Rapid Antimicrobial Susceptibility Testing Based on a Unique Spectral Intensity Ratio Analysis via Single Fluorescence Membrane Dye Staining and Flow Cytometry

Abstract

Single dye fluorescent staining and the combination of differences in both intensity and spectral emission permit determination of the minimum concentration of an antibiotic needed to inactivate bacteria (Minimum Inhibitory Concentration (MIC)), thereby providing a means for rapid Antibiotic Susceptibility Testing (AST). This allows for a quick and easy means for clinicians to determine a suitable treatment regimen for patients suffering from bacterial infections and those that eventually lead to sepsis.

1 .- 25 . (canceled) 
     
     
         26 . A method for treating a patient suffering from a bacterial infection comprising the steps of:
 obtaining a minimum inhibitory concentration of one or more bacteria in a sample from the patient from a process comprising:
 i. preparing a plurality of bacterial suspensions in a plurality of receptacles; 
 ii. adding to each of the plurality of bacterial suspensions different concentrations of an antimicrobial agent, thereby creating a plurality of suspensions comprising a combination of bacteria and the antimicrobial agent; 
 iii. incubating the plurality of suspensions comprising the combination of bacteria and the antimicrobial agent at a suitable temperature for a suitable period of time, thereby creating a plurality of incubated suspensions comprising a combination of bacteria and antimicrobial agent; 
 iv. adding to the suspensions of step iii. a single membrane-associated dye; 
 v. illuminating in a cytometer the suspensions of step iv. with an incident light at an excitation wavelength for the dye; 
 vi. measuring in the cytometer, intensity of emitted light at two emission wavelengths for the dye of each suspension; and 
 vii. using data obtained from the cytometer of the measurement of the intensity of emitted light at two emission wavelengths of each suspension, determining a spectral intensity ratio (SIR) based upon step vi. of each suspension, and determining the minimum inhibitory concentration of the antimicrobial agent for the bacteria by determining the SIR as a function of the antimicrobial concentration; and 
   treating the patient with the antimicrobial agent at a concentration based upon at least the obtained minimum inhibitory concentration.   
     
     
         27 . The method of  claim 26 , wherein the function of the antimicrobial concentration is a step function. 
     
     
         28 . The method of  claim 27 , wherein the step function of the antimicrobial concentration is in the form of: 
       
         
           
             
               
                 y 
                 ⁡ 
                 
                   ( 
                   x 
                   ) 
                 
               
               = 
               
                 a 
                 · 
                 
                   erf 
                   ( 
                   
                     
                       b 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       
                         π 
                         ⁡ 
                         
                           ( 
                           
                             x 
                             - 
                             c 
                           
                           ) 
                         
                       
                     
                     2 
                   
                   ) 
                 
               
             
           
         
         wherein a is a scaling parameter, b determines the step slope, and c is the minimum inhibitory concentration value. 
       
     
     
         29 . The method of  claim 26 , wherein the single membrane-associated dye is a styryl dye of a cyanine dye. 
     
     
         30 . The method of  claim 26 , wherein the single membrane-associated dye is N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide. 
     
     
         31 . The method of  claim 26 , wherein the single membrane-associated dye is N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide. 
     
     
         32 . The method of  claim 26 , wherein the excitation wavelength is between 360 nm and 570 nm. 
     
     
         33 . The method of  claim 26 , wherein the two emission wavelengths are between 520 nm and 850 nm. 
     
     
         34 . The method of  claim 26 , wherein the sample is a bodily fluid or a clinical isolate. 
     
     
         35 . The method of  claim 34 , wherein the sample is blood or urine. 
     
     
         36 . The method of  claim 26 , wherein the suitable incubation temperature is between 35° C. and 40° C. 
     
     
         37 . The method of  claim 26 , wherein the suitable period of incubation time is between 30 minutes and 5 hours. 
     
     
         38 . The method of  claim 26 , wherein by adding different concentrations of an antimicrobial agent it is meant that the antimicrobial agent is prepared by serial dilutions. 
     
     
         39 . The method of  claim 26 , wherein the sample is initially filtered to isolate the bacteria in a concentrated form and is then diluted to a fixed concentration of bacteria. 
     
     
         40 . The method of  claim 39 , wherein the dilution occurs with a liquid growth medium. 
     
     
         41 . The method of  claim 26 , wherein the sample is initially concentrated via centrifugation and then diluted to a fixed concentration of bacteria. 
     
     
         42 . The method of  claim 26 , wherein the different concentrations of the antimicrobial agent are made with a liquid growth medium. 
     
     
         43 . The method of  claim 26 , wherein the method includes removing a portion of each of the plurality of incubated suspensions comprising the combination of bacteria and the antimicrobial agent and placing the portions in new receptacles after step iii. 
     
     
         44 . The method of  claim 26 , wherein the illumination of step v. is at one wavelength. 
     
     
         45 . The method of  claim 26 , wherein the SIR is determined using mean values of the intensity of emitted light at each of the two emission wavelengths. 
     
     
         46 . A method of treating a patient suffering from a bacterial infection comprising:
 obtaining a minimum inhibitory concentration of one or more bacteria in a sample from the patient from a process comprising:
 preparing a plurality of bacterial suspensions from a sample of bacteria obtained from the patient in a plurality of receptacles; 
 adding to the plurality of bacterial suspensions varying concentrations of an antimicrobial agent, thereby creating a plurality of suspensions comprising a combination of bacteria and the antimicrobial agent; 
 incubating the plurality of suspensions comprising a combination of bacteria and antimicrobial agent at a suitable temperature for a suitable period of time, thereby creating a plurality of incubated suspensions comprising a combination of bacteria and antimicrobial agent; 
 adding to the plurality of incubated suspensions comprising the combination of bacteria and antimicrobial agent a single membrane-associated dye; 
 illuminating in a cytometer the suspensions comprising the single membrane-associated dye with an incident light at an excitation wavelength for the dye; 
 measuring in the cytometer, intensity of emitted light of each suspension at two emission wavelengths for the membrane-associated dye; 
 determining a spectral intensity ratio (SIR) based on data obtained from the cytometer of the measurement of the intensity of emitted light at two emission wavelengths of each suspension; and 
 determining the minimum inhibitory concentration of the antimicrobial agent for the bacteria by determining the SIR as a function of the antimicrobial concentration; and 
   treating the patient with the antimicrobial agent at a concentration based upon at least the obtained minimum inhibitory concentration.   
     
     
         47 . A method of treating a bacterial infection in a patient, comprising:
 obtaining a minimum inhibitory concentration of one or more bacteria in a sample from the patient from a process comprising:
 preparing a plurality of suspensions of the bacteria obtained from the patient in a plurality of receptacles with a different concentration of an antimicrobial agent in each suspension of the plurality of suspensions; 
 incubating the plurality of suspensions at a suitable temperature for a suitable period of time for bacteria growth in the suspensions; 
 adding a single membrane-associated dye to the incubated plurality of suspensions; 
 in a cytometer, illuminating the suspensions comprising the membrane-associated dye with an incident light at an excitation wavelength for the dye; 
 in the cytometer, measuring intensity of emitted light at two emission wavelengths for the membrane-associated dye for each suspension; 
 using data obtained from the cytometer of the measurement of the intensity of emitted light at two emission wavelengths for each suspension, determining a spectral intensity ratio (SIR) based upon the relative intensity of emitted light at two emission wavelengths for the membrane-associated dye for each suspension; and 
 determining the minimum inhibitory concentration of the antimicrobial agent for the bacteria by determining the SIR as a function of the antimicrobial concentration in the plurality of suspensions; and 
   treating the patient with the antimicrobial agent at a concentration based upon at least the obtained minimum inhibitory concentration.