Reactive oxygen and nitrogen species (RONS) are emerging as a novel antibacterial strategy to combat the alarming increase in antimicrobial resistance (AMR). RONS can inhibit bacterial growth through reactions with cellular molecules, compromising vital biological functions and leading to cell death. While their mechanisms of action have been studied, many remain unclear, especially in biologically relevant environments. In this study, we exposed Gram-positive and Gram-negative bacteria to varying RONS ratios, mimicking what microbes may naturally encounter. A ratio in favour of RNS induced membrane depolarization and pore formation, resulting in an irreversible bactericidal effect. By contrast, ROS predominance caused membrane permeabilization and necrotic-like responses, leading to biofilm formation. Furthermore, bacterial cells exposed to more RNS than ROS activated metabolic processes associated with anaerobic respiration, DNA & cell wall/membrane repair, and cell signalling. Our findings suggest that the combination of ROS and RNS can be an effective alternative to inhibit bacteria, but only under higher RNS levels, as ROS dominance might foster bacterial tolerance, which in the context of AMR could have devastating consequences.

Biomaterials capable of promoting wound healing and preventing infections remain in great demand to address the global unmet need for the treatment of chronic wounds. Phosphate-based glasses (PG) have shown potential as bioresorbable materials capable of inducing tissue regeneration, while being replaced by regenerated tissue and releasing therapeutic species. In this work, phosphate-glass-based fibers (PGF) in the system P2O5-CaO-Na2O added with 1, 2, 4, 6, and 10 mol % of the therapeutic metallic ions (TMI) Ag+, Zn2+, and Fe3+ were manufactured via electrospinning of coacervate gels. Coacervation is a sustainable, cost-effective, water-based method to produce PG. All TMI are effective in promoting wound closure (re-epithelialization) in living human skin ex vivo, where the best-performing system is PGF containing Ag+. In particular, PGF with ≥4 mol % of Ag+ is capable of promoting 84% wound closure over 48 h. These results are confirmed by scratch test migration assays, with the PGF-Ag systems containing ≥6 mol % of Ag+, demonstrating significant wound closure enhancement (up to 72%) after 24 h. The PGF-Ag systems are also the most effective in terms of antibacterial activity against both the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli. PGF doped with Zn2+ shows antibacterial activity only against S. aureus in the systems containing Zn2+ ≥ 10 mol %. In addition, PGF doped with Fe3+ rapidly accelerates ex vivo healing in patient chronic wound skin (>30% in 48 h), demonstrating the utility of doped PGF as a potential therapeutic strategy to treat chronic wounds.

Mesoporous glasses are a promising class of bioresorbable biomaterials characterized by high surface area and extended porosity in the range of 2 to 50 nm. These peculiar properties make them ideal materials for the controlled release of therapeutic ions and molecules. Whilst mesoporous silicate-based glasses (MSG) have been widely investigated, much less work has been done on mesoporous phosphate-based glasses (MPG). In the present study, MPG in the P2O5-CaO-Na2O system, undoped and doped with 1, 3, and 5 mol% of Cu ions were synthesized via a combination of the sol-gel method and supramolecular templating. The non-ionic triblock copolymer Pluronic P123 was used as a templating agent. The porous structure was studied via a combination of Scanning Electron Microscopy (SEM), Small-Angle X-ray Scattering (SAXS), and N2 adsorption-desorption analysis at 77 K. The structure of the phosphate network was investigated via solid state 31P Magic Angle Spinning Nuclear Magnetic Resonance (31P MAS-NMR) and Fourier Transform Infrared (FTIR) spectroscopy. Degradation studies, performed in water via Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES), showed that phosphates, Ca2+, Na+ and Cu ions are released in a controlled manner over a 7 days period. The controlled release of Cu, proportional to the copper loading, imbues antibacterial properties to MPG. A significant statistical reduction of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) bacterial viability was observed over a 3 days period. E. coli appeared to be more resistant than S. aureus to the antibacterial effect of copper. This study shows that copper doped MPG have great potential as bioresorbable materials for controlled delivery of antibacterial ions.

There is a great demand from patients requiring skin repair, as a result of poorly healed acute wounds or chronic wounds. These patients are at high risk of constant inflammation that often leads to life-threatening infections. Therefore, there is an urgent need for new materials that could rapidly stimulate the healing process and simultaneously prevent infections. Phosphate-based coacervates (PC) have been the subject of increased interest due to their great potential in tissue regeneration and as controlled delivery systems. Being bioresorbable, they dissolve over time and simultaneously release therapeutic species in a continuous manner. Of particular interest is the controlled release of metallic antibacterial ions (e.g. Ag + ), a promising alternative to conventional treatments based on antibiotics, often associated with antibacterial resistance (AMR). This study investigates a series of PC gels containing a range of concentrations of the antibacterial ion Ag + (0.1, 0.3 and 0.75 mol%). Dissolution tests have demonstrated controlled release of Ag + over time, resulting in a significant bacterial reduction (up to 7 log), against both non-AMR and AMR strains of both Gram-positive and Gram-negative bacteria ( Staphylococcus aureus , Enterococcus faecalis , Escherichia coli and Pseudomonas aeruginosa ). Dissolution tests have also shown controlled release of phosphates, Ca 2+ , Na + and Ag + with most of the release occurring in the first 24 h. Biocompatibility studies, assessed using dissolution products in contact with human keratinocyte cells (HaCaT) and bacterial strains, have shown a significant increase in cell viability ( p ≤ 0.001) when gels are dissolved in cell medium compared to the control . These results suggest that gel-like silver doped PCs are promising multifunctional materials for smart wound dressings, being capable of simultaneously inhibit pathogenic bacteria and maintain good cell viability.

There is an increasing demand for new biomaterials that both rapidly stimulate healing and prevent infections. Being bioresorbable, phosphate-glass fibres can simultaneously induce tissue regeneration and deliver therapeutic agents (e.g. antibacterial ions) in a controlled way. Here we present a series of copper-doped phosphate-based glass fibres in the P2O5-CaO-Na2O-(CuO)(x) system (x = 0, 1, 3, and 5 mol%) prepared for the first time via electrospinning of coacervate precursors. This method presents several advantages over the conventional high temperature melt spinning; the synthesis of the coacervate occurs at room temperature and in aqueous solution allowing the incorporation of temperature sensitive molecules. Moreover, electrospinning is an inexpensive, sustainable, easily scalable manufacturing process. The fibres produced are cotton-like, fully amorphous with an average diameter in the range 1-3 mu m. Dissolution studies on cations (Ca2+, Na+ and Cu2+) and phosphate anions (PO43-, P2O74-, and P3O105-) show a gradual release of all species in solution over time, ideal for controlled drug delivery applications. The antibacterial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria was found to increase with increasing Cu2+ content and was found to be more effective against E. coli than against S. aureus. Interestingly, Cu2+ content seem to have a positive effect on cytocompatibility. Tests performed on osteoblast-like MG63 osteosarcoma cells have shown that the copper doped phosphate-glass fibres have a significantly better capacity to regenerate bone tissue than the undoped glass fibres. These findings suggest that phosphate-based glass fibres are promising multifunctional biomaterials for both antibacterial activity and tissue regeneration.

Calcium phosphate glasses are a promising new generation of biomaterials that can simultaneously induce tissue regeneration and controlled release of therapeutic molecules. In this work, novel calcium phosphate glasses containing 0, 2, 4, and 6 mol % Cu2+ were synthesized via room temperature precipitation reaction in aqueous solution. The effect of Cu2+ addition on the glass properties and structure was investigated using thermal analysis, 31P solid-state MAS NMR, Raman spectroscopy, and X-ray diffraction. All glasses crystallize at temperature >500 °C and are mainly formed by Q1 groups. The release of P, Ca, and Cu in solution over time was monitored via inductively coupled plasma-optical emission spectroscopy. It was found that with increasing Cu content, the amount of P and Ca released decreases whereas the amount of Cu released increases. The effect of Cu2+ release on the antibacterial activity against S. aureus, a bacterial strain commonly found in postsurgery infections, has been investigated. The addition of copper has been shown to infer the glasses antibacterial properties. As expected, the antibacterial activity of the glasses increases with increasing Cu2+ content. Cytocompatibility was assessed by seeding human osteoblast-like osteosarcoma cells Saos-2 (HTB85) on the glass particles. A significant increase in cell number was observed in all the glasses investigated. The copper-doped calcium phosphate glasses have proven to be multifunctional, as they combine bone regenerative properties with antibacterial activity. Therefore, they have great potential as antibacterial bioresorbable materials for hard tissue regeneration.

The World Health Organization warns that the alarming increase in antibiotic resistant bacteria will lead to 2.7 million deaths annually due to the lack of effective antibiotic therapies. Clearly, there is an urgent need for short-term alternatives that help to alleviate these alarming figures. In this respect, the scientific community is exploring neglected ecological niches from which the prototypical antibiotic-producing bacteria Streptomycetes are expected to be present. Recent studies have reported that honeybees and their products carry Streptomyces species that possess strong antibacterial activity. In this study, we have investigated the antibiotic profile of two Streptomycetes strains that were isolated from beehives. One of the isolates is the strain Streptomyces albus AN1, which derives from pollen, and shows potent antimicrobial activity against Candida albicans. The other isolate is the strain Streptomyces griseoaurantiacus AD2, which was isolated from honey, and displays a broad range of antimicrobial activity against different Gram-positive bacteria, including pathogens such as Staphylococcus aureus and Enterococus faecalis. Cultures of S. griseoaurantiacus AD2 have the capacity to produce the antibacterial compounds undecylprodigiosin and manumycin, while those of S. albus AN1 accumulate antifungal compounds such as candicidins and antimycins. Furthermore, genome and dereplication analyses suggest that the number of putative bioactive metabolites produced by AD2 and AN1 is considerably high, including compounds with anti-microbial and anti-cancer properties. Our results postulate that beehives are a promising source for the discovery of novel bioactive compounds that might be of interest to the agri-food sector and healthcare pharmaceuticals.