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 . A method of preparing a phenotypically antibiotic-resistant subpopulation of stationary phase bacteria comprising at least the steps of: 
 (i) growing a bacterial culture to stationary phase; and    (ii) treating said stationary phase culture with one or more antibacterial agents at a concentration and for a time sufficient to kill growing bacteria, thereby selecting a phenotypically antibiotic-resistant subpopulation.    
     
     
         2 . A method as claimed in  claim 1  wherein said antibacterial agent is selected from the group consisting of: rifampicin, kanamycin, ampicillin and pyrazinamide.  
     
     
         3 . A method as claimed in  claim 1  or  2  wherein said antibacterial agent is used at a concentration of 25 to 150 μg/ml with bacteria present at a concentration of 10 5  to 10 9  bacteria/ml.  
     
     
         4 . A method as claimed in any one of  claims 1  to  3  wherein said bacteria are  Staphylococcus aureus, Eschericia coli, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus gordonii  or  Mycobacterium tuberculosis.    
     
     
         5 . A method as claimed in any one of  claims 1  to  4  wherein said bacteria are  Mycobacterium tuberculosis  and said antibacterial agent is rifampicin.  
     
     
         6 . A method as claimed in any one of  claims 1  to  4  wherein said bacteria are  Eschericia coli  and said antibacterial agent is kanamycin.  
     
     
         7 . A method as claimed in any one of  claims 1  to  4  wherein said bacteria are  Staphylococcus aureus  and said antibacterial agent is ampicillin.  
     
     
         8 . A phenotypically antibiotic-resistant subpopulation of stationary phase bacteria, obtainable by a method as defined in any one of  claims 1  to  7 .  
     
     
         9 . A process for assessing the antibacterial activity of a test compound or agent or for isolating a compound or agent having antibacterial activity against stationary phase bacteria comprising the steps of: 
 (i) preparing a phenotypically antibiotic-resistant subpopulation of stationary phase bacteria according to the method defined in an one of  claims 1  to  7 ;    (ii) incubating a sample of said phenotypically resistant subpopulation with one or more test compounds or agents; and    (iii) assessing any antibacterial effects against said phenotypically resistant subpopulation and optionally isolating a compound or agent exhibiting antibacterial activity.    
     
     
         10 . A process for preparing an agent or compound having antibacterial activity against stationary phase bacteria wherein said agent identified according to the process defined in  claim 9  is amplified.  
     
     
         11 . An antibacterial agent identified or prepared according to the process defined in  claim 9  or  10 .  
     
     
         12 . A chemical compound which exhibits antibacterial activity against a phenotypically antibiotic resistant subpopulation of bacteria as defined in claim.  
     
     
         13 . A composition comprising an antibacterial agent or chemical compound as defined in  claim 11  or  12  and a pharmaceutically acceptable excipient or diluent.  
     
     
         14 . A formulation comprising at least one antibacterial agent having activity against actively growing bacteria and at least one antibacterial agent or chemical compound having activity against a phenotypically antibiotic-resistant subpopulation of stationary phase bacteria as defined in  claim 12  or  13  wherein said formulation is presented as a combined preparation for simultaneous, separate or sequential use in the treatment of a bacterial infection.  
     
     
         15 . An antibacterial agent or chemical compound as defined in  claim 12  or  13  for use in the treatment of a bacterial infection.  
     
     
         16 . Use of an antibacterial agent or chemical compound as defined in  claim 12  or  13  in the preparation of a medicament for the treatment of a bacterial infection  
     
     
         17 . A method of treating of a bacterial infection comprising administering to a patient in need of such therapy an effective amount of an antibacterial agent or chemical compound as defined in  claim 12  or  13 .  
     
     
         18 . A method as claimed in  claim 17  further comprising administration of one or more antibacterial agents directed towards actively growing bacteria.  
     
     
         19 . A formulation, agent, compound, use of method as claimed in any one of  claim 14  to  18  where said bacterial infection is characterized by a subpopulation of persistent bacteria which may enter a dormant phase after infection.