Method for detecting escherichia coli

Abstract

Described herein are methods of detecting an infection and for detecting the presence or absence of microorganisms, for example, wound pathogens in a sample, by contacting a sample with an enzyme produced and/or secreted by the bacteria, and detecting modification or the absence of modification or the substrate, as an indicator of the presence or absence of the enzyme in the sample. The present invention also features a biosensor for detecting the presence or absence of bacteria in a sample.

1 . A method for detecting the presence or absence of an infection in a subject, comprising the steps of:
 a) contacting a sample obtained from a subject with a detectably labeled substrate for an enzyme produced and/or secreted by  Escherichia coli , under conditions that result in modification of said substrate by said enzyme; and   b) detecting the modification or the absence of the modification of said substrate,   wherein modification of said substrate indicates the presence of an infection in said subject, and wherein the absence of modification of said substrate indicates the absence of an infection in said subject.   
     
     
         2 . The method of  claim 1 , wherein said enzyme is a protease. 
     
     
         3 . The method of  claim 1 , wherein said sample is a body fluid. 
     
     
         4 . The method of  claim 1 , wherein said substrate is on a solid support. 
     
     
         5 . The method of  claim 4 , wherein said solid support comprises a material required to be free of microbial contaminants. 
     
     
         6 . The method of  claim 5 , wherein said solid support is selected from the group consisting of a wound dressing, a container for holding body fluids, a disk, a scope, a filter, a lens, foam, cloth paper, a suture, and a swab. 
     
     
         7 . The method of  claim 6 , wherein said container for holding body fluids is selected from the group consisting of a urine collection bag, a blood collection bag, a plasma collection bag, a test tube, a catheter, and a well of a microplate. 
     
     
         8 . A method for detecting the presence or absence of a wound infection in a subject, comprising the steps of:
 a) contacting a wound in a subject with a detectably labeled substrate for an enzyme produced and/or secreted by  Escherichia coli  under conditions that result in cleavage of said substrate by said enzyme, wherein the detectably labeled substrate provides a latent signal that is activated only when the substrate is cleaved; and   b) measuring the signal to detect the cleavage or the absence of cleavage of said substrate, wherein cleavage of said substrate indicates the presence of a wound infection in said subject, and wherein the absence of cleavage of said substrate indicates the absence of a wound infection in said subject.   
     
     
         9 . The method of  claim 8 , wherein said enzyme is a protease. 
     
     
         10 . The method of  claim 8 , wherein said substrate is on a solid support. 
     
     
         11 . The method of  claim 10 , wherein said solid support is a wound dressing. 
     
     
         12 . The method of  claim 8 , wherein the detectably labeled substrate is a peptide having a dabcyl moiety and an edans moiety attached at opposite ends of the peptide. 
     
     
         13 . The method of  claim 12 , wherein the substrate is cleaved between the dabcyl moiety and the edans moiety. 
     
     
         14 . The method of  claim 8 , wherein the detectably labeled substrate specific for  Escherichia coli  is a peptide selected from the group consisting of:
 (dabcyl-K)VSRRRRRGG(D-edans) (SEQ ID NO:2),   KKAS(E-edans)VSRRRRRGG(K-dabcyl) (SEQ ID NOS: 3 and 4), and   CHHHAS(E-edans)VSRRRRRGG(K-dabcyl) (SEQ ID NOS: 5 and 6).   
     
     
         15 . The method of  claim 14 , wherein the peptide has an affinity tag for the non-covalent or covalent attachment of the peptide substrate to a surface. 
     
     
         16 . The method of  claim 10 , wherein said solid support comprises a material required to be free of microbial contaminants. 
     
     
         17 . A method for detecting the presence or absence of a wound infection in a subject, comprising the steps of:
 a) contacting a wound in a subject with a detectably labeled substrate for an enzyme produced and/or secreted by  Escherichia coli , under conditions that result in modification of said substrate by said enzyme; and   b) detecting the presence of a known color resulting from the modification or the absence of the modification of said substrate, wherein modification of said substrate indicates the presence of a wound infection in said subject, and wherein the absence of modification of said substrate indicates the absence of a wound infection in said subject.   
     
     
         18 . The method of  claim 17 , wherein the known color is produced by the hydrolysis of the detectably labeled substrate. 
     
     
         19 . The method of  claim 18 , wherein the detectable label on the substrate is a chromogenic dye. 
     
     
         20 . The method of  claim 19 , wherein the chromogenic dye is para-nitrophenol.