Recent advances in therapeutic delivery systems of bacteriophage and bacteriophage-encoded endolysins

Antibiotics have been the cornerstone of clinical management of bacterial infection since their discovery in the early 20th century. However, their widespread and often indiscriminate use has now led to reports of multidrug resistance becoming globally commonplace. Bacteriophage therapy has undergone a recent revival in battle against pathogenic bacteria, as the self-replicating and co-evolutionary features of these predatory virions offer several advantages over conventional therapeutic agents. In particular, the use of targeted bacteriophage therapy from specialized delivery platforms has shown particular promise owing to the control of delivery location, administration conditions and dosage of the therapeutic cargo. This review presents an overview of the recent formulations and applications of such delivery vehicles as an innovative and elegant tool for bacterial control. </jats:p

Prevention of encrustation and blockage of urinary catheters by:Proteus mirabilis via pH-triggered release of bacteriophage

The crystalline biofilms of Proteus mirabilis can seriously complicate the care of patients undergoing long-term indwelling urinary catheterisation. Expression of bacterial urease causes a significant increase in urinary pH, leading to the supersaturation and precipitation of struvite and apatite crystals. These crystals become lodged within the biofilm, resulting in the blockage of urine flow through the catheter. Here, we describe an infection-responsive surface coating for urinary catheters, which releases a therapeutic dose of bacteriophage in response to elevated urinary pH, in order to delay catheter blockage. The coating employs a dual-layered system comprising of a lower hydrogel 'reservoir' layer impregnated with bacteriophage, capped by a 'trigger' layer of the pH-responsive polymer poly(methyl methacrylate-co-methacrylic acid) (EUDRAGIT®S 100). Evaluation of prototype coatings using a clinically reflective in vitro bladder model system showed that catheter blockage time was doubled (13 h to 26 h (P < 0.05)) under conditions of established infection (108 CFU ml-1) in response to a 'burst-release' of bacteriophage (108 PFU ml-1). Coatings were stable both in the absence of infection, and in the presence of urease-negative bacteria. Quantitative and visual analysis of crystalline biofilm reduction show that bacteriophage constitute a promising strategy for the prevention of catheter blockage, a clinical problem for which there is currently no effective control method

Development of an Infection-Responsive Fluorescent Sensor for the Early Detection of Urinary Catheter Blockage

Formation of crystalline biofilms following infection by <i>Proteus mirabilis</i> can lead to encrustation and blockage of long-term indwelling catheters, with serious clinical consequences. We describe a simple sensor, placed within the catheter drainage bag, to alert of impending blockage via a urinary color change. The pH-responsive sensor is a dual-layered polymeric “lozenge”, able to release the self-quenching dye 5(6)-carboxyfluorescein in response to the alkaline urine generated by the expression of bacterial urease. Sensor performance was evaluated within a laboratory model of the catheterized urinary tract, infected with both urease positive and negative bacterial strains under conditions of established infection, achieving an average “early warning” of catheter blockage of 14.5 h. Signaling only occurred following infection with urease positive bacteria. Translation of these sensors into a clinical environment would allow appropriate intervention before the occurrence of catheter blockage, a problem for which there is currently no effective control method

Limiting Pseudomonas aeruginosa Biofilm Formation Using Cold Atmospheric Pressure Plasma

We investigate the ability to disrupt and limit growth biofilms of Pseudomonas aeruginosa using application of cold atmospheric pressure (CAP) plasma. The effect of the bio-film's exposure to a helium (CAP) jet was assessed at varying time points during biofilm maturation. Results showed that the amount of time during biofilm growth that CAP pressure was applied has a crucial role on the ability of biofilms to mature and recover after CAP exposure. Intervention during the early stages of biofilm formation (0-8 h) results in a 4-5-log reduction in viable bacterial cells (measured at 24 h of incubation) relative to untreated biofilms. However, CAP treatment of biofilm at 12 h and above only results in a 2-log reduction in viable cells. This has potentially important implications for future clinical application of CAP to treat infected wounds

Thermally Triggered Release of the Bacteriophage Endolysin CHAPK and the Bacteriocin Lysostaphin for the Control of Methicillin Resistant Staphylococcus aureus (MRSA)

Staphylococcus aureus infections of the skin and soft tissue pose a major concern to public health, largely owing to the steadily increasing prevalence of drug resistant isolates. As an alternative mode of treatment both bacteriophage endolysins and bacteriocins have been shown to possess antimicrobial efficacy against multiple species of bacteria including otherwise drug resistant strains. Despite this, the administration and exposure of such antimicrobials should be restricted until required in order to discourage the continued evolution of bacterial resistance, whilst maintaining the activity and stability of such proteinaceous structures. Utilising the increase in skin temperature during infection, the truncated bacteriophage endolysin CHAPK and the staphylococcal bacteriocin lysostaphin have been co-administered in a thermally triggered manner from Poly(N-isopropylacrylamide) (PNIPAM) nanoparticles. The thermoresponsive nature of the PNIPAM polymer has been employed in order to achieve the controlled expulsion of a synergistic enzybiotic cocktail consisting of CHAPK and lysostaphin. The point at which this occurs is modifiable, in this case corresponding to the threshold temperature associated with an infected wound. Consequently, bacterial lysis was observed at 37°C, whilst growth was maintained at the uninfected skin temperature of 32°C.Data was collected through bacterial sampling and monitoring growth on agar plates (through colony counting). Data was collected through spectroscopic methods such as infrared spectroscopy and UV Vis spectroscopy including optical density.Spectroscopic data was processed using Microsoft excel and Prism software packages

Thermally Triggered Release of the Bacteriophage Endolysin CHAPK and the Bacteriocin Lysostaphin for the Control of Methicillin Resistant Staphylococcus aureus (MRSA)

Staphylococcus aureus infections of the skin and soft tissue pose a major concern to public health, largely owing to the steadily increasing prevalence of drug resistant isolates. As an alternative mode of treatment both bacteriophage endolysins and bacteriocins have been shown to possess antimicrobial efficacy against multiple species of bacteria including otherwise drug resistant strains. Despite this, the administration and exposure of such antimicrobials should be restricted until required in order to discourage the continued evolution of bacterial resistance, whilst maintaining the activity and stability of such proteinaceous structures. Utilising the increase in skin temperature during infection, the truncated bacteriophage endolysin CHAPK and the staphylococcal bacteriocin lysostaphin have been co-administered in a thermally triggered manner from Poly(N-isopropylacrylamide) (PNIPAM) nanoparticles. The thermoresponsive nature of the PNIPAM polymer has been employed in order to achieve the controlled expulsion of a synergistic enzybiotic cocktail consisting of CHAPK and lysostaphin. The point at which this occurs is modifiable, in this case corresponding to the threshold temperature associated with an infected wound. Consequently, bacterial lysis was observed at 37°C, whilst growth was maintained at the uninfected skin temperature of 32°C.Data was collected through bacterial sampling and monitoring growth on agar plates (through colony counting). Data was collected through spectroscopic methods such as infrared spectroscopy and UV Vis spectroscopy including optical density.Spectroscopic data was processed using Microsoft excel and Prism software packages

Study into the Kinetic Properties and Surface Attachment of a Thermostable Adenylate Kinase

Abstract: A thermostable adenylate kinase (tAK) has been used as model protein contaminant on surfaces, so used because residual protein after high temperature wash steps can be detected at extremely low concentrations. This gives the potential for accurate, quantitative measurement of the effectiveness of different wash processes in removing protein contamination. Current methods utilise non-covalent (physisorbtion) of tAK to surfaces, but this can be relatively easily removed. In this study, the covalent binding of tAK to surfaces was studied to provide an alternative model for surface contamination. Kinetic analysis showed that the efficiency of the enzyme expressed as the catalytic rate over the Michaelis constant (kcat/KM) increased from 8.45 ± 3.04 mM-1 s-1 in solution to 32.23 ± 3.20 or 24.46 ± 4.41 mM-1 s-1 when the enzyme was immobilised onto polypropylene or plasma activated polypropylene respectively. Maleic anhydride plasma activated polypropylene showed potential to provide a more robust challenge for washing processes as it retained significantly higher amounts of tAK enzyme than polypropylene in simple washing experiments. Inhibition of the coupled enzyme (luciferase/luciferin) system used for the detection of adenylate kinase activity, was observed for a secondary product of the reaction. This needs to be taken into consideration when using the assay to estimate cleaning efficacy.UV-Vis Luminescence spectroscopy IR spectroscopyData worked up in Microsoft Excel and presented in Origin Graphing Softwar