Furanone loaded aerogels are effective antibiofilm therapeutics in a model of chronic Pseudomonas aeruginosa wound infection.

Almost 80% of chronic wounds have a bacterial biofilm present. These wound biofilms are caused by a range of organisms and are often polymicrobial. Pseudomonas aeruginosa is one of the most common causative organisms in wound infections and readily forms biofilms in wounds. To coordinate this, P. aeruginosa uses a process known as quorum sensing. Structural homologues of the quorum sensing signalling molecules have been used to disrupt this communication and prevent biofilm formation by Pseudomonas. However, these compounds have not yet reached clinical use. Here, we report the production and characterisation of a lyophilised PVA aerogel for use in delivering furanones to wound biofilms. PVA aerogels successfully release a model antimicrobial and two naturally occurring furanones in an aqueous environment. Furanone loaded aerogels inhibited biofilm formation in P. aeruginosa by up to 98.80%. Further, furanone loaded aerogels successfully reduced total biomass of preformed biofilms. Treatment with a sotolon loaded aerogel yielded a 5.16 log reduction in viable biofilm bound cells in a novel model of chronic wound biofilm, equivalent to the current wound therapy Aquacel AG. These results highlight the potential utility of aerogels in drug delivery to infected wounds and supports the use of biofilm inhibitory compounds as wound therapeutics.

The Effect of High Protein Powder Structure on Hydration, Glass Transition, Water Sorption, and Thermomechanical Properties.

Poor solubility of high protein milk powders can be an issue during the production of nutritional formulations, as well as for end-users. One possible way to improve powder solubility is through the creation of vacuoles and pores in the particle structure using high pressure gas injection during spray drying. The aim of this study was to determine whether changes in particle morphology effect physical properties, such as hydration, water sorption, structural strength, glass transition temperature, and α-relaxation temperatures. Four milk protein concentrate powders (MPC, 80%, w/w, protein) were produced, i.e., regular (R) and agglomerated (A) without nitrogen injection and regular (RN) and agglomerated (AN) with nitrogen injection. Electron microscopy confirmed that nitrogen injection increased powder particles' sphericity and created fractured structures with pores in both regular and agglomerated systems. Environmental scanning electron microscopy (ESEM) showed that nitrogen injection enhanced the moisture uptake and solubility properties of RN and AN as compared with non-nitrogen-injected powders (R and A). In particular, at the final swelling at over 100% relative humidity (RH), R, A, AN, and RN powders showed an increase in particle size of 25, 20, 40, and 97% respectively. The injection of nitrogen gas (NI) did not influence calorimetric glass transition temperature (Tg), which could be expected as there was no change to the powder composition, however, the agglomeration of powders did effect Tg. Interestingly, the creation of porous powder particles by NI did alter the α-relaxation temperatures (up to ~16 °C difference between R and AN powders at 44% RH) and the structural strength (up to ~11 °C difference between R and AN powders at 44% RH). The results of this study provide an in-depth understanding of the changes in the morphology and physical-mechanical properties of nitrogen gas-injected MPC powders.

Exploiting a Rose Bengal-bearing, oxygen-producing nanoparticle for SDT and associated immune-mediated therapeutic effects in the treatment of pancreatic cancer.

Sonodynamic therapy (SDT) is an emerging stimulus-responsive approach for the targeted treatment of solid tumours. However, its ability to generate stimulus-responsive cytotoxic reactive oxygen species (ROS), is compromised by tumour hypoxia. Here we describe a robust means of preparing a pH-sensitive polymethacrylate-coated CaO2 nanoparticle that is capable of transiently alleviating tumour hypoxia. Systemic administration of particles to animals bearing human xenograft BxPC3 pancreatic tumours increases oxygen partial pressures (PO2) to 20-50 mmHg for over 40 min. RT-qPCR analysis of expression of selected tumour marker genes in treated animals suggests that the transient production of oxygen is sufficient to elicit effects at a molecular genetic level. Using particles labelled with the near infra-red (nIR) fluorescent dye, indocyanine green, selective uptake of particles by tumours was observed. Systemic administration of particles containing Rose Bengal (RB) at concentrations of 0.1 mg/mg of particles are capable of eliciting nanoparticle-induced, SDT-mediated antitumour effects using the BxPC3 human pancreatic tumour model in immuno-compromised mice. Additionally, a potent abscopal effect was observed in off-target tumours in a syngeneic murine bilateral tumour model for pancreatic cancer and an increase in tumour cytotoxic T cells (CD8+) and a decrease in immunosuppressive tumour regulatory T cells [Treg (CD4+, FoxP3+)] was observed in both target and off-target tumours in SDT treated animals. We suggest that this approach offers significant potential in the treatment of both focal and disseminated (metastatic) pancreatic cancer.

Microscopic Investigation of the Combined Use of Antibiotics and Biosurfactants on Methicillin Resistant Staphylococcus aureus.

One current strategy to deal with the serious issue of antibiotic resistance is to use biosurfactants, weak antimicrobials in their own right, with antibiotics in order to extend the efficacy of antibiotics. Although an adjuvant effect has been observed, the underlying mechanisms are poorly understood. To investigate the nature of the antibiotic and biosurfactant interaction, we undertook a scanning electron microscopy (SEM) and atomic force microscopy (AFM) microscopic study of the effects of the tetracycline antibiotic, combined with sophorolipid and rhamnolipid biosurfactants, on Methicillin-resistant Staphylococcus aureus using tetracycline concentrations below and above the minimum inhibitory concentration (MIC). Control and treated bacterial samples were prepared with an immersion technique by adsorbing the bacteria onto glass substrates grafted with the poly-cationic polymer polyethyleneimine. Bacterial surface morphology, hydrophobic and hydrophilic surface characters as well as the local bacterial cell stiffness were measured following combined antibiotic and biosurfactant treatment. The sophorolipid biosurfactant stands alone insofar as, when used with the antibiotic at sub-MIC concentration, it resulted in bacterial morphological changes, larger diameters (from 758 ± 75 to 1276 ± 220 nm, p-value = 10-4) as well as increased bacterial core stiffness (from 205 ± 46 to 396 ± 66 mN/m, p-value = 5 × 10-5). This investigation demonstrates that such combination of microscopic analysis can give useful information which could complement biological assays to understand the mechanisms of synergy between antibiotics and bioactive molecules such as biosurfactants.

Inactivation of the dnaK gene in Clostridium difficile 630 Δerm yields a temperature-sensitive phenotype and increases biofilm-forming ability.

Clostridium difficile infection is a growing problem in healthcare settings worldwide and results in a considerable socioeconomic impact. New hypervirulent strains and acquisition of antibiotic resistance exacerbates pathogenesis; however, the survival strategy of C. difficile in the challenging gut environment still remains incompletely understood. We previously reported that clinically relevant heat-stress (37-41 °C) resulted in a classical heat-stress response with up-regulation of cellular chaperones. We used ClosTron to construct an insertional mutation in the dnaK gene of C. difficile 630 Δerm. The dnaK mutant exhibited temperature sensitivity, grew more slowly than C. difficile 630 Δerm and was less thermotolerant. Furthermore, the mutant was non-motile, had 4-fold lower expression of the fliC gene and lacked flagella on the cell surface. Mutant cells were some 50% longer than parental strain cells, and at optimal growth temperatures, they exhibited a 4-fold increase in the expression of class I chaperone genes including GroEL and GroES. Increased chaperone expression, in addition to the non-flagellated phenotype of the mutant, may account for the increased biofilm formation observed. Overall, the phenotype resulting from dnaK disruption is more akin to that observed in Escherichia coli dnaK mutants, rather than those in the Gram-positive model organism Bacillus subtilis.