Sensitive and rapid determination of antimicrobial susceptibility

Abstract

The present invention relates to moving microorganisms to a surface, where they are grown in the presence and absence of antimicrobials, and by monitoring the growth of the microorganisms over time in the two conditions, their susceptibility to the antimicrobials can be determined. The microorganisms can be moved to the surface through electrophoresis, centrifugation or filtration. When the movement involves electrophoresis, the presence of oxidizing and reducing reagents lowers the voltage at which electrophoretic force can be generated and allows a broader range of means by which the target can be detected. Monitoring can comprise optical detection, and most conveniently includes the detection of individual microorganisms. The microorganisms can be stained in order to give information about their response to antimicrobials.

1 . (canceled) 
     
     
         2 . A method of determining a minimum inhibitory concentration (MIC) of an anti- organism agent in a sample comprising:
 loading a sample comprising a plurality of microorganisms into a detection chamber;   immobilizing an individual microorganism to a surface of the detection chamber;   adding an anti-organism agent to the detection chamber;   increasing the concentration of an anti-organism agent in steps over a period of time;   measuring the growth of the individual microorganism over time; and   determining the MIC of the anti-organism agent based on the concentration of the anti-organism agent that prevents growth of the individual microorganism.   
     
     
         3 . The method of  claim 1  further comprising, administering a constant dose of the anti-organism agent, and observing growth of the individual microorganism to determine a therapeutic dose. 
     
     
         4 . The method of  claim 1 , wherein the sample is loaded using an electrophoretic force, electroosmosis, positive displacement pumps, peristaltic pumps, magnetic fields or fluid flow. 
     
     
         5 . The method of  claim 1 , wherein the sample is loaded using an electrophoretic force. 
     
     
         6 . The method of  claim 1 , wherein the sample is immobilized using an electrophoretic force, fluid flow, filtration, electroosmosis, or centrifugal forces. 
     
     
         7 . The method of  claim 1 , wherein the sample is immobilized using an electrophoretic force. 
     
     
         8 . The method of  claim 1  further comprising, growing the individual microorganism in the detection chamber. 
     
     
         9 . The method of  claim 1 , wherein measuring the growth of an individual microorganism comprises using optical detection. 
     
     
         10 . The method of  claim 1  further comprising, determining viability of the microorganism. 
     
     
         11 . The method of  claim 1 , wherein immobilizing the individual microorganism comprises specific or nonspecific binding of a target microorganism to the detection chamber surface. 
     
     
         12 . The method of  claim 1  further comprising, determining the identity of the individual microorganism based on its response to the anti-organism agent. 
     
     
         13 . The method of  claim 1 , wherein the concentration of anti-organism agent increases no faster than half the doubling time of the individual microorganism under conditions of incubation lacking the anti-organism agent. 
     
     
         14 . A method of determining a minimum inhibitory concentration (MIC) of an anti-organism agent in a sample comprising:
 loading a sample comprising a plurality of microorganisms into a detection chamber;   immobilizing an individual microorganism to a surface of the detection chamber;   detecting growth of the individual microorganism at different concentrations of anti-organism agent; and   determining the MIC of the anti-organism agent based on the concentration of the anti-organism agent that prevents growth of the individual microorganism.   
     
     
         15 . The method of  claim 14  further comprising, determining that the individual microorganism growth in the absence of the anti-organism agent relative to the individual microorganism growth in the presence of the anti-organism agent is statistically relevant. 
     
     
         16 . The method of  claim 14 , wherein detecting individual microorganism growth comprises detecting doubling of less than 25% of the microorganisms in the sample. 
     
     
         17 . The method of  claim 14 , wherein detecting individual microorganism growth comprises observing the appearance of growth in the individual microorganism through its size, ability to accept additional staining agent, or the occurrence of nearby daughter microorganisms. 
     
     
         18 . The method of  claim 14  further comprising, determining the minimum bactericidal concentration (MBC) of the anti-organism agent based in the concentration of the anti-organism agent that caused the number of live microorganisms to decline. 
     
     
         19 . A method of determining a minimum inhibitory concentration (MIC) of an agent in a sample comprising:
 transporting a sample comprising a plurality of microorganisms to a first detection zone and a second detection zone;   attaching a first individual microorganism to a first detection surface in the first detection zone and a second individual microorganism to a second detection surface in the second detection zone;   determining the number of individual microorganisms in the first and second detection zones at a first time point;   adding a first concentration of agent to the first detection zone and a second concentration of agent to the second detection zone wherein the first concentration of agent is greater than the second concentration of the agent;   determining the number of individual microorganisms in the first and second detection zones at a second time point;   determining the MIC of the agent based on the concentration of the agent that prevents growth of the individual microorganism in either the first detection zone or the second detection zone.   
     
     
         20 . The method of  claim 19  further comprising, determining the persistent response of the individual microorganism once the agent has been removed. 
     
     
         21 . The method of  claim 19 , wherein the agent is an antibiotic, antifungal, hormone, drug, or environmental agent.