# US 12,180,461 B2 · Charcoal-free culture media for legionella

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- Number: US 12,180,461 B2
- Document ID: US12180461B2
- Status: Granted
- Kind: B2
- Application number: 17/270,620
- Filed: 2019-08-23
- Published: 2021-07-22
- Granted: 2024-12-31
- Earliest claimed: 2018-08-24
- Assignees: Secretary of State for Health and Social Care
- Applicants: Secretary of State for Health and Social Care
- Inventors: Victoria Jane Chalker; Owen Brad Spiller; Edward Portal
- Web page: https://patentgrep.com/patent/US12180461B2

## CPC

- C12N 1/20: Bacteria; Culture media therefor
- C12Q 1/045: {Culture media therefor}
- A61K 9/0078: {for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions}
- A61K 33/14: Alkali metal chlorides; Alkaline earth metal chlorides
- A61P 31/04: Antibacterial agents
- C12Q 1/18: Testing for antimicrobial activity of a material
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention provides in vitro methods for culturing Legionella, comprising incubating a Legionella bacterium on a charcoal-free solid agar medium comprising: serum, wherein the serum is present at a concentration of 1%-35% (v/v); a nitrogen source; and an iron source. Charcoal-free solid agar media for use in methods of the invention are also provided.

## Claims

### Claim 1

An in vitro method for culturing a Legionella bacterium, comprising incubating a Legionella bacterium on a charcoal-free solid agar medium comprising:
a) serum, wherein the serum is present at a concentration of 1%-35% (v/v);
b) a nitrogen source; and
c) an iron source.

### Claim 2

A screening method for identifying an antibiotic suitable for suppressing the growth of a Legionella bacterium, comprising:
a. contacting a Legionella bacterium with a charcoal-free solid agar medium to provide a test sample, wherein the charcoal-free solid agar medium comprises:
i. serum, wherein the serum is present at a concentration of 1%-35% (v/v);
ii. a nitrogen source; and
iii. an iron source;
b. incubating the test sample in the presence of a candidate antibiotic; and
c. identifying said candidate antibiotic as suitable for suppressing the growth of the Legionella bacterium when the bacterial load of said Legionella bacterium in the test sample subsequent to incubation is lower than a bacterial load of said Legionella bacterium in a control sample, wherein the control sample is incubated in the absence of said candidate antibiotic; or
d. identifying said candidate antibiotic as unsuitable for suppressing the growth of the Legionella bacterium when the bacterial load of said Legionella bacterium in the test sample subsequent to incubation is the same as or greater than the bacterial load of said Legionella bacterium in a control sample, wherein the control sample is incubated in the absence of said candidate antibiotic.

### Claim 3

A kit comprising a charcoal-free solid agar medium for culturing a Legionella bacterium, and/or reagents for preparing a charcoal-free solid agar medium for culturing the Legionella bacterium, wherein:
a. said charcoal-free solid agar medium comprises:
i. serum, wherein the serum is present at a concentration of 1%-35% (v/v);
ii. a nitrogen source; and
iii. an iron source; and/or
b. said reagents comprise:
i. agar;
ii. serum;
iii. a nitrogen source; and
iv. an iron source; and
wherein said kit further comprises instructions for culturing the Legionella bacterium on said charcoal-free solid agar medium.

### Claim 4 (depends on 2)

The screening method according to claim 2, wherein said method further comprises the step of recording data obtained in step (c) and/or step (d) on a suitable data carrier.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said nitrogen source is one or more of yeast extract, beef extract, liver extract, casein hydrolysate, plant cell extract, soy bean extract or any combination thereof.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the iron source is an iron chelate and/or ferric pyrophosphate.

### Claim 7 (depends on 1)

The method according to claim 1, wherein:
a. the agar is present at a concentration of 0.2%-5% (w/v);
b. the nitrogen source is present at a concentration of 0.2%-10% (w/v); and/or
c. the iron source is present at a concentration of 0.01%-0.1% (w/v).

### Claim 8 (depends on 1)

The method according to claim 1, wherein said charcoal-free solid agar medium further comprises one or more agent comprising:
a. a buffer;
b. alpha-ketoglutarate; and/or
c. cysteine.

### Claim 9 (depends on 1)

The method according to claim 1, wherein said charcoal-free solid agar medium further comprises one or more agent comprising:
a. a buffer that is present at a concentration of 1 mM-500 mM;
b. alpha-ketoglutarate that is present at a concentration of 0.001%-1% (w/v); and/or
c. cysteine that is present at a concentration of 1%-10% (w/v).

### Claim 10 (depends on 1)

The method according to claim 1, wherein said serum is one or more of horse serum, sheep serum, goat serum, bovine serum, fetal bovine serum, calf serum, mouse serum, rat serum, pig serum, guinea pig serum, porcine serum, or rabbit serum.

### Claim 11 (depends on 1)

The method according to claim 1, wherein said serum is horse serum.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the Legionella bacterium is one or more of the following species: L. adelaidensis, L. anisa, L. beliardensis, L. birminghamensis, L. bozemanae, L. brunensis, L. busanensis, L. cherrii, L. cincinnatiensis, L. donaldonsii, L. dresdeniensis, L. dumoffii, L. erythra, L. fairfieldensis, L. feeleii, L. geestiana, L. gormanii, L. gratiana, L. gresilensis, L. hackeliae sg1, L. impletisoli, L. israelensis, L. jamestowniensis, L. jordanis, L. lansingensis, L. londiniensis, L. longbeachae, L. maceachernii, L. micdadei, L. micdadei, L. moravica, L. pneumophila, L. nagasakiensis, L. taurinensis, L. wadsworthii, or L. yabuuchiae.

### Claim 13 (depends on 2)

The screening method according to claim 2, wherein said nitrogen source is one or more of yeast extract, beef extract, liver extract, casein hydrolysate, plant cell extract, soy bean extract or any combination thereof.

### Claim 14 (depends on 2)

The screening method according to claim 2, wherein the iron source is an iron chelate and/or ferric pyrophosphate.

### Claim 15 (depends on 2)

The screening method according to claim 2, wherein:
a. the agar is present at a concentration of 0.2%-5% (w/v);
b. the nitrogen source is present at a concentration of 0.2%-10% (w/v); and/or
c. the iron source is present at a concentration of 0.01%-0.1% (w/v).

### Claim 16 (depends on 2)

The screening method according to claim 2, wherein said charcoal-free solid agar medium further comprises one or more agent comprising:
a. a buffer;
b. alpha-ketoglutarate; and/or
c. cysteine.

### Claim 17 (depends on 2)

The screening method according to claim 2, wherein said charcoal-free solid agar medium further comprises one or more agent comprising:
a. a buffer that is present at a concentration of 1 mM-500 mM;
b. alpha-ketoglutarate that is present at a concentration of 0.001%-1% (w/v); and/or
cysteine that is present at a concentration of 1%-10% (w/v).

### Claim 18 (depends on 2)

The screening method according to claim 2, wherein said serum is one or more of horse serum, sheep serum, goat serum, bovine serum, fetal bovine serum, calf serum, mouse serum, rat serum, pig serum, guinea pig serum, porcine serum, or rabbit serum.

### Claim 19 (depends on 2)

The screening method according to claim 2, wherein said serum is horse serum.

### Claim 20 (depends on 2)

The screening method according to claim 2, wherein the Legionella is one or more of the following species: L. adelaidensis, L. anisa, L. beliardensis, L. birminghamensis, L. bozemanae, L. brunensis, L. busanensis, L. cherrii, L. cincinnatiensis, L. donaldonsii, L. dresdeniensis, L. dumoffii, L. erythra, L. fairfieldensis, L. feeleii, L. geestiana, L. gormanii, L. gratiana, L. gresilensis, L. hackeliae sgl, L. impletisoli, L. israelensis, L. jamestowniensis, L. jordanis, L. lansingensis, L. londiniensis, L. longbeachae, L. maceachernii, L. micdadei, L. micdadei, L. moravica, L. pneumophila, L. nagasakiensis, L. taurinensis, L. wadsworthii, or L. yabuuchiae.