aerosol delivery system has been developed that is consistently able to deliver doubling doses suitable for bronchial provocation testing in young children.