Improving the Thermal Stability of Bacteriophages Through the Novel Use of Ensilication®

Description

Ensilication is a sol-gel method whereby silanol groups are electrostatically bound to positively charged amino acid groups on the surface of the target biomolecule. The end result is the formation of a rigid silica shell around the target preventing it from physically unfolding due to temperature increases. This means that the ensilicated protein can be stored and transported without the cold chain. Here we propose the modification and use of our Ensilication method to improve the long-term thermal stability of bacteriophages K and MS2 at room temperature. Analytical techniques and in vitro bacterial culture will verify the ability and efficacy of the Ensilication method. We believe that the results from this study will prevent the need for cold chain of transportation and storage normally required for the use of bacteriophage therapeutics. In addition to this, we aim to visualise the Ensilication process for the first time, using electron microscopy. The data shows how bacteriophages were ensilicated and stored for long periods of time at room temperature while retaining their infectivity. Across the study phage K showed a loss of approximately 4 log in concentration. Considering how unstable the phages are at room temperature, both the ensilicated phages showed much greater resilience to extreme temperatures. No additional loss of titre after being heated at 90 ˚C for 30 minutes was observed when compared to loss already known from the Ensilication process. Although Ensilication results in an immediate loss of 4 log titre of the ensilicated bacteriophage, the survival of the phages at room temperature compared to native lysates proves that ensilicated samples can remain viable for use for extended periods of time without refrigeration.

Steps to reproduce

100µL/dish of bacterial overnight solution was added to 3mL/dish warm TSB containing 0.7%(w/v) TSA, left to stand for 10 minutes. 3 mL of inoculated 0.7% TSA solution added to each TS agar plate, left to set in sterile conditions. 100µL phage lysate added to 900µL SM buffer, for a 10x dilution; this was serially repeated to produce dilution range of 10-1>10-9. 10µL of each dilution pipetted, in triplicate, onto agar dishes with 3 dilutions per dish. Spots left to dry in sterile conditions before incubated (37˚C, 18 hours). After visible plaques were counted to obtain PFU/mL titre amount. Preparation of pre-hydrolysed silica: 40mL of a 1:1 mixture of tetraethyl orthosilicate and H2O, with 40µL of 32% HCl stirred at 700RPM for 40 minutes until solution became monophasic, stirring speed reduced to 125RPM for 20 minutes. 1 mL added to 10mL suspension of enriched filtered bacteriophage in 90mL of SM buffer. After stirring for 10 minutes reaction mixture vacuum filtered through 0.3µm glass fibre filters. Ensilicated phage powder then dried for 48 hours at room conditions before transferred to 1.5mL tubes for long term storage. A 10mg of ensilicated powder placed into 1mg/mL NaF in SM buffer, pH 2.90. Spun head over heels 1 hour before 2mL transferred to 40mL 1-hour-old bacteria. Incubated for 24 hours at 37˚C, 170RPM. Filtered through a 0.45µm filter before 4°C storage. SM buffer adjusted to pH6. 1:100 ratio of prehydrolysed silica to SM buffer. 1000 μL of enriched filtered phage K transferred to a 1.5 mL vial. 100 μL of prehydrolysed silica added. The vials gently inverted. After 45 seconds sample taken & blotted on freshly glow-discharged carbon grids. 1-2 drops of 1% uranyl acetate stain used. Each sample represented 60-seconds of Ensilication. Analysed with Tecnai 12 TEM with BioTwin Spirit objective lens & FEI Eagle 4kx4k CCD Camera at 120 kV acceleration voltage. Some sample times were repeated. Phage K heated at temperatures of 50, 55, 60, 65, 70 ˚C 1 hour. For each temperature point, 6 1mL samples of phage K placed into sealed vials and heated in furnace. At 10-minute intervals, a single vial was removed and stored at 4 ˚C until assayed. 1 mL phage and 50 mg of ensilicated phages placed in vials. Heated for 90°C/30 minutes. Ensilicated sample released and both samples plaque assayed. Ensilicated phage was ground and deposited on metal sample holders topped with carbon mica tape. Prepared samples and holders vacuum-dried overnight before imaging. Microscope used was JEOL FESEM6301F. Measured using DLS in solution to give hydrodynamic diameter in nm. All samples filtered through 0.22μm Millex syringe filters into polystyrene micro cuvettes that had been thoroughly rinsed with ddH2O and samples. Samples analysed using variety of sampling times and repeats at standard temperature and pressure, 173◦ measurement angle. Instrument used Malvern Zetasizer Nano ZS with 633 nm He-Ne laser.

Institutions

• University of Bath

Categories

Funders

• University of Bath Alumni Fund
• University of Bath

  United Kingdom

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