# US 8,785,148 B2 · Method and device for rapid detection of bacterial antibiotic resistance/susceptibility

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- Number: US 8,785,148 B2
- Document ID: US8785148B2
- Status: Granted
- Kind: B2
- Application number: 13/283,892
- Filed: 2011-10-28
- Published: 2012-05-17
- Granted: 2014-07-22
- Earliest claimed: 2009-05-05
- Assignees: Fraunhofer USA, Inc.; Trustees of Boston University
- Applicants: Alexis Sauer-Budge; Andre Sharon; Maxim Kalashnikov; Holger Wirz
- Inventors: Alexis Sauer-Budge; Andre Sharon; Maxim Kalashnikov; Holger Wirz
- Web page: https://patentgrep.com/patent/US8785148B2

## CPC

- C12Q 1/18: Testing for antimicrobial activity of a material
- A61P 31/04: Antibacterial agents
- G01N 33/582: {with fluorescent label}
- 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
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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

Described herein is a method and a device for expediting delivery of an agent to a damaged bacterial cell. In one embodiment, the methods and devices are useful for screening candidate antibiotics. In another embodiment, the methods and devices described herein are used to determine susceptibility of bacteria to an antibiotic. The methods also provide a method for determining an appropriate antibiotic to treat an individual having a bacterial infection.

## Claims

### Claim 1

A method for determining sensitivity of bacteria to an antibiotic, the method comprising:
(a) immobilizing the bacteria from a bacterial suspension by covalent attachment to a solid support,
(b) contacting said immobilized bacteria with an agent comprising a reporter moiety, which preferentially binds to damaged bacterial cells,
(c) subjecting said immobilized bacteria to a stressor in the presence of an antibiotic without the need for bacterial growth phase,
(d) detecting a signal from said reporter moiety in said immobilized bacteria wherein said detecting is performed between 1 and 30 minutes from contacting said covalently immobilized bacteria with the stressor in the presence of the antibiotic, and
(e) comparing the signal from said immobilized bacteria in the presence of the antibiotic to a control comprising immobilized bacteria subjected to the stressor in the absence of the antibiotic,
wherein detection of an increase in said signal in the presence of an antibiotic compared to a control indicates that said bacteria are susceptible to said antibiotic, and wherein a signal that is comparable to the control in the presence of an antibiotic indicates that said bacteria are resistant to said antibiotic.

### Claim 2 (depends on 1)

The method of claim 1, wherein said detecting is performed between 5 and 25 minutes from contacting said covalently immobilized bacteria with the agent and the stressor in the presence or absence of the antibiotic.

### Claim 3 (depends on 1)

The method of claim 1, wherein said detecting step comprises detecting fluorescence.

### Claim 4 (depends on 1)

The method of claim 1, wherein said reporter moiety comprises a fluorescent dye.

### Claim 5 (depends on 4)

The method of claim 4, wherein said fluorescent dye detects gram negative and/or gram positive bacteria.

### Claim 6 (depends on 4)

The method of claim 4, wherein said fluorescent dye is SYTOX green.

### Claim 7 (depends on 4)

The method of claim 4, wherein said fluorescent dye is DiBAC4(3).

### Claim 8 (depends on 1)

The method of claim 1, wherein said stressor comprises physical stress.

### Claim 9 (depends on 1)

The method of claim 1, wherein said stressor comprises chemical stress.

### Claim 10 (depends on 1)

The method of claim 1, wherein said stressor comprises physical and chemical stress.

### Claim 11 (depends on 1)

The method of claim 1, wherein said stressor comprises lysostaphin, an endolysin, lysozyme, oxidative stress or a porin.

### Claim 12 (depends on 1)

The method of claim 1, wherein said stressor comprises shear stress, osmotic stress, acidic pH or basic pH.

### Claim 13 (depends on 1)

The method of claim 1, wherein said method comprises a high-throughput method.

### Claim 14 (depends on 1)

The method of claim 1, wherein said bacteria are pathogenic bacteria.

### Claim 15 (depends on 14)

The method of claim 14, wherein said pathogenic bacteria is one that infects or causes disease in a human host.

### Claim 16 (depends on 1)

The method of claim 1, wherein the solid support is glass.