Scientists often study particular bacterial species separate from other species, yet this is seldom reality outside of the lab. Bacteria grow in complex ecosystems surrounded by multiple species working together or pitted against each other for survival. 

Staphylococcus aureus and Pseudomonas aeruginosa  are two human pathogens that often live in the same environments. Together, they can chronically infect the wounds of patients with diabetes, grow on implanted medical devices like catheters and heart valves, and invade the lungs of patients with cystic fibrosis. These species often form biofilms, fortress-like formations that protect the bacteria within, making antibiotics up to 1000 times less effective. This leads to long courses of antimicrobial therapies that are often ineffective at clearing the infection. 

In a new Cell Chemical Biology  study, researchers at the University of Nottingham identified a chemically modified inhibitor of P. aeruginosa that eradicates both P. aeruginosa and S. aureus  biofilms when used in combination with the antibiotic tobramycin (1). This new finding has the potential to change the way researchers think about treating chronic S. aureus and P. aeruginosa  mixed biofilm infections.

"There is really a gap in our antibiotic arsenal to target those chronic mixed biofilm infections, and anyone who is testing their antibiotics or new drugs against mixed species biofilms is tackling a really difficult problem, one for which we do not have a lot of good answers right now,” said George O’Toole a microbiologist at Dartmouth College who studies biofilms and was not involved in the study.  

P. aeruginosa bacteria communicate with each other through a process called quorum sensing (QS). This is a type of cell-to-cell communication that allows bacteria to detect how dense their local environment is and enhance their survival by mutual coordination. For example, some bacteria use QS to determine when there are enough bacterial cells in the population to form a biofilm. QS also helps bacteria decide when to produce antibiotics in self-defense against other species of bacteria. 

Paul Williams, a microbiologist at the University of Nottingham and author of the new study, has been fascinated by QS since the late 1980s when he and his colleagues accidentally discovered a molecule involved in bacterial communication that was widely used by multiple species of bacteria (2). Since then, he has been studying QS and searching for potent molecules to block it. 

Williams and his team previously synthesized a library of small molecules that inhibit QS in P. aeruginosa (3). They wanted to see if any of these inhibitors could kill other bacteria often found in the same environment as P. aeruginosa, such as S. aureus. 

He and his team identified a molecule that killed multiple gram-positive bacterial species living as single cells in liquid cultures, including S. aureus.  To determine if the new molecule had antibiofilm activity, the researchers treated S. aureus  biofilms or P. aeruginosa-S. aureus  mixed biofilms with the molecule alone, in combination with the antibiotic tobramycin, or with tobramycin alone. They discovered that in the mixed species biofilm, P. aeruginosa  protected S. aureus  from being killed by tobramycin.

"We didn't quite expect that. We just thought it would be a complete wipeout, but it was not,” said Williams.

However, they found that treating the mixed species biofilm with both the small molecule inhibitor and tobramycin killed both species and impaired their ability to form biofilms. The inhibitor acts against the two species of bacteria using two separate mechanisms. In P. aeruginosa, the compound inhibits a QS system that regulates the release of extracellular DNA, which it requires to form a biofilm. This softens up the biofilm and makes the bacteria within it more susceptible to antibiotics. In gram-positive bacteria, including S. aureus, the compound forms pores in the bacterial cell wall, leading to cell death.

O’Toole was impressed with the results of the study, but he is interested in a different test model. “The ideal experiment for me is letting that biofilm form with no drugs so that it reflects the biofilm forming in a patient,” said O’Toole. “They’re going to come into the clinic with that full blown biofilm infection already.”

Williams acknowledged the limitations of an in vitro  biofilm model, but because these compounds display some cytotoxicity, they would not be safe for use in humans or animal models. He and his colleagues are now testing similar compounds that have greater activity and lower cytotoxicity.

For now, Williams is excited that they identified a compound that both inhibits QS in P. aeruginosa  and kills gram-positive pathogens. “There’re a lot of different quorum sensing systems out there that we thought would be good targets for antimicrobials,” said Williams. “There's been an awful lot of basic science, but as of yet, nobody's actually got a new drug based on these systems in the clinic.”

References

1. Murray, E.J. et al. A Pseudomonas aeruginosa PQS quorum-sensing system inhibitor with anti-staphylococcal activity sensitizes polymicrobial biofilms to tobramycin. Cell Chem Biol  29, (2022).
2. Nigel, B.J. et al. A general role for the lux autoinducer in bacterial cell signalling: control of antibiotic biosynthesis in Erwinia. Gene  116, 87-91 (1992). 
3. Ilangovan F.A. et al. Structural basis for native agonist and synthetic inhibitor recognition by the Pseudomonas aeruginosa quorum sensing regulator PqsR (MvfR). PLoS Pathog  9, (2013).