Naing Tun Thet

Prototype Development of the Intelligent Hydrogel Wound Dressing and Its Efficacy in the Detection of Model Pathogenic Wound Biofilms

The early detection of wound infection in situ can dramatically improve patient care pathways and clinical outcomes. There is increasing evidence that within an infected wound the main bacterial mode of living is a biofilm: a confluent community of adherent bacteria encased in an extracellular polymeric matrix. Here we have reported the development of a prototype wound dressing, which switches on a fluorescent color when in contact with pathogenic wound biofilms. 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 static and dynamic models of wound biofilms, from clinical strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis, were established on nanoporous polycarbonate membrane for 24, 48, and 72 h, and the dressing response to the biofilms on the prototype dressing evaluated. The dressing indicated a clear fluorescent/color response within 4 h, only observed when in contact with biofilms produced by a pathogenic strain. The sensitivity of the dressing to biofilms was dependent on the species and strain types of the bacterial pathogens involved, but a relatively higher response was observed in strains considered good biofilm formers. There was a clear difference in the levels of dressing response, when dressings were tested on bacteria grown in biofilm or in planktonic cultures, suggesting that the level of expression of virulence factors is different depending of the growth mode. 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.

An in-situ infection detection sensor coating for urinary catheters

We describe a novel infection-responsive coating for urinary catheters that provides a clear visual early warning of Proteus mirabilis infection and subsequent blockage. The crystalline biofilms of P. mirabilis can cause serious complications for patients undergoing long-term bladder catheterisation. Healthy urine is around pH 6, bacterial urease increases urine pH leading to the precipitation of calcium and magnesium deposits from the urine, resulting in dense crystalline biofilms on the catheter surface that blocks urine flow. The coating is a dual layered system in which the lower poly(vinyl alcohol) layer contains the self-quenching dye carboxyfluorescein. This is capped by an upper layer of the pH responsive polymer poly(methyl methacrylate-co-methacrylic acid) (Eudragit S100®). Elevation of urinary pH (>pH 7) dissolves the Eudragit layer, releasing the dye to provide a clear visual warning of impending blockage. Evaluation of prototype coatings using a clinically relevant in vitro bladder model system demonstrated that coatings provide up to 12 h advanced warning of blockage, and are stable both in the absence of infection, and in the presence of species that do not cause catheter blockage. At the present time, there are no effective methods to control these infections or provide warning of impending catheter blockage.

Photopolymerization of polydiacetylene in hybrid liposomes: Effect of polymerization on stability and response to pathogenic bacterial toxins

Liposomes containing lipids and polydiacetylene (PDA) are hybrid systems encompassing both a fluid phospholipid membrane and a polymer scaffold (PDA). However, the biophysical role of PDA in such liposomes is not well understood. In this report, we studied the effects of photopolymerization of PDA on the stability of lipid-PDA liposomes, and their sensitivity to selected purified toxins and bacterial supernatants, using a fluorescence assay. Of the three different types of liposomes with variable lipid chain lengths that were chosen, the degree of polymerization had a significant impact on the long-term stability, and response, to external microbial exotoxins secreted by pathogenic bacteria, namely, Staphylococcus aureus and Pseudomonas aeruginosa. The degree of polymerization of TCDA played an important role in lipid-chain-length-dependent stabilization of lipid-PDA liposomes, as well as in their response to bacterial toxins of S. aureus and P. aeruginosa.

Development of a mixed-species biofilm model and its virulence implications in device related infections: MIXED-SPECIES BIOFILM INFECTION MODEL AND THEIR VIRULENCE

It is becoming increasingly accepted that to understand and model the bacterial colonization and infection of abiotic surfaces requires the use of a biofilm model. There are many bacterial colonizations by at least two primary species, however this is difficult to model in vitro. This study reports the development of a simple mixed-species biofilm model using strains of two clinically significant bacteria: Staphylococcus aureus and Pseudomonas aeruginosa grown on nanoporous polycarbonate membranes on nutrient agar support. Scanning electron microscopy revealed the complex biofilm characteristics of two bacteria blending in extensive extracellular matrices. Using a prototype wound dressing which detects cytolytic virulence factors, the virulence secretion of 30 single and 40 mixed-species biofilms was tested. P. aeruginosa was seen to out-compete S. aureus, resulting in a biofilm with P. aeruginosa dominating. In situ growth of mixed-species biofilm under prototype dressings showed a real-time correlation between the viable biofilm population and their associated virulence factors, as seen by dressing fluorescent assay. This paper aims to provide a protocol for scientists working in the field of device related infection to create mixed-species biofilms and demonstrate that such biofilms are persistently more virulent in real infections.

Development of a High-Throughput ex-Vivo Burn Wound Model Using Porcine Skin, and Its Application to Evaluate New Approaches to Control Wound Infection

Biofilm formation in wounds is considered a major barrier to successful treatment, and has been associated with the transition of wounds to a chronic non-healing state. Here, we present a novel laboratory model of wound biofilm formation using ex-vivo porcine skin and a custom burn wound array device. The model supports high-throughput studies of biofilm formation and is compatible with a range of established methods for monitoring bacterial growth, biofilm formation, and gene expression. We demonstrate the use of this model by evaluating the potential for bacteriophage to control biofilm formation by Staphylococcus aureus, and for population density dependant expression of S. aureus virulence factors (regulated by the Accessory Gene Regulator, agr) to signal clinically relevant wound infection. Enumeration of colony forming units and metabolic activity using the XTT assay, confirmed growth of bacteria in wounds and showed a significant reduction in viable cells after phage treatment. Confocal laser scanning microscopy confirmed the growth of biofilms in wounds, and showed phage treatment could significantly reduce the formation of these communities. Evaluation of agr activity by qRT-PCR showed an increase in activity during growth in wound models for most strains. Activation of a prototype infection-responsive dressing designed to provide a visual signal of wound infection, was related to increased agr activity. In all assays, excellent reproducibility was observed between replicates using this model.

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.