Modified bacterial pathogens and methods for effectuating rapid response to contamination by known or unknown bacterial pathogens
Inventors
Hassett, Daniel J. • Su, Shengchang • Lamkin, Thomas J. • Saldanha, Roland
Assignees
University of Cincinnati • United States Department of the Air Force
Publication Number
US-10724070-B2
Publication Date
2020-07-28
Expiration Date
2033-11-14
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Abstract
Stable, constitutively expressed, chromosomal fluorescent transcriptional fusions in bacterial pathogens and methods of using the same to screen candidate compounds for anti-bacterial efficacy.
Core Innovation
The invention relates to the development of stable, constitutively expressed chromosomal green and red fluorescent transcriptional fusions in bacterial pathogens for rapid identification of antibacterial agents. These fluorescent transcriptional fusions are integrated into the chromosome of Category A, B, or C bacterial pathogens, enabling a simple and fast read-out of bacterial viability via fluorescence signals that require extremely short exposure times, particularly less than 200 milliseconds. This technology facilitates high-throughput screening of candidate compounds for antibacterial efficacy, especially in response to contamination by antibiotic-resistant bacterial pathogens.
The problem addressed arises from the threat of bioterrorism using genetically engineered bacterial pathogens that may be resistant to existing antibiotics. Current methods for detecting bacterial contamination and screening compounds for antibacterial activity are not adequate for rapid response because existing fluorescent reporters in bacterial pathogens do not provide sufficiently fast or strong signals at single copy levels with minimal exposure time. Therefore, there is a critical need for bacterial pathogens engineered with fluorescent reporters capable of providing rapid, measurable signaling to screen large chemical libraries efficiently and inform counter-bioterrorism efforts in a timely manner.
Claims Coverage
The patent contains two main independent claims directed to methods involving modified bacterial pathogens with chromosomal fluorescent reporters and to the corresponding method of responding to bioterroristic dissemination using such modified bacteria. These claims introduce the inventive features of constitutive chromosomal expression of fluorescent fusion proteins in specific bacterial pathogens and their use in rapid antibacterial compound screening.
Use of modified bacterial pathogens with constitutive chromosomal fluorescent protein expression for rapid antibacterial screening
The method involves providing bacterial pathogens (specifically Bacillus anthracis Ames or Sterne strains) genetically modified via integration of vector plasmids engineered to constitutively express chromosomal transcriptional fusion fluorescent proteins resulting in sustained fluorescence while alive. Screening candidate compounds comprises contacting these modified pathogens and determining fluorescence extinction, indicating antibacterial efficacy.
High-throughput screening with rapid fluorescence identification
The screening method supports high throughput applications, with the step of identifying antibacterial efficacy requiring fluorescence measurement with exposure times less than 200 milliseconds, enabling rapid and time-effective compound screening.
Method of responding to intentional bioterrorist dissemination of bacterial pathogens using fluorescent modification
The method comprises isolating a sample of a known or unknown bacterial pathogen, modifying it by inserting a vector plasmid engineered for constitutive chromosomal fluorescent protein expression, screening antibacterial agent libraries by fluorescence extinction after contact with candidate compounds, and treating contamination with identified antibacterial compounds.
The independent claims focus on the novel use of genetically modified bacterial pathogens with stable, constitutive chromosomal fluorescent fusion proteins for rapid, high-throughput screening of antibacterial agents, particularly useful for counteracting bioterrorism threats via intentional dissemination of antibiotic-resistant pathogens.
Stated Advantages
Enables rapid identification of antibacterial compounds by providing a simple fluorescence read-out with exposure times less than 200 milliseconds.
Suitable for high-throughput screening of large chemical libraries, reducing time for effective antibacterial agent discovery.
Provides a time-effective rapid response platform for intentional contamination by antibiotic-resistant bacterial pathogens in bioterrorism scenarios.
Stable and constitutive expression of fluorescent proteins in chromosomal locations ensures sustained fluorescence during bacterial viability, improving reliability of screening assays.
Documented Applications
High-throughput screening of candidate compounds for antibacterial efficacy against Category A, B, or C bacterial pathogens with antibiotic resistance.
Rapid response methods to contamination resulting from intentional bioterrorism dissemination of known or unknown bacterial pathogens by screening modified pathogens expressing fluorescent reporters.
Use of fluorescently tagged bacterial pathogens for studies including virulence assessments, vaccine candidate testing, in vivo animal models, infective indices experiments, intramacrophage survival studies, and siRNA screening.
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