Screening process for antibacterial agents

Abstract

The present invention relates to a method of preparing a phenotypically antibiotic-resistant subpopulation of stationary phase bacteria by treating stationary phase bacteria with high doses of antibacterial agents, the subpopulation thus identified, a process for identifying new antibacterial agents by testing against the antibiotic-resistant subpopulation, the compounds thus identified and their uses, particularly in treating bacterial infections involving dormant bacteria.

1 - 19 . (Cancelled).  
     
     
         20 . A method for identifying a test compound having antibacterial activity against phenotypically antibacterial resistant bacteria, comprising the steps: 
 (a) providing a first antibacterial agent;    (b) providing a population of bacteria, wherein actively growing bacteria present in said population of bacteria are known to be killed upon exposure to a minimum inhibitory concentration (MIC) of the first antibacterial agent;    (c) growing the population of bacteria of step (b) in the absence of said first anti-bacterial agent to obtain actively growing bacteria that produce colonies;    (d) treating the resulting growing population of bacteria of step (c) with said first antibacterial agent such that all actively growing bacteria in the population are killed;    (e) identifying within the resulting population of bacteria obtained in step (d) a remaining subpopulation of live bacteria that are metabolically active but are either non-growing bacteria or slowly-growing bacteria that do not produce colonies (phenotypically antibacterial resistant bacteria);    (f) isolating the resulting subpopulation of phenotypically antibacterial resistant bacteria identified in step (e);    (g) treating a first sample of the subpopulation isolated in step (f) so that said live bacteria present in the subpopulation resume active growth to obtain a growing bacteria sample;    (h) contacting the growing bacteria sample obtained in step (g) with said MIC of said first antibacterial agent, and determining whether actively growing bacteria present in said growing bacteria sample are killed by said first antibacterial agent;    (i) when in step (h) the actively growing bacteria present in said growing bacteria sample are determined to be killed by said first antibacterial agent, thereafter contacting a second sample of the subpopulation isolated in step (f) with a test compound that is different from the first antibacterial agent; and    (j) assaying the resulting contacted subpopulation obtained in step (i) for a reduction in the number of bacteria, wherein when a reduction is found, said test compound is identified as having antibacterial activity against said phenotypically antibacterial resistant bacteria.    
     
     
         21 . The method according to  claim 20 , further comprising the step of: 
 (k) amplifying said test compound identified in step (j).    
     
     
         22 . The method according to  claim 20 , wherein the first antibacterial agent is selected from the group consisting of rifampicin, kanamycin, ampicillin and pyrazinamide.  
     
     
         23 . The method as claimed in  claim 20 , wherein the MIC of said first antibacterial agent is in the range of 25 to 150 μg/ml.  
     
     
         24 . The method as claimed in  claim 20 , wherein in step (b) the population of bacteria contains 10 5  to 10 9  bacteria/ml.  
     
     
         25 . The method as claimed in  claim 20 , wherein the bacteria are a strain of a species selected from the group consisting of the  Staphylococcus aureus, Escherichia coli, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus gordonii  and  Mycobacterium tuberculosis.    
     
     
         26 . The method as claimed in  claim 20 , wherein the bacteria are a strain of  Mycobacterium tuberculosis  and said first antibacterial agent is rifampicin.  
     
     
         27 . The method as claimed in  claim 20 , wherein the bacteria are a strain of  Escherichia coli  and said first antibacterial agent is kanamycin.  
     
     
         28 . The method as claimed in  claim 20 , wherein the bacteria are a strain of  Staphylococcus aureus  and said first antibacterial agent is ampicillin.