Jamison Walker and Sandra Hope, Microbiology & Molecular Biology
My project proposal included the use of Darwin’s theory of evolution by natural selection to determine if genetic mutations will arise in Bacteriophages that would allow them to infect related strains in a species of bacteria. Bacteriophages naturally have a target host range that they can infect. Within species of bacteria, the phage has tail receptors that allow it to infect specific strains. The other strains of the bacteria will be unaffected by the phage. In phage therapy, multiple phages with varied host ranges are chosen to combat this problem. Some phages are more effective than others in killing a larger range of bacteria strains. My proposed project is to take a phage with its existing host range and, through evolution, expand its infection capabilities. To successfully change the genome of a virus, mutations must take place allowing the virus to infect new strains.
The bacteriophages that were used in attempt to mutate their genes to infect strains that were originally thought to not be included in their host range were Paenibacillus larvae phages. This specific family of phages targets a pathogenetic bacteria that infects Honey Bees causing a disease known as American Foulbrood Disease. A current phage therapy treatment is being explored to treat American Foulbrood. In phage therapy, three or more phages are chosen to make up what is known as a phage cocktail. The purpose of the phage cocktail is to be target all possible strains of the pathogen. A phage cocktail is required for a successful phage therapy because phages are selective in the target host. Depending on the phage, a phage can infect a wide range of strains in a host or select few. One major disadvantage to phage is the selectiveness of bacteriophages. If there exists an unclassified strain to which the phage therapy does not kill the infection will persist often times leading to the death of the host. When this occurs, new phages needed to be isolated to combat the bacteria with the newly developed phage. My project was to determine if I could mutate a known phage to broaden its host range so that the process of isolating brand-new phages to infect unknown hosts would no longer be needed. Because I had access to the phages that would be included in the phage therapy, I chose one of the three phages, C1.2 as my test subject to determine if mutations would arise expanding its host range.
In testing my proposal to mutate phages to expand their host range I tested three different methods. The first method that I employed was inoculating C1.2 in a Paenibacillus larvae strain that had been previously been determined as one of the strains that C1.2 couldn’t infect. Bacterial strain that was chosen was PL 354. To retrieve PL 354 from freezer stock a sterile loop was used to remove a part of the frozen PL 354 solution and then streaked to isolation on PBHI (Pig Brain Heart Infusion) agar plates containing 18.5g/L PBHI broth powder, 15g/L agar, 10ml of 20% glucose and 10mg/ml thiamine. The plates were then incubated for 4 days to allow the Paenibacillus larvae spores to activate generating vegetative bacteria. Once the bacteria became vegetative another sterile loop was taken and inoculated into 25ml of PBHI broth containing, 18.5g/L PBHI broth powder, 10ml of 20% glucose and 10mg/ml thiamine, and incubated overnight at 37°C. The bacteria wouldn’t sometimes grow within 24 hours due to the concentration of bacteria initially inoculated, but when this occur if the culture was allowed to sit in the incubator for another 24 hours generally there would be a large enough pure culture that could be used to mutate the phages. Once the overnight culture was made 12.5ml of PL 354 overnight bacterial culture was removed and placed into a new flask. 12.5ml of a prepared C1.2 lysate was then added into the flask containing the 12.5ml of overnight culture and allowed to incubate at 37°C for another 24 hours. Following the 24-hour incubation period with phage and bacterial culture the sample was then centrifuged for 40minutes at 4000rpm to precipitate any vegetative bacteria leaving a supernatant containing phage only. The supernatant was then run through a .45μm or a .22μm filter to ensure that no vegetative bacteria was included in the supernatant. This filtered solution is known as a phage lysate. The phage lysate was then plated on PBHI agar plates. In order to successfully plate the phage lysate 50μl of the phage lysate was inoculated into 4.5ml of a PL 354 overnight culture and allowed to incubate for 30 minutes before plating on to the PBHI agar plates. The plates were then incubated at 37°C with 5% CO2 for 24-48 hours. This procedure yielded no results of any kind. The results that I was looking for was if there were any phage plaques that appeared following the plating of phage on the test bacterial strain.
The second method that was employed was similar to the first with modifications that would allow the phage to successfully grow inside a Paenibacillus larvae strain prior to plating on plates containing PL 354. This protocol was carried about by inoculating 1ml of C1.2 phage lysate into the PL ATCC strain and allowed to incubate at 37°C for 24 hours. Once the incubation period was finished the solution containing C1.2 and PL ATCC was centrifuged for 40 minutes at 4000rpm to precipitate any vegetative bacteria inside the solution and then filtered through a .45μm or a .22μm filter to ensure that no vegetative bacteria remained in the solution. 50μl of phage lysate was then inoculated into 4.5ml of a PL 354 overnight culture and then plated on to PBHI agar plates. The plates were allowed to incubate at 37°C with 5% CO2 for 24-48 hours. This protocol yielded no results despite repeating the protocol 20 individual times. Different strains were also tested to determine if I could mutate C1.2 to include them in its host range. The bacterial strains also tested included PL 309, PL 314, PL 307, and PL 304. When testing PL 304 I did discover plaques following the 24-48-hour incubation period. When I tested PL 304, I plated it over 20 times with the hopes that one of them included a mutated phage. All 20 plates contained phage plaques and upon further research into the genomic analysis that had been performed on the discovery of other Paenibacillus larvae phages I discovered that Bryan Merrill, a former student, had discovered multiple prophages from PL 304 bacterial strains. Although there was a slight possibility that my phage mutated, but because the concentration of phage plaques was so similar on all 20 plates my mentor and I concluded that I had most likely induced the prophage to be expressed rather than my phage mutating.