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US 11,952,611 B2Granted
Methods and systems for detection of antibiotic resistance
AssigneeLaboratory Corporation of America Holdings
InventorsMinh Mindy Bao Nguyen, Dwight L. Anderson, Jose S. Gil, Stephen E. Erickson, Matthew J. Brown
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Abstract
Disclosed herein are methods and systems for rapid detection of antibiotic resistance of a microorganism in a sample. A modified recombinant phage is also disclosed which comprises an indicator gene in the late gene region. The specificity of infectious agents allows a specific microorganism to be targeted, and an indicator signal may be amplified to optimize assay sensitivity.
Claims21
A method of detecting antibiotic-resistant bacteria in a sample comprising:
- (a) contacting the sample with an antibiotic,
- (b) contacting the sample with a recombinant bacteriophage specific to the bacteria, wherein the recombinant bacteriophage comprises a genetic construct inserted into a late gene region of the bacteriophage genome, the genetic construct comprising:
- (i) an indicator gene, wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein;
- and
- (ii) a promoter controlling transcription of the indicator gene, wherein the promoter is a bacteriophage late promoter,
- and wherein expression of the indicator gene results in an indicator protein product, and wherein the indicator protein product is a luciferase;
- (c) detecting a signal produced by the indicator protein product,
- wherein detection of the signal is used to determine the presence of antibiotic-resistant bacteria in the sample.
5 more elements
A method of determining effective dose of an antibiotic in killing bacteria comprising:
- (a) incubating each of one or more of antibiotic solutions separately with one or more samples comprising the bacteria, wherein the concentrations of the one or more of antibiotic solutions are different and define a range;
- (b) incubating the bacteria in the one or more samples with a recombinant bacteriophage specific to the bacteria, wherein the recombinant bacteriophage comprises a genetic construct inserted into a late gene region of the bacteriophage genome, the genetic construct comprising:
- (i) an indicator gene, wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein;
- and
- (ii) a promoter controlling transcription of the indicator gene, wherein the promoter is a bacteriophage late promoter,
- and wherein expression of the indicator gene results in an indicator protein product, and wherein the indicator protein product is a luciferase;
- and
- (c) detecting the indicator protein product, wherein detection of the indicator protein product in the one or more samples indicates the concentrations of antibiotic solutions used to treat the one or more samples are not effective, and the lack of detection of the indicator protein indicates the antibiotic is effective, thereby determining the effective dose of the antibiotic.
5 more elements
A kit for detecting antibiotic-resistant bacteria in a sample, wherein the kit comprises:
- (a) a recombinant bacteriophage specific to the bacteria, wherein the recombinant bacteriophage comprises a genetic construct inserted into a late gene region of the bacteriophage genome, the genetic construct comprising:
- (i) an indicator gene, wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein;
- and
- (ii) a promoter controlling transcription of the indicator gene, wherein the promoter is a bacteriophage late promoter, and wherein expression of the indicator gene results in an indicator protein product, and wherein the indicator protein product is a luciferase;
- and
- (b) an antibiotic.
3 more elements
A system for detecting antibiotic resistance of bacteria in a sample, comprising
- a recombinant bacteriophage specific to the bacteria, wherein the recombinant bacteriophage comprises a genetic construct inserted into a late gene region of the bacteriophage genome, the genetic construct comprising:
- (i) an indicator gene, wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein;
- and
- (ii) a promoter controlling transcription of the indicator gene, wherein the promoter is a bacteriophage late promoter,
- and wherein expression of the indicator gene results in an indicator protein product, and wherein the indicator protein product is a luciferase;
- a component for contacting the sample with the antibiotic,
- a component for contacting the sample with the recombinant bacteriophage specific to the bacteria;
- and
- a component for detecting the protein product in the sample that has contacted the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates the bacteria is resistant to the antibiotic.
6 more elements
Dependencies are inferred from the claim text.