Click4Tag working on pre-enrichment method for quicker market release
Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step, needed to reach the minimal number of cells for efficient identification.
To guarantee the absence of these bacteria, it can be necessary to block batches for 24 to 48 hours before they are put on the market, which is a limiting factor for batches of fresh, perishable products.
Researchers described a method that can reduce this step to five hours by concentrating the bacteria (E. coli) present in a sample.
Scientists in the Laboratoire de Chimie Bactérienne (CNRS/Aix-Marseille Université), the Institut de Chimie des Substances Naturelles (CNRS) and the Institut de Chimie Moléculaire et des Matériaux in Orsay (CNRS/Université Paris-Sud) said the technique is based on non-lethal click chemistry and the use of magnetic beads.
Release within a day
They said this process will enable the release of commercial batches within a day and be exploited by the start-up company Click4Tag.
Click chemistry is a method developed in 2001 which concerns the assembly of chemical substances that enables combination of two molecules.
The researchers describe a method allowing separation of culturable and non-culturable bacteria which could rely on surface functionalization of culturable bacteria to hook them onto magnetic beads using non-lethal copper free click chemistry. They worked with E. coli as a model of Gram negative bacterium.
Bacteria are fed a synthetic sugar that mimics a sugar naturally present on their surface, they said.
Cultivable bacteria assimilate the sugar, which is then found on their membranes only so they have been "labeled".
Then, using click chemistry, scientists were able to hook magnetic beads onto the bacteria which using a simple magnet made it possible to concentrate the labeled bacteria.
Results show this method can specifically detect cultivable bacteria of interest, even in the presence of dead bacteria or other organisms.
Around the magnet, the scientists collected more than 90% of the bacterial targets while concentrating them more than a thousand-fold - and within a shorter period of time.
Improvement on IMS
Extraction and concentration of a target organism from a sample by magnetic beads coated with specific monoclonal antibodies: ImmunoMagnetic Separation (IMS) is known.
Combined with IMS, many detection methods can be used as a second step, to quantify and/or identify the captured bacteria such as ELISA, PCR after an elution step.
Other methods involve a secondary antibody to form a sandwich complex for further use of detection tools such as ATP-bioluminescence or electrochemiluminescence .
However, these approaches tend to misestimate the actual number of culturable bacteria in a given sample, since IMS does not permit differentiation between culturable and non-culturable bacteria.
Direct immunomagnetic capture of specific bacteria can be drastically decreased in the presence of other organisms which can be critical and problematic if the pathogenic bacteria of interest is weakly present in the sample as it can lead to non-capture and false negative analysis.
Reduce pre-culture time
Researchers said the possibility to isolate and concentrate/enrich culturable Gram negative bacteria from a sample to be analyzed could reduce the pre-culture time required for subsequent PCR analysis or ELISA tests in routine assays.
“For instance, here we have been able to concentrate E. coli 100 fold (from 1ml to 10 μl),” they added.
“If we assume that the same procedure could be efficiently applied to all kinds of Gram negative bacteria and to the higher volume usually used for detection of microbial presence in food industry (250 ml), we would be able to concentrate bacteria up to 25,000 fold.
“Moreover, in the hypothesis where only 1 E. coli (doubling time 20 minutes) is present in the 250ml sample tested and that this cell starts to divide immediately (no latency), 6 hours are necessary to get the 103 bacteria.ml-1 required for further PCR identification analyses for example.”
The team said it was necessary to adapt the methodology to larger sample volumes and other bacteria, with start-up company Click4Tag to optimize and market the process within the next two years.
Source: PLOS ONE
Online ahead of print, DOI: 10.1371/journal.pone.0127700
“Rapid and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click Chemistry Coupled with Magnetic Beads Separation”
Authors: Emilie Fugier, Audrey Dumont, Annie Malleron, Enora Poquet, Jordi Mas Pons, Aurélie Baron, Boris Vauzeilles and Sam Dukan
