Composition And Method For Stabilizing And Maintaining The Viability Of Hardy Microorganisms

Abstract

The present application is to provide a composition and method for stabilizing and maintaining the viability of hardy microorganisms from sample collection to downstream analysis. In particular, there is a method for preserving viable hardy bacteria, such as Mycobacteria, Bacillus anthracis , or Clostridium difficile , in a biological sample, comprising contacting the biological sample with a stabilization composition, wherein the stabilization composition comprises a chelating agent, a denaturing, a salt and has a p H between about 6 and about 11.

1 . A method for stabilizing the microbiome within a biological sample comprising contacting the biological sample with a stabilization composition, wherein the stabilization composition comprises a chelating agent, a denaturing agent, a salt and has a pH between 6 and 11. 
     
     
         2 . The method of  claim 1 , wherein the biological sample is a mucoid biological fluid. 
     
     
         3 . The method of  claim 2  wherein the mucoid biological fluid is sputum or saliva. 
     
     
         4 . The method of  claim 1 , wherein the composition comprises one or more of the following characteristics:
 (a) the chelating agent is ethylene glycol tetraacetic acid (EGTA), (2-Hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), diethylene triamine pentaacetic acid (DTPA), nitrilotriacetic acid (NTA), ethylenediaminetriacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), N,N-bis(carboxymethyl)glycine, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof;   (b) the denaturing agent is an anionic detergent, a cationic detergent or a nonionic detergent; and   (c) the composition comprises: (i) 2% SDS, 12.5 mM CDTA, 250 mM LiCl, 50 mM glycine and has pH 10.5; or (ii) 4% SDS, 50 mM CDTA, 250 mM LiCl, 140 mM LiOH and has pH 6.8.   
     
     
         5 . The method of  claim 4  wherein the anionic detergent is sodium dodecyl sulfate (SDS), lithium dodecyl sulphate or sodium lauroyl sulfate (SLS). 
     
     
         6 . The method of  claim 4  wherein the cationic detergent is cetyltrimethyl ammonium bromide (CTAB). 
     
     
         7 . The method of  claim 4  wherein the nonionic detergent is Tween-20™, Triton X-100™, or Brij-58™. 
     
     
         8 . The method of  claim 1 , wherein the stabilization composition preserves, in a viable state, hardy bacteria in a biological sample and reduces or eliminates other microbial growth in the sample. 
     
     
         9 . A method for characterization of bacterial nucleic acid from viable hardy bacteria in a biological sample comprising said viable hardy bacteria, comprising:
 contacting the biological sample with a stabilization composition, wherein the stabilization composition comprises a chelating agent, a denaturing agent, a salt and has a pH between 6 and 11; and   amplifying the nucleic acid in the sample, wherein the level of amplified nucleic acid remains substantially unchanged if the amplification step occurs immediately after collection, or later.   
     
     
         10 . The method of  claim 9 , wherein the biological sample is a mucoid biological fluid. 
     
     
         11 . The method of  claim 10  wherein the mucoid biological fluid is sputum or saliva. 
     
     
         12 . The method of  claim 9 , wherein the composition comprises one or more of the following characteristics:
 (a) the chelating agent is ethylene glycol tetraacetic acid (EGTA), (2-Hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), diethylene triamine pentaacetic acid (DTPA), nitrilotriacetic acid (NTA), ethylenediaminetriacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), N,N-bis(carboxymethyl)glycine, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof;   (b) the denaturing agent is an anionic detergent, a cationic detergent or a nonionic detergent; and   (c) the composition comprises: (i) 2% SDS, 12.5 mM CDTA, 250 mM LiCl, 50 mM glycine and has pH 10.5; or (ii) 4% SDS, 50 mM CDTA, 250 mM LiCl, 140 mM LiOH and has pH 6.8.   
     
     
         13 . The method of  claim 12  wherein the anionic detergent is sodium dodecyl sulfate (SDS), lithium dodecyl sulphate or sodium lauroyl sulfate (SLS). 
     
     
         14 . The method of  claim 12  wherein the cationic detergent is cetyltrimethyl ammonium bromide (CTAB). 
     
     
         15 . The method of  claim 12  wherein the nonionic detergent is Tween-20™, Triton X-100™, or Brij-58™. 
     
     
         16 . A composition comprising:
 a chelating agent;   a denaturing agent; and   viable hardy microorganisms.   
     
     
         17 . The composition of  claim 16 , wherein the composition comprises one or more characteristics:
 (a) the chelating agent is ethylene glycol tetraacetic acid (EGTA), (2-Hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), diethylene triamine pentaacetic acid (DTPA), nitrilotriacetic acid (NTA), ethylenediaminetriacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), N,N-bis(carboxymethyl)glycine, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof;   (b) the denaturing agent is an anionic detergent, a cationic detergent, or a nonionic detergent; and   (c) the composition comprises: (i) 2% SDS, 12.5 mM CDTA, 250 mM LiCl, 50 mM glycine and has pH 10.5; or (ii) 4% SDS, 50 mM CDTA, 250 mM LiCl, 140 mM LiOH and has pH 6.8.   
     
     
         18 . The method of  claim 17  wherein the anionic detergent is sodium dodecyl sulfate (SDS), lithium dodecyl sulphate or sodium lauroyl sulfate (SLS). 
     
     
         19 . The method of  claim 17  wherein the cationic detergent is cetyltrimethyl ammonium bromide (CTAB). 
     
     
         20 . The method of  claim 17  wherein the nonionic detergent is Tween-20™, Triton X-100™, or Brij-58™. 
     
     
         21 . The composition of  claim 16 , wherein the hardy microorganism is a Mycobacteria,  Bacillus anthracis , or  Clostridium difficile.    
     
     
         22 . The composition of  claim 21 , wherein the Mycobacteria is  Mycobacterium tuberculosis  and wherein the  Bacillus anthracis  is in the form of spores.