Identification of bacterial species and subspecies using lipids

Abstract

The use of free, extractable lipids found in bacteria for identification of bacterial species and subspecies is described. Bacteria have been found to differ sufficiently in their extracted lipid compositions to effect identification using thin layer chromatographic techniques. Burkholderia pseudomallei, Burkholderia thailandensis , and Burkholderia mallei have been distinguished in this manner. Lipopeptides specific to Mycobacterium avium subspecies paratuberculosis , but not to the closely related bacterium Mycobacterium avium subspecies avium have also been used as a basis for bacterial subspecies identification using mass spectrometry and seroreactivity. Mass spectrometric analysis of total bacterial lipids of Burkholderia pseudomallei, Burkholderia thailandensis , and Burkholderia mallei , and mass spectrometric analysis of total bacterial lipids for Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subspecies avium , without further lipid separation, has shown that species and subspecies of bacteria may be identified using such analysis.

1 . A method for identifying a single bacterial species containing at least one extractable lipid, comprising the steps of: 
 extracting said at least one bacterial lipid using an organic solvent; and    identifying said at least one lipid by its polarity.    
     
     
         2 . The method of  claim 1 , further comprising the steps of: 
 removing the organic solvent;    dissolving the at least one lipid in a solvent having a chosen polarity; and    identifying said at least one lipid using two-dimensional thin-layer chromatography.    
     
     
         3 . The method of  claim 2 , wherein the two-dimensional thin-layer chromatography comprises silica-based gels.  
     
     
         4 . The method of  claim 2 , wherein the solvent having a chosen polarity is selected from the group consisting of chloroform, methanol, water, petroleum-ether, acetone, ethyl acetate, toluene, and mixtures thereof.  
     
     
         5 . The method of  claim 1 , further comprising the step of lyophilizing the bacterial cells before said step of extracting said at least one lipid.  
     
     
         6 . The method of  claim 1 , wherein said bacterial species is selected from the group consisting of  Burkholderia pseudomallei, Burkholderia thailandensis , and  Burkholderia mallei.    
     
     
         7 . A method for identifying a single bacterial subspecies containing at least one extractable lipid, comprising the steps of: 
 extracting said at least one bacterial lipid using an organic solvent; and    identifying said at least one lipid by its polarity.    
     
     
         8 . The method of  claim 7 , further comprising the steps of: 
 removing the organic solvent;    dissolving said at least one lipid in a solvent having a chosen polarity; and    identifying said at least one lipid using two-dimensional thin-layer chromatography.    
     
     
         9 . The method of  claim 8 , wherein the two-dimensional thin-layer chromatography comprises silica-based gels.  
     
     
         10 . The method of  claim 8 , wherein the solvent having a chosen polarity is selected from the group consisting of chloroform, methanol, water, petroleum-ether, acetone, ethyl acetate, toluene, and mixtures thereof.  
     
     
         11 . The method of  claim 7 , further comprising the step of lyophilizing the bacterial cells before said step of extracting said at least one lipid.  
     
     
         12 . The method of  claim 7 , wherein said bacterial subspecies is selected from the group consisting of  Mycobacterium avium  subspecies  paratuberculosis, Mycobacterium avium  subsp.  avium , and  Mycobacterium avium  subsp.  hominissuis.    
     
     
         13 . A method for identifying a single bacterial species containing at least one extractable lipid, comprising the steps of: 
 extracting said at least one bacterial lipid using an organic solvent; and    identifying said at least one lipid by its molecular weight.    
     
     
         14 . The method of  claim 13 , wherein said step of identifying said at least one lipid by its molecular weight is accomplished using mass spectrometry.  
     
     
         15 . The method of  claim 14 , wherein the mass peak corresponding to said at least one lipid and the fragmentation pattern thereof are observed.  
     
     
         16 . A method for identifying a single bacterial subspecies containing at least one extractable lipid, comprising the steps of: 
 extracting said at least one bacterial lipid using an organic solvent; and    identifying said at least one lipid by its molecular weight.    
     
     
         17 . The method of  claim 16 , wherein said step of identifying said at least one lipid by its molecular weight is accomplished using mass spectrometry.  
     
     
         18 . The method of  claim 17 , wherein the mass peak corresponding to said at least one lipid and the fragmentation pattern thereof are observed.  
     
     
         19 . The method of  claim 16 , wherein said bacteria subspecies is selected from the group consisting of  Mycobacterium avium  subspecies  paratuberculosis, Mycobacterium avium  subsp.  avium , and  Mycobacterium avium  subsp.  hominissuis.    
     
     
         20 . The method of  claim 16 , wherein said at least one lipid comprises a lipopeptide selected from the group consisting of Para-LP-01 and Para-LP-02, whereby the presence of either of said Para-LP-01 or Para-LP-02 confirms the presence of the bacterial subspecies  Mycobacterium avium  subspecies  paratuberculosis.    
     
     
         21 . A method for identifying a single bacterial subspecies containing at least one extractable lipid, comprising the steps of: 
 extracting said at least one bacterial lipid using an organic solvent; and    identifying said at least one lipid by its immunological properties.    
     
     
         22 . The method of  claim 21 , further comprising the steps of: 
 removing the organic solvent;    dissolving said at least one lipid in a solvent having a chosen polarity; and    separating said at least one lipid using two-dimensional thin-layer chromatography.    
     
     
         23 . The method of  claim 22 , wherein said step of identifying said at least one lipid is accomplished using an enzyme-linked immunosorbent assay for said separated at least one lipid.  
     
     
         24 . The method of  claim 23 , wherein said at least one lipid comprises a lipopeptide selected from the group consisting of Para-LP-01 and Para-LP-02, whereby the presence of either of said Para-LP-01 or Para-LP-02 confirms the presence of the bacterial subspecies  Mycobacterium avium  subspecies  paratuberculosis.    
     
     
         25 . The method of  claim 21 , wherein said step of identifying said at least one lipid is accomplished using thin layer chromatography, enzyme-linked immunosorbent assay.