title: Rapid and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click Chemistry Coupled with Magnetic Beads Separation.
abstract: Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N3), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope.
authors.affiliation: Aix Marseille Université, Laboratoire de Chimie Bactérienne (UMR 7283), Institut de Microbiologie de la Méditerranée (IMM), CNRS, 31 Chemin Joseph Aiguier-13402, Marseille, France.
authors.forename: Emilie
authors.initials: E
authors.lastname: Fugier
authors.affiliation: Aix Marseille Université, Laboratoire de Chimie Bactérienne (UMR 7283), Institut de Microbiologie de la Méditerranée (IMM), CNRS, 31 Chemin Joseph Aiguier-13402, Marseille, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Dumont
authors.affiliation: CNRS and Univ Paris-Sud, Laboratoire de Synthèse de Biomolécules, Institut de Chimie Moléculaire et des Matériaux d'Orsay, UMR 8182, 91405, Orsay, France.
authors.forename: Annie
authors.initials: A
authors.lastname: Malleron
authors.affiliation: Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles du CNRS, Avenue de la Terrasse, 91198, Gif-sur-Yvette, France.
authors.forename: Enora
authors.initials: E
authors.lastname: Poquet
authors.affiliation: Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles du CNRS, Avenue de la Terrasse, 91198, Gif-sur-Yvette, France.
authors.forename: Jordi
authors.initials: J
authors.lastname: Mas Pons
authors.affiliation: Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles du CNRS, Avenue de la Terrasse, 91198, Gif-sur-Yvette, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Baron
authors.affiliation: CNRS and Univ Paris-Sud, Laboratoire de Synthèse de Biomolécules, Institut de Chimie Moléculaire et des Matériaux d'Orsay, UMR 8182, 91405, Orsay, France; Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles du CNRS, Avenue de la Terrasse, 91198, Gif-sur-Yvette, France.
authors.forename: Boris
authors.initials: B
authors.lastname: Vauzeilles
authors.affiliation: Aix Marseille Université, Laboratoire de Chimie Bactérienne (UMR 7283), Institut de Microbiologie de la Méditerranée (IMM), CNRS, 31 Chemin Joseph Aiguier-13402, Marseille, France.
authors.forename: Sam
authors.initials: S
authors.lastname: Dukan
mesh_terms: D001386:Azides / Q000032:analysis; D018929:Cell Culture Techniques; D057930:Click Chemistry / Q000379:methods*; D006090:Gram-Negative Bacteria / Q000302:isolation & purification*; D018189:Immunomagnetic Separation / Q000379:methods*; D013400:Sugar Acids / Q000032:analysis
chemical_list: C000608137:8-azido-3,8-dideoxyoctulosonate; D001386:Azides; D013400:Sugar Acids