Method for specifically labelling living bacteria

Abstract

The present invention concerns a method for labeling specifically living bacteria of a given category of bacteria in a sample comprising bacteria, the method comprising the steps of: a) incubating said bacteria of said sample with at least one analog of a monosaccharide compound, said monosaccharide being an endogenous monosaccharide residue of glycans of the outer membrane of such given category of bacteria, the said endogenous monosaccharide residue comprising an ulosonic acid or ulosonate salt residue, the said analog of a monosaccharide compound being a modified monosaccharide substituted at a given position by a first reactive chemical group capable to react with a second reactive group of a labeling molecule, the said given position being preferably a position which comprises a free group in the said endogenous monosaccharide residue incorporated within said glycans of the outer membrane of the bacteria, b) contacting said bacteria with a said labeling molecule comprising a said second reactive group, for generating the reaction of said first reactive group of said analog residue incorporated within said glycans of the outer membrane of said living bacteria with said second reactive group of said labeling molecule.

1 . A method for labeling specifically living bacteria of a given category of bacteria in a sample comprising bacteria, the method comprising the steps of:
 a) incubating said bacteria of said sample with at least one analog of a monosaccharide compound, said monosaccharide being an endogenous monosaccharide residue of glycans of the outer membrane of such given category of bacteria, the said endogenous monosaccharide residue comprising an ulosonic acid or ulosonate salt residue, the said analog of a monosaccharide compound being a modified monosaccharide substituted at a given position by a first reactive chemical group capable to react with a second reactive group of a labeling molecule, the said given position being preferably a position which comprises a free group in the said endogenous monosaccharide residue incorporated within said glycans of the outer membrane of the bacteria,   b) contacting said bacteria with a said labeling molecule comprising a said second reactive group, for generating the reaction of said first reactive group of said analog residue incorporated within said glycans of the outer membrane of said living bacteria with said second reactive group of said labeling molecule.   
     
     
         2 . A method according to  claim 1  for labeling specifically bacteria capable of multiplying wherein said bacteria are incubated in a culture medium in or on which said bacteria are capable to multiply. 
     
     
         3 . A method according to  claim 1 , comprising the further step of:
 c) detecting living bacteria in detecting whether said bacteria comprise said labeling molecule bound to the glycans of their outer membrane and/or immobilizing said living bacteria bearing said labeling molecule onto a solid substrate.   
     
     
         4 . A method according to  claim 3  wherein said labeling molecule is a detectable molecule comprising a detectable substance or capable to react or to be bound to a detectable substance or said labeling molecule is a first molecule bearing a said second reactive group, said first molecule being capable to react or to be bound to a second molecule and/or to a solid substrate, preferably said second molecule comprising a detectable substance and/or said second molecule being bound to a said solid substrate. 
     
     
         5 . A method according to  claim 4  for specifically detecting living bacteria of a given category of bacteria in a sample comprising bacteria, wherein said labeling molecule is a detectable molecule comprising a detectable substance, the method comprising the step c) of detecting living bacteria in detecting whether said bacteria comprise said detectable molecule bound to the glycans of their outer membrane. 
     
     
         6 . A method according to  claim 4  wherein said labeling molecule is a first ligand or first binding protein bearing a said second reactive group and in step c) said living bacteria coupled to said first ligand or first binding protein is detected and/or immobilized by contacting said first ligand or first binding protein with a second ligand or second binding protein reacting or binding specifically to said first ligand or first binding protein. 
     
     
         7 . A method according to  claim 6  wherein said labeling molecule is a first ligand, preferably biotin, bearing a said second reactive group, and in step c) said living bacteria coupled to said first ligand are detected by reaction of said bacteria with an antibody specific to said first ligand, said antibody bearing a detectable substance, preferably a fluorochrome or luminescent molecule or an enzyme. 
     
     
         8 . A method according to  claim 4  wherein the said detectable substance is a fluorochrome or luminescent molecule detectable by fluorescence or luminescence. 
     
     
         9 . A method according to  claim 1  wherein the said analog of monosaccharide compound is an ulosonic acid having one of the following formulas (I) or (II), or an ulosonate salt thereof: 
       
         
           
           
               
               
           
         
         Wherein
 A, B and C can be independently H, OH, NH 2 , OH and NH 2  being substituted or not by protecting groups thereof, preferably substituted by alkyl, hydroxyalkyl, acyl, formyl or imidoyl groups, and 
 D is an alkyl chain in C 2  to C 4 , each carbon being substituted or not by OH or NH 2  substituted or not by protecting groups thereof, preferably by alkyl, hydroxyalkyl, acyl, formyl or imidoyl groups, and 
 at least one of A, B, C or D groups is substituted by a said first reactive group. 
 
       
     
     
         10 . A method according to any  claim 1  wherein the said analog of monosaccharide is a substituted octulosonic acid or octulosonate salt compound or a substituted nonulosonic acid or nonulosonate salt compound. 
     
     
         11 . A method according to  claim 10  for detecting specifically living Gram negative bacteria wherein the said given category of bacteria is the category of the Gram negative bacteria and said endogenous monosaccharide residue of said LPS layer of the outer membrane of the bacteria is a deoxyoctulosonic acid or deoxyoctulosonate residue, and said analog of monosaccharide compound is a substituted deoxyoctulosonic acid or deoxyoctulosonate compound. 
     
     
         12 . A method according to  claim 11  wherein the said analog of deoxyoctulosonic acid or deoxyoctulosonate compound is substituted by a said reactive group at one position selected among the positions 3, 4, 5, 7 and 8 of the monosaccharide, preferably 3, 7 and 8. 
     
     
         13 . A method according to  claim 9  wherein the said analog of deoxyoctulosonic acid or deoxyoctulosonate compound of formula (I) or (II) is substituted by a said first reactive group R 1  at the position 8 wherein D=—CHOH—CH 2 —R 1 , A=H, B=OH, C=OH in formula (I) or D=—CHOH—CHOH—CH 2 —R 1 , A=H, B=OH in formula (II). 
     
     
         14 . A method according to  claim 11  wherein the said Gram negative bacteria comprise  E. coli, Salmonella typhimurium, Legionella pneumophila  and  Pseudomonas aeruginosa.    
     
     
         15 . A method according to  claim 1  for labeling specifically living  Legionella pneumophila  bacteria and the said given category of bacteria is the category of the  Legionella pneumophila  bacteria and said endogenous monosaccharide residue of said LPS layer of the outer membrane of the bacteria is a 4-epilegionaminic acid (5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic acid) or 4-epilegionaminate residue, or a legionaminic acid (5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid) or legionaminate residue, and the said analog of a monosaccharide compound is respectively a substituted 4-epilegionaminic acid or 4-epilegionaminate compound, or a substituted legionaminic acid or legionaminate compound, preferably substituted at one position selected among the positions 3, 4, 5, 7, 8 and 9 of the monosaccharide cycle, preferably 5, 7 and 9. 
     
     
         16 . A method according to  claim 1  for labeling, preferably detecting, specifically living  Pseudomonas aeruginosa  bacteria and the said given category of bacteria is the category of the  Pseudomonas aeruginosa  bacteria and said endogenous monosaccharide residue of said LPS layer of the outer membrane of the bacteria is a 8-epilegionaminic acid (5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic acid) or 8-epilegionaminate residue, or a pseudaminic acid (5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid) or pseudaminate residue, and the said analog of a monosaccharide compound is respectively a substituted 8-epilegionaminic acid or 8-epilegionaminate compound, or a substituted pseudaminic acid or pseudaminate compound, preferably substituted at one position selected among the positions 3, 4, 5, 7, 8 and 9 of the monosaccharide cycle, preferably 5, 7 and 9. 
     
     
         17 . A method according to  claim 1  wherein the said first reactive group is selected among groups consisting in or bearing the group azido and groups consisting in or bearing the group alkyne, and the said second reactive group is selected among groups consisting in or bearing respectively the groups alkyne and azido, and reacting the said azido reactive group with the said alkyne reactive group is carried out in performing an azide alkyne cycloaddition. 
     
     
         18 . A method according to  claim 17  wherein the reaction is carried out in copper catalyzed conditions in the presence of a tris-triazolyl ligand, preferably TGTA. 
     
     
         19 . A method according to  claim 2  for numbering living bacteria wherein the said incubation of step a) and reaction of step b) are carried out on a solid substrate, preferably a membrane filter so that the cultivated bacteria emanating from a same original bacterium which has been multiplied are grouped together and can be visualized with a microscope and the said detectable molecule can be detected by visualization with a said microscope. 
     
     
         20 . A kit for carrying out the method of anyone of  claim 1  comprising:
 a said analog of a monosaccharide compound comprising an ulosonic acid or ulosonate compound substituted at a given position by a said first reactive chemical group, and 
 a said labeling molecule comprising a said second reactive group capable of reacting with said first reactive group, and 
 reactants for generating the reaction of said first reactive group of said analog residue incorporated within said glycans of the outer membrane of said bacteria with said second reactive group of said labeling molecule, and 
 a culture or incubation medium allowing the growth of a said given category of bacteria, preferably specific to the growth of said given category of bacteria.