# US 2005/0118567 A1 · Method for determining sensitivity to a bacteriophage

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- Number: US 2005/0118567 A1
- Document ID: US20050118567A1
- Status: Published application
- Kind: A1
- Application number: 10/498,428
- Filed: 2003-01-23
- Published: 2005-06-02
- Earliest claimed: 2002-01-23
- Inventors: Carl Merril; Sankar Adhya; Dean Scholl
- Web page: https://patentgrep.com/patent/US20050118567A1

## CPC

- G01N 33/56911: {Bacteria}
- A61K 35/76: Viruses; Subviral particles; Bacteriophages
- A61P 31/04: Antibacterial agents
- C12Q 1/6897: involving reporter genes operably linked to promoters
- G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures}
- A61K 2039/5256: {expressing foreign proteins}
- C12N 2710/00032: Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

This disclosure provides methods of selecting a therapeutic bacteriophage and identifying bacteria in a sample. The sample may be obtained from a plant or animal subject diagnosed with a disease caused by a bacterial infection or an object suspected of being exposed to a bacterium. The activity of reporter molecules, either encoded in the bacteriophage genome or added during sample analysis, is used to determine whether bacteriophages are capable of infecting assayed bacteria. Also provided are methods of selecting a bacteriophage for potential use in treating of bacterial infection, based upon the selectivity of the bacteriophage host range for the bacterium. The bacteriophages or bacteria may be immobilized in an array, such that multiple bacteriophages and/or bacteria may be assayed. Kits are also provided.

## Claims

### Claim 1

A method for selecting a bacteriophage capable of infecting a target bacterium, comprising:
contacting at least two different bacteriophages with a sample from a subject comprising the target bacterium in the presence of a reporter capable of generating a detectable signal in response to a lytic activity of a bacteriophage; and
detecting whether the signal is generated, the detection of the signal indicating that the bacteriophage is capable of infecting the target bacterium.

### Claim 2 (depends on 1)

The method of claim 1, wherein each bacteriophage comprises a nucleic acid encoding the reporter, wherein the reporter is expressed upon infection of the target bacterium by the bacteriophage such that the activity of the reporter is detected.

### Claim 3 (depends on 1)

The method of claim 1, wherein the reporter is added to the sample prior to detection of the reporter signal.

### Claim 4 (depends on 1)

The method of claim 1 wherein the at least two different bacteriophages are selected from the group consisting of: mycobacteriophage, K1, K5, K1-5, SP6, T4, T7, ENB6, A511, L5, and IRA.

### Claim 5 (depends on 1)

The method of claim 1 wherein the reporter is luciferase, green fluorescent protein, P-galactosidase, or chloramphenicol acetyl transferase.

### Claim 6 (depends on 1)

The method of claim 1 wherein the target bacterium comprises a bacterium of the genus Escherichia, Shigella, Salmonella, Erwinia, Yersinia, Bacillus, Vibrio, Legionella, Pseudomonas, Neisseria, Bordetella, Helicobacter, Listeria, Agrobacterium, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Corynebacterium, Mycobacterium, Treponema, Borrelia, Francisella, or Brucella.

### Claim 7 (depends on 1)

The method of claim 1, wherein contacting at least two different bacteriophages comprises contacting the sample with at least 10 different bacteriophages.

### Claim 8 (depends on 1)

The method of claim 1, wherein contacting at least two different bacteriophages comprises contacting the sample with at least 25 different bacteriophages.

### Claim 9 (depends on 1)

The method of claim 1, wherein contacting at least two different bacteriophages comprises contacting the sample with at least 50 different bacteriophages.

### Claim 10 (depends on 1)

The method of claim 1, wherein contacting at least two different bacteriophages comprises contacting the sample with at least 100 different bacteriophages.

### Claim 11 (depends on 1)

The method of claim 1, wherein contacting at least two different bacteriophages comprises contacting the sample with at least 500 different bacteriophages.

### Claim 12 (depends on 1)

The method of claim 1, wherein the sample is obtained from a subject.

### Claim 13 (depends on 12)

The method of claim 12 wherein the sample comprises a cell, tissue, secretion or exudate, fluid, gastric contents, blood, lymph, urine, a skin scrape or swab, serum, plasma, cerebrospinal fluid, saliva, sputum, stool, vomitus, milk, tears, or sweat.

### Claim 14 (depends on 12)

The method of claim 12, wherein the subject is a mammal.

### Claim 15 (depends on 14)

The method of claim 14, wherein the mammal is a human.

### Claim 16 (depends on 12)

The method of claim 12, wherein the subject is suspected of having a bacterial infection.

### Claim 17 (depends on 12)

The method of claim 12, wherein the subject has been diagnosed with a disease caused by a bacterial infection.

### Claim 18 (depends on 1)

The method of claim 1, wherein the sample is obtained from an object suspected to be contaminated with a biowarfare agent.

### Claim 19 (depends on 18)

The method of claim 18, wherein the sample is obtained by swabbing the object or collecting liquid wash applied to the object.

### Claim 20 (depends on 1)

The method of claim 1 wherein the different bacteriophages are contained in separate wells of a multi-well plate.

### Claim 21 (depends on 1)

The method of claim 1 wherein the number of bacteria in the sample is expanded in culture prior to selection.

### Claim 22 (depends on 16)

The method of claim 16, wherein the bacteriophage capable of infecting the target bacterium is selected for use in treating the subject's bacterial infection.

### Claim 23 (depends on 22)

The method of claim 22, further comprising administering to the subject a therapeutically effective amount of a bacteriophage of the same type as the selected bacteriophage.

### Claim 24 (depends on 23)

The method of claim 23, wherein the bacteriophage administered to the subject is a recombinant bacteriophage.

### Claim 25 (depends on 23)

The method of claim 23, wherein the bacteriophage administered to the subject is a parent bacteriophage of the selected recombinant bacteriophage.

### Claim 26 (depends on 23)

The method of claim 23, wherein the bacteriophage administered to the subject is a native variant of the selected recombinant bacteriophage.

### Claim 27 (depends on 23)

The method of claim 23, wherein the subject is a plant.

### Claim 28 (depends on 23)

The method of claim 23, wherein the subject is a mammal.

### Claim 29 (depends on 28)

The method of claim 28, wherein the mammal is a human.

### Claim 30

A kit for selecting a bacteriophage capable of infecting a target bacterium, comprising:
at least two different bacteriophages;
a reporter capable of generating a detectable signal in response to an activity of the bacteriophages; and
instructions for contacting each the bacteriophages with a sample and for detecting the signal.

### Claim 31 (depends on 30)

The kit of claim 30, wherein each bacteriophage comprises a nucleic acid encoding the reporter, wherein the reporter is expressed upon infection of the target bacterium by the bacteriophage such that the activity of the reporter is detected.

### Claim 32 (depends on 31)

The kit of claim 31 further comprising means for detecting expression of the reporter.

### Claim 33 (depends on 30)

The kit of claim 30, wherein the reporter is added to the sample prior to detection of the reporter signal.

### Claim 34 (depends on 30)

The kit of claim 30, further comprising a substrate upon which the different bacteriophages can be separated and contacted with the sample.

### Claim 35 (depends on 30)

The kit of claim 30 wherein the kit comprises at least 24 different bacteriophages.

### Claim 36 (depends on 30)

The kit of claim 30 wherein the kit comprises at least 48 different bacteriophages.

### Claim 37 (depends on 30)

The kit of claim 30 wherein the kit comprises at least 96 different bacteriophages.

### Claim 38 (depends on 30)

The kit of claim 30 wherein the kit comprises an array of bacteriophages at addressable locations in the array.

### Claim 39 (depends on 30)

The kit of claim 30, comprising one or more bacteria known to fall within the host range of a provided bacteriophage as a positive control.

### Claim 40

A method of identifying a bacterium that falls within the host range of a bacteriophage, comprising:
separately contacting at least two different recombinant bacteriophages with a sample comprising a bacterium, where each bacteriophage comprises a nucleic acid encoding a reporter capable of being expressed when the bacteriophage infects a bacterial host cell that is characteristically infected by the bacteriophage; and
detecting whether the reporter is expressed, where expression of the reporter indicates that the bacterium falls within the host range of the bacteriophage.

### Claim 41 (depends on 40)

The method of claim 40, comprising more than two different recombinant bacteriophages.

### Claim 42 (depends on 41)

The method of claim 41 wherein the at least two different recombinant bacteriophages have overlapping host ranges.

### Claim 43 (depends on 42)

The method of claim 42, comprising at least 25 different recombinant bacteriophages.

### Claim 44 (depends on 43)

The method of claim 43, comprising at least 50 different recombinant bacteriophages.

### Claim 45 (depends on 44)

The method of claim 44, comprising at least 100 different recombinant bacteriophages.

### Claim 46 (depends on 45)

The method of claim 45, comprising at least 500 different recombinant bacteriophages.

### Claim 47 (depends on 40)

The method of claim 40 where each recombinant bacteriophage is individually isolated.

### Claim 48 (depends on 47)

The method of claim 47 where each recombinant bacteriophage is contained in a separate well of a multi-well plate.

### Claim 49

A method of treating a bacterial infection in a host, comprising:
obtaining bacterial pathogen cells from a host;
contacting different bacterial pathogen cells with different recombinant bacteriophages, where each recombinant bacteriophage comprises a nucleic acid encoding a reporter capable of being expressed when the recombinant bacteriophage infects a bacterial host cell within its host range;
selecting a bacteriophage that expresses the reporter, where expression of the reporter indicates that the selected bacteriophage is capable of infecting a bacterial host cell obtained from the host; and
administering a therapeutically effective amount of the selected bacteriophage to the host, thereby treating the bacterial infection in the host.

### Claim 50 (depends on 49)

The method of claim 49 where the host is an animal or a plant.

### Claim 51 (depends on 50)

The method of claim 50 where the host is a mammal.

### Claim 52 (depends on 51)

The method of claim 51 where the mammal is a human.

### Claim 53 (depends on 49)

The method of claim 49 wherein obtaining a bacterial pathogen cell from the host comprises obtaining a bacterial pathogen cell of the genus Escherichia, Shigella, Salmonella, Erwinia, Yersinia, Bacillus, Vibrio, Legionella, Pseudomonas, Neisseria, Bordetella, Helicobacter, Listeria, Agrobacterium, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Corynebacterium, Mycobacterium, Treponema, Borrelia, Francisella, or Brucella.

### Claim 54 (depends on 49)

The method of claim 49 wherein administering a therapeutically effective amount of the selected bacteriophage to the host comprises administering a bacteriophage of the same native type as the selected bacteriophage.

### Claim 55

A device for selecting bacteriophages that are capable of infecting target bacteria, the device comprising:
an array of recombinant bacteriophages at addressable locations in the array, wherein the bacteriophages each include a signal sequence that provides a detectable signal when a bacteriophage infects a bacterium.

### Claim 56 (depends on 49)

The device of claim 49, further comprising bacteria in at least some of the addressable locations of the array.

### Claim 57 (depends on 55)

The device of claim 55, wherein at least some of the different addressable locations of the array contain different bacteriophage types that are capable of selectively infecting different bacterial strains or species, such that the signal indicates the ability of the bacteriophage to infect the bacterial strain or species that is located at the address from which the signal is provided.