New Cell Research Model Shows Promise Against P. Aeruginosa

A combination of bacteria-infecting viruses and antibiotics can be used to effectively protect lung cells against infection by Pseudomonas aeruginosa, a bacteria that commonly causes lung infections in people with cystic fibrosis (CF), according to a study led by researchers at the University of Geneva (UNIGE).

The model used to test this therapeutic approach — combining P. aeruginosa and epithelial lung cells in a laboratory setting — has been awarded the UNIGE 3R prize, which distinguishes UNIGE researchers for their contribution to the 3R principles. These principles aim to limit the use of animals in research through the “three Rs” — “reduce,” “refine,” and “replace.”

“At UNIGE, any scientific project involving animal experimentation is evaluated in order to improve the welfare of the animals during experiments, to reduce the number of animals needed to achieve the goals of the project, and to replace the use of animals with alternative methods when possible. Our commitment to the 3Rs principle is strong and valued through the 3Rs Award,” Daniele Roppolo, PhD, director of Animal Experimentation at UNIGE, said in a university press release.

The team’s model used both wild-type cells and cells harboring mutations in the CFTR gene (the gene defective in CF).

“Building on an existing technology, we created a new model adapted to a pulmonary setting in the context of a Pseudomonas infection. In addition, we used cells that reproduce cystic fibrosis in order to establish whether the treatment we propose could also effectively treat lung infections affecting these patients,” said Thilo Köhler, researcher at UNIGE, co-author of the study, and winner of the UNIGE’s 3R prize.

The study reporting the findings, “Combined Bacteriophage and Antibiotic Treatment Prevents Pseudomonas aeruginosa Infection of Wild Type and cftr- Epithelial Cells,” was published in Frontiers in Microbiology.

P. aeruginosa is an opportunistic pathogen that commonly causes lung infections in CF. Standard treatment against P. aeruginosa infections involves the use of antibiotics, which are medications that can kill bacteria. However, many bacteria develop resistance to specific antibiotics, which can make treatment challenging.

In recent years, there has been renewed interest in the use of bacteriophages — viruses that can infect and kill bacteria — to fight P. aeruginosa and other bacterial infections. Now, researchers at UNIGE conducted a series of lab experiments to further explore bacteriophages as a possible treatment for these infections.

Under normal conditions, human epithelial lung cells grown in laboratory conditions form a tight layer of cells that prevents compounds from passing through — similar to how these cells form a barrier between the body and air that is being inhaled and exhaled in the lungs.

The researchers found that, upon infection with P. aeruginosa, the trans-epithelial resistance (TER) of these cells decreased; in other words, the cell layer was disrupted and became “less tight.” Further experiments demonstrated that the reduction of TER was attributable to the death of lung cells by infection with P. aeruginosa.

The team then tested the effects of treatment of infected cells with one of three types of bacteriophages that can infect P. aeruginosa, or with an antibiotic called ciprofloxacin.

Treatment with individual bacteriophages or with ciprofloxacin reduced bacteria levels and lessened the reduction in TER.

However, researchers found that bacteria could escape these treatments over time. While bacterial numbers were substantially reduced at 24 hours after treatment, by 72 hours bacteria had regrown to levels close to what was seen in untreated cells, with concomitant reductions in TER. Experiments on bacteria that survived indicated they had mutations that made them resistant to the treatment.

Researchers then tested the effect of all three bacteriophages combined in a “cocktail.” While ciprofloxacin alone did not prevent a reduction of TER at 72 hours, the bacteriophage cocktail was partially protective. Combining the bacteriophage cocktail with the antibiotic fully preserved TER, suggesting “complete protection of the epithelial cell layer,” according to the researchers. These protective effects were seen in healthy cells and in cells with CFTR mutations.

“Our results are positive in the healthy cell model as well as in the model using cells with a [CFTR] gene mutation, which is very good news!” Köhler said. “The emergence of bacteria that are increasingly resistant to antibiotics is becoming a global scourge. It is therefore urgent to strengthen our therapeutic arsenal with other tools, including bacteriophages.”

In additional experiments, the team also demonstrated that the bacteriophage cocktail and antibiotic combination did not have inflammation-inducing effects on lung cells.

Overall, “combination of phage and ciprofloxacin efficiently protects wild type and cftr-epithelial cells from infection by P. aeruginosa and emergence of phage resistant mutants without inducing an inflammatory response. Hence, phage-antibiotic combination should be a safe and promising anti-Pseudomonas therapy,” the team concluded.