Quantifying endotoxin load in bacterial biofilms
AssigneeDePuy Synthes Products, Inc.
InventorsErica Shwarz, Wojciech Czaja
Life of this patent
Estimating the term…
Abstract
Disclosed herein are methods for quantifying total endotoxin load in a biofilm sample. Also provided are methods for identifying a gram-negative biofilm derived bacterial infection. The disclosed methods more accurately define actual total endotoxin levels and can detect the presence of endotoxin in a given biofilm volume at a higher resolution than current extraction techniques.
Claims22
A method for quantifying endotoxin concentration in a biofilm sample comprising cellulose from a gram negative bacteria comprising:
- digesting the gram negative biofilm sample with an enzyme for the digestion of extracellular matrix of the gram negative bacteria, wherein the enzyme comprises cellulose, to form a digested biofilm sample;
- separating the digested biofilm sample into a supernatant and a cell pellet;
- combining the cell pellet with a cell lysing agent to release endotoxins and form an endotoxin suspension;
- concentrating the endotoxin suspension into an endotoxin sample and a suspension supernatant;
- and,
- performing a bacterial endotoxin test (BET) assay on the endotoxin sample to obtain a sample endotoxin value.
A method of identifying an infection from pathogenic gram-negative bacteria within a gram-negative bacterial biofilm comprising cellulose comprising:
- enzymatically digesting a sample of the gram negative biofilm to form a digested biofilm sample using an enzyme comprising cellulase for the digestion of extracellular matrix of the pathogenic gram negative bacteria;
- separating the digested biofilm sample into a supernatant and a cell pellet;
- combining the cell pellet with a cell lysing agent to release a quantity of endotoxins, wherein the quantity of endotoxins has a lower limit of zero;
- concentrating the quantity of endotoxins, and,
- performing a bacterial endotoxin test (BET) assay on the concentration to obtain a sample endotoxin value, wherein a sample endotoxin value that is greater than zero is indicative of the presence of the pathogenic gram-negative bacteria in the biofilm.
Dependencies are inferred from the claim text.