A Colorful Way to Resist Antibiotics

By: Chandra Gravel '20 and Celia Slater '20

Ever since Alexander Fleming discovered penicillin in 1928, bacteria have had to find ways to adapt and develop new strategies in order to survive and resist new drugs. Because of this, antibiotic resistance is a growing problem and is one of the biggest public health challenges of our time. It is important for us to find new ways to combat this issue and to understand the underlying mechanisms that promote resistance.




One such organism with widespread antibiotic resistance is Pseudomonas aeruginosa. P. aeruginosa is a common Gram negative rod-shaped bacterium that lives primarily in water and soil. Like many environmental bacteria, P. aeruginosa live in slime-enclosed biofilms, which are like bacterial cities covered in protective ooze. Living in a biofilm has many advantages and it allows this organism to adhere to surfaces and to survive and replicate in more hostile environments, such as within human tissues and on medical devices. Because of this, P. aeruginosa, an opportunistic pathogen, can find its way into compromised tissues in human hosts and cause dangerous and often deadly infections. Pseudomonas aeruginosa infection is a serious problem in patients hospitalized with cancer, cystic fibrosis, and burns and the fatality rate in these patients is near 50 percent. 

P. aeruginosa Image: Shutterstock

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One reason Pseudomonas aeruginosa infections are so dangerous is that this bacterium is known to be resistant to many antibiotics due to its Gram negative outer membrane, which serves as a strong barrier that is impermeable to many antibiotics. Its ability to form biofilms also aids in its antibiotic resistance by making cells inside the biofilm impervious to many antibiotics- if the antibiotics can't get to the bacterium, they can't kill the bacterium. On top of all that, many strains of P. aeruginosa harbor antibiotic resistance plasmids, or small circular pieces of DNA that can hop from cell to cell and encode the genes for resistance to a particular antibiotic. In cystic fibrosis patients who acquire P. aeruginosa lung infections, most become infected with a strain that is so resistant it cannot be treated at all!

P. aeruginosa has very simple nutritional requirements, needing only acetate and ammonia as its most basic sources of carbon and nitrogen, but it can use a wide range of organic material for food. It is a facultative aerobe; meaning its preferred metabolism is respiration using oxygen but it can adapt to grow in low oxygen to no oxygen conditions. Like humans, it gains energy by transferring electrons from glucose to oxygen when oxygen is present. When oxygen is depleted or absent, however, it can use a wide variety of other molecules as the electron acceptor. One such substrate is a small, colorful metabolite called phenazine.

Image: Left-The phenazine 2-OHPCA turns the agar bright orange. Right- Aqueous solutions of some of the phenazines produced by various Pseudomonas strains. Price-Whelan et al.

Small molecules like phenazines can affect everything from reduction and oxidation, as described above, to what genes get turned on where, changing the bacterium's functions and mechanisms! In 2019, Schiessl et al. published a paper in Nature magazine where they made some very interesting discoveries about the role phenazines play in Pseudomonas biofilms and in antibiotic resistance.

Using deuterium, an isotope of hydrogen, Schiessl et al. observed the flow of metabolic pathways in Pseudomonas biofilms. Metabolically active areas of the biofilm would incorporate lots of deuterium into organic molecules and metabolic processes. They found that cells near the top of the biofilm, closest to the air, were metabolically active. However, they also found cells undergoing respiration much deeper down in the biofilm, where no oxygen was available. These deeper cells were using phenazines instead of oxygen as an electron acceptor! The researchers also cloned and tested a mutant strain that couldn’t produce phenazines and found that the mutants, Δphz, were not active at these depths.

Image: Sciessl et al figure 1.a) Biofilms of wild-type (normal) Pseudomonas aeruginosa (left) and Δphz Pseudomonas aeruginosa (right), a mutant strain that cannot produce phenazines.

Schiessl et al. soon found that phenazines don’t just help cells stay metabolically active without oxygen, they also contribute to Pseudomonas’s famous resistance to antibiotics. They found that when Pseudomonas cells were growing in a biofilm, they were resistant to several antibiotics, including ciprofloxacin, one of the antibiotics most commonly used to treat Pseudomonas infections. They tested biofilms of wild-type P. aeruginosa and the mutant Δphz strain in varying concentrations of ciprofloxacin. The biofilms were exposed to the antibiotic and then re-plated on fresh media to see how many colonies grew. Each colony that grew represented one cell from the original biofilm that was antibiotic resistant. The researchers found that more cells from the wild-type biofilm were antibiotic resistant than the Δphz biofilm (Figure 1D). Interestingly, when the same Pseudomonas strains were grown in liquid cultures, they were easily killed by these antibiotics. It appeared that growing in a biofilm gave these cells greater resistance to antibiotics. And what is one of the differences between growing in a liquid culture and growing in a biofilm? That’s right: phenazines. Clearly, these small, colorful molecules were playing an important role here. 

Image: Schiessl et al. Figure 1D) Biofilms of wild-type and Δphz Pseudomonas aeruginosa were exposed to four different concentrations of ciprofloxacin and then re-plated on fresh media to see how many colonies formed.

To understand what exactly phenazines were doing to make these bacteria more drug-resistant, they grew Pseudomonas cells on a medium containing only succinate, an organic molecule that enters metabolism downstream of glucose. These cells were no longer resistant to ciprofloxacin. It appeared that the oxidation of glucose by phenazines was vital to their ability to protect the cells from antibiotics. Additionally, they found that reduction of phenazines by Cco complexes, enzymes that donate electrons to oxygen (or in this case, phenazines) was important for antibiotic resistance, because mutant cells that couldn’t produce these enzymes could be easily killed by antibiotics.

In conclusion, Schiessl et al. found that something about the reduction of these small, colorful molecules by enzyme complexes in cells deep within biofilms was creating unique conditions in these cells that made them more resistant to antibiotics. This link between antibiotic resistance and the role phenazines play inside biofilms is of great importance considering that Pseudomonas aeruginosa biofilms are a major contributor to antibiotic-resistant infections. Pretty impressive, for such a little molecule!

About the authors:


Chandra Gravel ‘20

Chandra is a Biochemistry major and Francis Perkins scholar at Mount Holyoke College and works in the Berry lab on a newly discovered RNA chaperone protein called ProQ. In her free time, she enjoys spending time with her kids and dog outdoors.



Celia Slater ‘20
Celia Slater is a biochemistry major at Mount Holyoke College. She works in the Camp Lab, studying spore-forming bacteria. After graduating from Mount Holyoke, Celia plans to go to graduate school for genetics. In her free time, she enjoys drawing, playing video games, and playing Dungeons and Dragons with friends.