title: New fluorescein precursors for live bacteria detection.
abstract: Swiftness, reliability, and sensitivity of live bacteria detection in drinking water are key issues for human safety. The most widespread used indicator of live bacteria is a caged form of carboxyfluorescein in which 3' and 6' hydroxyl groups are masked as acetate esters (CFDA). This derivatization altogether abolishes fluorescein fluorescence and renders the molecule prone to passive diffusion through bacterial membranes. Once in the cytoplasm, acetate groups from CFDA are removed by bacterial hydrolases and fluorescence develops, rendering live but not dead cells detectable. Yet the reagent, carboxyfluorescein diacetate, still possesses a free carboxyl group whose ionization constant is such that the majority of the probe is charged at physiological pH. This unfavors probe permeation through membranes. Here, we prepare several chemical modifications of the carboxyl moiety of CFDA, in order to neutralize its charge and improve its passive diffusion through membranes. We show that the ethylamido derivative of the 5-carboxyl group from 5-carboxy-fluorescein diacetate or from Oregon green diacetate or from Oregon green diacetoxymethylester are stable molecules in biological media, penetrate into bacterial cells and are metabolized into fluorescent species. Only live bacteria are revealed since bleached samples are not labeled. Other derivatives with modification of the 5-carboxyl group with an ester group or with a thiourea-based moiety were almost inefficient probes. The most interesting probe, triembarine (5-ethylaminocarboxy-oregon green, 3',6'diacetoxymethyl ester) leads to 6-10 times more sensitive detection of bacteria as compared to CFDA. Addition of contrast agents (trypan blue or brilliant blue R) improve the signal-to-noise ratio by quenching extracellular fluorescence while bromophenol blue quenches both intracellular and extracellular fluorescence, allowing standardization of detections.
authors.affiliation: Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS/Université de Strasbourg, and Labex Medalis, ESBS, 300 Boulevard Sébastien Brant, 67412 Illkirch, France.
authors.forename: Celia
authors.initials: C
authors.lastname: Guilini
authors.affiliation: Laboratoire d'Innovation Thérapeutique, UMR 7200 CNRS/Université de Strasbourg, and Labex Medalis, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Baehr
authors.affiliation: Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS/Université de Strasbourg, and Labex Medalis, ESBS, 300 Boulevard Sébastien Brant, 67412 Illkirch, France.
authors.forename: Etienne
authors.initials: E
authors.lastname: Schaeffer
authors.affiliation: Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS/Université de Strasbourg, and Labex Medalis, ESBS, 300 Boulevard Sébastien Brant, 67412 Illkirch, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Gizzi
authors.affiliation: Bürkert Fluid Control Systems , BP21, 67220 Triembach-au-Val, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Rufi
authors.affiliation: Laboratoire d'Innovation Thérapeutique, UMR 7200 CNRS/Université de Strasbourg, and Labex Medalis, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch, France.
authors.forename: Jacques
authors.initials: J
authors.lastname: Haiech
authors.affiliation: Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS/Université de Strasbourg, and Labex Medalis, ESBS, 300 Boulevard Sébastien Brant, 67412 Illkirch, France.
authors.forename: Etienne
authors.initials: E
authors.lastname: Weiss
authors.affiliation: Laboratoire d'Innovation Thérapeutique, UMR 7200 CNRS/Université de Strasbourg, and Labex Medalis, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Bonnet
authors.affiliation: Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS/Université de Strasbourg, and Labex Medalis, ESBS, 300 Boulevard Sébastien Brant, 67412 Illkirch, France.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Galzi
mesh_terms: D001412:Bacillus subtilis / Q000302:isolation & purification; D001419:Bacteria / Q000302:isolation & purification*; D045247:Chryseobacterium / Q000302:isolation & purification; D016972:Enterobacter cloacae / Q000302:isolation & purification; D004926:Escherichia coli / Q000302:isolation & purification; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D050296:Microbial Viability; D011550:Pseudomonas aeruginosa / Q000302:isolation & purification
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; C024098:6-carboxyfluorescein