Use of Bacterial Beta-Lactamase for In Vitro Diagnostics and In Vivo Imaging, Diagnostics and Therapeutics

Abstract

Provided herein provided is an assay system for monitoring drug susceptibility of a pathogenic bacteria comprising color-producing substrates for a beta-lactamase of the pathogenic bacteria, an assay device for visibly detecting a product of the beta-lactamase on the substrate thereof and a reader configured to quantify the visibly detected product. Also provided is an in vitro method to determine susceptibility to a drug effective against a pathogenic bacteria, for example, a pathogenic Mycobacteria, that has a beta-lactamase activity. An excitation wavelength is delivered to a biological sample obtained from a subject having an infection from the pathogenic bacteria in the presence of a beta-lactamase substrate. The intensity of a signal, such as a fluorescent, luminescent or colorimetric signal, at an emission wavelength of a product of the beta-lactamase on the subject is correlated to drug susceptibility.

What is claimed is: 
     
         1 . An assay system for monitoring drug susceptibility of pathogenic bacteria, comprising:
 one or more color-producing substrates for a beta-lactamase of the pathogenic bacteria;   an assay device for visibly detecting a product of beta-lactamase activity on the substrate; and   a reader configured to quantify visible signals emitted by the detected product.   
     
     
         2 . The assay system of  claim 1 , said assay device comprising a platform having:
 means for receiving an incubation mixture comprising a biological sample of the pathogenic bacteria, a drug effective against the pathogenic bacteria, and the fluorescent, luminescent or color-producing substrate; and   means for capturing and concentrating a colored product produced by the beta-lactamase activity upon the substrate in fluid connection to the receiving means.   
     
     
         3 . The assay system of  claim 2 , said device further comprising means for allowing only the colored product to flow downstream from the receiving means. 
     
     
         4 . The assay system of  claim 2 , said device further comprising an internal control downstream from the receiving means. 
     
     
         5 . The assay system of  claim 2 , said device further comprising means for absorbing fluid downstream from the receiving means. 
     
     
         6 . The assay method of  claim 1 , wherein the substrate is a fluorogenic substrate CDG-OMe, CDC-1, CDC-2, CDC-3, CDC-4, CDC-5, CNIR5, CNIR5.2, CNIR5-QSY22, CNIR7, CNIR7-TAT, CNIR9, CNIR10, CNIR800, CNIR800.2, CNIR800-3, XHX2-81, XHX2-91, XHX3-1, XHX3-2, XHX3-26, or XHX3-32 or a derivative or analog thereof. 
     
     
         7 . The assay system of claim  25 , wherein the substrate comprises a colored dye or a chemical reagent. 
     
     
         8 . The assay system of claim  25 , wherein the substrate is linked to a particle or a microsphere or produces a detectable change in the mixture. 
     
     
         9 . The assay system of claim  25 , wherein the substrate comprises a chemical reagent, said device further comprising a second reagent as means to produce color from the chemical reagent. 
     
     
         10 . The assay system of claim  25 , wherein the substrate is linked to biotin, said device further comprising avidin as means to capture the biotin-linked substrate. 
     
     
         11 . An in vitro method for determining drug susceptibility of a pathogenic Mycobacteria in a subject infected by the same, comprising the steps of:
 a) obtaining a biological sample from the subject;   b) contacting said biological sample with an anti-mycobacterial drug;   c) contacting said biological sample with a fluorogenic substrate for Mycobacterial beta-lactamase;   d) delivering an excitation wavelength to the biological sample; and   e) measuring levels of fluorescence at an emission wavelength produced by a fluorescent product of the beta-lactamase action on the substrate in the biological sample over a period of time; wherein no increase or a decrease in fluorescence over the time period correlates to susceptibility of the pathogenic bacteria to the drug.   
     
     
         12 . The in vitro method of  claim 11 , wherein the measuring step comprises:
 detecting fluorescent signals emitted at intervals during a time period of about 24 hours; and   reading and recording an intensity thereof.   
     
     
         13 . The in vitro method of  claim 11 , further comprising:
 plating aliquots from the biological sample over the period of time to monitor colony formation of the pathogenic Mycobacteria.   
     
     
         14 . The in vitro method of  claim 11 , further comprising one or both of quantifying and differentiating infected cells from non-infected cells in the biological sample. 
     
     
         15 . The in vitro method of  claim 11 , further comprising monitoring for acquisition of resistance to the anti-Mycobacterial drug by the pathogenic Mycobacteria by the steps of:
 obtaining a biological sample after a treatment period with the anti-Mycobacterial drug;   repeating steps b) to e); wherein an increase in fluorescence levels over the time period correlates to resistance to the anti-Mycobacterial drug.   
     
     
         16 . The in vitro method of  claim 11 , wherein the biological sample is sputum, pleural fluid, urine, blood, saliva, stool, or a sample obtained by swabbing an area of interest on the subject. 
     
     
         17 . The in vitro method of  claim 11 , wherein the pathogenic Mycobacteria comprise a  Mycobacterium tuberculosis  complex, a  Mycobacterium avium  complex or  Mycobacterium marinum.    
     
     
         18 . The in vitro method of  claim 11 , wherein the fluorogenic substrate is CDG-OMe, CDC-1, CDC-2, CDC-3, CDC-4, CDC-5, CNIR5, CNIR5.2, CNIR5-QSY22, CNIR7, CNIR7-TAT, CNIR9, CNIR10, CNIR800, CNIR800.2, CNIR800-3, XHX2-81, XHX2-91, XHX3-1, XHX3-2, XHX3-26, or XHX3-32 or a derivative or analog thereof. 
     
     
         19 . The in vitro method of  claim 11 , wherein the excitation wavelength is about 540 nm to about 730 nm and the emission wavelength is about 650 nm to about 800 nm.