# US 2009/0258341 A1 · Compositions and Methods for Detecting Bacteria

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- Number: US 2009/0258341 A1
- Document ID: US20090258341A1
- Status: Published application
- Kind: A1
- Application number: 12/418,320
- Filed: 2009-04-03
- Published: 2009-10-15
- Earliest claimed: 2008-04-03
- Assignees: Colorado School of Mines
- Inventors: Kent J. Voorhees; Leah G. Luna
- Web page: https://patentgrep.com/patent/US20090258341A1

## 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)
- C12Q 1/18: Testing for antimicrobial activity of a material
- G01N 33/54306: {Solid-phase reaction mechanisms}
- G01N 33/56911: {Bacteria}
- C12N 2795/10211: Podoviridae
- C12N 2795/10245: Special targeting system for viral vectors
- 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
- 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)

## Abstract

Genetically modified bacteriophage and methods of using the same to detect bacterial types of interest are provided.

## Claims

### Claim 1

A method for determining the presence or absence of bacteria of a target bacterial type in a sample, comprising
(i) providing a sample;
(ii) contacting the sample with genetically modified bacteriophage that are selective for the target bacterial type under conditions that allow said genetically modified bacteriophage to infect said bacteria of the target bacterial type that may be present in said sample, thereby producing a bacteriophage exposed sample, wherein said genetically modified bacteriophage comprise a recombinant bacteriophage marker gene comprising a nucleic acid sequence encoding a bacteriophage marker operably linked to an expression control region that affects expression of said bacteriophage marker gene during the late eclipse or early latent period following infection of said bacteria of the target bacterial type by said genetically modified bacteriophage;
(iii) incubating said bacteriophage exposed sample for a period of time sufficient to allow said genetically modified bacteriophage to infect said bacteria of the target bacterial type that may be present in said sample; and
(iv) assaying said bacteriophage exposed sample for the bacteriophage marker encoded by said bacteriophage marker gene, wherein said assaying step does not detect the presence of said bacteriophage marker in the bacteriophage exposed sample in the absence of bacterial infection, and wherein the presence of said bacteriophage marker indicates the presence of said bacteria of the target bacterial type in said sample.

### Claim 2 (depends on 1)

The method according to claim 1, further comprising the use of a negative control, wherein steps (i)-(iv) are repeated with a control sample known to lack bacteria of the target bacterial type to confirm that said assaying step does not detect the presence of said bacteriophage marker in the bacteriophage exposed sample in the absence of bacterial infection.

### Claim 3 (depends on 1)

The method according to claim 1, wherein said nucleic acid sequence encoding a bacteriophage marker encodes an endogenous head assembly protein, and wherein said expression control region affects overexpression of said bacteriophage marker gene encoding said endogenous head assembly protein.

### Claim 4 (depends on 1)

The method according to claim 1, wherein said nucleic acid sequence encoding a bacteriophage marker encodes a heterologous product, and wherein said expression control region affects expression of said heterologous product during the late eclipse or early latent period following infection of bacteria of said target bacterial type.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said target bacterial type is selected from the group consisting of Yersinia pestis, Bacillus anthracis, Francisella tularensis, Burkholderia mallei, E. coli, and Staphylococcus.

### Claim 6 (depends on 1)

The method according to claim 1, wherein said genetically modified bacteriophage is a genetically modified φA1122.

### Claim 7 (depends on 1)

The method according to claim 1, wherein said genetically modified bacteriophage is a genetically modified φA1122, wherein said nucleic acid sequence encoding a bacteriophage marker encodes a φA1122 endogenous head assembly protein, and wherein said expression control region affects overexpression of said bacteriophage marker gene encoding said endogenous head assembly protein following infection of Yersinia pestis.

### Claim 8 (depends on 7)

The method according to claim 7, wherein said recombinant bacteriophage marker gene comprises upstream gene φ13-Laqlq-pTrc-rbs-gene φ13.

### Claim 9 (depends on 7)

The method according to claim 7, wherein said endogenous head assembly protein is the 15.8 kDa φA1122 head assembly protein.

### Claim 10 (depends on 1)

The method according to claim 1, wherein said bacteriophage marker is not surface exposed.

### Claim 11 (depends on 1)

The method according to claim 1, wherein said bacteriophage marker is not detectable on intact genetically modified bacteriophage by the use of an antibody that specifically binds to said bacteriophage marker.

### Claim 12 (depends on 1)

The method according to claim 1, wherein said assaying step involves phage lysis.

### Claim 13 (depends on 1)

The method according to claim 1, wherein said bacteria of the target bacterial type are present in said sample at a concentration of less than about 1000 cells/ml.

### Claim 14 (depends on 1)

The method according to claim 1, wherein said incubation step comprises a period of time less than the time required to initiate a second cycle of infection by progeny of said genetically modified bacteriophage.

### Claim 15 (depends on 1)

The method according to claim 1, wherein said incubation step comprises a period of time less than the replication time of said genetically modified bacteriophage.

### Claim 16 (depends on 1)

The method according to claim 1, wherein said incubation step comprises a period of time less than about 45 minutes.

### Claim 17 (depends on 1)

The method according to claim 1, wherein the period of time from said contacting step to the result of said assaying step comprises a period of time less about 45 minutes.

### Claim 18 (depends on 1)

The method according to claim 1, wherein said assaying step involves the use of at least one antibody to detect said bacteriophage marker.

### Claim 19 (depends on 18)

The method according to claim 18, wherein said antibody is a monoclonal antibody.

### Claim 20 (depends on 18)

The method according to claim 18, wherein said antibody is present on a lateral flow immunochromatography (LFI) apparatus or a SILAS surface.

### Claim 21

A genetically modified bacteriophage selective for a target bacterial type, comprising a recombinant bacteriophage marker gene, which marker gene comprises a nucleic acid sequence encoding a bacteriophage marker operably linked to an expression control region that is capable of affecting expression of said bacteriophage marker during the late eclipse or early latent period following infection of bacteria of said target bacterial type.

### Claim 22 (depends on 20)

A LFI device for detecting bacteria of a target bacterial type, comprising a first antibody that specifically binds to a bacteriophage marker, and a second antibody that specifically binds to said first antibody or to said bacteriophage marker, wherein said bacteriophage marker is produced during the late eclipse or early latent period following infection of bacteria of said target bacterial type by a genetically modified bacteriophage according to claim 20.

### Claim 23

A method for determining the resistance or susceptibility of a target bacterial type to an antibiotic, comprising:
(i) dividing a primary sample containing bacteria of said target bacterial type into a first and a second sample;
(ii) adding antibiotic to said first sample alone;
(iii) contacting said first and second samples with genetically modified bacteriophage that are selective for the target bacterial type under conditions that allow said genetically modified bacteriophage to infect said bacteria of the target bacterial type that are present in said second sample and may be present in said first sample, thereby producing first and second bacteriophage exposed samples, wherein said genetically modified bacteriophage comprise a recombinant bacteriophage marker gene comprising a nucleic acid encoding a bacteriophage marker operably linked to an expression control region that effects expression of said bacteriophage marker gene during the late eclipse or early latent period following infection of said bacteria of the target bacterial type by said genetically modified bacteriophage;
(iv) incubating said bacteriophage exposed samples for a period of time sufficient to allow said genetically modified bacteriophage to infect said bacteria of the target bacterial type present in said second sample;
(v) assaying said bacteriophage exposed samples for the bacteriophage marker encoded by said bacteriophage marker gene, wherein said assaying step does not detect the presence of said bacteriophage marker in the bacteriophage exposed sample in the absence of bacterial infection, wherein the presence of said bacteriophage marker indicates the presence of said bacteria of the target bacterial type in said samples; and
(vi) comparing the results of said assaying step for said first and second bacteriophage exposed samples, wherein decreased bacteriophage marker in said first bacteriophage exposed sample compared to said second bacteriophage exposed sample indicates that said target bacterial type is sensitive to said antibiotic.