ColorFlux - A visual indicator of bacterial efflux

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. 

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)
• AcrAB (Haemophilus influenzae)

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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 is primarily recommended for Gram+ bacteria as ColorFlux proved efficient at reflecting relative levels of efflux pump activity in all species tested so far. 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. 

Because Gram- bacteria tend to have a slow influx/fast efflux profile, the concentration of ColorFlux and incubation time might need to be adjusted for each particular bacterial species and efflux pump involved. So far, ColorFlux has been successfully used to visually distinguish relative efflux levels of the AcrAB pump in mutant Haemophilus influenzae bacterial strains using the recommended protocol of use. For other species, such as E.coli, 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. 

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.

ColorFlux has been validated using mutated strains, produced by genetic manipulation on homogeneous populations, and differing only by the efflux level of specific pumps. The detailed study is available in the Colorflux patent here. 

To further minimize the risk of other parameters interfering with the measured efflux levels, such as metabolic activity, the protocol has been thoroughly optimized. In particular, we recommend growing the bacteria in a basal medium (i.e. MH II) instead of a rish medium, which can lead to a rapid efflux of the ColorFlux compound.  

Although both assays can be used to measure bacterial efflux, ethidium bromide and ColorFlux differ a lot in their nature, mechanism of entry and fluorescence pattern. Therefore, the benefits of using one or the other will depend on your specific research goals. Main differences between both methods include:

- Mechanism of entry into bacterial cells: ColorFlux will quickly accumulate inside bacteria cells through passive diffusion instead of binding an intracellular compound as is the case with ethidium bromide. That is why ColorFlux is also quickly and efficiently expelled out of the cell upon efflux activity, while ethidium bromide requires a dissociation step.

- Data analysis: ColorFlux is naturally fluorescent, so it will fluoresce whether intracellular or extracellular contrary to ethidium bromide that fluoresces only upon binding to DNA. Therefore, the assay is done differently: ColorFlux is first allowed to accumulate inside cells until reaching a steady-state, after which cells are washed and the resulting efflux is assessed via colorimetric and/or fluorescence measurements. This has the advantage of giving a fast and reliable comparative assessment of efflux activity in different strains, even in absence of a plate reader. In flow cytometry, the approach is different too: efflux levels will be inferred by monitoring the kinetics of ColorFlux accumulation in a given strain. This is contrasting with the ethidium bromide method, in which efflux is directly reflected by the decrease in fluorescence signal.

- Toxicity: Contrary to ethidium bromide, ColorFlux is a non-toxic compound you can safely handle on the bench and dispose of.

To sum it up, the idea of ColorFlux is to provide a non-toxic method that can give a fast and reliable comparative assessment of efflux activity in different samples, visible by the naked eye. This can be particularly advantageous for quickly pre-selecting resistant strains, screen large amounts of efflux inhibitors, or measure changes when specific genes coding for efflux pumps or their regulators are deleted or overexpressed. In contrast, ethidium bromide will be most suited for direct measures of efflux kinetics.​