Methods, peptides and biosensors useful for detecting a broad spectrum of bacteria
View Patent ↗Described herein are methods of detecting a wound infection and for detecting the presence or absence of bacteria, for example, wound bacteria in a sample, by contacting a sample with a peptide substrate derived from the modification of the reactive site loop (RSL) domain of the α 1-proteinase inhibitor. In the current invention, we have demonstrated that these peptide substrates without the alpha 1 protein can be efficiently used as peptide substrates. The modification or the absence of modification of this peptide substrate by the enzyme produced and/or secreted by the bacteria, can serve as an indicator for the presence or absence of the bacteria 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 a wound-specific bacterium in a sample selected from a wound, a body fluid or fluid from a wound, said method comprising the steps of:
a) contacting said sample with a detectably labeled synthetic α 1-proteinase inhibitor reactive site loop domain peptide substrate selected from the group consisting of EAAGAMFLEAIPK (SEQ ID NO: 1), EGAMFLEAIPMSIPK (SEQ ID NO: 2), KGTEAAGAMFLEAIPMSIPPEVK (SEQ ID NO: 3), GAMFLEAIPMSIPPE (SEQ ID NO: 4), CGAMFLEAIPMSIPAAAHHHHH (SEQ ID NO: 5), and variants or fragments of any of said peptide substrates, wherein said variant or fragment of any of said peptide substrates has at least 40% sequence identity to any of said peptide substrates, under conditions that result in cleavage of said substrate by a protease enzyme produced in said sample by a wound-specific bacterium, wherein said peptide substrate is coupled to both a support and to at least one detectable moiety; and
b) detecting a cleavage or an absence of the cleavage of the substrate, the cleavage of the substrate indicating the presence of the wound-specific bacterium in the sample and absence of the cleavage of the substrate indicating absence of the wound-specific bacterium in the sample.
2. A method according to claim 1 , wherein the wound-specific bacterium is selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Pseudomonas aeruginosa, Enterococcus faecalis , Serratia marcescens, Proteus mirabilis, Enterobacter cloeae, Acetinobacter anitratus, Klebsiella pneumonia , and Escherichia coli .
3. A method according to claim 1 , wherein the at least one detectable moiety coupled to the substrate comprises a fluorescent probe and a quencher dye molecule.
4. A method according to claim 1 , wherein the at least one detectable moiety coupled to the substrate is a label selected from the group consisting of spin labels, antigen tags, epitope tags, haptens, enzyme labels, prosthetic groups, fluorescent materials, pH-sensitive materials, chemiluminescent materials, colorimetric components, bioluminescent materials, and radioactive materials.
5. A method according to claim 4 , wherein the substrate comprises at least one of the peptides selected from the group consisting of EGAMFLEAIPMSIPK (SEQ ID NO: 2) and variants fragments thereof, wherein said variant or fragment thereof has at least 40% sequence identity to SEQ ID NO: 2.
6. A method according to claim 1 , wherein the sample is selected from the group consisting of a surface of the wound and the fluid from the wound.
7. A method according to claim 1 , wherein the substrate is attached to a biosensor surface associated with the support.
8. A method according to claim 7 , wherein the support is a solid support selected from the group consisting of a wound dressing, a container for holding body fluids, a disk, a scope, a filter, a lens, a foam, a cloth, a paper, a suture, a dipstick, a swab, a urine collection bag, a blood collection bag, a plasma collection bag, a test tube, a catheter, and a well of a microplate.
9. A method according to claim 7 , wherein the solid support comprises a material required to be free of microbial contaminants.
10. A method according to claim 1 , wherein the at least one detectable moiety of the substrate comprises at least two dissimilar colorimetric components and the support comprises a solid support surface selected from a polymer, a membrane, a resin, a glass or a sponge, wherein modification of the substrate comprises cleaving at least a portion of the substrate that includes one of the colorimetric components, the cleaving resulting in a visible color change.
11. A method according to claim 10 , wherein the colorimetric components are covalently attached to the peptide.