Method for labeling specifically living microorganisms comprising the use of modified monosaccharide compounds

Abstract

The invention relates to a method for labeling specifically living microorganisms in a sample comprising microorganisms, the method comprising the steps of: a) incubating said microorganisms of said sample with at least one modified monosaccharide compound comprising a first reactive chemical group capable to chemically react with a second reactive group, so that a residue bearing said first reactive group is incorporated into such microorganisms, and b) contacting said residue incorporated in the microorganisms, with a labeling molecule comprising a said second reactive group, for generating the chemical reaction of said first reactive group of said residue incorporated within said living microorganisms with said second reactive group of said labeling molecule, resulting in a covalent link, characterized in that the said modified monosaccharide compound has the following formula (I′), or a salt thereof: —X can be O, NH or S, preferably O and NH, and —R1 and R2 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 —R3 is H or an alkyl chain in C.sub.1 to C.sub.4, each carbon being substituted or not substituted 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 X, R1, R2 and R3 groups, preferably R3, being substituted by a said first reactive group Ra. ##STR00001##

Claims

1. A method for labeling specifically living microorganisms in a sample comprising microorganisms, wherein said method comprises the steps of: a) incubating said microorganisms of said sample with at least one modified monosaccharide compound comprising a first reactive group Ra, which is capable to chemically react with a second reactive group, so that a residue bearing said first reactive group is incorporated into said living microorganisms, and b) contacting said residue incorporated into said microorganism with a labeling molecule comprising said second reactive group, for generating the chemical reaction of said first reactive group of said residue incorporated into said living microorganism with said second reactive group of said labeling molecule, resulting in a covalent link, wherein said modified monosaccharide compound has the following formula (I′), or a salt thereof: ##STR00013## X is O, and R1 and R2 are independently H, OH, or NH.sub.2, wherein the OH and NH2 are each being substituted or not by a protecting group selected from the group consisting of alkyl, hydroxyalkyl, acyl, formyl and imidoyl groups, and R3 is an alkyl chain of C.sub.1 to C.sub.4, each carbon being substituted or not by OH or NH.sub.2, wherein the OH or NH.sub.2 being substituted or not by a protecting group selected from the group consisting of alkyl, hydroxyalkyl, acyl, formyl and imidoyl groups, R3 group being substituted by said first reactive group Ra, and wherein the first reactive group Ra is selected from the group consisting of an azido group, a group bearing an azido, an alkyne group, and a group bearing an alkyne, and said second reactive group is selected from the group consisting of an alkyne group, a group bearing an alkyne, an azido group, and a group bearing an azido, wherein said first reactive group is capable of reacting with said second reactive group via an azide alkyne cycloaddition.

2. The method according to claim 1, wherein said modified monosaccharide compound is a stereoisomer having the formula (I) or a salt thereof ##STR00014##

3. The method according to claim 1, wherein: R1 and R2 are OH, which is substituted or not by said protecting group, and R3 is —CH.sub.3, —CH.sub.2OH, or —CH.sub.2NH.sub.2, which is substituted by said first reactive group Ra.

4. The method according to claim 1, wherein said modified monosaccharide compound is a compound having the following stereoisomer formula (Ia), or a salt thereof: ##STR00015##

5. The method according to claim 1, wherein Ra is —N.sub.3 or —C≡CH.

6. The method according to claim 1, wherein said microorganism is selected from the group consisting of: Prokaryotic microorganisms, and Unicellular eukaryotic microorganisms.

7. The method according to claim 6, for labeling one or more microorganisms selected from the group consisting of the following species microorganisms: a) bacteria: Acinetobacter baumannii, Bacillus cereus, Bacillus cereus, Bacillus cereus paris, Bacillus subtilis, Cronobacter sakazakii, Enterococcus durans, Enterococcus faecalis, Escherichia coli, Klebsiella pneumophila, Legionella pneumophila, Listeria monocytogenes, Micrococcus luteus, Neisseria gonorrhoeae, Neisseria meningitides, Proteus mirabilis, Providencia stuartii, Pseudomonas fluorescens Migula, Pseudomonas fluorescens Paris, Salmonella typhimurium, Serratia marcescens, Staphylococcus aureus, Staphylococcus aureus aureus, Staphylococcus epidermis, Staphylococcus saprophyticus, Stenotrophomonas maltophilia, Streptococcus agalactiae, Vibrio cholera, b) fungi: Aspergillus niger, Candida albicans, Fusarium and Geotrichum candidum, and c) amoebas: Acanthamoebae castellanii.

8. The method according to claim 1, comprising the further step of: c) detecting living microorganism in detecting whether said microorganism comprise said labeling molecule bound to said living microorganism and/or immobilizing said living microorganism bearing said labeling molecule onto a solid substrate, wherein said labeling molecule is a 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 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.

9. The method according to claim 8, wherein said labeling molecule is a detectable molecule comprising a detectable substance, wherein in step c), detecting living microorganism comprises detecting whether said microorganism comprise said detectable molecule bound to said microorganism.

10. The method according to claim 8, wherein said labeling molecule is a first ligand or first binding protein bearing said second reactive group and in step c) said living microorganism 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.

11. A kit for carrying out the method of claim 1, which comprises: said modified monosaccharide compound of formula (I), said labeling molecule comprising said second reactive group capable of reacting with said first reactive group, and optionally, reactants for generating the reaction of said first reactive group with said second reactive group of said labeling molecule.

12. The kit according to claim 11, which further comprises: said detectable molecule or said second molecule bearing a detectable substance comprising a fluorochrome or luminescent molecule or an enzyme, and/or a solid substrate bearing said second molecule capable of specifically reacting or binding with said labeling molecule.

13. The kit according to claim 12, which further comprises a culture or incubation medium allowing the growth of said microorganism.

Description

EXAMPLE 1: SYNTHESIS OF COMPOUND (IA): ARA-N.SUB.3

(1) In the synthesis the following reagents and conditions have been used: (i) TsCl, pyridine. (ii) pyridine, Ac.sub.2O. (iii) NaN.sub.3, DMF. (iv) CH.sub.3ONa, CH.sub.3OH.

(2) Thin layer chromatography was performed over Merck 60 F254 with detection by UV, and/or by charring with sulphuric acid or KMnO4 or phosphomolybdic acid solutions. Silica gel 60 40-63 μm was used for flash column chromatography.

(3) NMR spectra were taken on Bruker Avance 300 or 500 MHz spectrometers, using the residual protonated solvent as internal standard. Chemical shifts δ are given in parts per million (ppm) and coupling constants are reported as Hertz (Hz). Splitting patterns are designated as singlet (s), doublet (d), triplet (t), doublet of doublet (dd), doublet of doublet of doublet (ddd). Splitting patterns that could not be interpreted or easily visualized are designated as multiplet (m).

(4) Mass spectra were taken on a Thermo Scientific TSQ or on a Bruker micrOTOFq or on a Waters LCT Premier XE (ToF), with electrospray ionization in the positive (ESI+) mode of detection.

(5) IR-FT spectra were recorded on a Perkin Elmer Spectrum 100 spectrometer. Characteristic absorptions are reported in cm.sup.−1.

(6) Specific optical rotations were measured at 20° C. with an Anton Paar MCP 300 polarimeter in a 10-cm cell at 20° C. and 589 nm.

(7) Compound (Ia) was the compound (5) in the following scheme 1 showing the various compounds involved in the steps of the synthesis thereof.

(8) ##STR00009##

(9) The reagents and conditions in the various steps are: (i) TsCl (1.1 eq.), pyridine (1.0 M), 100° C..fwdarw.r. t., 18 h. (ii) pyridine/Ac.sub.2O (2:1, 0.7 M), 18 h. (iii) NaN.sub.3 (2.0 eq.), DMF (0.4 M), 80° C., 20 h, 15% over 3 steps. (iv) CH.sub.3ONa (0.1 eq.), CH.sub.3OH (0.2 M), r. t., 3 h, 99%

1) Preparation of 5-azido-5-deoxy-1,2,3-tri-O-acetyl-D-arabinofuranose (4)

(10) ##STR00010##

(11) Commercial D-arabinose (1) (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 (2), not isolated). 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 (3), not isolated). The residue was dissolved in DMF (100 mL), sodium azide (5.20 g, 80 mmol, 2.0 eq.) 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 (4) (1.83 g, 15%, α/β˜2:1).

(12) .sup.1H-NMR (360 MHz, CDCl.sub.3) δ (ppm): 6.41 (d, 0.33H, J.sub.1,2 3.5 Hz, H-1 β); 6.23 (d, 0.67H, J.sub.1,2˜1 Hz, H-1α); 5.40-5.37 (m, 1.34H, H-2β, H-3β); 5.23 (d, 0.67H, J.sub.1,2˜Hz, H-2α); 5.06 (d, 0.67H, J.sub.3,4 4.6 Hz, H-3α); 4.30 (ddd, 0.67H, H-4α); 4.16-4.10 (m, 0.33H, H-4β); 3.69 (dd, 0.67H, J.sub.5a,5b 13.5, J.sub.4,5a 3.1 Hz, H-5aα); 3.61 (dd, 0.33H, J.sub.5a,5b 13.1, Asa 3.6 Hz, H-Sa); 3.51-3.43 (m, 1H, H-Sba, H-Sb); 2.15, 2.13, 2.12, 2.11, 2.11, 2.09 (6s, 18H, 6 CH.sub.3CO).

(13) .sup.13C-NMR (62.5 MHz, CDCl.sub.3) δ (ppm): 170.3, 170.0, 169.1 (3 C═O); 99.2 (C-1α); 93.5 (C-1β); 84.1 (C-4α); 80.8 (C-4β); 80.6 (C-3α); 77.4 (C-2α); 75.1 (C-2β); 74.8 (C-3β); 53.0 (C-5β); 51.3 (C-5α); 20.9, 20.6, 20.3 (3 CH.sub.3).

(14) LRMS (ESI+): [M+H].sup.+ 324.0.

(15) HRMS (ESI+): [M+H].sup.+ (C.sub.11H.sub.15N.sub.3NaO.sub.7) Calc. m/z: 324.0802, found: 324.0802.

2) Preparation of 5-Azido-5-deoxy-D-arabinofuranose (5)

(16) ##STR00011##

(17) Protected 5-azido-1,2,3-tri-O-acetyl-D-arabinose (4) was then dissolved into anhydrous methanol (30 mL), treated with a methanolic solution of CH.sub.3ONa (0.2 mol.Math.L.sup.−1, 3 mL) and stirred at room temperature for 3 hours under an argon atmosphere. After neutralization (Dowex® 50 (H.sup.+)) filtration, and concentration, 5-azido-5-deoxy-D-arabinofuranose (5) was obtained in 99% yield (1.03 g).

(18) Rf (dichloromethane/methanol 92:8): 0.28.

(19) IR (cm.sup.−1): 3367, 2106, 1281, 1040.

(20) HRMS (ESI.sup.+): [M+H—N.sub.2].sup.+ (C.sub.5H.sub.10NO.sub.4) Calc. m/z: 148.0604, found: 148.0610.

(21) Anomer Alpha (5α):

(22) .sup.1H-NMR (500 MHz, D.sub.2O) δ (ppm): 5.24 (d, 1H, J.sub.1,2 2.9 Hz, H-1); 4.17 (ddd, 1H, J.sub.3,4 6.4, J.sub.4,5b 5.8, J.sub.4,5a 3.5 Hz, H-4); 4.01 (dd, 1H, J.sub.2,3 4.6, J.sub.1,2 2.9 Hz, H-2); 3.97 (dd, 1H, J.sub.3,4 6.4, J.sub.3,2 4.6 Hz, H-3); 3.64 (dd, 1H, J.sub.5a,5b 13.6, J.sub.4,5a 3.5 Hz, H-5a); 3.44 (dd, 1H, J.sub.5a,5b 13.6, J.sub.4,5b 5.8 Hz, H-5b).

(23) .sup.13C-NMR (125 MHz, D.sub.2O) δ (ppm): 101.0 (C-1); 81.3 (C-4); 81.2 (C-2); 76.3 (C-3); 51.5 (C-5).

(24) Anomer Beta (5 β):

(25) .sup.1H-NMR (500 MHz, D20) δ (ppm): 5.28 (br d, 1H, J.sub.1,2 3.1 Hz, H-1); 4.10-4.05 (m, 2H, H-2, H-3); 3.89 (ddd, 1H, J.sub.3,4 7.1, J.sub.4,5b 6.5, J.sub.4,5a 3.5 Hz, H-4); 3.59 (dd, 1H, J.sub.5a,5b 13.3, J.sub.4,5a 3.5 Hz, H-5a); 3.42 (dd, 1H, J.sub.5a,5b 13.3, J.sub.4,5b 6.5 Hz, H-5b).

(26) .sup.13C-NMR (125 MHz, D.sub.2O) δ (ppm): 95.2 (C-1); 79.6 (C-4); 75.8 (C-2); 74.7 (C-3); 52.6 (C-5).

EXAMPLE 2: COMPARISON OF LABELING OF LIVING MICROORGANISMS WITH COMPOUNDS

(27) Ara-N.sub.3 (Ia) of the present invention and compound Kdo-N.sub.3 (I-1) of the prior art.

(28) 1) Material and Methods.

(29) 1.1) Microorganism Strains and Growth Conditions.

(30) The bacteria and fungi strains listed in tables 1 and 3 are grown in the culture media and conditions listed in Tables 1 and 2. All strains were grown in a rotary shaker (160 rpm) at 30 or 37° C.

(31) TABLE-US-00001 TABLE 1 microorganisms REFERENCES Growth conditions BACTERIA Acinetobacter baumannii ATCC 17978 LB-24 H-37° C. Bacillus cereus Laboratory strain TSB-24 H-37° C. (FIEROBE team LCB/CNRS) Bacillus cereus CIP 66.24T TSB-24 H-37° C. Bacillus cereus paris Laboratory strain TSB-24 H-30° C. (N. HENRY team/CNRS) Bacillus subtilis Laboratory strain LB-24 H-37° C. (GUISEPPI team LCB/CNRS) Cronobacter sakazakii CIP 103183T TSB-24 H-37° C. Enterococcus durans Laboratory strain TSB-24 H-37° C. (FIEROBE team LCB/CNRS) Enterococcus faecalis Laboratory strain TSB-24 H-37° C. (FIEROBE team LCB/CNRS) Escherichia coli K12 MG1655 LB-24 H-37° C. Escherichia coli O86 Laboratory strain LB-24 H-37° C. (DENAMUR team/INSERM) Klebsiella pneumoniae CIP 101114 TSB + 5% sheep blood-24 H-30° C. Klebsiella pneumoniae Clinical strain TSB-24 H-37° C. (LASCOLA/Marseille, LaTimone) Kocuria varians Paris Laboratory strain TSB-24 H-30° C. (HENRY team/CNRS) Lactococcus lactis lactis Laboratory strain TSB-24 H-30° C. (FIEROBE team LCB/CNRS) Legionella pneumophila sg1 Paris CIP 33152 YEC + sup. Legionella 10%-24 H-37° C. Legionella pneumophila sg6 LG 0846 3022 YEC + sup. Legionella (CNRL, environmental strain) 10%-24 H-37° C. Listeria monocytogenes 1/2 CIP 82.110pT BHI-24 H-37° C. Listeria monocytogenes 1/2a CIP 100607 BHI-24 H-37° C. Micrococcus luteus Laboratory strain TSB-24 H-30° C. (FIEROBE team LCB/CNRS) Neisseria gonorrhoeae CIP 79.18T TSB + 10% Horse Blood-24 H-37° C. Neisseria meningitidis CIP 107858 TSB + 10% Horse Blood-24 H-37° C. Proteus mirabilis Clinical strain TSB-24 H-37° C. (LASCOLA/Marseille, La Timone) Providencia stuartii Clinical strain TSB-24 H-37° C. (LASCOLA/Marseille, La Timone) Pseudomonas fluorescens Migula ATCC 4927 TSB-24 H-37° C. Pseudomonas fluorescens Paris Laboratory strain TSB-24 H-37° C. (HENRY team/CNRS) Rhodocycclus Laboratory strain TSB-24 H-30° C. (HENRY team/CNRS) Salmonella typhimurium 12023 Laboratory strain LB-24 H-37° C. (BARRAS team LCB/CNRS) Serratia marcescens CIP 102446 TSB-24 H-30° C. Serratia marcescens Clinical strain TSB-24 H-37° C. (LASCOLA/Marseille, La Timone) Shewanella oneidensis Laboratory strain LB-24 H-37° C. (MEJEAN team LCB/CNRS) Staphylococcus aureus Clinical strain TSB-24 H-37° C. (DUKAN team LCB/CNRS) Staphylococcus aureus aureus IP 53-156 TSB-24 H-37° C. Staphylococcus epidermis Laboratory strain TSB-24 H-37° C. (FIEROBE team LCB/CNRS) Staphylococcus saprophyticus Clinical strain TSB-24 H-37° C. (LASCOLA/Marseille, La Timone) Stenotrophomonas maltophilia Clinical strain TSB-24 H-37° C. (LASCOLA/Marseille, La Timone) Streptococcus agalactiae Clinical strain TSB-24 H-37° C. (LASCOLA/Marseille, La Timone) Vibrio cholerae CIP 104151 TSB-24 H-37° C. EUKARYOTES Aspergillus niger Laboratory strain TSB-24 H-30° C. (FIEROBE team LCB/CNRS) Candida albicans Laboratory strain TSB-24 H-30° C. (FIEROBE team LCB/CNRS) Fusarium CNCM 1149-76 TSB-24 H-30° C. Geotrichum candidum CNCM 1447-83 TSB-24 H-30° C. Saccharomyces cerevisiae BY4741 YPD + 10% glucose Acanthamoebae castellanii ATCC 30234 PYG

(32) TABLE-US-00002 TABLE 2 Composition Provider Reference Tryptic Soy casein peptone (pancreatic) 17 g/l + Soya Sigma Aldrich 2209 Broth (TSB) peptone (papain digest.) 3 g/l + Sodium (USA) chloride 5 g/l + Dipotassium hydrogen phosphate 2.5 g/l + Glucose 2.5 g/l pH 7.3 Brain Heart brain infusion solids 12.5 g/l + beef heart Oxoid CM113 Infusion (BHI) infusion solids 5 g/l + proteose peptone (GB) 10 g/l + glucose 2 g/l + sodium chloride 5 g/l + di sodium phosphate 2.5 g/l YEC yeast extract 10 g/l + Casamino acid Becton Dickinson Bacto 212720 (USA) PYG Proteose peptone 20 g/L + yeast extract ATCC 712 PYG 1 g/L + glucose 18 g/L + calcium chloride + di sodium phosphate + potassium phosphate + magnesium sulphate + ° sodium citrate-PH = 6.5 Luria Bactotryptone 10 g/l+ Becton Dickinson 211699 Bertani (LB) (USA) Bacto yeast extract 5 g/l+ Becton Dickinson 212720 (USA) Sodium chloride+ Sigma Aldrich 55886 (USA) pastagar 15 g/l Biorad (USA) 64946 pH 7.2

(33) 1.2) Copper Catalyzed Click Chemistry

(34) Overnight cultures were diluted 100 times in fresh medium (final volume 100 μl) containing Kdo-N.sub.3(I-1) or Ara-N.sub.3(Ia) (10 mM). Microorganisms were incubated at 30 or 37° C. for 24 hours and then washed 3 times with phosphate buffer (0.05 M, pH 7.5) by centrifugation at 13,000×g for 2 min at room temperature.

(35) Two fluorochrome-alkyne probes of following formula A488-yne (6a) and A594-yne (6b) were used:

(36) ##STR00012##
CuSO.sub.4 and TGTA, at a final concentration of 2 mM and 4 mM respectively, were mixed overnight in phosphate buffer (0.05 M, pH 7.5) at 37° C. under vigorous shaking. Next, aminoguanidine, sodium ascorbate and A488-yne (6a) or A594-yne (6b) at a final concentration of 4 mM, 5 mM and 1 mM respectively were added to CuSO.sub.4/TGTA overnight mix. Finally, microorganisms were re-suspended in this solution and incubated for 30 minutes at 37° C. under shaking. Finally, cells were then washed 3 times with phosphate buffer by centrifugation at 14,000×g for 2 min at room and analyzed by microscopy.

(37) 1.3) Fluorescence Microscopy.

(38) Microorganisms were inoculated onto glass cover slips and covered with a thin (1 mm of thickness) semisolid 1% agar pad made with dilute LB (1/10 in phosphate buffer (0.05 M, pH 7.5)). 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).

(39) 2) Results

(40) 21.) Different Strains of Bacteria, Fungi and Amoebas have been Tested with Both Compounds Ara-N3 (Ia) of the Present Invention and Kdo-N.sub.3 (I-1) of the Prior Art in Comparison.

(41) These strains were grown first in the presence of compound (Ia) or (I-1) and the incorporation of the azido chemical reporter into the microorganism was monitored in a subsequent step, using copper-catalyzed azide-alkyne cycloaddition in the conditions previously described, with copper sulfate, sodium ascorbate, TGTA, a water-soluble tris(triazolyl) ligand for copper (I), and a fluorochrome-alkyne probe of above formula (6a) or (6b), for 30 minutes as above disclosed.

(42) In these experiments, strains showing highly distinctive fluorescence, indicative of an effective metabolic incorporation of the chemical reporter have been marked “+” in the table 3 below and the absence of labeling has been marked “−” and the not tested bacteria have been marked “NT” in table 3.

(43) TABLE-US-00003 TABLE 3 Microorganisms Kdo-N.sub.3 Ara-N.sub.3 BACTERIA Acinetobacter baumannii + + Bacillus cereus NT + Bacillus cereus NT + Bacillus cereus paris NT + Bacillus subtilis − + Cronobacter sakazakii + + Enterococcus durans NT + Enterococcus faecalis − + Escherichia coli K12 + + Escherichia coli O86 + + Klebsiella pneumoniae + + Klebsiella pneumoniae + + Legionella pneumophila sg1 Paris + + Legionella pneumophila sg6 + + Listeria monocytogenes 1/2 − + Micrococcus luteus − + Neisseria gonorrhoeae − + Neisseria meningitides + + Proteus mirabills + + Providencia stuartii + + Pseudomonas fluorescens Migula + + Pseudomonas fluorescens Paris + + Salmonella typhimurium 12023 + + Serratia marcescens + + Serratia marcescens + + Staphylococcus aureus − + Staphylococcus aureus aureus − + Staphylococcus epidermis − + Staphylococcus saprophyticus NT + Stenotrophomonas maltophilia + + Streptococcus agalactiae NT + Vibrio cholerae + + EUKARYOTES Aspergillus niger − + Candida albicans − + Fusarium − + Geotrichum candidum − + Acanthamoebae castellanii − +

(44) These experiments show that compound (Ia) of the present invention is assimilated by a broad range of bacteria and Fungi as well as amoebas (all tested microorganisms were labeled). Interestingly, compound (Ia) is assimilated by the following bacteria (all Gram positive bacteria) and Fungi strains (all the tested Fungi) which did not assimilate compound (I-1): a) Bacteria: Bacillus subtilis, Enterococcus faecalis, Neisseria gonorrhoeae, Listeria monocytogenes, Micrococcus luteus, Staphylococcus aureus, Staphylococcus aureus aureus, and b) Fungi: Aspergillus niger, Candida albicans, Fusarium and Geotrichum candidum, and c) amoebas: Acanthamoebae castellanii.