Methods for rapid antimicrobial susceptibility testing

Abstract

The present invention relates, in part, to methods and kits for rapidly determining antimicrobial susceptibility of microorganisms.

1 . A method for determining antimicrobial susceptibility of microorganisms comprising:
 determining relative surface areas of the microorganisms after culturing at a plurality of antimicrobial concentrations.   
     
     
         2 . The method of  claim 1  wherein the step of determining the relative surface areas comprises measuring an optical signal associated with surface area in each of the antimicrobial concentrations. 
     
     
         3 . The method of  claim 2  wherein the optical signal is provided by a signaling agent proportional to the surface area of the microorganisms in each of the plurality of antimicrobial concentrations. 
     
     
         4 . The method of  claim 3 , wherein the optical signal is amplified so as to resolve a surface area differential between a dividing microbe and a microbe undergoing filamentous growth. 
     
     
         5 . An automated method according to  claim 1 . 
     
     
         6 . A method for determining antimicrobial susceptibility of microorganisms which comprises
 (a) incubating a liquid suspension of microorganisms in the presence of an antimicrobial under conditions that promote growth of the microorganisms,   (b) adding a signaling agent comprising a signal amplifier and one or more chemical moieties that bind to a surface of the microorganisms;   (c) separating the microorganisms bound by the signaling agent from unbound signaling agent; and   (d) measuring a signal from the signaling agent bound to the surface of the microorganisms, wherein the signal is associated with a relative surface area of intact microorganisms.   
     
     
         7 . The method of  claim 6 , wherein adding said signaling is prior to, during or after the incubating step. 
     
     
         8 . The method of  claim 6 , wherein the one or more chemical moieties bind non-specifically to a surface of the microorganisms. 
     
     
         9 . The method of  claim 6 , wherein the signaling agent is capable of binding to a surface of the microorganisms by association and/or intercalation, and wherein the number of signaling agents that associate with and/or intercalate into the microorganism surface is proportional to microorganism surface area, thereby determining the antimicrobial susceptibility of the microorganisms. 
     
     
         10 . The method of  claim 6 , wherein multiple antimicrobials are tested in parallel. 
     
     
         11 . The method of  claim 6 , wherein the signaling agent comprises:
 an antibody, lectin, natural peptide, synthetic peptides, bacteriophage, synthetic and/or natural ligands, synthetic and/or natural polymers, synthetic and/or natural glycopolymers, carbohydrate-binding proteins and/or polymers, glycoprotein-binding proteins and/or polymers, charged small molecules, other proteins, bacteriophages, and/or aptamers.   
     
     
         12 . The method of  claim 6 , wherein the method further comprises a step of determining whether a microorganism is resistant, intermediately resistant or susceptible to one or more antimicrobials and/or determining one or more antimicrobial minimum inhibitory concentrations (MIC) based upon the signal from the signaling agent bound to the surface of the microorganisms. 
     
     
         13 . The method of  claim 6 , wherein the microorganisms are obtained from a biological sample from a subject having an infection of the microorganisms and/or are obtained from a culture derived from the biological sample; and wherein the biological sample is selected from the group consisting of blood or blood components, bronchoalveolar lavage, cerebrospinal fluid, nasal swabs, sputum, stool, throat swabs, vaginal swabs, urine, and wound swabs, or a combination thereof. 
     
     
         14 . A kit for determining antimicrobial susceptibility of microorganisms comprising:
 (a) a signaling agent capable of binding to a surface of the microorganisms of interest, wherein the signaling agent comprises
 (i) a linker group L and an amplifier group which comprises an Europium coordination complex; and wherein L forms a covalent bond to the amplifier group or L forms one or more non-covalent interactions with an amplifier group, 
 (ii) a signal amplifier and one or more chemical moieties that bind to a surface of the microorganisms, 
 (iii) an signaling agent capable of binding to a surface of the microorganisms by association and/or intercalation, and wherein the number of signaling agents that associate with and/or intercalate into the microorganism surface is proportional to microorganism surface area, or 
 (iv) a structure: 
   
       
         
           
           
               
               
           
         
         (b) a solution for incubating a sample containing microorganisms; and 
         (c) one or more reagents for generating signals from the signaling agent. 
       
     
     
         15 . The kit of  claim 14 , which further comprises magnetic beads to magnetically separate the microorganisms. 
     
     
         16 . The kit of  claim 14 , which further comprises a developer reagent to produce a measurable signal. 
     
     
         17 . The kit of  claim 16 , wherein the signal is optical and/or electrical. 
     
     
         18 . The kit of  claim 17 , wherein which the optical signal is a fluorescent, time-resolved fluorescent, absorbent, and/or luminescent signal. 
     
     
         19 . The kit of  claim 14 , wherein the kit further comprises a multiwell plate.