Investigation of the relative effects of Nano-emulsion and Bacteriophage / Nano-emulsion formulations on S. aureus and P. aeruginosa growth patterns in biofilms

The rise in antibiotic-resistant bacteria has received much attention over recent years, but the rate of development of new antibiotics to treat these emerging “superbugs” is very slow. The skin represents the primary defence mechanism against infection and hence injuries or burns are a significant pathway to bacterial infection. Burns are especially susceptible to colonisers such as Staphylococcus aureus, with 10% of all burns cases becoming infected. The consequences include significantly increased patient morbidity and mortality, and cost of treatment.
Bacteriophage therapy is a potential treatment that may help overcome the threat posed by antibiotic-resistant pathogenic bacteria, which are increasingly identified in hospitalised patients suffering from burn infections. The development of biocompatible and sustainable vehicles for incorporation of viable bacterial viruses into a responsive wound dressing is therefore of considerable interest.
This work has demonstrated that bacteriophage / nano-emulsion formulations have greater anti-microbial activity than freely suspended bacteriophage. These phage loaded emulsions cause rapid and complete bacterial death for three different strains of Staphylococcus aureus. The same effect is observed for preparations that are either stored at room temperature (18 – 20 °C), or chilled at 4°C, for up to 10 days of storage for two different strains of phage. Bacterial growth patterns are also slightly modified when nano-emulsions are present. Hence there is a need to understand the mechanisms by which these formulations cause enhanced antimicrobial activity, and separately their impact on bacterial growth.
A response surface design of experiments is used to gain initial understanding of the relative effects of the emulsion formulation on bacterial growth and phage lytic activity. We hypothesise that nano-emulsions have a double role in this complex system. Firstly, they prevent efficient uptake of nutrients by bacterial cells, although they do not completely inhibit growth. Secondly, they produce a “charge-shielding” effect on bacteriophages, leading to enhanced contact with bacterial cells, and also protecting the phage from adverse environmental conditions. The hypotheses are validated via measurements of size, concentration and zeta potential of the three different species in the system (bacteria, phage and emulsion droplets), as well as additional control experiments elucidating the effect of antibiotics when nano-emulsions were present. The system has been validated for three strains of Pseudomonas aeruginosa and its corresponding bacteriophages. Modelling approaches have also been developed for the analysis of the interactions in this complex system, and to inform the future application of such formulations in more realistic wound environments, such as biofilms.