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.

Fig 1. Schematic representation of metabolic lipopolysaccharide labeling using Kdo-N₃.

Culture of E. coli in the presence of Kdo-N₃ results in incorporation of the bioorthogonal azido function with the bacterial LPS. This incorporation can be further visualized by Copper-catalyzed Azide Alkyne Cycloaddition (CuAAC), in the presence of Cu(I) and a terminal alkyne. Alternatively, Strain-Promoted Alkyne Azide Cycloaddition (SPAAC) with a cyclooctyne-type reagent results in copper-free ligation.

Fig 2. Kdo-N₃ metabolically labels E. coli LPS.

(A) Metabolically incorporated Kdo-N₃ in E. coli was revealed by copper-free click chemistry (sulfo-DBCO-biotin followed by an anti-biotin A488 antibody). (B) Frequency distribution of the bacterial fluorescence values in the presence (green bars) or absence of Kdo-N₃ (black bars). Scale bar = 1 μm. More than ten independent experiments have been performed and a representative experiment is depicted.

Fig 3. Kdo-N₃ specifically labels culturable E. coli.

Detection of culturable E. coli (arrows) mixed with m-cherry dead E. coli (asterisk) (ratio = 10⁶:10⁶) by copper-free click chemistry (sulfo-DBCO-biotin followed by an anti-biotin A488 antibody) in the absence (A) or presence (B) of metabolically incorporated Kdo-N₃. Scale bar = 1 μm. Frequency distribution of each bacterial fluorescence values in the absence (A) or presence of Kdo-N₃ (B). Four independent experiments have been performed and a representative experiment is depicted.

Fig 4. Kdo-N₃ specifically labels E. coli culturable Gram negative bacterial membrane.

Detection of culturable E. coli (arrows) mixed with culturable GFP B. subtilis (asterisk) (ratio = 10⁶:3x10⁶) by copper-free click chemistry (sulfo-DBCO-biotin followed by an anti-biotin A594 antibody) in the absence (A) or presence (B) of metabolically incorporated Kdo-N₃. Scale bar = 1 μm. Frequency distribution of each bacterial fluorescence values in the absence (A) or presence of Kdo-N₃ (B). Five independent experiments have been performed and a representative experiment is depicted.

Fig 5. Specific isolation of culturable E. coli among a bacterial mix with magnetic beads.

Determination of culturable E. coli recovery in the supernatant (grey bars) and magnetic streptavidin bead fraction (white bars) with or without incorporation of Kdo-N₃ followed by copper-free click chemistry (sulfo-DBCO-biotin). Different amount of culturable E. coli alone or mixed with 10⁶ B. subtilis were tested: 10⁶ (A), 10³ (B), 10² (C), 10¹ (D). Pictures of culturable E. coli (arrows) recovered or not in the magnetic streptavidin bead fraction either in the presence or absence of Kdo-N₃, respectively (E). Bacteria were stained with DAPI and Kdo-N₃ incorporation within LPS was revealed with an anti-biotin A488 antibody. Scale bar = 1 μm. Data are means ± SD of four independent experiments.

Fig 6. Isolation of all culturable E. coli cells incubated with 25 mM of Kdo-N₃ within 2 h.

Determination of culturable E. coli recovery in the supernatant (grey bars) and magnetic streptavidin bead fraction (white bars) with or without incorporation of Kdo-N₃ 5mM or 25 mM for 2 and 4 h followed by copper-free click chemistry (sulfo-DBCO-biotin). Data are means ± SD of four independent experiments.