Triclosan depletes the membrane potential in Pseudomonas aeruginosa biofilms inhibiting aminoglycoside induced adaptive resistance

Michael M. Maiden, Christopher M. Waters

Preprint posted on 14 April 2020

Article now published in PLOS Pathogens at

Targeting antibiotic resistant bacteria in biofilms with synergistic killing methods

Selected by Amberley Stephens


The rise of antibiotic resistance and the formation of biofilms by Pseudomonas aeruginosa make them particularly hard to treat. The current treatment utilises the aminoglycoside family of antibiotics which cross the outer membrane, and inner membrane of this Gram-negative bacteria and inhibit protein translation by binding to the 30S subunit of the ribosome. P. aeruginosa form adaptive resistance to aminoglycosides by pumping them out of the cell using a type of efflux pump in the family called proton motive force (PMF) dependent resistance-nodule-cell division (RND) of efflux pumps. Despite the presence of these efflux pumps, aminoglycosides are still the first treatment for P. aeruginosa infections which are particularly prevalent in cystic fibrosis suffers.

Identifying new antimicrobials and getting them to market is a costly process both in time and money. Another approach is to find combination therapies which enhance the killing capacity of the antibiotics we currently have. This preprint explores the mechanism of a compound called triclosan which the authors show increases the killing effectivity of the aminoglycoside antibiotic tobramycin compared to tobramycin alone.


Combination treatment is better than single compound treatment

Inner membrane permeabilization is one factor contributing to cell death by aminoglycosides. Initial treatment of P. aeruginosa in a biofilm form for 2 hours with an aminoglycoside called tobramycin lead to only 11% cell permeabilization. In combination with the compound triclosan cell permeabilization increased to 50%. Triclosan is a protonophore, an exciting group of compounds which act to move protons across lipid membranes and can disrupt membrane potentials (Figure 1). A protonophore, bedaquiline, was approved by the Food and Drug Administration in the USA as the newest drug in 40 years to target multi-drug resistant Mycobacterium tuberculosis[1,2]. Triclosan is also a known fatty acid synthesis inhibitor, yet Pseudomonas is inheritably resistant to triclosan and can even be selectively isolated from a mixed bacteria population with triclosan.

Figure 1. Protonophores are hydrophobic molecules containing hydrogen donor and acceptor groups which allow them to transport protons across lipid membranes. Figure created with

Whole genome sequencing shows the aminoglycoside pulls the killing trigger

To investigate the synergistic mechanism and whether there was a dominant compound leading to cell death the authors purposefully induced resistance to identify mutations in key genes by gradually increasing the concentration of compounds over time. Whole genome sequencing identified single point mutations in a gene called fusA1 encoding EF-G1A, a protein involved in ribosome translocaion and recycling. This showed the main mode of cell killing was mediate by the aminoglycoside tobramycin.

Triclosan is only synergistic when aminoglycosides are present

Subsequently the authors investigated how triclosan was aiding the killing mechanism of tobramycin. Their previous work and high through put screening indicated that triclosan only enhanced killing when an aminoglycoside was present and the resistance mechanism, the efflux pump, was activated[3].

They tagged tobramycin with a fluorescent dye and incubated the P. aeruginosa biofilms with and without triclosan for 30 minutes, allowing time for the compounds to be internalised, but not to kill the cells. The bacteria were subsequently recovered and the quantity of tagged tobramycin released from the cells was quantified. The amount of tagged tobramycin released was higher from cells treated with triclosan than without, as the triclosan had prevented the efflux of the tobramycin leading to accumulation within the cells.

Triclosan disrupts the membrane potential that drives the RND efflux pump

RND efflux pumps utilise the proton gradient and move protons across the inner membrane in an opposite direction to the exported drug. The balance of protons across membranes creates a membrane potential (Δѱ). Protonophores can alter this membrane potential due to their ability to accept and donate protons and transfer them across lipid membranes (Figure 2).

To investigate changes in Δѱ in the presence of triclosan the P. aeruginosa bacteria were treated with a dye sensitive to Δѱ, DiOC2(3) dye. When untreated, 20% of cells maintained their Δѱ, when treated with triclosan this reduced to only 5%. When treated with tobramycin this increased to 37% due to other mechanisms increasing Δѱ and leading to increased activity of the efflux pumps. Yet treatment with both compounds lead to ~10% of cells with maintained Δѱ.

A derivative, methyl-triclosan without a dissociable proton did not have the same effects as triclosan, cells maintained their Δѱ and efflux pump activity, therefore showing the photonophore moiety of triclosan is key to its function.

Figure 2. Schematic of the synergistic mechanism of action of tobramycin and triclosan and the mechanism of adaptive resistance. 1. Tobramycin crosses cell membranes and inhibits translation by binding to ribosomes inside the bacteria. 2. The RND-type efflux pump is responsible for adaptive resistance and is formed of three main components, the outer membrane proteins (OMP), the membrane fusion proteins (MFP) and the inner membrane proteins (IMP) which work together to pump aminoglycosides out of the cell while pumping protons (H+) into the cytosol. 3. Triclosan disrupts the proton gradient by transferring protons into the cytosol. This prevents the functioning of the efflux pump and allows tobramycin concentrations to increase in the cytosol leading to cell death. Taken from Figure 7 in preprint.

A combination of triclosan and tobramycin is effective in reducing P. aeruginosa in wounds in vivo

To test the effect of synergistic combination treatment in vivo, mice with open wounds were infected with bioluminescent P. aeruginosa. A hydrogel of triclosan only, tobramycin only and a combination of triclosan and tobramycin was applied. The combination treatment lead to a 4-fold decrease in P. aeruginosa when imaged (Figure 3.). Tobramycin alone lead to a 2.5 fold decrease and triclosan alone lead to no decrease in infection.

Figure 3. Treatment with a triclosan and tobramycin loaded hydrogel lead to a decrease in P. aeruginosa infection. The luminescent P. aeruginosa in a wound model were imaged before and after treatment with a hydrogel containing triclosan and tobramycin. The black arrow indicates where the hydrogel was placed on the wound. A decrease in luminescence radiance indicated a reduction in live bacteria after treatment.


The addition of the photonophore, triclosan, with the aminoglycoside tobramycin to treat P. aeruginosa biofilm infection lead to a 99% increase in cell death in vitro after 6-hrs of treatment and a reduction of P. aeruginosa infection in an in vivo model compared to tobramycin alone. The mechanism of action was determined to based upon the protonphore property of triclosan which transports protons across the inner membrane into the cytosol. This disrupts the proton gradient that the RND-efflux pump relies on to pump out the tobramycin. The result is a greater accumulation of the tobramycin inside the cell, arrest of translation as the drug binds to ribosomes, and eventually cell death.

Why I chose this preprint

The ability of bacteria to resist antibiotics is both fascinating and scary. I’d never heard of a protonophores before so I wanted to learn more about their mechanism of action. The paper had a good flow during reading and well thought out experiments that followed a logical order.

Questions for authors

Have you tested these compounds on bacteria not in biofilm formation? Does it make a difference?

How is this different/better than oxyclozanide which is also mentioned in the preprint as a protonophore uncoupler?

How far into biofilm do these compounds penetrate? Does it make a difference how mature the biofilm is as to how effective the compounds are?

Why are hydrogels used to administer the treatment? How do you determine how much compound is taken into the hydrogel?

Will hydrogels likely be used to treat wound infected humans?


  1. Andries K, Verhasselt P, Guillemont J, Göhlmann HWH, Neefs JM, Winkler H, Van Gestel J, Timmerman P, Zhu M, Lee E, et al.: A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science (80- ) 2005, 307:223–227.
  2. Mahajan R: Bedaquiline: First FDA-approved tuberculosis drug in 40 years. Int J Appl Basic Med Res 2013, 3:1.
  3. Maiden MM, Agostinho Hunt AM, Zachos MP, Gibson JA, Hurwitz ME, Mulks MH, Waters CM: Triclosan is an aminoglycoside adjuvant for eradication of pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 2018, 62.



Posted on: 27 April 2020 , updated on: 28 April 2020


Read preprint (1 votes)

Author's response

Michael M. Maiden and Christopher M. Waters shared

Thank you very much for choosing our article to highlight! We appreciate it.

Have you tested these compounds on bacteria not in biofilm formation? Does it make a difference?

Yes, we have tested triclosan alone and in combination with tobramycin on cells growing planktonically (not as a biofilm). We found that the combination did not act synergistically. On its face this seems counter intuitive. However, planktonic cells are incredibly sensitive to tobramycin. Without the defense biofilms provide (reduce diffusion of drug, increase efflux pump activity, increase mutation rate, and slowed metabolism… to name a few), planktonic cells are no match for tobramycin. So, it wasn’t surprising that we could not enhance tobramycin when it is already so active.

How is this different/better than oxyclozanide which is also mentioned in the preprint as a protonophore uncoupler?

This combination is more potent and effective than oxyclozanide. However, it appears they are both working by targeting the same cellular processes.

How far into biofilm do these compounds penetrate? Does it make a difference how mature the biofilm is as to how effective the compounds are?

This is an interesting question. From our first paper, we examined the effect of the combination on 3-dimensional biofilms growing in a flow cell (Maiden et al., 2018), and it appears that the combination is able to diffuse throughout the biofilm. It is expected that tobramycin has reduced diffusion, as has been tested previously, however as portions of the biofilm lyse, new layers are opened and access is gained to deeper communities within the biofilm.

We have also tested biofilms of various ages and found that combination remains effective, even against biofilms that are days old.

Why are hydrogels used to administer the treatment? How do you determine how much compound is taken into the hydrogel?

Great question! Hydrogels are a routine treatment modality for chronic wounds, and in-fact chronic wounds are manged with a variety of packing techniques to maintain hydration and supply antimicrobials. We decided to use hydrogels to as a vehicle to deliver both compounds simultaneously over a sustained period, rather than applying liquid droplets that could easily wash away or be removed by the mice themselves. In our approach, the hydrogels were covered and the mice could not access them. This is akin to the now available antibiotic infused band-aids that can be bought at your local grocery store (in the USA).

The hydrogels were made in a specific way so that they would have a specific concentration of each compound diffused throughout it. Hydrogels start off as a liquid and as they cool, they become a semi-solid. This allows you to mix various compounds together as you would add sugar into coffee and mix with a spoon. However, unlike coffee, as the solution cools it becomes a semi-solid rather than a bitter drink.

Will hydrogels likely be used to treat wound infected humans?

Hydrogels are already routinely used for the treatment of wounds in humans! It is also an emerging area of research. Here is an excerpt from Koehler et al., 2018 on hydrogels:

“Hydrogel-based wound dressings are one of the most promising materials in wound care, fulfilling important dressing requirements, including: (a) keeping the wound moist whilst absorbing extensive exudate, (b) adhesion-free coverage of sensitive underlying tissue, (c) pain reduction through cooling and (d) a potential for active intervention in the wound healing process.”


Maiden MM, Agostinho Hunt AM, Zachos MP, Gibson JA, Hurwitz ME, Mulks MH, Waters CM: Triclosan is an aminoglycoside adjuvant for eradication of pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 2018, 62.

Koehler J, Brandl FP, Goepferich AM: Hydrogel wound dressings for bioactive treatment of acute and chronic wounds. Eur Polym J 2018, 100:1–11.

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