Methods and systems for detection of antibiotic resistance
Inventors
Nguyen, Minh Mindy Bao • Anderson, Dwight L. • Gil, Jose S. • Erickson, Stephen E. • Brown, Matthew J.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11952611-B2
Publication Date
2024-04-09
Expiration Date
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.
Core Innovation
The disclosed invention provides methods for detecting antibiotic-resistant bacteria in a sample by contacting the sample with an antibiotic and contacting the sample with a recombinant bacteriophage specific to the bacteria. The recombinant bacteriophage includes a genetic construct inserted into a late gene region of the bacteriophage genome, and the genetic construct comprises an indicator gene that is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein, together with a promoter controlling transcription of the indicator gene, wherein the promoter is a bacteriophage late promoter.
Expression of the indicator gene results in an indicator protein product, and the indicator protein product is a luciferase. A signal produced by the indicator protein product is detected, and detection of the signal is used to determine the presence of antibiotic-resistant bacteria in the sample. In related aspects, the detection signal is compared with an experimentally determined value derived from a control sample, including defining a background threshold based on average background signal and standard deviation terms.
The invention further includes using the same recombinant bacteriophage indicator approach to determine effective dose of an antibiotic in killing bacteria. Antibiotic solutions are incubated separately with samples comprising the bacteria over a range of different concentrations, and the bacteria in the samples are incubated with the recombinant bacteriophage. Detecting the indicator protein product indicates that the concentrations used are not effective, while lack of detection indicates that the antibiotic is effective, thereby determining the effective dose.
Claims Coverage
The independent claims cover three main areas: detecting antibiotic-resistant bacteria in a sample, determining an effective dose of an antibiotic, and providing kit and system formats. Across the independent claims, there are inventive features centered on a recombinant bacteriophage genetic construct in a late gene region that drives expression of a luciferase indicator protein product for signal-based interpretation.
Late gene region luciferase indicator phage genetic construct
A recombinant bacteriophage specific to the bacteria comprising a genetic construct inserted into a late gene region of the bacteriophage genome, the genetic construct comprising an indicator gene that is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein, and a promoter controlling transcription of the indicator gene wherein the promoter is a bacteriophage late promoter, wherein expression of the indicator gene results in an indicator protein product that is a luciferase.
Antibiotic contact and signal detection to indicate resistance
A method comprising contacting the sample with an antibiotic; contacting the sample with the recombinant bacteriophage specific to the bacteria; 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.
Control-derived background threshold signal calling
The method comparing the signal produced by the sample to an experimentally determined value produced by a control sample, and defining a background threshold calculated from an average background signal and a standard deviation multiplied by 1–3 times, wherein a signal greater than a background threshold indicates the presence of one or more antibiotic-resistant bacteria in the sample.
Effective dose determination by antibiotic concentration range and indicator detection
A method comprising incubating each of one or more antibiotic solutions separately with one or more samples comprising the bacteria wherein the concentrations define a range; incubating the bacteria in the one or more samples with a recombinant bacteriophage specific to the bacteria comprising the late gene region luciferase indicator genetic construct; and detecting the indicator protein product wherein detection indicates the concentrations are not effective and the lack of detection indicates the antibiotic is effective, thereby determining the effective dose of the antibiotic.
Kit comprising late gene region luciferase indicator phage and antibiotic
A kit for detecting antibiotic-resistant bacteria in a sample comprising a recombinant bacteriophage specific to the bacteria comprising the late gene region luciferase indicator genetic construct; and an antibiotic.
System with antibiotic, late gene region luciferase indicator phage, and indicator detection component
A system for detecting antibiotic resistance of bacteria in a sample comprising a recombinant bacteriophage specific to the bacteria comprising the late gene region luciferase indicator genetic construct; 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.
Across the independent claims, the core inventive concept is a recombinant bacteriophage engineered with an indicator gene (non-fusion and not contiguous with a structural phage protein gene) under a bacteriophage late promoter, inserted into a late gene region and expressing luciferase as an indicator protein product. Detection of the luciferase indicator protein product, and in refinements signal calling using control-derived thresholds, is used either to determine the presence of antibiotic-resistant bacteria or to determine whether antibiotic concentrations are effective, with kit and system formulations that include the antibiotic and the recombinant bacteriophage plus detection components.
Stated Advantages
Enables determination of the presence of antibiotic-resistant bacteria in a sample based on detection of a luciferase indicator protein product signal.
Enables determination of an effective dose of an antibiotic by interpreting detection versus lack of detection of the luciferase indicator protein product.
Provides control-sample-based signal calling using a background threshold calculated from average background signal and standard deviation terms.
Provides kit and system formats for detecting antibiotic resistance using a recombinant bacteriophage and an antibiotic, with indicator detection.
Documented Applications
Not explicitly described in patent.
Interested in licensing this patent?