Choline Lysis Buffer for Rapid Microorganism Isolation
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Solution Overview
Problem
Current methods for isolating viable microorganisms from positive blood culture samples are time-consuming and often result in non-viable organisms, making it difficult to perform antimicrobial susceptibility testing and identification, especially for challenging organisms like Streptococcus pneumoniae, which can lead to inappropriate antibiotic treatment in septicemia patients.
Innovation Solution
The use of a choline-containing solution in conjunction with a lysis buffer to lyse blood cells while maintaining the viability of microorganisms, allowing for rapid isolation and subsequent downstream analyses such as mass spectrometry and antimicrobial susceptibility testing without interfering with identification methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If liquid separation methods with lysis buffers containing detergents are used to isolate microorganisms from blood cells, then blood cells are effectively lysed and removed, but the microorganisms become non-viable and cannot be used for AST testing
Solution Approach 1:
The patent changes the chemical parameters of the lysis buffer by specifying precise concentrations of non-ionic detergent (0.1-1% v/v), ionic detergent (0.01-0.5% w/v), and EDTA (0.1-10 mM), along with controlled incubation time (5-30 minutes) and temperature (20-37°C). These parameter optimizations enable effective blood cell lysis while preserving microorganism viability, resolving the contradiction between ease of isolation and reliability of microorganism viability.
2Reliability
If sub-culturing methods are used to obtain plated pure culture of viable microorganisms, then viable microorganisms can be obtained for AST testing, but the process takes up to 48 hours which delays appropriate antibiotic treatment
Solution Approach 1:
The patent performs preliminary optimization of the lysis buffer treatment conditions before AST testing is required. By pre-establishing the optimal combination of detergent concentrations, incubation time, and temperature that preserves microorganism viability, the method eliminates the need for time-consuming sub-culturing steps later, reducing the total time to obtain viable microorganisms for AST testing from 48 hours to a much shorter duration.
Solution Approach 2:
The patent skips the traditional sub-culturing step by rushing through the isolation process using optimized lysis buffer treatment. This allows direct progression from blood sample to viable microorganism isolation without the intermediate plating and re-culturing steps, significantly reducing the time required while maintaining microorganism viability.
3Productivity
If traditional lysis buffers are used to rapidly isolate microorganisms, then isolation time is reduced, but the microorganisms become non-viable making AST testing impossible
Solution Approach 1:
The patent creates a composite lysis buffer system combining three key components: non-ionic detergent (0.1-1% v/v), ionic detergent (0.01-0.5% w/v), and EDTA (0.1-10 mM). This composite formulation works synergistically to efficiently lyse blood cells while the EDTA chelates divalent cations that would otherwise damage microorganisms, preserving their viability. The composite approach enables rapid isolation without sacrificing microorganism viability for AST testing.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables the rapid isolation of viable microorganisms from a single blood culture sample, enabling accurate identification and antimicrobial susceptibility testing, including for difficult-to-identify organisms like S. pneumoniae, thereby improving treatment efficacy and reducing the time to appropriate antibiotic administration.
Implementation Method 1
combining at least a portion of the biological sample with a choline-containing solution and a lysis buffer to lyse the blood cells
Data Source
AI summary
Methods of the invention include the isolation of intact, viable microorganism(s) from positive blood culture (“PBC”) samples for use in downstream analyses such as identification and antimicrobial susceptibility testing (“AST”). The methods involve collecting a portion of the PBC sample, adding a choline-containing solution, lysing the blood cells, isolating the viable microorganism, and performing downstream analysis of the isolated, viable microorganism. The methods can be applied to a variety of gram-positive bacteria, gram-negative bacteria, and/or yeast, and particularly to strains of S. pneumoniae.