Methods and compositions for reducing bacterial tolerance to antibacterials, disinfectants and organic solvents

Abstract

The invention relates to methods and compositions for manipulating bacterial resistance to non-antibiotic antibacterial compositions, disinfectants and organic solvents. The invention provides methods for rendering bacterial cells susceptible to non-antibiotic antibacterial compositions. Also provided are methods to reduce the selection of bacterial mutants having an multiple antibiotic resistance phenotype by non-antibiotic antibacterial compositions. The invention also provides methods for testing the ability of non-antibiotic antibacterial compositions to select for or induce a multiple antibiotic resistance phenotype in bacteria. Also provided are methods for increasing or decreasing bacterial tolerance to organic solvents by increasing or decreasing the activity of bacterial organic solvent efflux pumps. Compositions useful in the foregoing methods are also provided.

We claim:  
     
         1 . A method for inhibiting the selection or propagation of a bacterial mutant that overexpresses an efflux pump comprising: 
 contacting bacteria with an agent that binds to a gene locus or an expression product thereof, wherein the expression of the gene locus enhances expression of the efflux pump, in an amount effective to inhibit the gene locus-enhanced expression of the efflux pump.    
     
     
         2 . The method of  claim 1 , wherein the gene locus is selected from the group consisting of a mar locus, a sox locus and a rob locus.  
     
     
         3 . The method of  claim 2 , wherein the gene locus is marA.  
     
     
         4 . The method of  claim 2 , wherein the gene locus is soxS.  
     
     
         5 . The method of  claim 2 , wherein the gene locus is robA.  
     
     
         6 . The method of  claim 1 , wherein the efflux pump is acr-like.  
     
     
         7 . The method of  claim 6 , wherein the efflux pump is acrAB.  
     
     
         8 . The method of  claim 1 , wherein the agent is selected from the group consisting of antisense nucleic acids, antibodies, ribozymes, chemicals and proteins which repress expression of the gene locus.  
     
     
         9 . The method of any of claims  1 - 8 , wherein the agent is an antisense nucleic acids.  
     
     
         10 . A method for rendering bacterial cells more susceptible to a non-antibiotic bactericidal or bacteriostatic agent that is a substrate of an efflux pump comprising: 
 administering to the bacterial cell an inhibitor of a gene locus or an expression product thereof, wherein the expression of the gene locus enhances expression of an efflux pump.    
     
     
         11 . The method of  claim 10 , wherein the gene locus is selected from the group consisting of a mar locus, a sox locus and a rob locus.  
     
     
         12 . The method of  claim 11 , wherein the gene locus is marA.  
     
     
         13 . The method of  claim 11 , wherein the gene locus is soxS.  
     
     
         14 . The method of  claim 11 , wherein the gene locus is robA.  
     
     
         15 . The method of  claim 10 , wherein the efflux pump is acr-like.  
     
     
         16 . The method of  claim 15 , wherein the efflux pump is acrAB.  
     
     
         17 . The method of  claim 10 , wherein the inhibitor is selected from the group consisting of antisense nucleic acids, antibodies, ribozymes, chemicals and proteins which repress expression of the gene locus.  
     
     
         18 . The method of any of claims  10 - 17 , wherein the inhibitor is an antisense nucleic acid.  
     
     
         19 . A method for rendering bacterial cells more susceptible to a non-antibiotic bactericidal or bacteriostatic agent that is a substrate of an efflux pump comprising: 
 administering to the bacterial cell an inhibitor of the efflux pump.    
     
     
         20 . The method of  claim 19 , wherein the efflux pump is acr-like.  
     
     
         21 . The method of  claim 20 , wherein the efflux pump is acrAB.  
     
     
         22 . The method of  claim 19 , wherein the inhibitor is selected from the group consisting of L-phenylalanyl-L-arginyl-β-naphthylamide, 4% ethanol, methanol, hexane, minocycline.  
     
     
         23 . The method of any of claims  19 - 22 , wherein the inhibitor is L-phenylalanyl-L-arginyl-β-naphthylamide.  
     
     
         24 . A method for increasing the ability of bacterial cells to survive in an organic solvent comprising: 
 enhancing expression in the bacterial cells of an organic solvent bacterial efflux pump by growing the bacterial cells in the presence of a non-mar/sox/rob agent that induces the overexpression of the organic solvent bacterial efflux pump.    
     
     
         25 . The method of  claim 24 , wherein the agent is a gene encoding an acr-like pump or an expression product thereof.  
     
     
         26 . The method of  claim 25 , wherein the acr-like pump is acrAB.  
     
     
         27 . The method of  claim 24 , wherein the agent is selected from the group consisting of an antibiotic, and a non-antibiotic antibacterial compound.  
     
     
         28 . A method for decreasing the ability of bacterial cells to survive in an organic solvent comprising: 
 reducing expression in the bacterial cells of an organic solvent bacterial efflux pump by growing the bacterial cells in the presence of an agent that reduces the expression of the organic solvent bacterial efflux pump.    
     
     
         29 . The method of claims  28 , wherein the agent is an inhibitor of a gene locus or an expression product thereof, wherein the expression of the gene locus enhances expression of an efflux pump.  
     
     
         30 . The method of  claim 29  wherein the gene locus is selected from the group consisting of a mar locus, a sox locus and a rob locus.  
     
     
         31 . The method of  claim 30  wherein the gene locus is marA.  
     
     
         32 . The method of  claim 30  wherein the gene locus is soxS.  
     
     
         33 . The method of  claim 30  wherein the gene locus is robA.  
     
     
         34 . The method of  claim 29  wherein the efflux pump is acr-like.  
     
     
         35 . The method of  claim 34  wherein the efflux pump is acrAB.  
     
     
         36 . The method of  claim 29  wherein the inhibitor is selected from the group consisting of antisense nucleic acids, antibodies, ribozymes, chemicals and proteins which repress expression of the gene locus.  
     
     
         37 . The method of any of claims  28 - 36  wherein the inhibitor is an antisense nucleic acid.  
     
     
         38 . A method for testing the ability of a non-antibiotic composition to induce a multiple antibiotic resistance phenotype in a bacterium comprising 
 (a) contacting the bacterium with the non-antibiotic composition,    (b) determining the expression of a bacterial gene locus, the altered expression of which is indicative of induction of the multiple antibiotic resistance phenotype in the bacterium, and    (c) comparing the result of (b) with a control, wherein altered expression of the bacterial gene locus indicates that the non-antibiotic composition induces the multiple antibiotic resistance phenotype in the bacterium.    
     
     
         39 . The method of  claim 38 , wherein the gene locus is selected from the group consisting of a mar locus, a sox locus, a rob locus and an acr-like efflux pump locus.  
     
     
         40 . The method of  claim 39 , wherein the gene locus is marA.  
     
     
         41 . The method of  claim 39 , wherein the gene locus is soxS.  
     
     
         42 . The method of  claim 39 , wherein the gene locus is robA.  
     
     
         43 . The method of  claim 39 , wherein the efflux pump is acr-like.  
     
     
         44 . The method of  claim 43 , wherein the efflux pump is acrAB.  
     
     
         45 . The method of  38 , wherein the composition is an inactive ingredient.  
     
     
         46 . The method of  claim 45 , wherein the inactive ingredient is a non-bactericidal ingredient.  
     
     
         47 . The method of  claim 45 , wherein the inactive ingredient is a non-bacteriostatic ingredient.  
     
     
         48 . The method of  claim 38 , wherein step (b) is performed by determining the enzymatic activity of an expression product of a marker gene fused to the bacterial gene locus.  
     
     
         49 . The method of  claim 48 , wherein the marker gene is lacZ.  
     
     
         50 . A composition comprising: 
 a non-antibiotic bactericidal or bacteriostatic first agent and    a second agent that inhibits the expression of or activity of an efflux pump.    
     
     
         51 . The composition of  claim 50 , wherein the second agent inhibits the expression of a gene locus or an expression product thereof, wherein the expression of the gene locus enhances expression of the efflux pump.  
     
     
         52 . The composition of  claim 51 , wherein the second agent is selected from the group consisting of antisense nucleic acids, antibodies, ribozymes, chemicals and proteins which repress expression of the gene locus.  
     
     
         53 . The composition of  claim 52 , wherein the second agent is an antisense nucleic acid.  
     
     
         54 . The composition of  claim 50 , wherein the second agent inhibits an acr-like efflux pump.  
     
     
         55 . The composition of  claim 54 , wherein the second agent is selected from the group consisting of L-phenylalanyl-L-arginyl-β-naphthylamide, 4% ethanol, methanol, hexane, minocycline.  
     
     
         56 . The method of  claim 55 , wherein the second agent is L-phenylalanyl-L-arginyl-β-naphthylamide.  
     
     
         57 . The composition of  claim 50 , wherein the first agent is selected from the group consisting of triclosan, pine oil, quaternary amine compounds including alkyl dimethyl benzyl ammonium chloride, chloroxylenol, triclocarbon, disinfectants and organic solvents.  
     
     
         58 . A method for identifying an antibacterial composition which does not select or induce a multiple antibiotic resistance phenotype in a bacterium, comprising 
 (a) contacting the bacterium with the antibacterial composition,    (b) determining the expression of a bacterial gene locus, the altered expression of which is indicative of induction of the multiple antibiotic resistance phenotype in the bacterium, and    (c) comparing the result of (b) with a control, wherein a lack of altered expression of the bacterial gene locus indicates that the antibacterial composition induces the multiple antibiotic resistance phenotype in the bacterium.    
     
     
         59 . The method of  claim 58 , wherein the gene locus is selected from the group consisting of a mar locus, a sox locus, a rob locus and an acr-like efflux pump locus.  
     
     
         60 . The method of  claim 59 , wherein the gene locus is marA.  
     
     
         61 . The method of  claim 59 , wherein the gene locus is soxS.  
     
     
         62 . The method of  claim 59 , wherein the gene locus is robA.  
     
     
         63 . The method of  claim 59 , wherein the efflux pump is acr-like.  
     
     
         64 . The method of  claim 63 , wherein the efflux pump is acrAB.  
     
     
         65 . The method of  claim 58 , wherein step (b) is performed by determining the enzymatic activity of an expression product of a marker gene fused to the bacterial gene locus.  
     
     
         66 . The method of  claim 65 , wherein the marker gene is lacZ.