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

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.

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.

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.