Anti-microbial biotherapeutic agents: alternatives to conventional pharmaceutical antibiotics

Abstract

Novel antimicrobial agents that can serve as replacements to conventional pharmaceutical antibiotics are disclosed. The antimicrobial agents comprise conjugatively transmissible plasmids that kill targeted pathogenic bacteria, but are not harmful to donor bacteria. Two types of lethal transmissible plasmids are disclosed. One type kills recipient bacteria by unchecked (“runaway”) replication in the recipient cells and is prevented from occurring in donor cells. Another type kills recipient bacteria by expressing a gene that produces a product detrimental or lethal to recipient bacterial cells, that gene being prevented from expression in donor cells.

1 - 30 . (canceled)  
     
     
         31 . A method for treating a recipient bacterium, comprising: conjugatively transferring into said recipient bacterium a recombinant transmissible plasmid configured to undergo runaway replication in said recipient bacterium wherein said runaway replication in said recipient bacterium is lethal to said recipient bacterium.  
     
     
         32 . The method of  claim 31 , wherein said conjugatively transferring comprises exposing said recipient bacterium to a donor bacterium comprising said recombinant transmissible plasmid, wherein said donor bacterium is configured to conjugatively transfer said recombinant transmissible plasmid to a recipient bacterium.  
     
     
         33 . The method of  claim 32 , wherein said donor bacterium comprises one or more transfer genes.  
     
     
         34 . The method of  claim 33 , wherein said one or more transfer genes are contained on said recombinant transmissible plasmid.  
     
     
         35 . The method of  claim 33 , wherein said donor bacterium further comprises a helper plasmid, wherein said one or more transfer genes are contained on said helper plasmid.  
     
     
         36 . The method of  claim 33 , wherein said transfer genes are those of a plasmid selected from the group consisting of F, R6K and Ti.  
     
     
         37 . The method of  claim 31 , wherein said recombinant transmissible plasmid comprises an origin of transfer.  
     
     
         38 . The method of  claim 31 , wherein said recombinant transmissible plasmid comprises a portion of a plasmid selected from the group consisting of RK2, R6K, pCU1, p15A, pIP501, pAMβ1 and pCRG1600.  
     
     
         39 . The method of  claim 32 , wherein said donor bacterium comprises a gene encoding a plasmid replication repressor.  
     
     
         40 . The method of  claim 39 , wherein said recombinant transmissible plasmid comprises an origin of replication that is negatively controlled by said plasmid replication repressor.  
     
     
         41 . The method of  claim 40 , wherein said origin of replication of said recombinant transmissible plasmid is from a plasmid selected from the group consisting of R6K, RK2, rts1, p15A, RSF1010, F, and P1.  
     
     
         42 . The method of  claim 39 , wherein said gene encoding a plasmid replication repressor comprises a gene encoding a bifunctional protein, wherein said bifunctional protein comprises a plasmid replication activator function and a plasmid replication inhibitor function.  
     
     
         43 . The method of  claim 42 , wherein said gene encoding a bifunctional protein comprises a pir gene from plasmid R6K, wherein said pir gene encodes a π protein.  
     
     
         44 . The method of  claim 42 , wherein said recombinant transmissible plasmid further comprises a gene encoding a mutant variant of said bifunctional protein, wherein said mutant variant of said bifunctional protein comprises an alteration that reduces said plasmid replication inhibitor function.  
     
     
         45 . The method of  claim 44 , wherein said gene encoding a mutant variant of said bifunctional protein comprises a mutant variant of a pir gene from plasmid R6K, wherein said mutant variant of a bifunctional protein is a mutant variant of a π protein.  
     
     
         46 . The method of  claim 45 , wherein said mutant variant of a π protein comprises at least one amino acid deletion or substitution relative to the amino acid sequence of a non-mutant π protein.  
     
     
         47 . The method of  claim 46 , wherein said at least one amino acid deletion or substitution comprises a deletion or substitution at amino acid 105, 106 or 107 of the amino acid sequence of a non-mutant π protein.  
     
     
         48 . The method of  claim 46 , wherein said at least one amino acid deletion or substitution comprises a deletion or substitution at amino acids 106 and 107 of the amino acid sequence of a non-mutant π protein.  
     
     
         49 . The method of  claim 32 , wherein said donor bacterium is non-pathogenic.  
     
     
         50 . The method of  claim 32 , wherein said donor bacterium is selected from the group consisting of  Escherichia coli, Lactobacillus  spp.,  Lactococcus, Bifidobacteria, Eubacteria , and bacterial minicells.  
     
     
         51 . The method of  claim 31 , wherein said recipient bacterium is a pathogenic bacterium.  
     
     
         52 . The method of  claim 31 , wherein said recipient bacterium is Gram-negative.  
     
     
         53 . The method of  claim 31 , wherein said recipient bacterium is Gram-positive.  
     
     
         54 . The method of  claim 31 , wherein said recipient bacterium is a selected from the group consisting of  Campylobacter  spp.,  Enterobacter  spp.,  Enterococcus  spp.,  Escherichia coli, Gardnerella vaginalis, Haemophilis  spp.,  Helicobacter pylori, Mycobacterium tuberculosis, Propionobacter acnes, Pseudomonas aeruginosa  and other  Pseudomonas  spp.,  Salmonella typhimurium, Shigella  spp. and  Staphylococcus  spp.  
     
     
         55 . A method for treating a recipient bacterium, comprising: conjugatively transferring into said recipient bacterium a recombinant transmissible plasmid configured to express a killer gene in said recipient bacterium wherein expression of said killer gene in said recipient bacterium is lethal to said recipient bacterium.  
     
     
         56 . The method of  claim 55 , wherein said conjugatively transferring comprises exposing said recipient bacterium to a donor bacterium comprising said recombinant transmissible plasmid, wherein said donor bacterium is configured to conjugatively transfer said recombinant transmissible plasmid to a recipient bacterium.  
     
     
         57 . The method of  claim 55 , wherein said recombinant transmissible plasmid comprises at least one killer gene.  
     
     
         58 . The method of  claim 57 , wherein said donor bacterium is unaffected by expression of said at least one killer gene.  
     
     
         59 . The method of  claim 58 , wherein said donor bacterium represses expression of said at least one killer gene.  
     
     
         60 . The method of  claim 57 , wherein said at least one killer gene comprises a gene of a bacteriophage.  
     
     
         61 . The method of  claim 60 , wherein said bacteriophage is selected from the group consisting of T-series phages, P1, p22 and λ.  
     
     
         62 . The method  claim 56 , wherein said donor bacterium comprises one or more transfer genes.  
     
     
         63 . The method of  claim 62 , wherein said one or more transfer genes are contained on said recombinant transmissible plasmid.  
     
     
         64 . The method of  claim 62 , wherein said donor bacterium further comprises a helper plasmid, wherein said one or more transfer genes are contained on said helper plasmid.  
     
     
         65 . The method of  claim 62 , wherein said one or more transfer genes are transfer genes of a plasmid selected from the group consisting of F, R6K and Ti.  
     
     
         66 . The method of  claim 55 , wherein said recombinant transmissible plasmid comprises an origin of transfer.  
     
     
         67 . The method of  claim 56 , wherein said donor bacterium is non-pathogenic.  
     
     
         68 . The method of  claim 56 , wherein said donor bacterium is selected from the group consisting of  Escherichia coli, Lactobacillus  spp.,  Lactococcus, Bifidobacteria, Eubacteria , and bacterial minicells.  
     
     
         69 . The method of  claim 55 , wherein said recipient bacterium is a pathogenic bacterium.  
     
     
         70 . The method of  claim 55 , wherein said recipient bacterium is Gram-negative.  
     
     
         71 . The method of  claim 55 , wherein said recipient bacterium is Gram-positive.  
     
     
         72 . The method of  claim 55 , wherein said bacterium is a selected from the group consisting of  Campylobacter  spp.,  Enterobacter  spp.,  Enterococcus  spp.,  Escherichia coli, Gardnerella vaginalis, Haemophilis  spp.,  Helicobacter pylori, Mycobacterium tuberculosis, Propionobacter acnes, Pseudomonas aeruginosa  and other  Pseudomonas  spp.,  Salmonella typhimurium, Shigella  spp. and  Staphylococcus  spp.