Methods of expanding bacteriophage host-range and bacteriophage produced by the methods
View Patent ↗A method of producing novel bacteriophages with expanded host-range and bacteriophages with expanded host ranges are disclosed. The method produces mutant phage strains which are infectious to a second host and can be more infectious to their natural host than in their natural state. The method includes repeatedly passaging a selected phage strain into bacterial cultures that contain varied ratios of its natural host bacterial strain with a bacterial strain that the phage of interest is unable to infect; the target-host. After each passage the resulting phage are purified and screened for activity against the target-host via double-overlay assays. When mutant phages that are shown to infect the target-host are discovered, they are further propagated in culture that contains only the target-host to produce a stock of the resulting mutant phage.
1. A method of producing bacteriophages with expanded host-range, comprising:
determine that a selected phage strain infects a host bacterial strain and not a target-host bacterial strain, wherein the selected phage strain is a Clostridium sporogenes B1 phage or a Clostridium sporogenes B3 phage, the host bacterial strain is a first Clostridium sporogenes strain, and the target-host bacterial strain is a second Clostridium sporogenes strain;
culture the host bacterial strain and the target-host bacterial strain separately to obtain a host culture and a target-host culture, respectively;
mix the host culture and the target-host culture into various co-culture ratios of each, thereby obtaining a series of a plurality of first co-cultures;
add the selected phage strain to each of the plurality of first co-cultures;
add a predetermined mutagen to each of the plurality of first co-cultures, wherein the predetermined mutagen comprises mitomycin C;
incubate under bacterial culture conditions;
harvest a resulting phage from each of the plurality of first co-cultures and purify; and
apply the resulting phage to a subsequent set of co-cultures identical to the first co-cultures but naive to the phage.
2. A method of producing bacteriophages with expanded host-range, comprising:
determine that a selected phage strain infects a host bacterial strain and not a target-host bacterial strain, wherein the selected phage strain is a Clostridium sporogenes B1 phage or a Clostridium sporogenes B3 phage, the host bacterial strain is a first Clostridium sporogenes strain, and the target-host bacterial strain is a second Clostridium sporogenes strain;
subject the selected phage in bacterial co-cultures consisting of various ratios of the host bacterial strain and the target-host bacterial strain;
separate the host bacterial strain, the target-host bacterial strain, and a resulting phage;
assay the resulting phage against naïve host and target-host to determine if a host-expansion event has occurred;
repeat phage purification, passaging, and assaying for host-expansion into target-host until there is evidence of infecting activity against target-host bacterial strain; and
isolate a mutant phage and cultivate in naïve target-host culture to produce a population of phage mutant.
3. The method of claim 2 , further comprising:
combining one or more populations of mutant phages to produce a mutant phage cocktail.
4. The method of claim 1 , wherein the series of the plurality of first co-cultures comprises a ratio of 1:0, 9:1, 1:1, 1:9, and 0:1 of the host bacterial strain:the target-host bacterial strain.
5. The method of claim 1 , wherein the incubate step is performed under static, anaerobic conditions.
6. The method of claim 1 , wherein the resulting phage is a mutant phage comprising ssDNA genome.
7. The method of claim 1 , further comprising, after the apply step:
isolate the resulting phage; and
ensure the resulting phage infects the target-host bacterial strain, thereby identifying a host-expanded mutant phage.
8. The method of claim 7 , further comprising, after the ensure step:
propagate further the host-expanded mutant phage in a target-host culture.
9. The method of claim 1 , further comprising, after the apply step:
passaging the resulting phage cultivated in each co-culture ratio.
10. The method of claim 2 , wherein the bacterial co-cultures comprises a ratio of 1:0, 9:1, 1:1, 1:9, and 0:1 of the host bacterial strain:the target-host bacterial strain.
11. The method of claim 2 , wherein the subject step is performed under static, anaerobic conditions.
12. The method of claim 2 , wherein the mutant phage comprises ssDNA genome.
13. The method of claim 2 , further comprising, after the isolate step:
propagate further the mutant phage in a target-host culture.