Mass Spectrometric β-Lactamase Resistance Detection via Substrate Hydrolysis
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Solution Overview
Problem
Current methods struggle to directly determine β-lactamase resistances in bacteria using mass spectrometry, as the presence of new or modified proteins is difficult to identify in the protein profiles measured, hindering rapid disease control and effective antibiotic treatment.
Innovation Solution
A method using mass spectrometry to measure the hydrolytic breakdown of β-lactam substrates by β-lactamases in bacteria, where the enzymatic cleavage of the β-lactam ring is detected, with customized substrates and MALDI time-of-flight mass spectrometry to identify the type and strength of β-lactamases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mass spectrometry is used to identify microbial proteins, then identification accuracy is improved, but the ability to directly detect resistance-related proteins deteriorates
Solution Approach 1:
The patent uses β-lactam substrates as intermediary molecules that interact with β-lactamase enzymes. These substrates serve as mediators between the enzyme activity and the mass spectrometer detection, allowing indirect measurement of resistance through substrate hydrolysis products rather than direct protein detection
Solution Approach 2:
The patent replaces direct mechanical/protein-based detection with a chemical reaction-based detection system. Instead of detecting resistance proteins directly, the system uses chemical substrates that undergo hydrolysis reactions catalyzed by β-lactamases, converting the detection problem from protein identification to chemical product analysis
2Measurement precision
If conventional resistance determination methods are used, then resistance type can be identified, but measurement time and complexity increase
Solution Approach 1:
The patent performs preliminary action by pre-selecting specific β-lactam substrates with known mass differences that correspond to different β-lactamase types. This preliminary preparation of differentiated substrates allows rapid classification of resistance types without time-consuming sequential testing
Solution Approach 2:
The patent segments the resistance determination process into distinct mass spectral peaks corresponding to different substrate hydrolysis products. Each peak represents a specific β-lactamase type, allowing parallel detection and classification of multiple resistance types in a single measurement
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
Enables rapid and sensitive determination of β-lactamase resistances, allowing for targeted antibiotic treatment by measuring the hydrolyzed cleavage product and reaction speed, providing insights into resistance type and strength.
Implementation Method 1
The catalytic effect of the bacterially produced β-lactamases on the substrate causes the β-lactam ring to hydrolytically split open
Implementation Method 2
ionization by matrix-assisted laser desorption
Implementation Method 3
MALDI time-of-flight mass spectrometry
Data Source
AI summary
The invention relates to the determination of resistances of microorganisms which produce β-lactamases, in particular “extended spectrum β-lactamases” (ESBL). The invention provides a method whereby the microbial resistance can be measured very simply and quickly by means of the catalytic effect of the microbially produced β-lactamases on β-lactam antibiotics, which consists in a hydrolytic cleavage of the β-lactam ring. The method determines the resistance of the bacteria a few hours after a suitable substrate, either a β-lactam antibiotic or a customized β-lactam derivative, has been added to a suspension of the microbes, by direct mass spectrometric measurement of the substrate breakdown caused by the β-lactamases.