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Methods to determine sensitivity profile of bacterial strain to therapeutic composition

A therapeutic composition and sensitivity technology, applied in the field of determining the sensitivity spectrum of bacterial strains to therapeutic compositions, can solve problems such as treatment delay and complications

Pending Publication Date: 2020-04-21
ADAPTIVE PHAGE THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This delay in obtaining sensitive results may lead to treatment delays and complications in patients with systemic bacterial infections

Method used

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  • Methods to determine sensitivity profile of bacterial strain to therapeutic composition
  • Methods to determine sensitivity profile of bacterial strain to therapeutic composition
  • Methods to determine sensitivity profile of bacterial strain to therapeutic composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142] Example 1: Phage Isolation / Characterization from Environmental Sources.

[0143] Powdered TSB medium (Becton, Dickinson and Company) can be mixed with raw sewage to a final concentration of 3% w / v. Different bacterial strains can be grown to exponential phase and 1 mL of each strain is added to a 100 mL aliquot of the TSB-sewage mixture and incubated overnight at 37°C and 250 rpm. The next day, 1 mL of the infected TSB-sewage mixture was harvested and centrifuged at 8,000 xg for 5 minutes to pellet cells and debris. Transfer the supernatant to a sterile 0.22 µm Centrifuge tube filters (Coming, NY) and centrifuge at 6,000 xg to remove any residual bacteria. A 10 μL aliquot of the filtrate was mixed with 100 μL of an exponentially growing culture of the bacterial strain, incubated at 37°C for 20 minutes, mixed with 2.5 mL of molten top agar (0.6% agar) tempered to 50°C, and poured over TSB agar plate (1.5% TSB agar). Plates were incubated overnight at 37°C and subseq...

Embodiment 2

[0147] Example 2: Assays for generating phage-host susceptibility profiles.

[0148] In order to perform the disclosed methods, it is necessary to compare the genomes of multiple different bacterial strains with similar or identical phage-host susceptibility profiles. If the phage-host susceptibility profile of the bacteria is known, then the following assays need not be performed. However, if the phage-host susceptibility profile of the bacterium is unknown, such a profile can be determined or experimentally derived using any of the following assays.

[0149] One method of determining the susceptibility / resistance profile of bacteria relies on automated indirect liquid lysis assays. Briefly, overnight cultures of bacterial strains were inoculated with 1% v / v tetrazole Dye mix wells of TSB 96-well plate. Phage was then added to each well, and the plate was placed in the OmniLog TM System (Biolog, InC, Hayward, CA) was incubated overnight at 37°C. See Henry, Bacteriophage 2...

Embodiment 3

[0156] Example 3: Genome Sequencing, Assembly and Annotation

[0157] Phage and / or bacterial genomes can be sequenced using standard sequencing techniques and assembled using contig analysis well known in the art. For example, 5 μg of DNA isolated from phage or bacteria can be extracted and shipped to a contract sequencing facility. 40x to 65x sequencing coverage was obtained using pyrosequencing technology on a 454FLX instrument. Files generated by the 454FLX instrument were assembled with GS assembler (454, Branford, Conn.) to generate consensus sequences. Quality improvement of the genomic sequence may involve sequencing 15-25 PCR products across the genome to ensure correct assembly, double-strand formation, and resolution of any remaining base conflicts occurring within identical polynucleotide tracts. The open reading frame (ORF) of the encoded protein can be predicted using standard programs known in the art (eg, BLASTP), followed by manual evaluation and correction i...

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Abstract

Methods and systems for pattern search and analysis to identify and select therapeutic molecules that can be used to treat bacterial infections or contaminations are disclosed. Examples include methods and systems for pattern search and analysis to identify and select bacteriophage based on comparison of the genomes of a query bacterium and / or a query phage strain to a therapeutic molecule-host training set of bacterial strains and / or phage strains in which the phage strains (or other therapeutic molecules) have been shown to have the capacity to act as an antibacterial agent by either killing, replicating in, lysing and / or inhibiting the growth of the bacterial strains in the training set. Therapeutic compositions, including phage, identified using the methods described herein can then beused to treat bacterial infections in a subject and / or contamination in the environment.

Description

technical field [0001] The present invention relates to cell-free methods and kits useful for predicting the susceptibility of bacteria to therapeutic compositions comprising phages, antibiotics and / or other bactericidal compounds. Synergistic bactericidal activity between therapeutic compositions can also be predicted using the cell-free methods and kits described herein. Background technique [0002] In the following discussion, for purposes of background and introduction, certain articles and methods will be described. Nothing contained herein should be construed as an "admission" of prior art. Applicant expressly reserves the right, where appropriate, to demonstrate that articles and methods referenced herein do not constitute prior art, where appropriate and in accordance with applicable legal provisions. [0003] Multidrug resistant (MDR) bacteria are emerging at an alarming rate. Currently, it is estimated that at least 2 million infections are caused by MDR organi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16B20/00G16B40/00
CPCG16B20/00G16B40/00Y02A90/10G06N20/00G16B40/30G16B40/20
Inventor 卡尔·梅里尔
Owner ADAPTIVE PHAGE THERAPEUTICS INC
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