# US 12,110,511 B2 · Methods and culture media for production, quantification and isolation of bacteriophages

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- Number: US 12,110,511 B2
- Document ID: US12110511B2
- Status: Granted
- Kind: B2
- Application number: 16/966,472
- Filed: 2019-01-31
- Published: 2021-02-18
- Granted: 2024-10-08
- Earliest claimed: 2018-01-31
- Assignees: JyväSkyläN Yliopisto
- Applicants: Jyväskylän yliopisto
- Inventors: Lotta-Riina Sundberg; Gabriel Almeida; Elina Laanto
- Web page: https://patentgrep.com/patent/US12110511B2

## CPC

- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 2795/00021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2795/00051: Methods of production or purification of viral material
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention is directed to a method for producing bacteriophages infecting a bacterial host such as Flavobacterium columnare or Aeromonas sp, the method comprising the steps of adding the bacterium to a sterile culture medium supplemented with mucin or another mucus component, incubating said culture medium, thereby obtaining a bacterial culture, optionally collecting the supernatant containing floating (planktonic) bacterial cells from said culture and transferring the supernatant to a new container in order to discard most of the biofilm and adding a bacteriophage infecting the bacterium to said bacterial culture obtained from a previous step, and incubating the culture until the phage yield increases or peaks. The method can also be used for preparing modified enrichment cultures for optimized phage isolation. Addition of mucin to soft-agar culture medium is proposed as an optimized technique for viral titration. This invention also covers all relevant steps in setting up a phage therapy product: phage production, phage quantification and phage isolation.

## Claims

### Claim 1

A method for producing bacteriophages comprising the steps of:
a) adding bacterial cells to a sterile culture medium supplemented with mucin,
b) incubating said culture medium, thereby obtaining a bacterial culture modified by mucin exposure,
c) optionally removing a biofilm from said bacterial culture,
d) adding a bacteriophage capable of infecting the bacterial cells to the bacterial culture obtained from step b) or c), and
e) incubating the bacterial culture obtained in step d) until the culture is ready for phage collection.

### Claim 2 (depends on 1)

The method according to claim 1, further comprising:
f) preparing a bacteriophage extract from the bacterial culture obtained in step e), thereby producing a stock of bacteriophages.

### Claim 3 (depends on 2)

The method according to claim 2, further comprising:
g) preparing a sterile semi-solid culture medium supplemented with mucin,
h) adding aliquots of bacterium grown in a culture medium and a dilution of the bacteriophage stock obtained in step f) to an aliquot of the semi-solid culture medium obtained in step g) and plating the mix on an agar plate or a Petri dish; or alternatively adding an aliquot of bacterium grown in a culture medium to an aliquot of the semi-solid culture medium obtained in step g) and plating the mix on an agar plate or a Petri dish and adding a dilution of the bacteriophage stock obtained in step f) on top of the plated mix;
i) repeating the previous step with a different dilution of said bacteriophage stock, wherein each mix is plated on a separate agar plate or Petri dish;
j) incubating the plates obtained in steps h) and i); and
k) determining the number of bacteriophage particles in the stock by counting plaques on the plates.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the bacteriophage added to said bacterial culture in step d) is provided by an environmental sample or any sample suspected of containing bacteriophages infecting the tested bacterial host.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said bacterial cells represent a strain of Flavobacterium columnare or Aeromonas sp.

### Claim 6

A method for determining the number of plaque-forming phage particles in a bacteriophage stock or a sample comprising or suspected to comprise bacteriophages capable of infecting a bacterial host, the method comprising the steps of:
a) preparing a sterile semi-solid culture medium supplemented with mucin,
b) adding aliquots of the bacterial host grown in a culture medium and a dilution of a bacteriophage stock or sample to an aliquot of the semi-solid culture medium obtained in step a) and plating the mix on an agar plate or a Petri dish; or alternatively adding an aliquot of bacterium grown in a culture medium to an aliquot of the semi-solid culture medium obtained in step a) and plating the mix on an agar plate or a Petri dish and adding a dilution of the bacteriophage stock or sample on top of the plated mix;
c) repeating the previous step with a different dilution of said bacteriophage stock or sample, wherein each mix is plated on a separate agar plate or Petri dish;
d) incubating the plates obtained in steps b) and c); and
e) determining the number of bacteriophage particles in the stock or sample by counting plaques on the plates.

### Claim 7 (depends on 6)

The method according to claim 6, wherein said bacterial host is a strain of Flavobacterium columnare or Aeromonas sp.

### Claim 8

A culture medium for producing a stock of bacteriophages comprising essential nutritional ingredients for the growth of a bacterial host and mucin as a supplement, wherein said culture medium is a liquid medium, a solid medium, or a soft-agar medium, and wherein said culture medium is prepared according to Shieh recipe or Reasoner's 2A agar recipe.

### Claim 9 (depends on 8)

The culture medium according to claim 8 comprising 0.001-1% (w/v) mucin.

### Claim 10 (depends on 9)

The culture medium according to claim 9 comprising 0.01-0.2% (w/v) mucin.

### Claim 11 (depends on 8)

The culture medium according to claim 8, wherein said mucin is purified mucin.