Andrew Jenkins

Enhancement of the antimicrobial properties of bacteriophage-K via stabilization using oil-in-water nano-emulsions

Bacteriophage therapy is a promising new treatment that may help overcome the threat posed by antibiotic‐resistant pathogenic bacteria, which are increasingly identified in hospitalized patients. The development of biocompatible and sustainable vehicles for incorporation of viable bacterial viruses into a wound dressing is a promising alternative. This article evaluates the antimicrobial efficacy of Bacteriophage K against Staphylococcus aureus over time, when stabilized and delivered via an oil‐in‐water nano‐emulsion. Nano‐emulsions were formulated via thermal phase inversion emulsification, and then bacterial growth was challenged with either native emulsion, or emulsion combined with Bacteriophage K. Bacteriophage infectivity, and the influence of storage time of the preparation, were assessed by turbidity measurements of bacterial samples. Newly prepared Bacteriophage K/nano‐emulsion formulations have greater antimicrobial activity than freely suspended bacteriophage. The phage‐loaded emulsions caused rapid and complete bacterial death of three different strains of S. aureus. The same effect was observed for preparations that were either stored at room temperature (18–20°C), or chilled at 4°C, for up to 10 days of storage. A response surface design of experiments was used to gain insight on the relative effects of the emulsion formulation on bacterial growth and phage lytic activity. More diluted emulsions had a less significant effect on bacterial growth, and diluted bacteriophage‐emulsion preparations yielded greater antibacterial activity. The enhancement of bacteriophage activity when delivered via nano‐emulsions is yet to be reported. This prompts further investigation into the use of these formulations for the development of novel anti‐microbial wound management strategies.

Development of an amperometric assay for phosphate ions in urine based on a chemically modified screen-printed carbon electrode.

An amperometric assay for the determination of inorganic phosphate (Pi) in urine has been developed without the need for sample preparation. A screen-printed carbon electrode modified with the electrocatalyst cobalt phthalocyanine (CoPC-SPCE) and covered with a cellulose acetate membrane (CAM) serves as the sensor. The sensor detects hydrogen peroxide (H(2)O(2)), which is produced as a result of the oxidative decarboxylation of pyruvate, catalyzed by pyruvate oxidase (PyOd), in the presence of Pi, oxygen, and cofactors. Following optimization of solution conditions, and in the presence of a urine sample, a linear range was found to exist between the rate of current increase and phosphate concentration over the range of 2.27 x 10(-5) to 1.81 x 10(-4)M, and the limit of detection was found to be 4.27 x 10(-6)M. The assay was applied to the determination of phosphate ions in the urine of a normal subject, and the mean concentration in unspiked urine was found to be 3.40 x 10(-5)M with a coefficient of variation of 8.0% (n=5). The mean recovery of phosphate added to urine samples was 98.7% with a coefficient of variation of 5.5% (n=3). To the authors knowledge, this is the first report of an amperometric assay for Pi that incorporates a CoPC-SPCE as the sensing device.

Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model.

The Galleria mellonella infection model was used to assess the in vivo efficacy of phage therapy against laboratory and clinical strains of Pseudomonas aeruginosa. In a first series of experiments, Galleria were infected with the laboratory strain P. aeruginosa PAO1 and were treated with varying multiplicity of infection (MOI) of phages either 2h post-infection (treatment) or 2h pre-infection (prevention) via injection into the haemolymph. To address the kinetics of infection, larvae were bled over a period of 24h for quantification of bacteria and phages. Survival rates at 24h when infected with 10 cells/larvae were greater in the prevention versus treatment model (47% vs. 40%, MOI=10; 47% vs. 20%, MOI=1; and 33% vs. 7%, MOI=0.1). This pattern held true when 100 cells/larvae were used (87% vs. 20%, MOI=10; 53% vs. 13%, MOI=1; 67% vs. 7%, MOI=0.1). By 24h post-infection, phages kept bacterial cell numbers in the haemolymph 1000-fold lower than in the non-treated group. In a second series of experiments using clinical strains to further validate the prevention model, phages protected Galleria when infected with both a bacteraemia (0% vs. 85%) and a cystic fibrosis (80% vs. 100%) isolate. Therefore, this study validates the use of G. mellonella as a simple, robust and cost-effective model for initial in vivo examination of P. aeruginosa-targeted phage therapy, which may be applied to other pathogens with similarly low infective doses.

Rapid electrochemical identification of pathogenic Candida species

This study describes the development of a novel assay to detect fungal DNA and identify the most clinically relevant invasive human pathogenic fungi to the species level using oligonucleotide probes, labelled with electrochemically active groups, and solid-state electrodes. A panfungal probe designed against the 18S rRNA gene region, capable of detecting all fungal pathogens tested, and species-specific probes, designed against the ITS2 region for detection of the five Candida species most commonly encountered in the clinical setting (Candida albicans, Candida glabrata, Candida parapsilosis species complex, Candida krusei and Candida tropicalis), are described. When tested with PCR-amplified DNA from both type and clinical strains of the relevant species, the probes were able to positively identify the relevant fungi, indicated by production of a current significantly elevated above the background reading. No cross-reactivity was observed with any of the species-specific probes when compared with nine non-target Candida species or in the presence of human DNA equivalent to an equal number of ITS2 targets. The panfungal probe gave results that were similarly positive against 15 other fungal species and also did not cross-react with human DNA. The limit of detection of the assay was shown to be approximately 1 genome equivalent for all probes using extracted genomic DNA.

Intelligent Wound Dressing for Therapeutic and Diagnostic Management of Wound Infection

Wound infection is a global problem and approximately 13,000 patients with burns required treatment in hospitals in England and Wales every year. Diagnosis of burn infection is problematic and currently diagnosed by clinical observation and judgement. Standard microbiological culture to identify causative pathogens usually take several days. If pathogens present, this will causes tissue damage by further colonization, extensive infection and formation of difficult-to-treat wound biofilm in wounds which inevitably require aggressive antibiotic treatments. Early indication of infection at point-of-care and ability to rapidly distinguish between infected and non-infected states of wound will help in clinical decision making, prevent over-management by inappropriate use of antibiotics, improve patient outcomes and reduce costs of treatment. Here we have develop an intelligent wound dressing that can detect the infection in wounds. The dressing is made of a hydrated agarose film in which the fluorescent dye containing vesicles were mixed with agarose and dispersed within the hydrogel matrix. The release of dye is triggered by interaction of vesicles with virulence factors, secreted in population-density-dependent fashion via quorum sensing, from pathogenic bacteria. Efficacy of dressing was tested with developed static wound biofilm model using clinical strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis. The dressing indicated a clear response when in contact with biofilms produced only by pathogenic strains of bacteria. Colorimetric detection on wound biofilms of prevalent pathogens (S. aureus, P. aeruginosa and E. faecalis) is also demonstrated using an ex-vivo porcine skin model of burn wound infection.