Phage-mediated immunoassay and methods for determining susceptibility of bacteria to antibiotic or probiotic agents
Inventors
BELENKY, Alexander Solomon • Schofield, David A.
Assignees
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Publication Number
US-11560584-B2
Publication Date
2023-01-24
Expiration Date
Abstract
Methods for determining the susceptibility or resistance of bacteria to antibiotic agents are provided. In one embodiment, the methods include culturing the bacteria in the presence or absence or the antimicrobial agent to generate a primary culture which is then cultured in the presence or absence of transforming phages. The recombinant phages are specific to the bacteria and comprise a heterologous marker (e.g., a nucleic acid that is expressible as a detectable product such as an RNA or a protein). The susceptibility or resistance of the bacteria to the antimicrobial agent may be determined by assaying the culture for the presence or absence of the heterologous marker, wherein a reduction in the level or activity of the marker in the culture compared to the level or activity of the marker in a comparative culture indicates that the bacteria is sensitive to the antibiotic agent.
Core Innovation
The invention relates to phage-mediated, multiplex formats for bacterial identification and antimicrobial susceptibility testing in which a plurality of recombinant bacteriophage are each host-specific for one of a plurality of bacterial species of interest. Each recombinant bacteriophage comprises a nucleic acid encoding a heterologous protein marker that is expressible upon bacteriophage transduction into the bacterial species of interest, where the heterologous protein marker comprises a chemiluminescent label.
In multiplex workflows, portions of a sample are generated into primary culture aliquots and are cultured in the presence or absence of one or more antimicrobial agents having known antimicrobial activity specific for the bacterial species of interest. Each primary culture aliquot is exposed to a different recombinant bacteriophage from the plurality specific to a bacterial species of interest to create secondary cultures, and susceptibility or resistance is determined by detecting the presence or absence of the chemiluminescent label directly in a cell lysate from each secondary culture.
The invention further includes methods for screening to identify a test agent having antimicrobial activity for a bacterial species of interest by culturing the bacterial species of interest with and without the test agent to form primary cultures, exposing the primary cultures to a transducible recombinant bacteriophage, and detecting a signal correlated with expression of the chemiluminescent label in cell lysates from the secondary cultures. Signals from first and second secondary cultures are compared to identify test agents having antimicrobial activity, including detection on a lateral flow immunoassay.
Claims Coverage
The document provides three independent claims covering multiplex bacterial species presence testing with antimicrobial susceptibility determination, a related primary/secondary culture workflow, and screening methods to identify antimicrobial test agents. Across the independent claims, the core inventive elements include host-specific recombinant bacteriophage transduction, expressible heterologous protein markers with chemiluminescent labels, cell-lysate detection, and lateral flow immunoassay readouts in a multiplex or comparative screening context.
Multiplex bacterial species testing and antimicrobial susceptibility via host-specific recombinant bacteriophages with chemiluminescent protein markers
A multiplex method that simultaneously tests for the presence of a plurality of bacterial species of interest in a sample and determines susceptibility of the bacterial species of interest to an antimicrobial agent using a plurality of recombinant bacteriophages, each host-specific for one bacterial species and comprising a nucleic acid encoding an expressible heterologous protein marker with a chemiluminescent label, where marker detection in a cell lysate from secondary cultures indicates presence of the bacterial species and resistance or susceptibility outcomes are inferred, including placing the cell lysate directly on a lateral flow immunoassay for signal detection.
Multiplex primary and secondary culture workflow with chemiluminescent label detection
A multiplex method generating primary cultures by culturing sample aliquots in the presence and absence of the antimicrobial agent, generating secondary cultures by exposing portions of the primary cultures to host-specific recombinant bacteriophage, and detecting presence or absence of the chemiluminescent label directly in cell lysates by placing the cell lysate directly on a lateral flow immunoassay, where the detected signal is correlated with susceptibility outcomes.
Screening test agent antimicrobial activity using transducible recombinant bacteriophage delivery of chemiluminescent protein marker
A screening method that identifies a test agent having antimicrobial activity for a bacterial species of interest using a test agent, a culture of the bacterial species, and a transducible recombinant bacteriophage comprising a nucleic acid encoding a heterologous protein marker with a chemiluminescent label expressible upon transduction, where primary cultures with and without the test agent are generated, secondary cultures are generated by exposing the primary cultures to the recombinant bacteriophage, signals correlated with expression are detected directly in cell lysates via lateral flow immunoassay, and signals from first and second secondary cultures are compared to identify the test agent having antimicrobial activity.
Across the independent claims, the multiplex and screening coverage centers on host-specific recombinant bacteriophages that transduce bacterial species to express heterologous protein markers with chemiluminescent labels, followed by direct cell-lysate detection using lateral flow immunoassays, with susceptibility or antimicrobial activity inferred from signal comparisons between treated and control cultures.
Stated Advantages
Provides simultaneous testing for the presence of multiple bacterial species in a sample in a multiplex format.
Enables determination of susceptibility, including resistance, of bacterial species to an antimicrobial agent using chemiluminescent label detection.
Uses cell lysate and lateral flow immunoassay to detect chemiluminescent label signals from secondary cultures.
Supports screening to identify a test agent having antimicrobial activity by comparing detected signals correlated with expression of the chemiluminescent label.
Documented Applications
Multiplex bacterial identification and antimicrobial susceptibility or resistance testing in a sample by detecting chemiluminescent marker signals after recombinant bacteriophage exposure in primary and secondary culture workflows.
Screening to identify a test agent having antimicrobial activity for a bacterial species of interest by culturing with and without the test agent, transducing with a recombinant bacteriophage, detecting chemiluminescent label signals in cell lysates, and comparing signals between treated and untreated conditions.
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