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Phage-mediated immunoassay and methods for determining susceptibility of bacteria to antibiotic or probiotic agents

A bacteriophage, antimicrobial technology for use in the field of bacteriophage-mediated immunoassays and methods for determining the susceptibility of bacteria to antibiotics or probiotics, capable of addressing severe infections, few clinicians, and inability to satisfy a variety of Issues such as the need for efficient and effective means of diverse bacteria

Pending Publication Date: 2020-08-28
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, clinicians rarely, if ever, use them alone due to concerns about drug resistance and their long-term side effects
[0012] Tetracyclines are not a first-line treatment option for severe Gram-negative infections; however, due to limited efficacy of other drug classes, they are considered an option for the treatment of severe infections
Also, because the assay includes the detection of fluorescent or luminescent markers expressed in small samples, the assay is limited in the types of samples that can be analyzed
[0018] In conclusion, current methods for identifying drug-resistant bacteria do not meet today's need for an efficient and effective means of phenotyping a wide variety of bacteria, including mixtures thereof, e.g., based on the type of resistance they possess

Method used

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  • Phage-mediated immunoassay and methods for determining susceptibility of bacteria to antibiotic or probiotic agents
  • Phage-mediated immunoassay and methods for determining susceptibility of bacteria to antibiotic or probiotic agents
  • Phage-mediated immunoassay and methods for determining susceptibility of bacteria to antibiotic or probiotic agents

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0238] Construction of recombinant phage for expression of heterologous markers

[0239] In this study, molecules (markers) that are not naturally produced by target cells or by phage vectors or by bacteria-infected hosts are prepared and then heterologous molecules (markers) are specifically detected by immunoassays. Marker molecules may be peptides or proteins, optionally labeled with, for example, a polyhistidine (His) tag or the like.

[0240] Construction of recombinant phage : Recombinant phage are constructed via recombinant plasmid-mediated homologous recombination by inserting a DNA sequence encoding a heterologous marker into a strongly expressed region of the phage genome downstream of a nucleic acid sequence encoding a capsid protein (cps). Strong promoters located upstream of cps are selectively activated during expression of the phage genome following infection, resulting in the production of many copies of the corresponding mRNA transcript. Construction of re...

Embodiment 2

[0246] Generation of recombinant phage using DNA translocation

[0247] Use commercially available EZ::TN TM A transposase system (Epicenter Technologies, Madison, WI) was used to construct phage containing a heterologous reporter nucleic acid as described in Goryshin et al., J. Biol. Chem., 273(13):7367-7374, 1998. Then, the terminal ME-bound EZ::TN TM Transposomes were electrotransformed into electrocompetent E. coli K1 strain (ATCC strain 23503). Strains were made electrocompetent by growth in LB medium at 37°C to an optical density (OD) of 0.8, followed by several washes in 15% glycerol. Electroporation was accomplished using a BIORAD GENE PULSER obtained from Bio-Rad Laboratories (Hercules, CA).

[0248] The transformed E. coli K1 strain was infected with prototype K1-5 phage. Transformed bacteria were grown to an OD of 0.4 in LB-ampicillin medium at 37°C. Phage K1-5 was added at a multiplicity of infection of approximately 1 phage per 10 bacteria and the OD was moni...

Embodiment 3

[0251] Screening of samples obtained from subjects using recombinant phage

[0252] A subject (eg, a human patient) exhibiting symptoms of bacterial infection (eg, fever, headache, abdominal pain, and nausea) is identified, and the following samples are collected from the subject: 0.01 mL cerebrospinal fluid (CSF) sample, 1.0 mL sputum Liquid samples and 1.0mL blood samples. Each sample was diluted with 4.0 mL of LB broth to promote the growth of all bacteria present in the corresponding sample and incubated at 37°C for 4 hours. After incubation, distribute each sample in 100 µL aliquots into 30 wells of a 96-well plate. Aliquots of blood samples were added to wells 1-30, aliquots of CSF samples were added to wells 31-60, and aliquots of sputum samples were added to wells 61-90. Wells 91-93 were used as positive controls, and wells 94-96 were used as negative controls.

[0253] The following five recombinant phages were obtained: K1-5::luxAB phage, which infects Escherichia...

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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 theantimicrobial 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 determinedby 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.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 62 / 440,971, filed December 30, 2016, which is hereby incorporated by reference in its entirety. technical field [0003] The subject matter described herein relates to methods for determining the susceptibility of bacteria to a test agent, and methods for determining whether a target bacterial species is resistant to one or more antimicrobial agents. Further embodiments relate to methods of screening new test compounds for antimicrobial or probiotic activity, including identifying the presence of these agents in biological samples, including food and environmental samples. Background technique [0004] Since the first practical use of the antibiotic penicillin, many other antimicrobial agents have been developed, and antimicrobial therapy has greatly contributed to the advancement of modern medicine and the increase in average life expectancy. Howev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04G01N33/569
CPCC12Q1/04G01N33/56911Y02A50/30C12Q1/18G01N2333/195C12Q1/689G01N2333/01
Inventor A·贝伦基D·A·斯科菲尔德
Owner QUIDEL
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