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Rapid and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click Chemistry Coupled with Magnetic Beads Separation.

Fugier E, Dumont A, Malleron A, Poquet E, Mas Pons J, Baron A, Vauzeilles B, Dukan S - PLoS ONE (2015)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0127700.g006: 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.

Mentions: In all previous experiments, E. coli were cultured overnight in the presence of Kdo-N3. We finally investigated the minimal period of Kdo-N3 assimilation allowing retention of the majority of E. coli cells onto magnetic beads after azide-cyclooctyne ligation. For this purpose, approximately 5 culturable E. coli per ml of medium were incubated for 2 to 4 hours in the presence of various concentrations of Kdo-N3 (0 to 25 mM, 25 mM being the maximal Kdo concentration allowing normal E. coli cell growth as indicated in S4 Fig) and E. coli cells trapped by magnetic beads were eventually quantified. Analyses depicted in Fig 6 demonstrates that 2 h of 25 mM Kdo-N3 incubation was sufficient to isolate the majority of culturable E. coli cells present in a sample.


Rapid and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click Chemistry Coupled with Magnetic Beads Separation.

Fugier E, Dumont A, Malleron A, Poquet E, Mas Pons J, Baron A, Vauzeilles B, Dukan S - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465638&req=5

pone.0127700.g006: 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.
Mentions: In all previous experiments, E. coli were cultured overnight in the presence of Kdo-N3. We finally investigated the minimal period of Kdo-N3 assimilation allowing retention of the majority of E. coli cells onto magnetic beads after azide-cyclooctyne ligation. For this purpose, approximately 5 culturable E. coli per ml of medium were incubated for 2 to 4 hours in the presence of various concentrations of Kdo-N3 (0 to 25 mM, 25 mM being the maximal Kdo concentration allowing normal E. coli cell growth as indicated in S4 Fig) and E. coli cells trapped by magnetic beads were eventually quantified. Analyses depicted in Fig 6 demonstrates that 2 h of 25 mM Kdo-N3 incubation was sufficient to isolate the majority of culturable E. coli cells present in a sample.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus