Carbapenemases for use with antibiotics for the protection of the intestinal microbiome

Abstract

This invention relates, in part, to various compositions and methods for protecting the gastrointestinal microbiome from antibiotic disruption.

1 . A method of protecting a subject's gastrointestinal microbiome, comprising administering an effective amount of a pharmaceutical composition comprising a broad spectrum carbapenemase to a subject in need thereof, wherein:
 the subject is undergoing treatment or has recently undergone treatment with a carbapenem antibiotic, and   the broad spectrum carbapenemase is capable of degrading or inactivating the carbapenem in the subject's gastrointestinal tract.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein the broad spectrum carbapenemase is a metallo-β-lactamase. 
     
     
         4 .- 6 . (canceled) 
     
     
         7 . The method of  claim 3 , wherein the broad spectrum carbapenemase has an amino acid sequence having at least 95% sequence similarity with SEQ ID NO: [37] 68. 
     
     
         8 . The method of  claim 7 , wherein the broad spectrum carbapenemase has an amino acid sequence having at least 99% sequence similarity with SEQ ID NO: 68. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the protection of the subject's microbiome comprises treatment or prevention of a microbiome-mediated disorder. 
     
     
         11 . (canceled) 
     
     
         12 . The method of claim [ 1 ]  10 , wherein the microbiome-mediated disorder is one or more of an antibiotic-induced adverse effect,  C. difficile  infection (CDI), and a  C. difficile -associated disease. 
     
     
         13 . The method of  claim 12 , wherein the antibiotic-induced adverse effect and/or CDI or  C. difficile -associated disease is one or more of: antibiotic-associated diarrhea,  C. difficile  diarrhea (CDD),  C. difficile  intestinal inflammatory disease, colitis, and pseudomembranous colitis. 
     
     
         14 . The method of  claim 1 , wherein the protection of the subject's microbiome comprises maintenance of a normal intestinal microbiota. 
     
     
         15 . The method of  claim 14 , wherein the method treats and/or prevents the overgrowth of one or more pathogenic microorganisms in the GI tract of a subject. 
     
     
         16 . The method of  claim 15 , wherein the method treats or prevents a nosocomial infection and/or a secondary emergent infection. 
     
     
         17 . The method of  claim 1 , wherein the broad spectrum carbapenemase does not substantially interfere with blood or plasma levels of the antibiotic. 
     
     
         18 . The method of  claim 1 , wherein the broad spectrum carbapenemase degrades or inactivates excess or residual antibiotic in the subject's gastrointestinal tract. 
     
     
         19 - 21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the carbapenem is selected from meropenem, imipenem, ertapenem, and doripenem. 
     
     
         23 - 60 . (canceled) 
     
     
         61 . A method for producing an antibiotic-degrading agent in  Escherichia coli  ( E. coli ), comprising:
 (a) providing a host  E. coli  cell transformed with a vector comprising a sequence encoding the antibiotic-degrading agent;   (b) culturing the  E. coli  cell to induce expression of the antibiotic-degrading agent; and   (c) recovering the antibiotic-degrading agent from a soluble fraction prepared from the  E. coli  cell,   wherein:
 the antibiotic-degrading agent is broad spectrum carbapenemase having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 68 or SEQ ID NO: 53 and 
 the culturing is in the presence of an amount of zinc sufficient to substantially increase the amount of antibiotic-degrading agent protein in a soluble fraction and reduce the amount of antibiotic-degrading agent protein in inclusion bodies relative to culturing in the absence of zinc. 
   
     
     
         62 - 76 . (canceled) 
     
     
         77 . The method of  claim 61 , wherein the broad spectrum carbapenemase has an amino acid sequence of SEQ ID NO: 68. 
     
     
         78 . The method of  claim 61 , wherein the broad spectrum carbapenemase has an amino acid sequence of SEQ ID NO: 53. 
     
     
         79 . The method of  claim 8 , wherein the broad spectrum carbapenemase has an amino acid sequence having at least 99% sequence similarity with SEQ ID NO: 68 and lacks a leader sequence. 
     
     
         79 . The method of  claim 79 , wherein the broad spectrum carbapenemase consists essentially of an amino acid sequence of SEQ ID NO: 68. 
     
     
         80 . The method of  claim 1 , wherein the broad spectrum carbapenemase has an amino acid sequence having at least 95% sequence similarity with one of SEQ ID NOs: 53 and 54. 
     
     
         81 . The method of  claim 1 , wherein the broad spectrum carbapenemase has an amino acid sequence having at least 95% sequence similarity with SEQ ID NO: 38.