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ColorFlux - A visual indicator of bacterial efflux

https://www.idylle-labs.com/web/image/product.template/423/image_1920?unique=53144e7
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Fast, reliable and non-toxic solution for measuring bacterial efflux

150.00 € 150.0 EUR 150.00 €

150.00 €

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A technology developed by Olivier Siri, Jean-Michel Bolla, Michel Camplo & Mrunal Patil (Aix-Marseille Université, France)

​ColorFlux is a fast, reliable and non-toxic solution for assessing bacterial efflux for your research on antibiotic resistance. This coloured fluorescent compound quickly accumulates within bacteria as a function of their efflux pump activity. Simply add ColorFlux to your bacteria and watch them change colour ! 

How does it work?

ColorFlux staining was shown to reflect the activity of well-characterized efflux pumps from the major facilitator superfamily and ATP-binding cassette families in a variety of Gram+ bacteria:
- NorA, MepA, MepB, PatA, PatB (Staphylococcus aureus & Streptococcus pneumoniae)
- BmrA (Bacillus subtilis)
The list will be updated regularly according to the feedback of users. 

Technical information

Bacterial species compatibility : Gram +
Tested and validated on : Staphylococcus aureus, Bacillus subtilis, Streptococcus pneumoniae. 
Colour: Red
Form: liquid
Detection methods: visual & fluorescence. Also compatible with single-cell approaches (live imaging and flow cytometry) 
Peak excitation/emission wavelengths: Ex:530nm/Em:650nm
Storage: at 4°C protected from light for 8 months

1 kit contains 1mL of an aqueous solution with a concentration of 1 mg/mL for a final volume of 1L of screening medium


Additional Resources:

ColorFlux staining of MepA&B mutants in Staphylococcus aureus


  Left graph shows the fluorescence signals detected for each strain using an Ex=530nm, and right graph shows corresponding fluorescence intensity values obtained for each strain at Em=645nm. WT = Wild Type strains, MepA1B+++ = MepA&B overexpressing strains.
Credits: Mrunal Patil & Jean-Michel Bolla, Aix-Marseille Université – 2023

ColorFlux staining of NorA mutants in Staphylococcus aureus


Top picture shows pellet colour variations according to NorA expression. Bottom figures show the fluorescence signals detected for each strain using an Ex=530nm. 
Credits: Mrunal Patil & Jean-Michel Bolla, Aix-Marseille Université – 2023

ColorFlux staining of PatA/PatB mutants in Streptococcus pneumoniae


Left pictures show pellet colour variations according to PatA/PatB expression. The right graph show fluorescence signals detected for each strain using an Ex=530nm. 
Credits: Mrunal Patil & Jean-Michel Bolla, Aix-Marseille Université – 2023

ColorFlux staining of BmrA mutants in Bacillus Subtilis


Top picture shows pellet colour variations according to BmrA expression. Bottom figures show the fluorescence signals detected for each strain using an Ex=530nm. 
Credits: Mrunal Patil & Jean-Michel Bolla, Aix-Marseille Université – 2023

Bacillus subtilis bacteria (moderate activity) stained with ColorFlux

 Credits: Mrunal Patil & Jean-Michel Bolla, Aix-Marseille Université – 2023
 ​

How to use ColorFlux bacterial efflux probe:



How to use ColorFlux in pictures

Find below advice & tips for ColorFlux use:

ColorFlux is a cationic compound that quickly penetrates Gram + bacteria through passive diffusion and is efficiently expelled out of the cell depending on efflux activity.  

After incubating bacteria with ColorFlux for 15 minutes, a steady-state level is reached and relative levels of efflux activity can be quickly assessed by looking at cell pellet color. It is also possible to read fluorescent signals for a precise quantification of efflux levels. Alternatively, kinetics of ColorFlux accumulation can be monitored by flow cytometry. 

ColorFlux is a substrate for efflux pumps that are well-known as key players in bacterial efflux-mediated resistance. In particular, NorA involved in first-line response in S. aureus, PatA/B involved in fluoroquinolone resistance in S. pneumoniae or BmrA, a well-characterized multidrug ABC transporter in B. subtilis.

In a pilot study conducted at IHMT-NOVA, Lisboa on 101 S. aureus clinical strains from humans and animals, ColorFlux detected efflux-resistant bacteria simply by colorimetric assessment. This study demonstrated that ColorFlux can be used as a quick & reliable tool to detect clinically relevant resistant bacteria. 

Which efflux pumps is ColorFlux a substrate of?

ColorFlux staining was shown to reflect the activity of well-characterized efflux pumps from the major facilitator superfamily and ATP-binding cassette families in a variety of Gram+ bacteria:

  • NorA, MepA, MepB, PatA, PatB (Staphylococcus aureus & Streptococcus pneumoniae)
  • BmrA (Bacillus Subtilis)

We would greatly appreciate your valuable inputs by helping us complementing this list with any of your positive research experiences. Drop us a message through our Contact page!

Contrary to ethidium bromide, ColorFlux is a non-toxic compound that you can safely handle on the bench and dispose of. It is a particularly useful tool for training purposes & on-field applications. 

The total procedure to stain bacteria and visualise efflux levels by colorimetric assessment should take no more than 30 minutes. 

ColorFlux has been initially developed for Gram+ bacteria because the resulting bacterial coloration after incubation with ColorFlux reflects relative levels of efflux pump activity. Indeed, the fast influx/slow efflux properties of Gram+ bacteria allow ColorFlux to be quickly internalized and then expelled as a function of efflux, resulting in different shades of red that are representative of relative levels of efflux activity. 

In Gram- bacteria, the situation is a bit different: because they tend to have a slow influx/fast efflux profile, ColorFlux will be instantaneously expelled from the bacteria as soon as some pumps are present. Therefore, although bacteria without pumps will turn red, it won't always be possible to distinguish between WT strains and those with enhanced efflux as they will always turn white. This was tested in E.coli, in which WT strain turned white and mutants with ArcAB pump deleted turned red. Some preliminary tests combining ColorFlux with compounds to reduce efflux pump activity, i.e. CCCP, have shown promising results and might help getting relative efflux level measurements in Gram- bacteria. However, precise concentrations & timing to use are yet to be determined for each particular bacterial species and efflux pump involved. 

To sum up, the applicability of ColorFlux in Gram- bacteria will depend on the intended application. While ColorFlux can be useful as a tool to quickly check for the presence/absence of efflux pumps (i.e. to validate efflux pump knock-out mutants), some protocol optimization will be required if you are aiming at comparing relative efflux activity levels between strains, or to detect resistant bacteria.

Any other questions? Feel free to contact us.


ColorFlux has been designed by Olivier Siri, Jean-Michel Bolla, Michel Camplo and Mrunal Patil.

Publication coming soon.

 

Everspark technology has been intially developed by Karine Monier, Arnaud Favier and Christophe Place and and published in Scientific Reports:
Provost, A., Rousset, C., Bourdon, L. et al. Innovative particle standards and long-lived imaging for 2D and 3D dSTORM. Sci Rep 9, 17967 (2019). https://doi.org/10.1038/s41598-019-53528-0
 

Publications:

Nanoscale engagement and clusterization of Programmed death ligand 1 (PD-L1) in the membrane lipid rafts of Non-Small Cell Lung Cancer cells 
Martina Ruglioni, Simone Civita, Tiziano Salvadori, Sofia Cristiani, Vittoria Carnicelli, Serena Barachini,  Iacopo Petrini, Irene Nepita, Marco Castello, Alberto Diaspro, Paolo Bianchini, Barbara Storti, Ranieri Bizzarri,  Stefano Fogli and Romano Danesi   bioRxiv 2022.08.09.503318; doi: https://doi.org/10.1101/2022.08.09.503318

HIV-1 diverts actin debranching mechanisms for particle assembly and release in CD4 T lymphocytes
Rayane Dibsy, Erwan Bremaud, Johnson Mak, Cyril Favard, Delphine Muriaux
BioRxiv, December 16, 2022. doi. 10.1101/2022.12.15.520580

Fluorescent Polymer-AS1411-Aptamer Probe for dSTORM Super-Resolution Imaging of Endogenous Nucleolin
Fabre L, Rousset C, Monier K, Da Cruz-Boisson F, Bouvet P, Charreyre MT, Delair T, Fleury E, Favier A. Biomacromolecules. 2022 May 12. doi: 10.1021/acs.biomac.1c01706. PMID: 35549176

Comparative analysis of ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vector vaccines.
Michalik S, Siegerist F, Palankar R, Franzke K, Schindler M, Reder A, Seifert U, Cammann C, Wesche J, Steil L, Hentschker C, Gesell-Salazar M, Reisinger E, Beer M, Endlich N, Greinacher A, Völker U. Haematologica. 2022 Apr 1;107(4):947-957. doi: 10.3324/haematol.2021.280154. PMID: 35045692

Metabolic biorthogonal labeling and dSTORM imaging of peptidoglycan synthesis in Streptococcus pneumoniae 
Jennyfer Trouve, Oleksandr Glushonkov and Cecile Morlot
Star Protocols, December 13, 2021. doi: 10.1016/j.xpro.2021.101006

Insights in ChAdOx1 nCoV-19 vaccine-induced immune thrombotic thrombocytopenia.
Greinacher A, Selleng K, Palankar R, Wesche J, Handtke S, Wolff M, Aurich K, Lalk M, Methling K, Völker U, Hentschker C, Michalik S, Steil L, Reder A, Schönborn L, Beer M, Franzke K, Büttner A, Fehse B, Stavrou EX, Rangaswamy C, Mailer RK, Englert H, Frye M, Thiele T, Kochanek S, Krutzke L, Siegerist F, Endlich N, Warkentin TE, Renné T. 
Blood. 2021 Dec 2;138(22):2256-2268. doi: 10.1182/blood.2021013231. PMID: 34587242

Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres.
Szikora S, Novák T, Gajdos T, Erdélyi M, Mihály J. Bio Protoc. 2020 Jun 20;10(12):e3654. doi: 10.21769/BioProtoc.3654. eCollection 2020 Jun 20. PMID: 33659324

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