This easy to perform protocol evaluates the antibacterial efficacy of diverse bacteriophage combinations, also known as phage cocktails, under varying conditions in a high-throughput setting. The main advantages of the technique is the ability to stream different biotics and abiotic factors, such as temperature, that may affect phage efficacy under a range of environmental conditions. This method can facilitate the design of appropriate phage cocktails to enhance biocontrol and therapeutic outcomes and determine the interaction between bacterial hosts and bacteriophages.
To begin with, set up the microplate assay using filtered tips to avoid cross contamination. First, add 180 microliters of mTSB in the wells of columns one to 12 of the 96-well microplate, then prepare serial tenfold dilutions of each phage in columns one to eight of the sterile 96-well microplates to set up the assay. Add 20 microliters of individual phages or phage cocktails to wells one to eight of the top row A of the microplate.
For phage-free and blank control, add 20 microliters of mTSB to the top well of columns nine to 12. While pipetting, mix the well contents with gentle and repeated aspiration at least five times and changing tips between dilutions. Remove 20 microliters from the last row H, then set up a reservoir for the tested bacterial strain by transferring two to three milliliters of the diluted culture into the reservoir using a one milliliter pipette.
Using a multichannel pipette, add 20 microliters of the diluted bacterial culture to each well in columns one to 10 while changing tips between each addition. Cover and incubate the microplate at 37 degree Celsius. At two hour intervals, remove the microplate from the incubator and read the plate using a microplate reader.
Alternatively, incubate the plate in a microplate reader and read it over time. To examine the optical density at 600 nanometers using a microplate reader, open the program on a microplate reader. Select the Simple Mode in the startup options window and then select Create a New Protocol in Task Manager.
From the Select Plate Type window, choose 96-well plate from the dropdown list. As detection method, select Absorbance for the read type, select Endpoint/Kinetic option for optics type, select Monochromators, and then click OK.For Read Step, enter 600 nanometers for wavelengths, select Normal for read speed and click OK.To set the temperature for the assay, click on the Incubate check box and select Incubator On.Enter the desired temperature in the temperature box and then click OK.Click on the Preheat before continuing with the next step check box for the 37 degree Celsius incubation temperature, though the option is not required for the test conditions at 22 degrees Celsius. To prevent the condensation on the plate lid during incubation, set the temperature gradient by entering a value in the gradient box and then click OK.Next, click on the Kinetic check box to open the kinetic setup.
Set the Run Time for 10 hours for 37 degree Celsius incubation or 22 hours for room temperature and enter two hours as the reading interval, then click on OK.Click on the Shake check box in the procedure window to set up the shake condition, if needed for each kinetic read. Select Linear as Shake Mode and change the Duration to 30 seconds. Set Linear Frequency value to 731 cycles per minute, and then click on OK.In the Protocol Summary Dialogue window, click on Plate Layout, select Blanks, Assay Controls, and Samples, and click Next.
After defining the settings for each well type, click on Finish. Select a well ID in the left-hand side interface, assign it to the matrix shown in the Plate Layout window, then click on OK.Click on the Read Plate button, save the protocol as a prt file and click on Save. Insert the plate and click on OK.Once the experiment is completed, save the experiment as a xpt file and click on Save.
Export the data to Excel by clicking on the Yes button in the message window box for further analysis. Click on the Save Yes/No selection and the don't ask again check box to save preference. Following the protocol, a comparison of phage efficacy with various phage combinations, temperatures, times, and multiplicity of infections, or MOIs, was performed.
Based on this analysis, anti-O157 phage efficacy was maximized after 14, 16, or 18 hours of incubation at 22 degrees Celsius. For understanding the phage killing kinetics of bacteria, OD values at 600 nanometers were plotted against sampling time, MOIs, and phage treatment. The representative bacterial growth curves at 37 degrees Celsius treated are shown.
T4 completely inhibited the bacterial growth at each sampling time even at low MOI. To analyze the phage inhibited bacterial growth, the mean OD values from all the MOIs were plotted against each sampling time and phage treatment. The representative graphs at two temperatures revealed that phage killing efficacy, for instance, for T1 plus T4 was enhanced at 22 degree Celsius compared to 37 degree Celsius.
The comparison of overall phage effectiveness is shown, which facilitates identifying the best phase treatment. For example, for Escherichia coli strain 3081, treatment with phages T4 and T5 plus T4 was the most effective treatment at 37 degree Celsius. However, at 22 degree Celsius, in addition to T4, phage T1 plus T4, T1 plus T4 plus rV5, and four phage cocktails were effective.
When performing this procedure, extremely accurate pipetting, particularly when using a multichannel pipette and uniformity of approach are essential to obtain comparable and interpretable results. Follow up experiments are required to assess the further performance of the most influential cocktail in the screening, preventing the emergence of antiphage mutants in an extensive broth culture system and other biological matrices.
