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.

Claims

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: ##STR00018## Wherein A, B and C can be independently H, OH, NH.sub.2, OH and NH.sub.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.sub.2 to C.sub.4, each carbon being substituted or not by OH or NH.sub.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.sub.1 at the position 8 wherein D=CHOHCH.sub.2R.sub.1, A=H, B=OH, C=OH in formula (I) or D=CHOHCHOHCH.sub.2R.sub.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.

Description

[0126] Other characteristics and advantages of the present invention will be more apparent in the light of the following detailed description referring to the following figures wherein:

[0127] FIG. 1A shows the Structure of the major component of E. coli K12 lipopolysaccharide, with the KDO sugar residue incorporated within IC, the Inner core of the LPS of Gram negative bacteria between OC, the outer core and LA, the lipid A, showing the anchoring site A for O-antigen and the possible site of modification B of KDO in position 8; Glc: D-glucose; GlcN: 2-amino-2-deoxy-D-glucose; KDO: 3-deoxy-D-manno-octulosonate; Hep: L-glycero-D-manno-heptose; Gal: D-galactose;

[0128] FIG. 1B shows the KDO biosynthetic pathway (KDO pathway);

[0129] FIG. 2A shows the molecules used in this example (1=KDO-N.sub.3, 2=TGTA, 3=A488-yne);

[0130] FIG. 2B shows a schematic representation of metabolic LPS labelling of E. coli with 1=KDO-N.sub.3;

[0131] FIG. 3A represents photography of E. coli K12 with metabolically incorporated KDO-N.sub.3 (+KDO-N.sub.3) and without metabolically incorporated KDO (KDO-N.sub.3) before it is revealed via A488-yne-Cu(I) catalyzed click chemistry after 5 min. (in grey) and after it is revealed (in black);

[0132] FIG. 3B represents a graphic of frequency distribution of the bacterial fluorescence values generated and plotted with (grey bars) or without (black bars) adding KDO-N.sub.3, the arbitrary unit of fluorescence (a.u.f.) range being from 0 to 1300 in abscissa and the relative frequency (r.f.) range being from 0 to 1;

[0133] FIG. 3C is a photography showing fluorescence concentrated at the cellular surface of E. coli labelled via A488-yne - Cu(I) catalyzed click chemistry during 5 min, the image being deconvolved using Richardson-Lucy algorithm with an experimental point spread fusion;

[0134] FIG. 4 are photographs representing the detection of metabolically incorporated KDO-N.sub.3 by various bacterial strains, metabolically incorporated KDO-N.sub.3 by various bacteria was revealed via A488-yne-Cu(I) catalyzed click chemistry after 60 min. Phase contrast and fluorescence images (Left panel) without adding KDO-N.sub.3; (right panel) with adding KDO-N.sub.3. Scale bar 1 m as in FIG. 3 A (bacteria with added KDO-N3 (+KDO-N3) and without added KDO (KDO-N3) before it is revealed via A488-yne-Cu(I) catalyzed click chemistry after 5 min in grey and after it is revealed in black; and

[0135] FIG. 5 represents the results (photographs and graphics) of detection of metabolically incorporated KDO-N.sub.3 by various bacterial strains. Metabolically incorporated KDO-N.sub.3 by various bacterial strains was revealed via A488-yne-Cu(I) catalyzed click chemistry after 60 min. Frequency distribution of the bacterial fluorescence values was generated and plotted with (grey bars) or without (black bars) adding KDO-N.sub.3.

[0136] The following description is the description of an illustrative example of click labeling of bacterial membranes of Gram negative bacteria via metabolic modification of the LPS inner-core with modified KDO.

[0137] Viable Gram-negative bacteria can specifically incorporate a modified KDO into their Lipopolysaccharides, decorating the cell surface with a bio orthogonal chemical reporter. Click-chemistry allows further labeling of viable cells, in an overall rapid process as schematically shown in FIG. 1.

Example 1

Assimilation of 8-azido-3,8-dideoxy-D-manno-octulosonic Acid (KDO-N.SUB.3.) and Specific Detection of Gram Negative Bacteria

[0138] 1) Within all potential targets, 3-deoxy-D-manno-octulosonic acid (KDO) appears to be a very attractive candidate. Indeed, KDO is a specific and essential component of the inner core of LPS,[7,8] and has long been considered as being present in the LPS of almost all Gram negative species (as well as higher plants and algae), where at least one residue is directly connected to lipid A (FIG. 1A).[9] Due to its vital importance, KDO has been considered as a determinant for the characterization of Gram negative bacteria, and the KDO pathway as a potential target for the development of new antibacterials.[10] In this pathway (FIG. 1B), arabinose-5-P is condensed with PEP, leading to the formation of KDO-8-P, which is then transformed into free KDO, and further activated in the form of the CMP-KDO donor, prior to LPS elaboration.

[0139] For all these reasons, it has been sought whether this KDO pathway, as a LPS-specific pathway, could be tolerant enough to incorporate a modified KDO, such as 8-azido-8-deoxy-KDO (1, FIG. 2A), into the core of E. coli LPS, and potentially other Gram negative bacteria. Given the presence of free KDO as an intermediate in the pathway, we postulated that if cell penetration of this analogue of KDO could be sufficient,[11] it could then potentially be directly activated, partially replace endogenous KDO into LPS, and be detected on the cell surface by azide-alkyne click chemistry (FIG. 2B).[12] Moreover, modification of the C-8 position of KDO by a bio orthogonal azido group should prevent reverse metabolism by KDO-8-P phosphatase (3.1.3.45), limiting the potential dissemination of the chemical reporter into other carbohydrates and metabolites.

[0140] Amongst the many potential multi-step synthetic strategies available to access 8-azido-3,8-dideoxy-D-manno-octulosonic acid 1, [13] this compound has been prepared in a straightforward manner (Scheme 1) [14] adapted from the approach described in 1963 by Ghalambor and Heath for direct KDO synthesis.[15] Namely 5-azido-5-deoxy-D-arabinofuranose[16] (6) was condensed with sodium oxaloacetate (7), leading after decarboxylation in slightly acidic conditions to KDO-N.sub.3 (1), which was isolated as its ammonium salt in 57% yield (86% based on recovered 6). The 5-azido-5-deoxy-D-arabinofuranose precursor 6 could be obtained in a very direct, simple and time-saving strategy from commercial D-arabinose, as described herein after.

1a) Synthesis of ammonium 5-azido-5-deoxy-D-arabinofuranose 6

[0141] The 5-azido-5-deoxy-D-arabinofuranose precursor 6 could be obtained in a very direct, simple and time-saving strategy, avoiding the alternative use of a bulky temporary trityl protection: commercial D-arabinose was directly tosylated on the primary position of its furanose forms, further acetylated, and subjected to nucleophilic displacement by azide anion without intermediate purification. At this step, the product could be easily separated from any other byproduct by flash chromatography. Final deacetylation afforded 6 in 15% overall yield.

##STR00012##

5-azido-5-deoxy-D-arabinofuranose (6)

[0142] Commercial D-Arabinose 5 (6.00 g, 40 mmol) was heated at 100 C. for 2 hours in pyridine (40 ml). The solution was allowed to cool down, further treated with tosyl chloride (8.38 g, 44 mmol; 1.1 equiv.), and stirred for 16 hours at room temperature. Acetic anhydride (20 ml) was then added. After complete acetylation, as determined by TLC, solvents were evaporated, and residual traces were co-evaporated several times with toluene. The residue was dissolved in DMF (100 ml), NaN.sub.3 (5.20 g, 80 mmol, 2 equiv.) was added, and the suspension was heated at 80 C. for 20 hours. After dilution with ethyl acetate and washing with water, the organic layer was dried over anhydrous magnesium sulfate and concentrated. The residue was purified by flash chromatography (Petroleum ether/Ethyl acetate 7:3). The first eluted product was determined to be the expected 5-azido-1,2,3-tri-O-acetyl-D-arabinofuranose (1.83 g, 15%, /2:1). LRMS (ESI.sup.+) 324.0 [M+Na].sup.+; HRMS (ESI+) calculated for [C.sub.11H.sub.15N.sub.3NaO.sub.7].sup.+324.0802, found: 324.0802; .sup.1H NMR (CDCl.sub.3, 360 MHz) (ppm): 6.41 (d, 0.33H, J.sub.1,2 3.5 Hz, H-1); 6.23 (d, 0.67H, H-1); 5.40-5.37 (m, 1.34H, H-2, H-3); 5.23 (d, 0.67H, J.sub.1,2 1 Hz, H-2); 5.06 (d, 0.67H, J.sub.3,4 4.6 Hz, H-3); 4.30 (ddd, 0.67H, H-4); 4.16-4.10 (m, 0.33H, H-4); 3.69 (dd, 0.67H, J.sub.4,5a 3.1 Hz, J.sub.5a,5b 13.5 Hz, H-5a); 3.61 (dd, 0.33H, J.sub.4,5a 3.6, J.sub.5a.5b 13.1 Hz, H-5a); 3.51-3.43 (m, 1H, H-5b, H-5b); 2.15, 2.13, 2.12, 2.11, 2.11, 2.09 (6s, 18H, 6 CH.sub.3CO); .sup.13C NMR (CDCl.sub.3, 62.5 MHz) (ppm): 170.3, 170.0, 169.1 (OC(O)CH.sub.3), 99.2 (C-1), 93.5 (C-1), 84.1 (C-4), 80.8 (C-4), 80.6 (C-3), 77.4 (C-2), 75.1 (C-2), 74.8 (C-3), 53.0 (C-5), 51.3 (C-5), 20.9, 20.6, 20.3 (OC)(O)CH.sub.3).

[0143] 1b) Protected 5-azido-1,2,3-tri-O-acetyl-D-arabinose was then dissolved into anhydrous methanol (30 ml), treated with a methanolic solution of MeONa (0.2 mol.l.sup., 3 ml) and stirred at room temperature for 3 h under an argon atmosphere. After neutralization (Dowex 50 (H.sup.+)) filtration, and concentration, 5-azido-5-deoxy-D-arabinofuranose 6 was obtained in 99% yield (1.03 g). LRMS (ESI.sup.+) 198.0 [M+Na].sup.+, 40%, 230 [M+MeOH+Na].sup.+, 100%; HRMS (ESI.sup.+) calculated for [C.sub.5H.sub.9N.sub.3NaO.sub.4].sup.+198.0485, found: 198.0485; .sup.1H NMR (D.sub.2O, 300 MHz) (ppm): 5.20 (d, 0.45H, J.sub.1,2 3 Hz, H-1); 5.16 (d, 0.55H, J.sub.1,2 3 Hz, H-1); 4.12-4.05 (m, 0.55H, H-4); 4.02-3.85 (m, 2H, H-2, H-2, H-3, H-3); 3.84-3.76 (m 0.45H, H-4); 3.56 (dd, 0.55H, J.sub.5a,5b 13.5, J.sub.4,5a 3.0 Hz, H-5a); 3.51 (dd, 0.45H, J.sub.5a,5b 13.0 Hz, J.sub.4,5a 3.5 Hz, H-5a); 3.35 (dd, 0.55H, J.sub.4,5b 6.5 Hz, H-5b); 3.33 (dd, 0.45H, J.sub.4,5b 6.5 Hz, H-5b); .sup.13C NMR (D.sub.2O, 75 MHz) (ppm): 101.1 C-1; 95.3 C-1; 81.4, 81.3, 79.7, 76.4, 75.9, and 74.8 C-2, 2, 3, 3, 4, 4; 52.7 C-5; 51.6, C-5.

1c) Synthesis of Ammonium 8-azido-3,8-dideoxy-D-manno-octulosonate (1.NH.SUB.3.)

[0144] A cool (4 C.) solution of 5-azido-5-deoxy-D-arabinofuranose 6 (437 mg, 2.5 mmol) in water (2.1 mL) was added to a solution of oxaloacetic acid (528 mg, 4.0 mmol) in water (2.5 mL), the pH of which has been adjusted to 11 by addition of aqueous NaOH (10M). After being stirred for two hours at room temperature, the solution was neutralized (Dowex 50 (H.sup.+)), filtrated, and heated 20 min. at 80 C.

[0145] After its pH had been adjusted to 7 with AcOH (0.5M), the mixture was purified by anion exchange chromatography (Dowex 18 (HCO.sub.2.sup.)). Initial elution with water gave unreacted 6 (150 mg, 34%). Further elution with a concentration gradient of formic acid (0.5 mol.l.sup.1.fwdarw.2 mol.l.sup.1), freeze-drying, treatment with a Dowex 50 (H.sup.+) resin, and neutralization by ammonia (0.2 mol.l.sup.1), gave after concentration, ammonium 8-azido-3,8-dideoxy-D-manno-octulosonate (1.NH.sub.3, 400 mg, 57%).

[0146] Rf 0.38 (isopropyl alcohol/water 9:1). LRMS (ESI.sup.) 262.1 [MH].sup., 100%; 525.1 [2MH].sup., 5%; 547.1 [2M2H+Na].sup., 10%; HRMS (ESI.sup.) calculated for [C.sub.8H.sub.12N.sub.3O.sub.7].sup.262.0681, found: 262.0667. IR (cm.sup.1)=3210, 2111 (N.sub.3), 1604, 1401, 1077. NMR of 1, like free KDO and derivatives, is complicated due to the presence of multiple forms (e.g. -pyranose (p, 58%), -pyranose (p, 4%), -furanose (f, 24%), -furanose (f, 14%)). Selected NMR data: .sup.1H NMR (D.sub.2O, 400 MHz) (ppm): 3.56 (dd, J.sub.8a,8b 13.2, J.sub.7,8a 2.4 Hz, H-8ap); 3.39 (dd, J.sub.7,8b 6.0 Hz, H-8bp); 2.55 (dd, J.sub.3a,3b 14.3 Hz, J.sub.3a,4 7.2 Hz, H-3af); 2.33 (dd, J.sub.3a,3b 13.1, J.sub.3a,4 6.6 Hz, H-3af); 2.26 (dd, J.sub.3b,4 7.3 Hz, H-3bf); 2.03 (dd, J.sub.3b,4 3.6, H-3bf); 1.94 (dd, J.sub.3a,3b=12.7, J.sub.3a,4 12.7 Hz, H-3ap); 1.84 (dd, J.sub.3b,4=4.9 Hz, H-3bp); 1.71 (dd, J.sub.3a,3bJ.sub.3b,4 12.0 Hz, H-3bp); .sup.13C NMR (D.sub.2O, 100 MHz) (ppm): 176.4 (C-1p), 104.1 (C-2f), 96.2 (C-2p), 85.3 (C-5f), 71.5, 68.0, 66.3 and 66.0 (C-4p, 5p, 6p, 7p), 53.8 (C-8p), 44.6 (C-3f), 33.5 (C-3p).

##STR00013##

[0147] 2) Non-pathogenic E. coli K12, which lacks an O-antigen, [17] was cultured overnight in the presence of KDO-N.sub.3 (1) as described in the following paragraph 3), and further treated, during a time course experiment, using optimized copper-catalyzed click conditions [18] as described in the following paragraph 2a), in the presence of a glucose-derived tris-triazolyl ligand (2) [19] and an Alexa Fluor 488 fluorophore bearing a terminal alkyne group (3). After 5 min of incubation, a very bright labeling of bacteria was observed, while control experiments (in the absence of the KDO-N.sub.3 analogue) did not show any signal (FIG. 3A, B). Fluorescence was carried out as described in the following paragraph 2b). Fluorescence was mostly evident around the cell periphery suggesting that membrane were preferentially labeled as expected (FIG. 3C).

[0148] 2a) Copper Catalyzed Click Chemistry.

[0149] Overnight cultures were diluted 1000 times in fresh medium (final volume 100 l) containing KDO-N.sub.3 (4 mM). Bacteria were incubated at 37 C. for 12 hours and then washed 3 times with phosphate buffer (0.05 M, pH 7.5) by centrifugation at 13000g for 1 min at room temperature.

[0150] CuSO.sub.4 and TGTA, at a final concentration of 2 mM and 4 mM respectively, were mixed overnight in a phosphate buffer (0.05 M, pH 7.5) at 37 C. under vigorous shaking. Next, aminoguanidine, sodium ascorbate and A488-yne at final concentrations of 4 mM, 5 mM and 0.13 mM respectively were added to CuSO.sub.4/TGTA overnight mix. Finally, bacteria were re-suspended in this solution. After 5, 30, 60 or 180 minutes, cells were washed 3 times with phosphate buffer by centrifugation at 13000g for 1 min at room temperature and analyzed by microscopy.

[0151] 2b) Fluorescence Microscopy.

[0152] Bacteria were inoculated onto glass cover slips and then covered with a thin (1 mm of thickness) semisolid 1% agar pad made with dilute LB ( 1/10 in phosphate buffer). Images were recorded with epifluorescence automated microscope (Nikon TE2000-E-PFS, Nikon, France) equipped with a CoolSNAP HQ 2 camera (Roper Scientific, Roper Scientific SARL, France) and a 100/1.4 DLL objective. Excitation light was emitted by a 120 W metal halide light and signal was monitored using appropriate filters. Digital analysis and image processing were conducted by a custom automation script (Visual Basic) under Metamorph 7.5 (Molecular Devices, Molecular Devices France, France), as previously described [50,51].

[0153] 3) To further consolidate this approach, its efficiency and specificity have been tested on three other Gram negative bacteria that use KDO (O86 E. coli, Salmonella typhimurium, Legionella pneumophila strain Paris) as well as three negative controls, including Shewanella oneidensis, which has recently been shown to use 8-amino-8-deoxy-KDO instead of KDO [21] and two Gram positive bacteria (Bacillus subtilis, Staphylococcus aureus) which do not produce KDO.[22]

[0154] The bacterial strains and growth conditions were as follows. E. coli 1(12, E. coli O86 and S. typhimurium 12023 were grown in M9 medium (containing also: casamino acid 0.2%, Glucose 0.2%, CaCl.sub.2 1mM, MgSO.sub.4 5 mM), S. oneidensis, B. subtilis and S. aureus were grown in Luria-Bertani (LB) medium and L. pneumophila sp. Paris was grown in yeast extract medium supplemented with L-cysteine, ferric pyrophosphate and -ketoglutarate (YEC). All strains were grown in a rotary shaker (160 rpm) at 37 C. except S. oneidensis which was grown at 28 C.

[0155] As expected, when the two E. coli strains, S. typhimurium and L. pneumophila Paris showed efficient and well defined cell-surface labeling, and no labeling was observed with S. oneidensis or Gram positive bacteria because these bacteria do not present KDO at their cell surface (FIG. 4 and FIG. 5).

[0156] 4) Incubation and Reaction on Membrane Filter.

[0157] Samples containing bacteria were filtered through 25-mm, 0.45-mm-pore-size black polyester membrane filters. Individual membranes were placed on cellulose pads (25 mm) soaked in 650 l of nutritive broth (depending of the bacteria of interest, different nutritive broth can be used) supplemented with 0.5% pyruvate or catalase (protect against oxygen toxic effect) and KDO-N.sub.3 (4 mM) in Petri dishes. Petri dishes containing the samples were incubated at 37 C. for a certain time, (depending the bacteria of interest).

[0158] Next, CuSO.sub.4 and TGTA, at a final concentration of 2 mM and 4 mM respectively, were mixed overnight in a phosphate buffer (0.05 M, pH 7.5) at 37 C. under vigorous shaking. Next, aminoguanidine, sodium ascorbate and A488-yne at final concentrations of 4 mM, 5 mM and 0.13 mM respectively were added to CuSO.sub.4/TGTA overnight mix.

[0159] Finally, individual membranes were placed on cellulose pads soaked in 650 l of this solution in Petri dishes and incubated at room temperature for 30 minutes.

[0160] Images were recorded with epifluorescence automated microscope (Nikon TE2000-E-PFS, Nikon, France) equipped with a CoolSNAP HQ 2 camera (Roper Scientific, Roper Scientific SARL, France) and a 100/1.4 DLL objective. Excitation light was emitted by a 120 W metal halide light and signal was monitored using appropriate filters. Digital analysis and image processing were conducted by a custom automation script (Visual Basic) under Metamorph 7.5 (Molecular Devices, Molecular Devices France, France), as previously described [50,51].

[0161] 5. In conclusion, it has been demonstrated that the KDO analogue can be metabolically assimilated and incorporated into the LPS without the necessity to use genetically modified bacteria. More interestingly the fact that the modified KDO needs first to be metabolically assimilated led to the use of this method for fast detection (the overall process taking less than one day) of metabolically active/viable Gram negative bacteria. This last application is very powerful in regard to the fact that detection of viable bacteria needs normally between 2 days and more than one month depending on the bacterial strain.

[0162] Severe pathogens are hiding amongst Gram negative bacteria, and the rapid identification of viable cells represents a major sanitary challenge. The present invention therefore provides a simple strategy to label the cell surface of metabolically active Gram negative bacteria, via metabolic incorporation of a modified ulosonic acid or ulosonate residue such as KDO into their lipopolysaccharide, followed by further conjugation using click-chemistry.

[0163] Of course, KDO-N.sub.3 assimilation can be subsequently coupled to the fluorescence in situ hybridization (FISH) [24] or any other well-known procedure allowing the specific detection of viable bacteria of interest.

[0164] The ribosomal-RNA (rRNA) approach to microbial evolution and ecology has become an integral part of environmental microbiology. Based on the patchy conservation of rRNA, oligonucleotide probes can be designed with specificities that range from the species level to the level of phyla or even domains. When these probes are labelled for instance with fluorescent dyes or the enzyme horseradish peroxidase, they can be used to identify single microbial cells directly by fluorescence in situ hybridization (FISH) [38]. A new approach has been proposed using specifically phage [39].

Example 2

Assimilation of 8-O-(4-ethynylbenzyl)-3-deoxy-D-manno-octulosonic acid (KDO-CCH)

[0165] This compound 8-O-(4-ethynylbenzyl)-3-deoxy-D-manno-octulosonic acid or its salt (8-O-(4-ethynylbenzyl)-3-deoxy-D-manno-octulosonate), analog of compound (Ia-1) can be prepared by the following process:

##STR00014##

[0166] A cool solution of 5-O-(4-ethynylbenzyl)-D-arabinofuranose in water can be added to a solution of oxaloacetic acid in water, the pH of which being adjusted to 10.5-11 by addition of aqueous NaOH. After being stirred at room temperature, the solution can be neutralized, and shortly heated at 80 C.

[0167] After its pH being adjusted to 7 with AcOH, the mixture can purified by anion exchange chromatography on formate resin. After initial elution with water, further elution with a concentration gradient of formic acid, freeze-drying, treatment with an acidic resin, neutralization by ammonia, and concentration, can give ammonium 8-O-(4-ethynylbenzyl)-3-deoxy-D-manno-octulosonate.

[0168] The incorporation of the compound in the bacterial LPS and the labeling can be then carried out with an azido-derived labeling molecule, using the same reagents and methods as for KDO-N.sub.3 (example 1).

[0169] KDO-CCH (before neutralization by ammonia) :

##STR00015##

Example 3

Assimilation of 9-azido-5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic Acid and Specific Detection of Legionella Bacteria

[0170] This compound 9-azido-5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic acid or its salt (9-azido-5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonate), analog of compound (Ib-1) can be prepared by the following process [52]:

##STR00016##

[0171] 6-azido-2,4-diacetamido-2,4,6-trideoxy-D-mannopyranose can be added to a solution of oxaloacetic acid in water, the pH of which being adjusted to 10.5-11 by addition of aqueous NaOH, and the mixture being stirred at room temperature in the presence or not of sodium tetraborate. Further additions of oxaloacetic acid might be necessary to ensure a good conversion. After neutralization with an acidic resin, filtration and concentration to a small volume, the solution could be applied to a formate resin, washed with water, and eluted with formic acid. Further purification, for example by reversed-phase HPLC, might be necessary.

[0172] The incorporation of the compound in the bacterial LPS and the labeling can be then carried out with the same reactive groups, the same reagents and methods as for KDO (example 1).

Example 4

Assimilation of 5,7-diazidoacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic Acid and Specific Detection of Legionella Bacteria

[0173] This compound 5,7-diazidoacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic acid or its salt (5,7-diazidoacetamido-3,5,7,9 -tetradeoxy-D-glycero-D-talo-non-2-ulosonate), analog of compound (Ib-1), can be prepared like in the previous example 2, by reaction of 2,4-diazidoacetamido-2,4,6-trideoxy-D-mannopyranose with oxaloacetic acid.

##STR00017##

[0174] The incorporation of the compound in the bacterial LPS and the labeling thereof can be then carried out as for KDO.

Example 5

Use of Various Culture Media

[0175] The kind of culture medium is not limitative as both M9 and LB E coli culture media have been successfully tested.

[0176] 1) Materials and Methods

[0177] 1.1) Bacterial Strains and Growth Conditions

[0178] E. coli K12 wild type strain grown in M9 medium (containing also: casamino acid 0.2%, glucose 0.2%, CaCl2 1 mM, MgSO4 5 mM) or in Luria-Bertani (LB) medium [54]. Cells were grown in a rotary shaker (200 rpm) at 37 C.

[0179] 1.2) Copper Catalysed Click Chemistry

[0180] Overnight cultures were diluted 100 times in fresh medium (final volume 200 l) containing KDO-N.sub.3 (1mM). Bacteria were incubated at 37 C. for 12 hours and then washed 3 times with phosphate buffer (0.05 M, pH 7.5) by centrifugation at 14000g for 2 min at room temperature.

[0181] A click pre-mix composed of CuSO4 and TGTA, at a final concentration of 2 mM and 4 mM respectively, is incubated overnight in a phosphate buffer (0.05 M, pH 7.5) at 37 C. under vigorous shaking. Next, amino guanidine, sodium ascorbate and A488-yne at final concentrations of 4 mM, 5 mM and 0.13 mM respectively were added to the overnight click pre-mix. Bacteria were then re-suspended in this solution and incubated 30 minutes at 37 C. under shaking. Finally, cells were washed 3 times with phosphate buffer by centrifugation at 14000g for 2 min at room temperature.

[0182] 1.3) Fluorescence Microscopy

[0183] Microscopic analysis were performed after Copper click chemistry on bacterial inoculum placed onto glass cover slips and covered with a thin (1 mm thick) semisolid 1% agar pad made with dilute LB ( 1/10 in phosphate buffer). Images were recorded with an epifluorescence automated microscope (Nikon TE2000-E-PFS, Nikon, France) equipped with a Cool SNAP HQ 2 camera (Roper Scientific, Roper Scientific SARL, France) and a 100/1.4 DLL objective. Excitation light was emitted by a 120 W metal halide light and signal was monitored using appropriate filters. Digital analysis and image processing were conducted by a custom automation script (Visual Basic) under Metamorph 7.5 (Molecular Devices, Molecular Devices France, France).

[0184] 2) Results

[0185] KDO assimilation occurs whatever the culture medium used.

[0186] Analysis has been performed on 616 E. coli cells from LB medium and 598 E. coli cells from minimum medium (M9).

[0187] A higher KDO assimilation is obtained when cells were grown in LB medium compared to M9 medium. However, both media are convenient and sufficient to detect and quantify cultivable bacteria.

Example 6

Detection and Counting of Cultivable/Living and Dead E. coli Cells

[0188] Known numbers of both cultivable E coli capable of multiplying and dead E. coli have been contacted with KDO-N3. KDO-N3 was incorporated only in living E coli. KDO-N.sub.3 was incorporated in all of the living E. coli.

[0189] 1) Materials and Methods

[0190] 1.1) Bacterial Strains and Growth Conditions

[0191] E. coli K12 was grown in Luria-Bertani (LB) medium in a rotary shaker (200 rpm) at 37 C. Dead E. coli K12 were obtained after heating at 120 C. for 15 minutes.

[0192] 1.2) Copper catalysed click chemistry (as disclosed in example 5).

[0193] 1.3) Bacterial counting and fluorescence microscopy

[0194] 1.3.1) For total bacterial scoring, cells were fixed and stained in a solution of paraformaldehyde 3%-DAPI 2 g/ml and filtered on an isopore polycarbonate membrane (Milipore).

[0195] 1.3.2) Colony forming units (CFU) were also monitored by plating dilutions before Copper click chemistry.

[0196] 1.3.3) Microscopic analyses were performed after Copper click chemistry on bacterial inoculum placed onto glass cover slips and covered with a thin (1 mm thick) semisolid 1% agar pad made with dilute LB ( 1/10 in phosphate buffer). Images were recorded with an epifluorescence automated microscope (Nikon TE2000-E-PFS, Nikon, France) equipped with a Cool SNAP HQ 2 camera (Roper Scientific, Roper Scientific SARL, France) and a 100/1.4 DLL objective. Excitation light was emitted by a 120 W metal halide light and signal was monitored using appropriate filters. Digital analysis and image processing were conducted by a custom automation script (Visual Basic) under Metamorph 7.5 (Molecular Devices,

[0197] Molecular Devices France, France).

[0198] 2) Results

[0199] 2.1) the total cell concentration calculated by the total scoring disclosed in 1.3.1) was 9 10.sup.9+/0.1 10.sup.9 cells/ml while the cultivable cell concentration counted by the colonies counting disclosed in 1.3.2) was 4 10.sup.9+/0.15 10.sup.9 cfu/ml. These result indicates that 44.5% (+/4%) of all cells present in this sample are cultivable.

[0200] 2.2) the counting by fluorescence after click chemistry as disclosed in paragraphs 1.2/1.3.3) provided the following results. One Sample was incubated without KDO-N3, and one sample was incubated with KDO-N3. Fluorescence analysis has been performed on 1878 E. coli cells with KDO-N3 and 3039 E. coli cells without KDO-N3. It enabled to determine a threshold of arbitrary fluorescence unit of 30 below which the cells are dead and above which the cells are cultivable. Next, applying this threshold (30), it was possible to count cell identified as dead (below 30) and identified as cultivable (above 30) on bacteria having reacted through click chemistry as in 1.3.3) Doing this evaluation, 1587 dead cells (KDO-N.sub.3) and 1452 cultivable cells (KDO-N3+) leading to a 47.7% of cultivable cell were found. This value appears statistically identical to the one obtained using dead and cultivable cells, 44.5% demonstrating that only cultivable cells are detected by the method of the present invention.

Example 7

Detection of only Gram Cultivable Cells

[0201] A mixture of Gram+ bacteria (B. subtilis) and Gram bacteria (E coli) have been contacted with KDO-N3. Only cultivable Gram bacteria was labeled with KDO-N.sub.3 and detected.

[0202] 1) Material and Method

[0203] 1.1) Bacterial Strains and Growth Conditions

[0204] E. coli K12 wild type strain rendered fluorescent via m-cherry [55], E. coli K12 sodA-mCherry [55] and B. subtilis were grown in Luria-Bertani (LB) medium in a rotary shaker (200 rpm) at 37 C.

[0205] 1.2) Copper catalysed click chemistry (as disclosed in example 6)

[0206] 1.3) Bacterial counting and fluorescence microscopy (as disclosed in example 5)

[0207] 2) Results

[0208] B. subtilis and E. coli were grown in LB medium during 12 hours. After that time cultivable cell concentration from E. coli (1.66 10.sup.9 cfu/ml) and B. subtilis (5.9 10.sup.8 cfu/ml) have been identified by counting disclosed in paragraph 1.32), indicating that 26% of the cultivable cells were B. subtilis and 74% were E. coli.

[0209] Moreover, because an E. coli sodA-mCherry strain was used, it was possible to differentiate E. coli and B. subtilis using fluorescence as disclosed above but before click chemistry. Among the 696 analyzed cells, 170 were mCherry negative (24.4%) representing by consequence B. subtilis, and 526 mCherry positive (75.6%) representing by consequence E. coli. These results are confirmed with the percentages obtained from the cfu values.

[0210] Then, all of the bacteria of the same sample have been subjected to the above click chemistry reaction and the results of counting via fluorescence are given in the following table 1.

[0211] Within the KDO-N.sub.3 negative cells, the number of mCherry negative cells representing by consequence B. subtilis (166) and the number of mCherry positive cells representing by consequence the number of E. coli cells (9) were evaluated. Within the KDO-N.sub.3 positive cells, the number of mCherry negative cells representing by consequence the number of B. subtilis (4) and the number of mCherry positive cells representing by consequence the number of cultivable E. coli (517) have been evaluated.

[0212] Using all these values, it was possible to identify 4 B. subtilis as false positive (about 2%). By contrasts the 9 E. coli KDO negative (about 2%) can be either false positive or false negative since the standard method error range is about 10%.

TABLE-US-00001 KDO-N3+ KDO-N3 Total mCherry + (E. coli) 517 9 526 mCherry (B. subtilis) 4 166 170 Total 521 175 696

[0213] These above results demonstrate therefore that KDO-N.sub.3 has well been assimilated in substantially only cultivable E. coli cells and not in cultivable B. subtilis cells.

Example 8

Detection via Biotin-Alkyne

[0214] After Kdo-N3 assimilation, click chemistry has been performed using biotin-alkyn and viable E. coli cells were detected using an anti-biotin antibody coupled to fluorochome Alexa Fluor A494. As observed by comparing cultivable counting and Kdo positive cell counting, all viable E. coli bacteria were detected by the following experimental procedure.

[0215] 1) Bacterial Strains and Growth Conditions

[0216] E. coli 1(12 wild type strain is grown in Luria-Bertani (LB) medium in a rotary shaker (200 rpm) at 37 C.

[0217] 2) Copper Catalyzed Click Chemistry:

[0218] Overnight cultures were diluted 100 times in fresh medium (final volume 200 l) containing KDO-N.sub.3 (1 mM). Bacteria were incubated at 37 C. for 12 hours, washed 3 times with phosphate buffer (0.05 M, pH 7.5) by centrifugation at 14000g for 2 min at room temperature. A click pre-mix composed of CuSO4 and TGTA, at a final concentration of 2mM and 4 mM respectively, was incubated overnight in phosphate buffer at 37 C. under vigorous shaking. Next, aminoguanidine, sodium ascorbate at final concentrations of 4 mM and 5 mM respectively were added to the overnight pre-mix click. This click-mix was added to the washed culture and supplemented or not with biotin-alkyn (Carbosynth) (1 to 100 g) and incubated for 1 to 60 min at 37 C. under shaking.

[0219] 3) Bacterial Counting and Fluorescence Microscopy:

[0220] For total bacterial scoring both before and after the Copper click chemistry, cells were fixed and stained in a solution of paraformaldehyde 3%-DAPI 2 g/ml and filtered on an isopore polycarbonate membrane (Milipore).

[0221] An anti-biotin antibody coupled to Alexa Fluor A594 (Jackson Immuno Researsh) was used diluted 10 to 1000 times to label bacteria. Colony forming units (CFU) were also monitored by plating dilutions before Copper click chemistry.

[0222] Microscopic analyses were performed after Copper click chemistry on bacterial inoculum placed onto glass cover slips and covered with a thin (1 mm thick) semisolid 1% agar pad made with dilute LB ( 1/10 in phosphate buffer). Images were recorded with an epifluorescence automated microscope (Nikon TE2000-E-PFS, Nikon, France) equipped with a CoolSNAP HQ 2 camera (Roper Scientific, Roper Scientific SARL, France) and a 100/1.4 DLL objective. Excitation light was emitted by a 120 W metal halide light and signal was monitored using appropriate filters. Digital analysis and image processing were conducted by a custom automation script (Visual Basic) under Metamorph 7.5 (Molecular Devices, Molecular Devices France, France).

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